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Research
Differential DNA methylation in discrete developmental stages of the parasitic nematode Trichinella spiralis
Fei Gao1,2, Xiaolei Liu1, Xiu-Ping Wu1, Xue-Lin Wang1, Desheng Gong2, Hanlin Lu2, Yudong Xia2, Yanxia Song1, Junwen Wang2, Jing Du1, Siyang Liu2, Xu Han2, Yizhi Tang1, Huanming Yang2, Qi Jin1*, Xiuqing Zhang2* and Mingyuan Liu1*
Author Affiliations
1 Key Lab for Zoonosis Research, Ministry of Education, Institute of Zoonosis, Jilin University; Zoonosis Research Centre of State Key Lab for Molecular Virology and Genetic Engineering, Chinese Academy of Medical Sciences, 5333 Xi An Road, Changchun, 130062, China
2 Science and Technology Department, BGI-Shenzhen, Beishan Industrial Zone, Yantian District, Shenzhen 518083, China
For all author emails, please log on.
Genome Biology 2012, 13:R100 doi:10.1186/gb-2012-13-10-r100
The electronic version of this article is the complete one and can be found online at: http://genomebiology.com/2012/13/10/R100
Received:12 April 2012
Revisions received:10 September 2012
Accepted:17 October 2012
Published:17 October 2012
© 2012 Gao et al.; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background
DNA methylation plays an essential role in regulating gene expression under a variety of conditions and it has therefore been hypothesized to underlie the transitions between life cycle stages in parasitic nematodes. So far, however, 5'-cytosine methylation has not been detected during any developmental stage of the nematode Caenorhabditis elegans. Given the new availability of high-resolution methylation detection methods, an investigation of life cycle methylation in a parasitic nematode can now be carried out.
Results
Here, using MethylC-seq, we present the first study to confirm the existence of DNA methylation in the parasitic nematode Trichinella spiralis, and we characterize the methylomes of the three life-cycle stages of this food-borne infectious human pathogen. We observe a drastic increase in DNA methylation during the transition from the new born to mature stage, and we further identify parasitism-related genes that show changes in DNA methylation status between life cycle stages.
Conclusions
Our data contribute to the understanding of the developmental changes that occur in an important human parasite, and raises the possibility that targeting DNA methylation processes may be a useful strategy in developing therapeutics to impede infection. In addition, our conclusion that DNA methylation is a mechanism for life cycle transition in T. spiralis prompts the question of whether this may also be the case in any other metazoans. Finally, our work constitutes the first report, to our knowledge, of DNA methylation in a nematode, prompting a re-evaluation of phyla in which this epigenetic mark was thought to be absent.
Background
Developmental regulation of gene expression plays a crucial role in the transitions between significantly differentiated life-history stages, such as is the case in parasitic nematodes; however, the underlying mechanisms of this gene regulation are poorly understood. Although DNA methylation has been established in other organisms as an important method for altering chromatin structure and regulating the expression of genes, its contribution to nematode development has not been adequately assessed given that so far no 5' cytosine methylation has been identified in any stage of Caenorhabditis elegans [1]. Most vertebrate cell types have approximately 60 to 90% of the CpG dinucleotides modified to 5-methylcytosine (5mC) [2], whereas invertebrate genomes vary extensively in the extent of DNA methylation, and some genomes have undetectable levels of methylation [3]. Recently, technological progress has enabled high-resolution detection of 5mC, opening the way for more detailed examination of the role of DNA methylation in a greater variety of eukaryotic genomes [4].
Parasitic nematodes are a good example of the biological importance of developmental regulation of genes, including the principal agent of human trichinellosis, Trichinella spiralis. This food-borne agent infects a wide variety of vertebrate hosts through their ingestion of meat containing encysted muscle larvae (ML). ML are released by the host's gastric juices, after which they grow substantially and mature into sexually active adults (Ad) in the host's intestines. New-born larvae (NBL) are released from mature females and then disseminate through the bloodstream, invade skeletal muscles, and encyst in a collagen capsule to form a new generation of ML [5]. The host muscle cells proliferate as they are transformed into 'nurse cells' for the parasite [6]. The major clinical symptoms of trichinellosis (myopathy) derive from inflammation directed against the encysted ML. Thus, successful nematode development entails a series of physically and functionally distinct stages that require accurate recognition of specific biological cues. In this way, the life cycle of parasitic nemotodes is distinct from that of free-living nematodes, such as Caenorhabditis elegans, that live in a more homogeneous environment.
Stage-specific expression has been observed for genes in Trichinella spp. [7]. Differential expression was especially obvious for genes encoding the excretory-secretory (E-S) proteins released from the larvae. For example, a gene encoding a 43-kDa glycoprotein is expressed in precapsular and postcapsular muscle larvae, but not in adults [8]. E-S proteins may therefore contribute to capsule formation [9]. Stage-specific gene expression may also assist parasitic evasion or forestalling of immune reactions that would inhibit continued transmission. Thus, how stage-specific transcriptional regulation is accomplished in these organisms might prove useful for understanding and preventing infection.
Recent innovations in high-throughput sequencing have enabled researchers to infer methylation patterns at single-base resolution [10]. MethylC-seq enables methylation analyses with unprecedented precision, and the recently released draft genome sequence of T. spiralis [11] provided us the means to evaluate the methylome of its three distinct stages. Our work here describes the first comprehensive study that confirms the existence of DNA methylation in T. spiralis and characterizes the differential methylomes of the organism during these life stages. We further identified sets of genes whose DNA methylation status varied between the developmental stages. Our data shed light on the developmental biology of an important food-borne zoonosis, and our approach opens the way for future assessment of methylation as a mechanism of developmental regulation in this and other metazoans that undergo similar life cycle transitions.
Results
The presence of DNA methylation in the T. spiralis genome
To understand whether T. spiralis possesses the ability to methylate DNA, we conducted reciprocal Blast searches to identify genes that might be related to known DNA (cytosine-5)-methyltransferases. Our data revealed the existence of several relevant orthologous genes annotated in the draft T. spiralis genome [11] (Table S1 in Additional data file 1). We found that EFV54759.1 and EFV58204.1 were homologous to dnmt3 de novo methyltransferases and to the maintenance methyltransferase dnmt1, respectively, in species that are known to have DNA methylation, such as human and mouse. Of additional interest, T. spiralis appeared to be the only nematode, compared to 11 other nemotodes, that possessed de novo methylation machinery (dnmt3). The other nematodes only contained orthologs to maintenance methyltransferase dnmt1, including Caenorhabditis elegans. We also identified an ortholog to dnmt2 (EFV60295.1), but it was more similar to a previously identified tRNA methylase [12-14], which suggests the potential existence of RNA methylation in T. spiralis. We used the sequences of these dnmt-like proteins to reconstruct a phylogenetic tree (Figure 1). This analysis indicated that T. spiralis dnmt3 was not a close relative of orthologs in its host mammals, suggesting that T. spiralis dnmt3 did not originate from its host through horizontal gene transfer.
Additional file 1. Supplemental Tables S1 to S6 and corresponding captions.
Format: XLSX Size: 4.5MB Download file
Figure 1. Phylogenetic tree of dnmt proteins. Multiple sequence alignment was performed by ClusterW, then ClusterW with the neighbor-joining method based on JTT+ G (Jones-Taylor-Thornton and Gamma Distribution) model was applied to reconstruct the phylogenetic tree. The species with best hits to T. spiralis dnmts were used as representatives that span the phylum and were analyzed in this study.
We performed PCR with reverse transcription (RT-PCR) and found that T. spiralis dnmt2 and dnmt3 genes were differentially expressed among three life stages, but that dnmt1 expression remained at about the same level (Figure S1a in Additional data file 2). Correspondingly, enzymatic data using nuclear protein extracts also showed differential catalytic activity of the T. spiralis dnmts (Figure S1b in Additional data file 2). We also carried out ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), which further confirmed the existence of DNA methylation in T. spiralis, showing that the total amount of DNA methylation in the Ad stage was significantly higher than in the NBL stage (Figure S2 in Additional data file 2) [15].
Additional file 2. Information on all primer sequences, and Supplemental Figures S1 to S6 and corresponding legends.
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This file can be viewed with: Adobe Acrobat Reader
Given these results, we assessed the genome-wide DNA methylation profiles in the three life stages of T. spiralis (Ad, ML, and NBL) using MethylC-Seq. We generated 61.65, 23.52 and 55.77 million raw reads, respectively. We aligned the reads to the T. spiralis reference sequence [16] and mapped approximately 96.36% of the reads to Ad, 91.30% to ML and 99.27% to NBL, yielding 2.91, 1.05 and 2.71 Gb of DNA sequence to Ad, ML, and NBL, respectively. The average read depth was 21.36, 10.80 and 26.21 per strand, respectively. On average, over 81.6% of each strand of the 64 Mb T. spiralis reference sequence was covered by at least one sequence read in each of the three stages. Because of the potential for the occurrence of non-conversion and thymidine-cytosine sequencing errors, we estimated the false-positive rate as the percentage of cytosines sequenced at cytosine reference positions in the Lambda genome, which are normally unmethylated (Materials and methods). We then applied the error rates for each stage (0.0060, 0.0064 and 0.0025 for Ad, ML, and NBL, respectively) to correct mC identification according to a method described by Lister et al. [10] that is based on a binomial test and false discovery rate constraints. Corrected estimates resulted in approximately 0.31 million and 0.24 million mCs in the Ad and ML genomes (comprising 1.59% and 1.22% of their sequenced cytosines, respectively). In contrast, methylation was nearly undetectable in NBL (0.002 million; 0.01%; Table S2 in Additional data file 2). We validated the results using two different methods: (1) bisulfite-PCR (BSP), cloning, and conventional sequencing by the Sanger method; and (2) methylated DNA immunoprecipitation (MeDIP) combined with quantitative PCR (QPCR). For BSP, we assessed six randomly selected genomic regions that varied in their estimated amount of methylation, and obtained strong agreement between the two experimental results (P-value < 0.05 using double t-test; Figure S3 in Additional data file 2; Table S3 in Additional data file 1) [15]. For MeDIP with QPCR, we assessed three randomly selected genomic regions and confirmed the existence of DNA methylation in all three regions (Figure S4 in Additional data file 2).
Characterization of overall methylation patterns in the three life stages
We further characterized the global patterns of DNA methylation in the genomes of the different T. spiralis stages. The highest amount of detected mCs (82.25% and 89.06%) in Ad and ML were located in CG regions, indicating a dominant role of CpG methylation in these stages. Due to the very low level of DNA methylation in NBL, the distribution of CG and non-CG methylation was very similar to the background (Figure 2a). The average methylation level of specific cytosine residues could be estimated from the fraction of methylated sequence reads at that site. Here we found that the average methylation level of specific cytosine residues was estimated from the fraction of methylated sequence reads at that site (Figure 2c).
Figure 2. DNA methylation patterns and chromosomal distribution of three life stages of T. spiralis. (a) The fraction of mCs identified in each sequence context in the three life stages in comparison with the fraction of all Cs in each sequence context in the genome. (b) Distribution of MRs identified on the two DNA strands (Watson and Crick) throughout the whole genome. The value refers to the average percentage of methylation of the MRs, as shown on the y-axis. (c) Distribution of mCs (y-axis) across the percentage methylation levels (x-axis).
Since the mCs in the T. spiralis genome are relatively sparse compared to vertebrate genomes, we identified methylation regions (MRs) of the genome using relatively dense mCs (Materials and methods). Different CG and non-CG methylation might be subject to distinct forms of genetic control; therefore, MR identification was performed independently for CG and non-CG contexts. Across the genome, we observed an increase in CG methylation as the parasites matured from the NBL to the ML stage and, to a lesser extent, in the transition from ML to Ad. In addition, CG methylation levels fluctuated drastically across the genome, indicating a mosaic methylation pattern where relatively dense methylated domains are interspersed with regions that are not methylated (Figure 2b). Such a pattern has been observed in previous studies on other invertebrates [3]. In contrast, we identified only a small number of non-CG MRs (Table S4 in Additional data file 1).
In all types of genomic elements, we saw a methylation increase from NBL to ML and from ML to Ad as well as the global pattern. We then examined patterns of methylation in distinct genomic elements, including genes, tandem repeats, and transposable elements. Genes were methylated more frequently than the genome average (Figure 3a, b). Within genes, the coding sequences were more methylated than the flanking DNA or promoter regions, while introns were the least methylated (Figure 3c, d). Notably, repeat elements, including tandem repeats and transposable elements, exhibited much higher DNA methylation than the genome average (Figure 3a, b). Previous studies have indicated that the methylation level of transposons across different phylogenetic units may vary. It has been reported that transposable elements are highly methylated in mammals, plants and zebrafish (Danio rerio), and moderately methylated in Ciona intestinalis, but are usually unmethylated in the honey bee Apis mellifera and silkworm Bombyx mori [17,18]. In T. spiralis, we observed higher methylation on transposons relative to the immediate flanking regions as well (Figure S5 in Additional file 2), which is similar to what is seen in Ciona intestinalis [17].
Figure 3. Average methylation levels of different genomic regions of the three life stages of T. spiralis. (a, b) Average density of methylation levels (a) and percentage of methylation levels (b) (y-axis; Materials and methods) at different functional regions (x-axis). (c, d) Average density of methylation levels (c) or percentage of methylation levels (d) (y-axis) of intervals around genic regions (x-axis). Two-kilobase regions upstream and downstream of each gene were divided into 100-bp (bp) intervals. Each coding sequence or intron was divided into 20 intervals (5% per interval).
The relationship between stage-dependant methylation and gene expression
We evaluated differential gene expression among the three life stages using Illumina high-throughput RNA-seq technology. Most of the raw reads (numbering 28,662,704, 26,128,346 and 28,962,820, respectively, for the Ad, ML and NBL stages) could be uniquely mapped to previously annotated genes (62.26%, 64.38%, and 64.34%). We detected 12,675, 12,683 and 12,909 annotated genes out of the total 16,379 with at least one unique read. The majority of these genes (11,636) were expressed in all three life stages, and we saw 234 Ad-stage specific, 183 ML-specific and 445 NBL-specific genes. Of note, we also detected stage-dependent expression of methyltransferases that were concordant with prior RT-PCR results (Figure S2 in Additional data file 2). Finally, among genes that were expressed in more than one stage, we identified differential expression in 1,752 pair-wise comparisons (Table S5 in Additional data file 1).
We characterized the changes in DNA methylation among the three distinct life stage methylomes and the relationship between methylation and differential gene expression. For this, we divided expressed genes with at least one sequencing read into quartiles of expression levels, and examined the expressed genes together with another category composed of genes exhibiting no expression. We found that DNA methylation levels of gene upstream regions had a negative correlation with gene expression levels, and non-expressed genes in particular had different patterns of DNA methylation as the methylation levels in their upstream regulatory regions were higher than in the coding sequences (Figure 4a, b). Based on this, it is likely that methylated promoters induce silencing in T. spiralis, akin to the widely accepted role for hypermethylation of promoters as a means of repressing gene expression in plants and mammals [19,20]. In contrast, the gene-body methylation levels in our analysis showed a bell-shaped, rather than monotonic, relationship with gene expression levels. Generally in the gene body, the non-expressed and most highly expressed genes had the lowest DNA methylation levels, whereas the mid-level expressed genes had the highest percentage of DNA methylation (Figure 4a-c). A bell-shaped relationship between gene-body methylation and expression levels has been observed previously in plants (Arabidopsis thaliana and Oryza sativa), invertebrates (Ciona intestinalis and Nematostella vectensis), and humans as well [4,21,22], indicating conservation of the role of methylation across phylogenetically diverse species.
Figure 4. Relationship between DNA methylation and expression levels of genes in Ad and ML stages of T. spiralis. (a, b) Average density (a) or percentage of methylation levels (b) within genes that were classified based on expression levels. The first class includes silent genes with no sequencing read detected, and the second to fifth classes cover expressed genes from the lowest 25% to highest 25%. In the curve graph, 2-kb regions upstream and downstream of each gene are divided into 100-bp intervals, and each gene was divided into 20 intervals (5% per interval). In the histogram graph, overall average (± standard error) methylation levels for genes are indicated.
Biological implications of stage-dependant methylation in T. spiralis
We examined the correspondence of methylation status to divergent gene expression in different stages. Due to the overall low level of genome methylation in general, we limited this analysis to MRs exhibiting high levels of methylation where we had at least 5× depth of coverage. Using this criteria, we found a total of 652 ML and 767 Ad MRs enriched for methylation in CG regions, but MRs in non-CG regions were rare. In contrast, we found no MRs in the NBL stage. As shown in Figure 5a, 389 MRs were shared between Ad and ML. These MRs were located in 486 and 551 genes in the ML and Ad stages, respectively, with the majority located in gene-body regions (Table S4 in Additional data file 1).
Figure 5. Analysis of highly enriched MRs and annotation of the genes containing MRs. (a) Venn diagram of shared and stage-specific MRs in different sequence contexts of Ad and ML stages. (b) Venn diagram of shared and disparate genes containing MRs in Ad and ML stages, expressed (red) and silent (blue) genes are separated.
We carried out a Gene Ontology analysis to functionally characterize those genes with CG MRs in Ad and ML using GOstat [23]. Enrichment of GO terms defined by a significant false discovery rate-corrected P-value (≤0.01) in the 'molecular function' category was indicated in 'DNA integration', 'DNA metabolic process' and so on. In the 'biological process' category, 'nucleic acid metabolic process' and 'endopeptidase activity' and so on were enriched. Of note, we found that many genes were shared among different molecular pathways and constituted a central focus of study in parasitic nematodes (Table S6 in Additional data file 1). Given this, we explored the potential for DNA methylation regulating genes that are related to parasitic activities. For example, the protein EFV53263.1 is encoded by a DNase II gene in the 'DNA metabolic process' category that is expressed in a stage-specific manner and important for T. spiralis parasitism [24], and we found that this gene was uniquely transcribed in the NBL stage, whereas it was not expressed and had hypermethylated promoter regions in the Ad and ML stages. Considering the globally monotonic and negative correlation between promoter methylation and gene expression, this finding strongly suggested that promoter methylation plays a role in the regulation of stage-specific expression of certain DNase II genes in T. spiralis (Figure 6a).
Figure 6. Stage-dependant DNA methylation in relation to gene repression and alternative splicing. (a) Graphical representation of differential methylation profiles in the NBL-specific DNase II gene encoding protein EFV53263.1. Ad (red line) and ML (blue line) show hypermethylation in their gene upstream regions. (b) Distribution of methylation on both sides of donor and acceptor sites in 20-nucleotide sliding windows of 200 nucleotides total length centered at the splice junctions (jxn). The exon-intron boundaries are shown in vertical dashed lines. (c) Graphical representation of methylation densities in constitutive and skipped exons, introns and 100-nucleotide 5', 3' intronic sites. The relative widths of the shaded regions correspond to methylation frequencies.
In addition to a role for promoter methylation in expression, recent studies have suggested that gene-body methylation is involved in alternative splicing regulation. Our RNA-seq results indicated that many T. spiralis genes were alternatively spliced (Figure S6 in Additional data file 2), and in relation to this, we saw a steep change in the methylation frequency across splice junctions on both sense and antisense strands in the MR genes (Figure 6b). Moreover, there were considerable methylation differences between skipped and constitutive exons or between retained and spliced introns (Figure 6c). Particularly prominent was also the 'infiltration of methylation' in intronic sequences of neighboring splice junctions (Figure 6b, c). These results are in agreement with previous studies [25-27]. Taken together, our data suggest that DNA methylation status has a relationship with the donor/acceptor sequence context around the splice junctions, indicating the potential influence of DNA methylation on alternative splicing of MR genes.
Discussion
The status of DNA methylation in nematode genomes has been unclear. Research on Caenorhabditis elegans, which reportedly lacked mC in age-synchronous senescent populations, indicated it had negligible influence [1]. With regard to Caenorhabditis elegans, our computational searches here indicate that it has dnmt1, but not dnmt2 [12] or dnmt3. As dnmt3 is essential for de novo methylation [28], the lack of dnmt3 in Caenorhabditis elegans might explain the absence of de novo methylation in this nemotode. In contrast, we identified three methyltransferase genes in the T. spiralis genome that were orthologs to known dnmts in vertebrates, including dnmt1, dnmt2 and dnmt3. Intriguingly, of 11 species of nematodes tested, T. spiralis appeared to be the only nematode possessing de novo methylation machinery (dnmt3). Moreover, we found that expression levels of dnmt2 and dnmt3 increased during development from larvae to adulthood, but that dnmt1 did not. We note that our analysis indicated dnmt2 was more similar to a tRNA methylase instead of a DNA methyltransferase [12-14], which might indicate that RNA methylation also plays a role in T. spiralis development, and is therefore worth more detailed analysis in the future.
We further carried out the first comprehensive, high-resolution analysis of methylation in T. spiralis, to assess the intriguing possibility, given the presence of de novo DNA methyltransferase orthologs, that epigenetic control might help govern the development of its distinct life history stages via temporally regulated gene expression. Methylome sequencing revealed a mosaic methylation pattern in T. spiralis, typical of other invertebrates [29,30]. DNA methylation increased drastically during maturation from NBL to ML, and adults exhibited the highest observed DNA methylation level. This finding contrasts with a trend seen in some other species where methylation patterns remain stable throughout the life cycle [29]. For instance, in the sea urchin, which also has distinct life stages, methylated and non-methylated regions in its genome retain the same overall methylation composition throughout all tested stages [31]. The relative overall constancy of methylation patterns is also a feature of vertebrate genomes. However, the extent of DNA methylation may reflect changes in both intrinsic and environmental exposure [32]. For instance, studies in humans indicated that total genomic 5-methylcytosine has been found to typically decrease during aging [33,34], in concordance with declining Dnmt1 activity with age [35]. Parasites such as T. spiralis certainly undergo more drastic lifespan changes in response to environmental cues, that is, metamorphosis critical to their survival and reproduction. Our findings here provide evidence to indicate that these DNA methylation changes might play an important role in regulating such transformations in T. spiralis.
Previous studies have indicated that methylation may be an evolutionarily ancient means of transcriptional control as it is maintained in phylogenetically diverse lineages. In both plants and vertebrates, the notion that methylation in promoters primarily represses genes by impeding transcriptional initiation has been widely accepted [19,20], whereas intermediate levels of expression have been associated with genes experiencing the greatest extent of methylation in the gene body, indicating a bell-shaped relationship [4,21,22,36]. However, in the fungus Neurospora crassa [37] and the silkworm Bombyx mori [18], transcription initiation is unaffected. Thus, DNA methylation shows remarkable diversity in its extent and function across eukaryotic evolution. Here, our results indicate that the presence of promoter methylation correlates with reduced gene expression levels. Promoter hypermethylation may regulate a portion of stage-specific genes by repressing their transcription initiation in non-expressed stages, as exemplified by a NBL-specific DNase II gene (Figure 6a). Our assessment of gene-body methylation, which had a bell-shaped relationship between methylation and gene expression, indicates there was no overt relationship between expression and methylation levels. We did, however, see evidence for a relationship between methylation within the gene-body and alternative splicing of these genes in T. spiralis, indicating these regulatory mechanisms of gene and protein activity are an area of interest for future study as well; and the presence of a tRNA methylase ortholog makes further study of RNA regulation in general in the organism of interest.
In relation to the notion that complex regulatory machinery of DNA methylation has been developed in T. spiralis with species-specific characteristics, probably in response to environmental cues, we found that many of the MR genes are enriched in pathways that are functionally important for parasitic nematodes. Such genes modulate the interaction between the parasite and its host so as to protect the parasite against host immune responses. There was also enrichment in pathways that are important to parasitic activity, including previously reported catalytically active E-S proteins. Of note, hydrolases are among the most abundant proteins secreted by parasites and facilitate host tissue invasion [38]. Also important to T. spiralis is the conversion of muscle cells to nurse cells, and DNA-binding proteins [39], which are often affected by methylation changes, are believed to interfere with host cell signaling in ways that promote this conversion. Additionally, many such proteins are encoded by large, developmentally regulated gene families and assume different isoforms, which is also relevant to our findings that MRs were primarily distributed in gene bodies rather than promoter regions and the DNA methylation status was related to the donor/acceptor sequence context around their splice junctions.
Conclusions
We describe the first comprehensive study confirming the existence of DNA methylation in three life stages of T. spiralis. Our data also provide support for DNA methylation being associated with the regulation of genes that are closely related to the parasitism of the organism. In this context, in T. spiralis and in other organisms that experience discrete and highly specialized development forms, further consideration should be given to mechanisms where DNA methylation is involved in suppression of spurious transcriptional initiation of infrequently transcribed genes, promotes transcriptional termination, or mediates alternative splicing, as has been shown for other model organism systems.
Materials and methods
Collection of T. spiralis muscle larvae, adults and new-born larvae
Infective T. spiralis ML were obtained from infected mice at 35 days post-infection by digestion of minced skeletal muscle in 1% pepsin and 1% HCl for 45 minutes at 42°C with agitation, as previously described [40]. Seventy male 6-week-old Wistar rats were then orally inoculated with a dose of 8,000 infective ML. Adult worms (Ad1) were obtained from the intestine of ten rats at 30 h post-infection. The remaining 60 rats were sacrificed at 6 days post-infection, and the adult worms (Ad6) were recovered and incubated in Iscove's Modified Dulbecco's Medium (IMDM) in 75-cm2 cell culture plates at 37°C. Newborn larvae were harvested every 6 h. All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (publication no. 85-23, revised 1996). The protocol was approved by the Ethical Committee of the Institute of Zoonosis, Jilin University, China (reference number 20080106).
Enzymatic activity analysis of Dnmts
To test the dnmt enzymatic activity of T. spiralis, 11 μg of nuclear extracts for each assay were incubated in 37°C for 2 h using a EpiQuik™ DNMT Activity/Inhibition Assay Ultra Kit (Epigentek, Farmingdale, NY, USA) according to the manufacturer's instructions.
BlastP searches and phylogenetic analysis of Dnmts
Reciprocal BlastP comparisons were first performed to identify dnmt orthologs. Significant hits were defined as those satisfying the following criteria: E-value < 10-5 and the aligned segments covering at least 30% of the sequence length of the hit. For phylogenetic analysis, multiple sequence alignment was performed by ClusterW [41]. The ClusterW with the neighbor-joining method [42] based on JTT+ G (Jones-Taylor-Thornton and Gamma Distribution) model was applied to reconstruct the phylogenetic tree.
UPLC-MS/MS analysis of global DNA methylation
UPLC-MS/MS analysis was performed according to a previously published method [43]. Genomic DNA (0.2 μg) extracted from Ad and NBL was digested with 1U DNase I, 2U Alkaline Phoaphatase, Calf Intestinal (CIP) and 0.005U snake venom phosphodiesterase I at 37°C for 24 h. A microcon centrifugal filter device with a 3,000 D cutoff membrane was used to remove protein from the digested DNA samples by centrifuging at 12,000 rpm for 60 minutes. The mobile phase, consisting of 5.0% methanol and 95% water (plus 0.1% formic acid), was used for UPLC separation of the nucleotides at a flow rate of 0.25 ml/minute. Enzymatically digested DNA samples (10 μl each) were injected for UPLC-MS/MS analysis and each run took 10 minutes. Mass spectrometry conditions were as follows: ionizationmode, ESI-positive; capillary voltage, 3,500 V; nitrogen drying gas temperature, 300°C; drying gas flow, 9 L/min; nebulizer, 40 psi. For MS/MS analysis of nucleotides, the fragmentor voltage was 90 V, collision energy was performed at 5 eV and scan time was 100 ms. Multiple-reaction monitoring (MRM) mode was used for the UPLC-MS/MS analysis by monitoring transition pairs of m/z 242.1/126.0 corresponding to 5mdC. The isotope labeled internal standard (5mdC-d3) was used to quantify genomic DNA methylation level, whose m/z was 245.4/129.0.
MethylC-seq library construction and sequencing
Prior to library construction, 5 μg of genomic DNA spiked with 25 ng unmethylated Lambda DNA (Promega, Madison, WI, USA) was fragmented using a Covarias sonication system to a mean size of approximately 200 bp. After fragmentation, libraries were constructed according to the Illumina Pair-End protocol with some modifications. Briefly, purified randomly fragmented DNA was treated with a mix of T4 DNA polymerase, Klenow fragment and T4 polynucleotide kinase to repair, blunt and phosphorylate the ends. The blunt DNA fragments were subsequently 3' adenylated using Klenow fragment (3'-5' exo-), followed by ligation to adaptors synthesized with 5'-methylcytosine instead of cytosine using T4 DNA ligase. After each step, DNA was purified using the QIAquick PCR purification kit (Qiagen, Shanghai, China). Next, a ZYMO EZ DNA Methylation-Gold Kit™ (ZYMO Research, Irvine, CA, USA) was employed to convert unmethylated cytosine into uracil, according to the manufacturer's instructions, and 220 to 250 bp converted products were size selected. Finally, PCR was carried out in a final reaction volume of 50 μl, consisting of 20 μl of size-selected fractions, 4 μl of 2.5 mM dNTP, 5 μl of 10× buffer, 0.5 μl of JumpStart™ Taq DNA Polymerase, 2 μl of PCR primers and 18.5 μl water. The thermal cycling program was 94°C for 1 minute, 10 cycles of 94°C for 10 s, 62°C for 30 s, 72°C for 30 s, and then a 5-minute incubation at 72°C, before holding the products at 12°C. The PCR products were purified using the QIAquick gel extraction kit (Qiagen). Before analysis with Illumina Hiseq2000, the purified products were analyzed by the Bioanalyser analysis system (Agilent, Santa Clara, CA, USA) and quantified by real time PCR. Raw sequencing data were processed using the Illumina base-calling pipeline (Illumina Pipeline v1.3.1). The sodium bisulfite non-conversion rate was calculated as the percentage of cytosines sequenced at cytosine reference positions in the Lambda genome.
RNA-sequencing and real-time PCR validation
Total RNA was extracted using the Invitrogen TRIzol® Reagent and then treated with RNase-free DNase I (Ambion, Guangzhou, China) for 30 minutes. The integrity of total RNA was checked using an Agilent 2100 Bioanalyser. cDNA libraries were prepared according to the manufacturer's instructions (Illumina). The poly(A)-containing mRNA molecules were purified using Oligo(dT) Beads (Illumina) from 20 μg of total RNA from each sample. Tris-HCl (10 mM) was used to elute the mRNA from the magnetic beads. To avoid priming bias when synthesizing cDNA, mRNA was fragmented before the cDNA synthesis. Fragmentation was performed using divalent cations at an elevated temperature. The cleaved mRNA fragments were converted into double-stranded cDNA using SuperScript II, RNaseH and DNA Pol I, primed by random primers. The resulting cDNA was purified using the QIAquick PCR Purification Kit (Qiagen). Then, cDNA was subjected to end repair and phosphorylation using T4 DNA polymerase, Klenow DNA polymerase and T4 Polynucleotide Kinase (PNK). Subsequent purifications were performed using the QIAquick PCR Purification Kit (Qiagen). These repaired cDNA fragments were 3'-adenylated using Klenow Exo- (Illumina) and purified using the MinElute PCR Purification Kit (Qiagen), producing cDNA fragments with a single 'A' base overhang at the 3' end for subsequent ligation to the adapters. Illumina PE adapters were ligated to the ends of these 3'-adenylated cDNA fragments and then purified using the MinElute PCR Purification Kit (Qiagen). To select a size range of templates for downstream enrichment, the products of the ligation reaction were purified on a 2% TAE- Certified Low-Range Ultra Agarose (Bio-Rad, Hercules, CA, USA). cDNA fragments (200 ± 20 bp) were excised from the gel and extracted using the QIAquick Gel Extraction Kit (Qiagen). Fifteen rounds of PCR amplification were performed to enrich the adapter-modified cDNA library using primers complementary to the ends of the adapters (PCR Primer PE 1.0 and PCR Primer PE 2.0; Illumina). The 200 ± 20 bp PCR products were purified using QIAquick Gel Extraction Kit (Qiagen), using the MinElute spin columns (Qiagen). Finally, after detection on an Agilent Technologies 2100 Bioanalyser using the Agilent DNA 1000 chip kit and quantification on a StepOne plus qPCR (ABI, Woodlands, Singapore), the cDNA library products were sequenced using the Illumina Genome Analyser. Real-time PCR validation was conducted using the Maxima® SYBR® Green qPCR Master Mix kit (Fermentas, Beijing, China), according to the manufacturer's instructions, in an ABI Prism 7500 Sequence Detection System machine (Applied Biosystems Inc., CA, USA). All real-time RT-PCR data were normalized to the NBL stage (see Additional data file 1 for primer information).
Sequence alignment of MethylC-seq
The reads generated by Illumina sequencing were aligned onto the T. spiralis reference genome [11]. The Lambda genome was also included in the reference sequence as an extra chromosome so that reads originating from the unmethylated control DNA could be aligned. Because DNA methylation has strand specificity, the plus strand and the minus strand of the T. spiralis genome were separated to form alignment target sequences. To do this, each cytosine in the genome was converted to a thymine, termed the T-genome, which represented the plus strand. Meanwhile, each guanine in genome sequences was converted to adenosine, termed the A-genome, which represented the minus strand. Additionally, the original forms of the reads were also transformed to deal with the bisulfite treatment nucleotide conversion in the alignment process. First, the observed cytosines on the forward read of each read pair were replaced in silico by thymines, and secondly, the observed guanines on the reverse read of each read pair were replaced in silico by adenosines. We then mapped the 'alignment form' reads to the 'alignment form' target sequence using SOAPaligner with default parameters [16]. Every hit of a single placement with a minimum number of mismatches and a clear strand assignment was defined as an unambiguous alignment, and each alignment was used for mC ascertainment.
Gene annotation and functional analysis
For gene annotation, the BLAST algorithm was applied to further annotate the genes defined in the available T. spiralis genome annotation because the current annotation is incomplete. All the predicted protein sequences of T. spiralis genes were aligned using BLAST with known annotated protein sequences from three databases, including SWISS-Prot, TrEMBL and InterPro. A cutoff E-value < 1e-05 was applied for annotation, and a best alignment term for each query protein sequence was selected if more than query sequence was aligned based on this cutoff E-value from BLAST.
For function analysis, GO analysis was performed based on the annotated genes by GOstat software [23]; 8,286 genes with annotation out of 16,380 genes were used as background, and 287 and 242 genes with annotation out of the 540 and 454 genes containing MRs in Ad and ML, respectively, were used as input genes. Fisher's exact test was performed and the P-values generated for each GO category were adjusted according to the Benjamini and Hochberg correction method.
Identification of methylcytosines and methylation regions and determination of methylation level
For mC identification, we transformed each aligned read and the two strands of the T. spiralis genome back to their original forms to build an alignment. In the unique part of the genome, cytosines that were covered by cytosines from reads on the same strand or guanines from those on the opposite strand (hereafter, referred to as ascertainment bases) were called as potential methylated sites. To account for the inefficient conversion of the bisulfite treatment and for sequencing errors, a correction method based on binomial tests and false discovery rate constraints [10] was applied to the data to build a high-quality methylome for each of the three stages. The probability p in the binomial distribution B(n, p) was estimated from the number of cytosine bases sequenced in reference cytosine positions in the unmethylated Lambda genome (referred to as the error rate: non-conversion plus sequencing error frequency). The bisulfite conversion rates for all samples were over 99%, and the error rates were as follows: Ad, 0.0060; ML, 0.0064; NBL, 0.0025. Then, the mCs from the binomial distribution analysis were selected for determination of MRs, which were defined as being under a threshold of more than five continuous mCs (either CpG or non-CpGs in at least one strand) and having a distance between adjacent mCs of less than the median value (18 bp) of all distance values. Stage-specific MRs were defined as containing more than five continuous mCs and no overlap between two samples.
Percentage methylation was computed as the fraction of reads number of 'C' in the total reads number of 'C' and 'T' for each covered CpG site, and herein average percentage methylation of all cytosine residues for any genomic region covered was computed as the fraction of reads number of 'C' in the total reads number of 'C' and 'T' for each genomic region. Density methylation (mC/C) was determined as the number of mCs divided by total number of C sites in any genomic region.
Validation of DNA methylation
Two strategies were applied to validate the methylation status of randomly selected genomic regions. BSP combined with cloning Sanger sequencing. BSP primers were designed by the online MethPrimer software (Additional data file 1). Genomic DNA (500 ng) was converted using the ZYMO EZ DNA Methylation-Gold Kit™ according to the manufacturer's instructions. PCR amplification was carried out with a thermal cycling program of 94°C for 1 minute, 30 cycles of 94°C for 10 s, 58°C for 30 s, 72°C for 30 s, and then 5 minutes at 72°C. The products were then held at 12°C. Following amplification, PCR products were gel selected and purified using the QIAquick gel extraction kit (Qiagen), and the purified PCR products were subcloned. The colonies from each region were sequenced on a 3730 genetic analyxer (Applied Biosystems) to determine the methylated cytosine levels.
MeDIP followed by QPCR (see Additional data file 1 for primer information) was performed on 300 to 400 ng of original genomic DNA for each sample, which was randomly sheared to an average length of 200 to 500 bp by sonication. A MeDIP assay was then performed using the Magnetic Methylated DNA Immunoprecipitation Kit (Diagenode, Liege, Belgium) according to the instructions. The immunoprecipitated products and 10% amount of original input DNA were purified with ZYMO DNA Clean & Concentrator-5 kit (ZYMO) in parallel. The purified DNA was analyzed by QPCR on an ABI StepOne Plus Real Time PCR System (Applied Biosystems Inc.) using Eva Green (Biotium, Shanghai, China). The relative methylation levels of particular genomic loci among samples were compared by measuring the amount of immunoprecipitated DNA after normalization to the 10% of input DNA: %(MeDNA-IP/Total input) = 2^[Ct(10%input) - 3.32 - Ct(MeDNA-IP)] × 100%.
Data availability
Sequence data and processed data are available under the Gene Expression Omnibus accession GSE39328. UPLC-MS/MS and BS-PCR data have been deposited in GigaDB, the GigaScience database, with the unique identifier doi:10.5524/100043 [15].
Abbreviations
Ad: adult; BSP: bisulfite-PCR; E-S: excretory-secretory; Gb: gigabase; GO: Gene Ontology; mC: methylcytosine; MeDIP: methylated DNA immunoprecipitation; ML: muscle larvae; MR: methylation region; NBL: new-born larvae; QPCR: quantitative PCR; UPLC-MS/MS: ultra-performance liquid chromatography-tandem mass spectrometry.
Authors' contributions
FG, ML, QJ, XZ and HY conceived the project. FG designed experiments and interpreted data. XL, XPW, XLW, YS, JW, JD, XH, and YT performed experiments. FG, DG, HL, YX and SL conducted bioinformatic analysis. FG prepared and wrote the manuscript. All authors have read and approved the manuscript for publication.
Acknowledgements
The authors would like to thank Laurie Goodman and Benjamin M. Rosenthal for help in editing the manuscript for grammar and writing style, thank Hailin Wang for help in UPLC/MS analysis, and thank Bo Li for help in phylogenetic analysis. This study was supported by the Ministry of Science and Technology of China (MOST: 2008ZX10004-11 and 2011AA10A200) and by the National Natural Science Foundation of China (NSFC: 30825033, 31030064, 30972177, 30950110328, 81070311 and 31072124).
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wejqcsuq5wvnvos33hv6yriyg2qqo5dd
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An unofficial blog that watches Google's attempts to move your operating system online.
Send your tips to gostips@gmail.com.
December 8, 2012
Funny Google Flights Messages
Google Flights shows some custom messages after you select your favorite flights. Depending on your destination, Google shows messages like "London, baby!", "Ahh, Paris... bon voyage!", "Have a great time in the Eternal City!" (Rome), "Have a great time in the Emerald Isle!" (Dublin), "Enjoy your trip to the Windy City!" (Chicago), "Have a great time in Music City, USA!" (Nashville), "Have a great time in Baltimore, hon!".
For most destinations, Google shows generic messages like "Hope you have fun in Frankfurt!".
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login ask-a-question questions unanswered tags faq
It's been a while that I've gone it alone in researching investment options, but I'm ready to start driving on my own rather than pay hefty fees to an advisor. I'd love to hear from folks where they go to be informed and research investment options on the web. I'm thinking forums, tools (asset allocation in particular), and research services such as Morningstar. Particularly interested in funds.
I used to read Barrons, Worth, and even Money and Kiplingers of the world quite a bit but they may not fit me anymore. Worth are geared now to very high net worth folks, Money/Kiplingers of the world are almost past-quarter performance chasers. Only Barrons seem worthwhile to me these days.
BTW, I am old school and believe mostly in value investing, Graham and Dodd style.
asked Aug 17 '11 at 19:46
wlai
1
As a former high paid advisor (average acct: >$1 million) I think two worthy resources are the Value Line Investment Survey http://www.valueline.com/ for stocks but they're $550 per year (1800 stocks). Their mutual fund reports are $116 per year online (12,000 mutual funds). Your local library may have a subscription to either of those and if there's a university nearby with a business school they probably have one as well. If that's too much to spend you can read the SEC filings for funds and companies online at http://www.sec.gov/edgar.shtml
Also its helpful to realize that CNBC is more like SportsCenter than anything that might convey actionable or useful information and doesn't really help you at all for anything except practicing blocking out distractions and focusing.
link
answered Sep 13 '11 at 16:10
pdebruic
1
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Shops 4 27a is on 4/27a, J; is near 27/27b, J; is near 28/27b, J; is near 29/27b, J; is near 30/27b, J; is near 34/27b, J; is near 31/27b, J; is near 33/27b, J; Shops 4 27a is geographically located at latitude(3.2107 degrees) 3° 12' 38" North of the Equator and longitude (101.7323 degrees) 101° 43' 56" East of the Prime Meridian on the Map of Kuala Lumpur.
The locations related to Shops 4 27a are represented by the line of sight between two points and may not be nearest by road. For example, Shops 4 27a is located 250 metres from SJK Desa Setapak. Shops 4 27a is located 327 metres from Mosque Wangsa Maju B. Shops 4 27a is located 331 metres from SJK Wangsa Maju Seksyen 2. Shops 4 27a is located 373 metres from Park Wangsa Maju 27b. Shops 4 27a is located 395 metres from Madrasah Taman Bunga Raya.
Ujana Harmonis Resort 2.9km, Makeshift Resort Kg Kemensah 5.1km, Sucasa Corp Apartment 5.5km, are places to stay (hotel, service apartment, inn) located near Shops 4 27a.
Shops Jalan 27f 0.4km, Shops Taman Bunga Raya 0.4km, Shops Wangsa Maju Jalan 2 27b 0.5km, are places to shop (shopping mall, shop houses) located near Shops 4 27a.
Zoo Negara 3km, P Ramlee Memorial 3.2km, Shell Taman Greenwood 4.1km, are places of interest (attraction) located near Shops 4 27a.
SJK Desa Setapak 0.2km, SJK Wangsa Maju Seksyen 2 0.3km, SMK Wangsa Maju 0.4km, are places of learning (school, college, university) located near Shops 4 27a.
Park Wangsa Maju 27b 0.4km, Field L Mahsuri 1 0.5km, Park Jalan Masria 5 0.6km, are parks, playgrounds, open fields or commons located near Shops 4 27a.
Shops 4 27a
SJK Desa Setapak
Mosque Wangsa Maju B
SJK Wangsa Maju Seksyen 2
Park Wangsa Maju 27b
Madrasah Taman Bunga Raya
SMK Wangsa Maju
Shops Jalan 27f
Shops Taman Bunga Raya
Menara Alpha
Shops Wangsa Maju Jalan 2 27b
Shops Wangsa Maju 4a 27a
Field L Mahsuri 1
Putra Wangsa Maju
Hall Taman Bunga Raya
Park Jalan Masria 5
Caltex Jalan Genting Klang
Park Wangsa Maju 6 27b
Univ Tunku Abdul Rahman
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Shops 4 27a
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Zimbabwe pushes for open education despite oppression
Image credits: photo
(5 votes)
Zimbabwe (formerly Rhodesia) is a landlocked country located in Southern Africa. For many years, it was regarded as the breadbasket of Africa. But since Zimbabwe gained independence from Britain in 1980, Robert Mugabe has been the leader, and the fate of the country has largely been tied to him and his policies.
Starting in the mid-1990s, Mugabe began to usher a land redistribution policy where white-owned farms were seized to benefit landless black Zimbabweans, and the economy soon plummeted. Hyperinflation and massive unemployment, food and fuel shortages ensued. Millions were left destitute. Freedom of speech and freedom of the press were suppressed. Thousands emigrated. Some of my Zimbabwean friends and former neighbors in the United Kingdom and New York City described harrowing tales of government-led oppression and violence where they were lucky to escape with their lives and the clothing on their backs. These were nurses, teachers, administration personnel, academics, and accountants—not farmers.
Today, organizations such as the Computer Society for Zimbabwe and Practical Action in Harare, and projects, like the Ubuntu Zimbabwe Team, are helping to create a fledgling open source movement in Zimbabwe despite state-sponsored oppression, economic collapse, and a crackdown on the dissemination of news.
With a population of 12.5 million, the country has the highest literacy level (85-90%) in Africa. Unsurprisingly, however, printed newspaper readership has been declining for years. This is partly due to the cost of buying a newspaper and partly due to many dominant newspapers—as well as radio and television stations—being state-controlled and misreporting the accurate state of the country.
Human rights and democracy groups have declared the need for a more open society through an independent media, but Zimbabweans are increasingly turning to the Internet and mobile phone usage as alternative sources for information and communication, as they are relatively free of government restrictions. As a result, Zimbabweans are turning to the Internet to use:
• open source materials, such as Ubuntu, as a means to organize and disseminate news and knowledge
• open content as a means to organize and marshal support for human rights and democracy.
The organization, Women (and men) of Zimbabwe Arise (WOZA) is one example of a civic movement using the Internet to inform others about their political activities and human rights.
Other organizations, like the Zimbabwe Advertising Research Foundation (ZARF) and Zimbabwe All Media Products and Services Survey (ZAMPS), conduct studies on Internet usage every quarter. Between 2005 and 2008, Internet usage increased over 165% with the country having one of the highest rates of usage in Africa. During the last quarter of 2011, Internet usage grew 3%, from 31% to 34%. It is clear that Internet access is expanding tremendously in Zimbabwe, but there is still a vast digital divide between urban and rural areas.
Most people use the Internet at cybercafes or at work in the capital of Harare or in the city of Bulawayo where electricity is still rationed but where an electrical and telephone infrastructure exists because a dial-up connection rather than broadband is the norm. In rural, isolated areas or in poor townships where most Zimbabweans live, few, if any, residents can afford Internet access or have the infrastructure to access it. With unemployment at 94%, Internet usage is restricted to those with a job. And because most Zimbabweans who are employed make around $250 USD per month, few can afford the monthly Internet service plan of $50 USD per month, the modem, computer, or other fees.
Few also can afford the $25 USD monthly fee for a 3G mobile phone connection. Only the wealthy or people with access to money (i.e. expatriates often financially support family members who remain in Zimbabwe) can afford to own their home computer, modem, and pay the monthly Internet service plans. And yet, Internet access was close to 17% in 2011, up from 0.3% in 2000.
Interestingly, artists and musicians are posting messages on Ubuntu to attract a following and have their messages heard; they're pushing the open source movement and open content in Zimbabwe. However, Zimbabwe's open source movement has been severly hampered by a lack of government interest, funding, and provisions (such as electrical rationing, even in urban areas). Additionally, the digital divide has been further worsened from a lack of skilled persons and professionals—due to immigration and forced migration to place like South Africa, the United Kingdom, Australia, and other countries as the result of the political and economic crisis.
In 2008 and 2009, Zimbabwe was ranked the third worst country in the world for its national information communication technology (ICT) status by the WorldEconomic Forum. Yet, before the country's political and economic turmoil, hyperinflation, and record unemployment, Zimbabwe's education system was one of the best in Africa.
Despite these obstacles, some Zimbabweans are working to increase open access in higher, secondary, and primary education. As the United Nations states, "despite an appreciation of the Open Access (OA) concept by most Zimbabwean institutions of higher learning, there is a paucity of OA repositories in Zimbabwe on the web."
At the university level, open access, such as at the University of Zimbabwe and 13 other higher institutions, means free links to peer-reviewed literature and allowing users to read, download, copy, print, or search them. The aim is to increase knowledge and information through open access. At the secondary and primary levels, organizations, such as World Links Zimbabwe, have been distributing computers and working to promote access to open source software in schools with the Open Society Initiative for Southern Africa, OSISA.
This year, for instance, Bindura University of Science Education Library will be promoting open access from October 22nd through the 28th. A national eLearning programme is being considered for the 8,000 primary and secondary schools in Zimbabwe in an effort to overhaul the state of education (98% of schools were closed in 2009). Until recently, UNICEF has been paying the school fees for over 400,000 students (primary school only). In 2010, school attendance bounced back, but many still lack the money to pay for school.
Some Zimbabweans participating in the country's open source movement recognize that the way to overhaul possibly the world's worst education system and advance the arts and sciences is through promoting open source practices and principles in the schools, and minds of the children. And these open source supporters have been networking online.
Children truly are the future for Zimbabwe. Organizations such as the Open Society Initiative for Southern Africa (OSISA) are working to improve access to alternative education for out-of-school children and youth in Zimbabwe. And school grant programmes, like the Basic Assistance Module (BEAM), allow disadvantaged children to stay in school or return to school when their education is at risk due to a family's reluctance to spend limited resources on their daughter, for instance. Parents in this situation often chose to send a son to school rather than a daughter.
In Zimbabwe, a poor child needs $525 USD to pay for school fees (for primary school: grades 1st through 7th). Many families simply cannot afford this cost; civil servants barely earn $100 USD per month, and the majority of the populace is unemployed.
Teachers, especially in rural areas, witness first hand the lack of educational opportunity afforded to so many children, and yet their own physical security while trying to promote open source practices needs to be considered and addressed as well. These teachers are often fearful of encouraging free thinking and of political intimidation and violence against them—especially during elections, which are expected in 2012 or 2013.
The protection of teachers is critical to democracy and also to national development in Zimbabwe. As one Zimbabwean stated:
A major goal of any education system must be the encouragement of the learner to think and explore.
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A bold experiment this August: One new Linux distribution every day
Image credits: Kirill_M
(7 votes)
In open source software communities, few events are as exciting as the release of a new operating system. Community members may wait for months—even years—as fresh versions of their favorite Linux distributions are collectively and meticulously prepared, debugged, and packaged for the world.
Next month, Todd Robinson will release a Linux-based desktop operating system in a single day. Thirty-one times in a row.
Robinson, co-founder of Webpath Technologies and systems development engineer at On-Disk.com, is about to launch a project that he hopes will powerfully demonstrate the value and versatility of open source software. Dubbed "31 Flavors of Fun"—a nod to the retired Baskin Robbins slogan—the effort will involve assembling a different operating system every day during the month of August.
Robinson is no stranger to the art of customizing Linux distributions. For On-Disk, he produces, remixes, and personalizes Linux variants on numerous media—products he sells to home users, educators, and IT professionals alike. (Robinson assures us that operations at On-Disk will continue uninterrupted during the "31 Flavors" experiment.)
Expect the first release on Wednesday. And when the project concludes, Robinson will report on his experience in a presentation entitled "The Road to 31 Flavors" at this year's Ohio LinuxFest.
As Robinson prepares to launch his ambitious initiative, he graciously answered some of our questions about his goals, his concerns, and his love of open source.
How did you develop the idea for this project?
Initially the idea came in response to comments from many On-Disk.com customers and to negative articles floating around concerning Linux not having a "standard" desktop since Gnome 2. For me, it's the flip side of the coin that has always made things interesting: the flexibility of almost endless combinations of applications and desktop setups that can be used to accomplish incredible things. Over the years I've had many different desktop setups designed around the specific tasks I needed to perform. In fact, at the moment, it would be impossible for me to perform my daily functions using any existing proprietary solutions. Even the desktop I'll use to create the distributions in August will be different from what I'm using now. When the idea first came to me it was, "I should create a bunch of desktop releases to show off the variety of Linux desktop options," quickly followed by, "but that sounds like way too much work," and finally, "but I can't think of a better way to actually demonstrate the variety in Linux and advantages of open source development at the same time." And that last one got me hooked.
You see, at all the Linux and open source events we (my wife and I) attend, people boast about how superior open source development is, and even give personal reasons for their opinions. On the other side of the fence there are those who feel differently, and who also argue to present their case. But both sides, logical in their own reasoning, lack any sort of "real world" experiment as proof of concept.
I was going to try 30 days of releases, but August has 31 days and the Baskin Robins slogan, "31 Flavors of Fun", popped into my mind. I decided to go with it. Besides, I think I'll have an easier time of it if I remember that it's supposed to be fun.
What benefits of open source software and the open source development model is the project designed to highlight and demonstrate?
When all is said and done, and I run the numbers, I suspect it'll show developing under open source costs less and requires less manpower. But a huge benefit, which I think is often overlooked, is the much higher success rate. Lets face it: a lot of development attempts outright fail. By freely sharing our knowledge we're not dependent upon just what we ourselves, or a small development team, can come up with. Through online forums, blogs, and source listings everywhere, anything we need to know is literally at our fingertips. Open source is more about shared knowledge than just the source code itself. But it's precisely because the code is open and available to all that we can ask others about it, get opinions on the best way(s) to proceed, and quite often get free assistance along the way. Yes, I am making these 31 releases myself, but just like any other developer using open source, I do so standing on the giant shoulders of the open source community. If nothing else, I'd like to do my part to help stomp out the myth that open source solutions are unreliable. What better sense of security can a company have than knowing your techs and admins have the resources and expertise of the entire open source community to call upon should problems arise?
On a personal level, I'd like to help draw in as many developers as I can to participate in open source development. Only through openly sharing our knowledge and expertise does mankind have any chance of solving the problems we have created.
Producing an entire operating system in a single day will certainly be a challenge. What hurdles to success do you anticipate?
Time. Time is the biggest hurdle. An average ISO build can take anywhere from a 1/2 hour to three hours depending on the approach. It's when something in the build fails and it needs to be debugged and re-built that will seriously eat up the clock. Just like any system administrator or software developer out there with a deadline approaching, I simply won't have the luxury of time to redo something so I need to make sure every build has the best possible chance of success. And obviously there is only so much testing I can get done before needing to commit to the build, so I'm sure some releases are going to be a lot more functional than others.
The most challenging of the proposed releases will be for the Raspberry Pi. Chances are good that the release for it may not come until after August. It's going to take a lot of trial and error to come up with something truly usable while only having a maximum of 233MB of RAM to work with. My plan is to start working on it throughout August, and if it gets done during the month and can be included as one of the 31 flavors then that's great. If not, that'll be OK as well. Even if it's released during August, I'll most likely do some more work on it later because it's a custom release I committed to some time ago that we're planning to maintain.
What do you hope will happen to your operating systems after their initial releases?
I don't really have any hope for them one way or the other. It would be great if they can remain available on the mirrors for a few years to allow people to experience some of the variety that Gnu/Linux has to offer. I'm sure that at least a couple will be maintained and made available through On-Disk.com, like I do with Unite!, and through occasional limited releases.
How can others get involved in the project?
The biggest way is just to spread the word and tune in as we move forward. The more people watching as I do the impossible using open source solutions, the louder the message will be. And of course try out some of the Releases as they become available. Have fun with it. Think of it as a month long Linux/open source fest. I'm still taking requests, and the feedback has been very helpful. So, please keep them coming.
There is a private contact form or public forum for requests. The following have been set up for those who wish to follow along:
Sponsors are needed so that publicly available downloads of the 31 releases can be offered. As much as 60GB of server space may be required, so please contact me if you are able to help. Your sponsorship will be public and mentioned at all talks and publications in regards to this project.
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It's easy! Just pick the product you like and click-through to buy it from trusted partners of Quotations Book. We hope you like these personalized gifts as much as we do.
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It's easy! Just pick the product you like and click-through to buy it from trusted partners of Quotations Book. We hope you like these personalized gifts as much as we do.
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I know well the coequal role of the Congress in our constitutional process. I love the House of Representatives. I revere the traditions of the Senate despite my too-short internship in that great body. As President, within the limits of basic principles, my motto toward the Congress is communication, conciliation, compromise, and cooperation. Ford, Gerald R.
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The one extra degree makes the difference. This simple analogy reflects the ultimate definition of excellence. Because it's the one extra degree of effort, in business and life, that can separate the good from the great. This powerful book by S.L. Parker and Mac Anderson gives great examples, great quotes and great stories to illustrate the 212° concept. A warning - once you read it, it will be hard to forget. Your company will have a target for everything you do ... 212°
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Doc: “There’s things we have to do better”
RedsArmyAdmin December 17, 2009 Uncategorized 1 Comment
Doc made his weekly stop on the Dennis & Callahan show. Among the highlights… why the Celtics keep getting blasted on the offensive boards:
We have to guard the ball better in front of us. The reason we’ve given
up so many offensive rebounds is partly due to the dribble penetration
and that’s something that has hurt us, especially against quicker
teams. Atlanta beat us off the dribble. Phoenix beat us off the dribble. Orlando
beat us off the dribble and it led to points, it led to 3’s and it led
to the offensive rebounds. We have to control these things.
When you get beat off the dribble, a big has to step up and help… which means the guy who just got toasted has to bust-ass back to recover. How often is that happening? The biggest culprit getting beat off the dribble has to be Ray Allen, followed by Rajon Rondo when he gambles or takes a play off. He's doing less of that right now… but he's still doing it.
Personally… I don't like the thought of playing off a guy who can beat you off the dribble. I say you get up in his jock and make him make a mistake. Set up shop about 2 inches from him and let him try to blow past you. That takes a lot of effort… but against the Atlantas and Orlandos of the world, it's that or watch them drill 3's all night.
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Your Morning Dump… Where Doc will watch the minutes tonight
John - Red's Army February 11, 2013 Celtics News, The Morning Dump 16 Comments
Every morning, we compile the links of the day and dump them here… highlighting the big storyline. Because there’s nothing quite as satisfying as a good morning dump.
If Celtics coach Doc Rivers elected to give Pierce and Garnett a night off when the team travels to Charlotte for the tail end of a back-to-back on Monday, he could be forgiven (heck, with no national TV broadcast, the league might not even make the team pay for it). As Boston prepped to board its midnight flight, Rivers was noncommittal about how he’ll proceed.
“I’ll let you know after the game [Monday],” Rivers said. “I mean Paul played 54 minutes, and he’s the guy that I’m most concerned with to be honest. And they just played so hard. … If we have to rest guys, play them shorter minutes tomorrow — the only way I can do it is by my eyes. You’ll never know how guys feel until [Monday].”
ESPN Boston: Eyes on the clock
WWGPD?
(What Would Gregg Popovich Do?)
I’m willing to take my chances against the Bobcats with Paul Pierce and Kevin Garnett taking a break. Go ahead and start Jeff Green and Chris Wilcox, leave Pierce and KG on the bench… or in a box that reads “break glass in case of emergency or foul trouble.”
I’d say just leave them home, but that would give the C’s just 11 guys, and one of them would be Fab Melo. So Pierce and KG have to make the trip. But I wouldn’t play them unless they absolutely had to.
Knowing Doc, though, they’ll probably both play, but he’ll pull them after just a few minutes. It’ll probably be more of a modified pre-season type of thing. Personally, I’m not even too concerned about tonight’s result. Last night was enough of a win to get me through to Wednesday’s Bulls game.
The rest of the links:
CSNNE: Losses have turned C’s into winners | Celtics-Nuggets by the numbers | Garnett saves best for last in 3 OT win | Pierce, KG defy age | Pierce makes mark in win | ESPN Boston: All part of the experience | Captain clutch works overtime | WEEI: Pierce: This is the type of win that can give us confidence | Terry: My gut tells me keep shooting | Kris Joseph traded by Red Claws | Pierce leads C’s to 7th win | These Celtics have reinvented themselves, and their potential is intriguing | Extra rest works out well | Greatest of Threes | Herald: Celtics outlast Nuggets | Terry continues to provide veteran touch | Celtics enjoy snow day | Pierce fills stat sheet in Rondo’s absence | MWDN: After turbulence, Jet back on course | An endless night to remember at the Garden
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Breath of Fire III
From StrategyWiki, the video game walkthrough and strategy guide wiki
Jump to: navigation, search
Breath of Fire III
Developer(s) Capcom
Publisher(s) Capcom
Release date(s)
PlayStation
PlayStation Portable
Genre(s) RPG
System(s) PlayStation, PlayStation Portable
Mode(s) Single player
Rating(s)
PlayStation
ELSPA: Ages 3+
ESRB: Teen
OFLC: Parental Guidance
PlayStation Portable
CERO: All ages
OFLC: Parental Guidance
PEGI: Ages 7+
Preceded by Breath of Fire II
Followed by Breath of Fire IV
Series Breath of Fire
Neoseeker Related Pages
Breath of Fire III (Japanese: ブレスオブファイアIII "Buresu obu Faia Surī"?) is an RPG developed and published by Capcom for the PlayStation game console. Originally released in Japan in 1997, the game was later released for North America and the PAL region in 1998. It is the third video game in the Breath of Fire series, and the first to feature three-dimensional environments and effects, as well as several new gameplay elements including an expanded combat system, the ability to learn enemy skills, and environment interaction. In 2005, Breath of Fire III was ported to the PlayStation Portable handheld system in Japan, with an English version released exclusively in Europe in 2006.
Set in a fantasy world, the story concerns Ryu, a young boy with the mysterious ability to transform into powerful dragons who must discover the truth behind his origins, as well as locate his lost friends and surrogate family, Rei and Teepo. The game's plot is presented in two parts: half concerning Ryu as a child, and the other as an adult. He is accompanied by a number of supporting characters who aid him on a journey that leads them across the world and eventually confront a mad goddess. Though modestly successful, the game is described as a thoroughly traditional yet classic role-playing game with a unique Jazz-inspired soundtrack.
Table of Contents
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Thread Tools Search this Thread
Posts: 4,022 | Thanked: 1,611 times | Joined on Jul 2007 @ nd usa
#11
Happy birthday to Nokia tablet, happy birthday to Nokia tablet........hope many more happy birthdays to come.
Cant help but noticing the coincidence, today:
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Join Date: Aug 2007
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Default Re: N800 supply and price
its dead jim...
____________________________________________
bun
Streamed 100plus TV: USA:http://www.internettablettalk.com/fo...ad.php?t=18769
Streamed 100plus TV: rest of the world, http://www.internettablettalk.com/fo...ad.php?t=19044
Last edited by bunanson; 05-23-2008 at 01:01 AM.
Posts: 631 | Thanked: 1,120 times | Joined on Sep 2005 @ Helsinki
#12
Happy birthday Maemo. The boy is growing up and being taught new skills all the time.
Posts: 701 | Thanked: 21 times | Joined on Feb 2006 @ Italy
#13
Happy birthday to all of the "internettablets" family.
Hope this will last longer in time even more than what we are now celebrating.
I have gone through all of the OS, starting from 2005 when I received my Nokia 770 during November 2005, then all of the others and now I'm waiting for the next tablet to see whether it is worth buying (I decided to remain with my N800 for the moment), it was a great user and community experience and I'm sure it will be as well in the future since everyday new users are joining and new softwares, hacks and ideas are appearing.
Long life to the internettablets (and to ITT )
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Posts: 89 | Thanked: 24 times | Joined on Jun 2006
#14
Originally Posted by Texrat
Those comments were meant in a broader sense, ie, platform.
Yeah, I know. Sorry for the negativity, but I guess I kinda feel a bit burned from being one of the early adopters. Obviously I believe in the concept enough to have put hard cash in for the effort. It's just frustrating to be able to get the latest updates for my 9 year old Pentium II GNU/Linux boxen (which I use daily) and not be able to do the same for my 770 after 1 year of ownership. (Yes, Nokia made an effort to make the hacker edition, but after reading all the reports of things broken and it not being supported, I can't say that counts as an update to the latest and greatest.) I do look forward to the next tablet though, in hopes of snatching one of the current ones for cheap, but there's always that lingering fear of software obsolescence...
Unlike a phone, it seems that tablets need constant software upgrades to keep up with the web experience, because that's what a tablet is supposed to do. For a phone, as long as it makes a phone call, I don't mind that it is lacking in other features, like a full browser.
Posts: 89 | Thanked: 24 times | Joined on Jun 2006
#15
Okay, I don't feel so abandoned... I installed the latest OS2007HE and have to say that it works quite nicely! Very fast! Since I mostly use the N770 as a browser when traveling and as a photo album, it's exactly the kind of upgrade I needed. It's a shame that it's not officially supported, thus, making it unlikely that someone like myself, who's interested in a tool, not a tinkering box, will install it.
Unfortunately the reason why I installed it in the first place is because of a bug. I'm getting "No connections available" in my rich wi-fi access point area. One speculation is that the connection list is getting overflowed. OS2007HE and OS2006 don't work but OS2005 does. Anyway, if anyone else is afflicted with this same bug, vote for bug #2082 on Bugzilla via www.maemo.org.
Kudos to Nokia for making an effort to keep us N770 users up-to-date. I'll more likely install the unsupported updates now.
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"A REALLY INTELLIGENT INTERVIEWER." -- Lance Henriksen
"QUITE SIMPLY, THE BEST HORROR-THEMED BLOG ON THE NET." -- Joe Maddrey, Nightmares in Red White & Blue
**Find The Vault of Horror on Facebook and Twitter, or download the new mobile app!**
**Check out my other blogs, Standard of the Day, Proof of a Benevolent God and Lots of Pulp!**
Tuesday, July 7, 2009
First the Blogosphere, Now Fiction! The VoH Revolution Continues
I don't usually make The Vault of Horror about me--but dammit, this is a red letter date in B-Sol history. That's because yours truly has had his very first horror short story accepted for publication in an honest-to-goodness magazine!
The mag is Midnight Echo, and its published by the Australian Horror Writers Association (AHWA). My story, "Hell Hath No Fury", will be included in Issue #3, which I believe will be out in the fall.
To say I'm excited would be the understatement of the century. This is the culmination of years of aspiration to be a published fiction writer, and many months of work on this story alone. The word "vindication" scarcely does it justice.
I want to heap loads of thanks on to Ms. Harker of Musings Across a Continuum for pointing me in the direction of Midnight Echo in the first place, and of course, to the one and only BJ-C of Day of the Woman, my amazing protege, for taking the time out to look the story over and provide some very valuable feedback.
Check out the Midnight Echo website here. I'll be letting everyone know about issue availability once I know more.
Made it, Ma! Top of the world!
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Australian Bureau of Statistics
Celebrating the International Year of Statistics 2013
ABS Home > Statistics > Newsletters by Release Date
Newsletters by Release Date
September, 1999
10/09/1999 Statistics News NSW, Sep 1999 (cat no. 1300.1.55.001)
08/09/1999 Demography News, Sep 1999 (cat no. 3106.0)
06/09/1999 Statistical Update Queensland (Newsletter), Jul 1999 (cat no. 1316.3)
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Australian Bureau of Statistics
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Image copyright: Copyright expired - public domain
This image is in the Public Domain
ID number
P01016.004
Photographer
Hamilton, George Fullard
Object type
Black & white
Place made
Ottoman Empire: Turkey, Gallipoli, Anzac Area (Gallipoli)
Date made
25 April 1915
Physical description
Black & white
Collection
Photograph
Description
Soldiers of the 1st Battalion, Australian Imperial Force, awaiting orders from the southern edge below Plugges Plateau. Colonel Dobbin is in the centre mid-ground, to the left of the branch.
Permalink: http://www.awm.gov.au/collection/P01016.004
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Research article
Fungal-associated NO is involved in the regulation of oxidative stress during rehydration in lichen symbiosis
Myriam Catalá1*, Francisco Gasulla2, Ana E Pradas del Real1, Francisco García-Breijo2,3, Jose Reig-Armiñana2 and Eva Barreno2
Author Affiliations
1 Universidad Rey Juan Carlos, Biología Celular, Dpto. Biología y Geología, (ESCET), Madrid, Spain
2 Universitat de València, Botánica & ICBIBE-Jardí Botànic, Fac. CC. Biológicas, C/Dr. Moliner 50. 46100-Burjassot. Valencia, Spain
3 U. Politécnica de Valencia. Dpto. Ecosistemas Agroforestales. Camino de Vera s/n. 46022-Valencia, Spain
For all author emails, please log on.
BMC Microbiology 2010, 10:297 doi:10.1186/1471-2180-10-297
The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1471-2180/10/297
Received:28 May 2010
Accepted:22 November 2010
Published:22 November 2010
© 2010 Catalá et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background
Reactive oxygen species (ROS) are normally produced in respiratory and photosynthetic electron chains and their production is enhanced during desiccation/rehydration. Nitric oxide (NO) is a ubiquitous and multifaceted molecule involved in cell signaling and abiotic stress. Lichens are poikilohydrous organisms that can survive continuous cycles of desiccation and rehydration. Although the production of ROS and NO was recently demonstrated during lichen rehydration, the functions of these compounds are unknown. The aim of this study was to analyze the role of NO during rehydration of the lichen Ramalina farinacea (L.) Ach., its isolated photobiont partner Trebouxia sp. and Asterochloris erici (Ahmadjian) Skaloud et Peksa (SAG 32.85 = UTEX 911).
Results
Rehydration of R. farinacea caused the release of ROS and NO evidenced by the fluorescent probes DCFH2-DA and DAN respectively. However, a minimum in lipid peroxidation (MDA) was observed 2 h post-rehydration. The inhibition of NO in lichen thalli with c-PTIO resulted in increases in both ROS production and lipid peroxidation, which now peaked at 3 h, together with decreases in chlorophyll autofluorescence and algal photobleaching upon confocal laser incidence. Trebouxia sp. photobionts generate peaks of NO-endproducts in suspension and show high rates of photobleaching and ROS production under NO inhibition which also caused a significant decrease in photosynthetic activity of A. erici axenic cultures, probably due to the higher levels of photo-oxidative stress.
Conclusions
Mycobiont derived NO has an important role in the regulation of oxidative stress and in the photo-oxidative protection of photobionts in lichen thalli. The results point to the importance of NO in the early stages of lichen rehydration.
Background
Lichens are symbiogenetic organisms composed of fungi (mycobionts) and their photosynthetic partners (photobionts). They are poikilohydrous, subject to repeated desiccation/rehydration cycles, and able to survive in extreme, frequently very dry environments, such as deserts or the arctic tundra. Reactive oxygen species (ROS) are known to be a major cause of damage during desiccation, especially in photosynthetic organisms [1]. In some species, rehydration provokes an extracellular oxidative burst (reviewed in [2]) and it has been shown that the status of the antioxidant glutathione (GSH) is correlated with the ability of lichens to tolerate desiccation [3-5]. Furthermore, there is evidence of effective communication between mycobionts and photobionts, in which one partner up-regulates the antioxidant system of the other, endowing the symbiotic association with an important adaptive advantage and evolutionary success [6]. Nonetheless, much remains to be learned about lichen metabolism of ROS during dehydration/rehydration cycles, since it has been recently reported that classical antioxidant mechanisms play a limited role in the strategies that facilitate transition of photobionts to the desiccated state [7].
Reactive oxygen species are produced in the respiratory and photosynthetic electron chains of many organisms. In photosynthetic organisms, the production of ROS is enhanced during desiccation and/or rehydration because carbon fixation is impaired, whereas chlorophyll electrons continue to be excited. ROS result from the uncontrolled donation of electrons from electron transport chains in chloroplasts and mitochondria to molecular oxygen, initiating an indiscriminate chain reaction. If antioxidant defenses are overcome by ROS production, the uncontrolled free radicals cause widespread cellular damage by provoking protein alterations, lipid peroxidation, and the formation of DNA adducts [8].
The bioactive gas nitric oxide (NO) has multiple biological functions in a very broad range of organisms. These functions include signal transduction, cell death, transport, basic metabolism, ROS production and degradation [9,10], among others (reviewed in [11]). It is well-known that NO exerts both pro-oxidant and antioxidant effects, depending on the ambient redox status, the presence of other reactants, and the nature of the reaction (for a review of the antioxidant actions of NO, see [12]). In plants, ROS and reactive nitrogen species have been shown to be involved in the defensive response of plants to biotic or abiotic stresses such as pathogens [13], drought [14], and air pollutants or UV-B radiation [15]. In the latter study, the authors found support for the hypothesis that NO reactive species, together with the glutathione system, play a key role in the coordination of gene expression during plant symbiosis. NO has been postulated as one of the first antioxidant mechanisms to have evolved in aerobic cells [16,17]. This idea builds on the work of Feelisch and Martin [18], who suggested a role for NO in both the early evolution of aerobic cells and in symbiotic relationships involving NO efficacy in neutralizing ROS. In addition, NO is involved in the abiotic stress response of green algae such as Chlorella pyrenoidosa Pringsheim, by reducing the damage produced by photo-oxidative stress [19].
The first work that focused on NO production in lichens was published in 2005, by Weissman and co-workers [20], who carried out a microscopy study of Ramalina lacera (With.) J.R. Laundon. These authors described the occurrence of intracellular oxidative stress during rehydration together with the release of NO by the mycobiont, but not by the photobiont. We have recently reported evidence that NO is involved in oxidative stress in lichens exposed to the oxidative pollutant cumene hydroperoxide [21]. However, there have been no further studies on NO function, or on the occurrence of NO in other lichens.
Real-time imaging of cellular function in vivo and of cell/tissue localization can be achieved with high sensitivity and specificity by using fluorescent probes together with fluorescence and confocal microscopy. For example, following entry of the probe DCFH2-DA into the cell it is converted by intracellular esterases to DCFH2, which upon oxidation by free radicals, mainly OH, CO3•-, NO2, and thyl radicals (such as GS), yields the fluorescent product (DCF) (reviewed by [22]).
Nitrogen oxide is produced at low concentrations and has a short half-life, which makes it difficult to detect in vivo. Interest in NO, due to its ubiquity and physiological relevance, has therefore led to the generation of several techniques for measuring its production. For example, the rapid reaction of 2,3-diaminonaphthalene (DAN) with NO to form the fluorescent product 1-(H)-naphthotriazole (NAT) is the basis for a very sensitive analytical method to measure NO production. DAN does not react directly with NO and therefore does not inhibit its actions. The high sensitivity of this technique allows its use in the quantification of NO production in living cells [23-25]. However, perhaps the most commonly employed methods for the analysis of NO in aqueous solutions is by measuring NO2- using the Griess reagent [23]. Alternatively, inhibitors of NO function can also be used to understand the physiological roles of this molecule. Carboxy-PTIO (c-PTIO) is a water-soluble and stable free radical that reacts stoichiometrically with NO. In vivo, c-PTIO inhibits the physiological effects mediated by NO, whereas in vitro it can be used to quantitate NO levels by ESR spectrometry [11].
The lichen Ramalina farinacea (L.) Ach. is a widespread species with large environmental tolerance. This green-greyish lichen is a fruticose, pendulous, epiphytic species that is very common in Mediterranean sclerophyllous oak forests. It lives on a great variety of substrates and different habitats such as plant bark, decomposing wood and rocks [26]. In the Iberian Peninsula it occurs at all altitudes, more frequently in areas with regular fogs being absent in maritime habitats. It shows especial preference for places with a high atmospheric humidity. This lichen is the Ramalina species with lower sensitivity to SO2 and is considered as toxitolerant [27].
The aim of this work is to investigate the release and role of NO in the oxidative stress caused by rehydration in the lichen Ramalina farinacea (L.) Ach. NO and ROS specific fluorescent probes will be used to morphologically localize these molecules in vivo with fluorescence and confocal microscopy. Furthermore, ROS kinetics and chlorophyll autofluorescence will be recorded during the first minutes after rehydration. Lipid peroxidation and NO-endproducts will be quantified at different time points. NO especific inhibitor c-PTIO will be used in order to elucidate NO functions. Likewise, NO production and relation with photosynthesis will be studied in different models of isolated photobionts: Ramalina farinacea (L.) Ach. isolated Trebouxia sp. photobionts, and in Asterochloris erici (Ahmadjian) Skaloud et Peksa, SAG 32.85 = UTEX 911.
Methods
Chemicals
The chemicals 2,6-di-tert-buthyl-4-methylphenol trichloroacetic acid (BHT), 2-thiobarbituric acid (TBA), 1,1,3,3, tetraethoxypropane (TEP), cumene hydroperoxide 88% (CP), and bisbenzimide H (Hoechst) were provided by Sigma Aldrich Química S.A (Tres Cantos, Spain); 2,7-dichlorodihydrofluorescein diacetate (DCFH2-DA), hydrochloric acid (HCl) and ethanol (etOH) were purchased from Panreac Química S.A.U (Barcelona, Spain); 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO) and 2,3-diaminonaphthalene (DAN) were from Invitrogen S.A (El Prat de Llobregat, Spain); and Triton X-100 was from VWR Prolabo (Barcelona, Spain).
Lichen material
Ramalina farinacea (L.) Ach. was collected in the air-dried state from Quercus rotundifolia Lam. at Sierra de El Toro (Castellón, Spain; 39°54'16"N, 0°48'22"W). Samples were maintained in a silica gel atmosphere during 24 h and frozen at -20°C until the experiment, 1 month after collection.
Epifluorescence probes
2,7-Dichlorodihydrofluorescein diacetate (DCFH2-DA) was used as probe in the detection of ROS (DCF, λexc = 504 nm, λem = 524 nm). DCFH2-DA is not appreciably oxidized to the fluorescent state without prior hydrolysis inside the cell.
2,3-Diaminonaphthalene (DAN) reacts with the nitrosonium cation that forms spontaneously from NO to yield the fluorescent product 1H-naphthotriazole which emits blue fluorescence (λexc = 375 nm, λem = 425 nm). Since the selectivity of DAN for the nitrosonium cation is high, NO can be detected without the inhibition of its function [25].
Fluorometric Kinetics of Free Radical Production and Chlorophyll Autofluorescence
Dry fragments of lichen thalli were placed in black flat bottom 96 multiwell plates and kept at -20°C until use. One of the plates was rehydrated with deionised water 24 h before the experiment and kept at 17°C, PAR 35 μmol m-2 s-1 16 h photoperiod.
Both dry and hydrated lichens were submerged during 5 minutes in deionised water 10 μM DCFH2-DA with or without c-PTIO 200 μM. The excess of solution was eliminated and the kinetics of DCF and chlorophyll emitted fluorescence were simultaneously measured in a SPECTRAFluor Plus microplate reader (Tecan Group Ltd., Männedorf, Switzerland). Excitation of both substances was performed at λexc 485 nm, emission of DCF fluorescence was recorded at λem 535 nm and chlorophyll autofluorescence at λem 635 nm, during one hour. Twelve replicates were analyzed by treatment and all values are referred to the weight of sample.
Microscopy
Fragments of lichen thalli were rehydrated for 5 min with either deionized water or 200 μM c-PTIO, and the corresponding fluorescence probe (10 μM DCFH2-DA or/and 200 μM DAN). The samples were then placed in a freezing microtome (CM 1325; Leica, Germany) and cut in sections of 30 microns. The slices were washed with deionized water and mounted on slides prior to their observation by fluorescence microscopy (OLYMPUS Provis AX 70 fluorescence microscope) or confocal laser scanning microscopy (TCS Leica SP Confocal Laser Scanner Microscope, Leica, Heidelberg, Germany) at the SCSIE (UVEG, Valencia).
Isolated photobionts of Ramalina farinacea
The photobiont R. farinacea (Trebouxia sp.) was isolated following the protocol described by Gasulla et al. [28]. Basically, it involves homogenization of lichen thalli (from 15 mg to 2 g), a one-step centrifugation through Percoll (r), followed by washing with Tween 20 and sonication. Algae were grown in 3N Bold's basal medium (BBM3N) containing 10 g casein and 20 g glucose per liter [29] with a 16:8 h light:dark photoperiod and at a temperature of 15°C. The medium was changed every 2 weeks and the concentration of algae set at 105 cells/ml.
Physiology of photosynthesis
An axenic strain of the lichen photobiont Asterochloris erici (Ahmadjian) Skaloud et Peksa (SAG 32.85 = UTEX 911) was used for this study. Algae were grown on cellulose-acetate discs on agar BBM3N containing 10 g casein and 20 g glucose per liter [29,30]. Cultures were maintained at 20°C under a 12 h photoperiod with 30 μmol m-2s-1 white-light illumination.
After 21 days, the discs were removed from the culture medium and dried in a closed container with a saturated solution of ammonium nitrate (R.H. 62%), and maintained under culturing conditions. The samples remained in the dried state for 24 h, were then rehydrated with distilled water or 200 μM c-PTIO and returned to culture conditions for 24 h.
In vivo chlorophyll a fluorescence was measured with a modulated light fluorometer (PAM-2000, Walz, Effeltrich, Germany). The samples were kept in the dark for 30 min and the minimum (dark) fluorescence yield (Fo) measured after excitation of the algae with a weak measuring beam from a light-emitting diode. The maximum fluorescence yield (Fm) was determined with an 800 ms saturating pulse of white light (SP, 8000 μmol m-2 s-1). Variable fluorescence (Fv) was calculated as Fm-Fo, and the maximum quantum yield of photosystem II (PSII) as Fv/Fm. The samples were allowed to re-adapt in the dark for 2 min, after which actinic light (AL, 200 μmol m-2 s-1, unless otherwise stated) was switched on, and SPs were applied at 1 min intervals to determine: (1) the maximum fluorescence yield during actinic illumination (F'm), (2) the level of modulated fluorescence during a brief (3 s) interruption of actinic illumination in the presence of 6 μmol m-2 s-1 far red (FR, 730 nm) light (F'o), and (3) steady-state chlorophyll a fluorescence yield after 11 pulses (Fs). Photochemical quenching (qP), and the quantum efficiency of PSII photochemistry (ФPSII) were estimated following the methods of Genty et al. [31] and Kramer et al. [32].
Measurement of malondialdehyde
Lipid peroxidation was evaluated as malondialdehyde (MDA) by the method of Reilly and Aust [33], modified as described below [34,35]. Working standards were made by diluting a 2 mM stock solution of the malondialdehyde precursor TEP with 80% ethanol supplemented with 2% of the antioxidant BHT to suppress the decomposition of lipid peroxides during the assay. Working concentrations of 0-50 μM were prepared for the lichens and 0-8 μM for the algae.
Lichen thalli were homogenized on ice with 1 ml of deionized water and centrifuged at 16,060 × g for 10 min. Supernatants were frozen at -20°C for NOx analysis, and the pellets resuspended in 500 μl ethanol-BHT. Algae were homogenized directly in 500 μl of ethanol-BHT with glass fragments (approx. 1 mm diameter) and strong vortexing for 30 min. Subsequently, 900 μM of TBA (2.57 × 10-2M), TCA (9.18 × 10-1M), and HCl (3.20 M) working solution was added to each sample and to the standards. The samples and standards were vortexed in a Vortex Labnet ×100 for 5 min at 3,000 rpm and then placed in a 70°C water bath for 30 min. Afterwards, the samples and standards were vortexed again, cooled on ice, and centrifuged at 10,060 × g for 10 min.
The absorbance of supernatants was measured at 532 nm (A532) in a Spectronic Genesys8 spectrophotometer. The absorbance at 600 nm (A600) was then measured and this value was subtracted from the A532 to eliminate the interferences of soluble sugars in the samples [35].
NO end-products determination
To estimate NO generation, NO oxidation end-products (nitrate and nitrite) were measured in the soluble fraction of the samples using a Skalar autoanalyzer, model SAN++. The automated determination of nitrate and nitrite is based on the cadmium reduction method: the sample is passed through a column containing granulated copper-cadmium to reduce nitrate to nitrite. The nitrite (that originally present plus that obtained from the reduction of nitrate) concentration is determined by its diazotization with sulfanilamide followed by coupling with N-(1-naphthyl)ethylenediamine dihydrochloride to form a highly colored azo dye, the absorbance of which is measured at 540 nm. This is the most commonly used method to analyze NO production and is known as the Griess reaction [23].
Statistics
At least three samples for each treatment and each incubation time were prepared. Four assays were carried out on four different days for the lichens and on three different days for the algae. Data were analyzed for significance with Student's t-test or by ANOVA.
Results
Bright-field micrographs showing the general anatomy of Ramalina farinacea are presented in Figure 1. The photobiont layer is located in the medulla and is surrounded by dispersed fungal hyphae, which become densely packed in the cortex of the lichen.
Figure 1. Anatomy of Ramalina farinacea. Thalli of R. farinacea: A, B Bright-field microscopy of slices cut in a freezing microtome (magnification 200× and 1000×, respectively); C, D ultramicrotome survey sections (10 μm) stained with toluidine blue (magnification 200× and 1000×, respectively). C cortex, PL photobiont layer, Pho photobiont, M medulla, Hy fungal hyphae
ROS generation, chlorophyll autofluorescence and lipid peroxidation during lichen rehydration
Although several works have described an extracellular oxidative burst during rehydration in some lichen species, virtually nothing is known about intracellular ROS production and its relationship to abiotic stress. In order to determine whether intracellular ROS release follows the rehydration of R. farinacea thalli, 10 μM of the fluorescent probe DFCH2-DA was added to the deionized water used for rehydration. The samples were observed by fluorescence and confocal microscopy 3-4 h after rehydration.
The presence of 2',7'- dichlorofluorescin (DCF), the fluorescent oxidation product of DCFH2, indicated the intracellular production of free radicals during lichen rehydration (Figure 2B-D). DCF was especially concentrated in the lichen cortex. No significant green autofluorescence was detected in the absence of the probe (Figure 2A). Confocal microscopy showed discrete points of green fluorescence around several large photobionts (Figure 2E), probably due to mycobiont hyphae tips.
Figure 2. ROS in rehydrated R. farinacea thalli. Thalli of R. farinacea rehydrated with deionized water and 10 μM DCFH2-DA and observed 3-4 h post-rehydration. A, B, C, D ROS content, as revealed by the green fluorescence emission of DCF under a fluorescence microscope (magnification: 400× for A, B and 1000× for C, D); E overlay of confocal microscopy images reveals ROS distribution around some of the photobionts (green fluorescence); F overlay of confocal microscopy images of ROS content of R. farinacea thalli that had been rehydrated with c-PTIO 200 μM, arrows point to photobionts photobleached by the confocal laser during the observation (oxPho). Red fluorescence is due to the photobiont's chlorophyll in all cases. Each micrograph is representative of several images corresponding to independent samples. C cortex, M medulla, PL photobiont layer, Pho photobiont, oxPho photobleached photobiont, Hy fungal hyphae
A fluorometric kinetics of intracellular free radical production in Ramalina farinacea thalli was performed in order to confirm microscopical data. Figure 3A demonstrates that the rate of intracellular free radical production in recently rehydrated thalli was much higher than the rate of intracellular free radical production in thalli kept in the hydrated state during the previous 24 h. Furthermore, intracellular release of free radicals during rehydration under physiological conditions was biphasic with an initial exponential phase of 20 min followed by a linear phase (Figure 3B). Chlorophyll autofluorescence was simultaneously recorded since this parameter is a surrogate of the levels and integrity of this molecule and therefore of the photosynthetic status of the cell. A slight increase in chlorophyll autofluorescence both in thalli hydrated for 24 h (Figure 3C, solid squares) and in thalli recently rehydrated (Figure 3D, solid squares) could be measured.
Figure 3. Fluorometric kinetics of free radical production and chlorophyll autofluorescence in R. farinacea thalli. A, Kinetics of intracellular free radical production evidenced by DCF fluorescence in recently rehydrated thalli (solid squares) compared with thalli hydrated for 24 h (solid circles); B, Kinetics of intracellular free radical production evidenced by DCF fluorescence in thalli rehydrated with deionised water (solid squares) or c-PTIO 200 μM (solid triangles); C, chlorophyll autofluorescence in lichens rehydrated with deionised water (solid squares) or c-PTIO 200 μM (solid triangles); D, chlorophyll autofluorescence in thalli hydrated 24 h before, and treated for 5 min with deionised water (solid squares) or c-PTIO 200 μM (solid triangles). Fluorescence units are arbitrary and comparisons of relative magnitudes can only be made within the same graph. Bars represent means and error bars the standard error of 12 replicates.
To determine whether the observed increase of ROS caused oxidative stress during rehydration, lipid peroxidation in R. farinacea was quantified in the first 24 h of rehydration under physiological conditions. After 1 h of rehydration, MDA levels dropped significantly to a minimum (Figure 4A). After 2 h, the levels began to increase such that they were slightly elevated at 4 h, at which time the maximum value was reached. This latter amount was unchanged at 24 h post-rehydration.
Figure 4. MDA content and NO end-products of rehydrated Ramalina farinacea thalli. MDA content: A rehydration with deionized water, B rehydration with c-PTIO (200 μM) in deionized water. NO end-products: C rehydration with deionized water, D rehydration with c-PTIO (200 μM) in deionized water. Student t test: * p < 0.05. The error bars stand for the standard error of at least 9 replicates
NO release during lichen rehydration
The release of NO in a lichen species was recently demonstrated for the first time. In order to confirm these results in another lichen species, R. farinacea, two approaches were used: fluorescence visualization of the released NO and quantification of the NO end-products. Accordingly, thalli were rehydrated in deionized water containing 200 μM DAN for the visualization of NO release and in deionized water alone for the quantification of NO end-products.
Microscopic analysis of blue fluorescence evidenced the production of NO, which was intimately associated with the fungal hyphae. Staining was especially intense in the medulla (Figure 5).
Figure 5. NO content of rehydrated R. farinacea thalli. Fluorescence microscopy of thalli of R. farinacea rehydrated with deionized water and 200 μM DAN. Blue fluorescence evidence NO presence, red fluorescence is due to the photobiont's chlorophyll in all cases. Micrographs are representative of several images corresponding to independent samples. C cortex, M medulla, PL photobiont layer, Pho photobiont, Hy fungal hyphae
Air oxidation of NO in an aqueous environment results in the near exclusive generation of NO2-, which is further oxidized to NO3 = [23]. NO end-products (NOx) were quantified by the classical method of Griess. NOx levels increased over 2 h to reach a maximum (Figure 4C). By 4 h, NOx levels had decreased to slightly below the initial levels, reaching a minimum, after which the levels remained constant for up to 24 h.
Effect of NO scavenging during lichen rehydration on ROS production, chlorophyll autofluorescence and lipid peroxidation
To study the role of NO during rehydration, R. farinacea thalli were rehydrated with 200 μM of the membrane-permeable compound c-PTIO, which specifically reacts with NO to inhibit its biological actions.
NO scavenging with c-PTIO completely suppressed DAN fluorescence emission (image not shown). It also produced a remarkable increase in ROS production in both the cortex and the medulla (Figure 2F). The confocal laser beam produced an oxidative burst in the photobionts, leading to chlorophyll photo-oxidation and DCF fluorescence onset within seconds (Figure 2F).
The kinetics study (Figure 3B, solid triangles) confirmed that NO inhibition during rehydration multiplies the levels of intracellular free radicals at 0 min (52.1 ± 2.85 versus 18.4 ± 1.67 a.u.). Moreover, inhibition of NO eliminates the initial exponential phase of free radical production seen during physiological rehydration of thalli (Figure 3B, solid squares). Chlorophyll autofluorescence was simultaneously measured and no evident differences between physiological and NO-inhibited rehydration could be observed (Figure 3C, solid triangles). However, NO inhibition in 24h-hydrated thalli resulted in an important decrease in chlorophyll autofluorescence that tends to recover normal values after 1 h (Figure 3D, solid triangles).
Lipid peroxidation during NO-specific inhibition with c-PTIO was measured quantitatively; the results are presented in Figure 4B. MDA levels reach a maximum at 2 h and a minimum at 4 h. The MDA levels measured following rehydration with cPTIO were the opposite of those obtained under physiological conditions. Figure 4D shows that, overall, NO end-products decreased in amount when c-PTIO was used.
Microscopy studies of isolated algae
Confocal studies clearly showed that NO deprivation caused photo-oxidative damage in the photobiont (Figure 2F). NO is known to reduce photo-oxidative stress in some species of green algae. A specific role for NO in the prevention of photo-oxidation in Trebouxia algae was confirmed in the following studies.
A suspension of axenically cultured Trebouxia sp., the photobiont isolated from R. farinacea, was treated with 200 μM c-PTIO in the presence of both DCFH2-DA and DAN. The images of control cells are presented in Figure 6A. NO inhibition by c-PTIO resulted in chlorophyll bleaching in some algae (Figure 6B). DAN fluorescence could not be detected by this method but the oxidative burst caused by c-PTIO provided indirect evidence of endogenous NO production in the algae. Direct measurements of NO end-products in the supernatant of photobiont suspensions at different time periods of culture (0-24 h) showed that these algae were able to produce NO in the low-nanogram range. NO levels reached a peak of 567 ng per million cells 2 h after preparation of the suspension (Table 1).
Figure 6. ROS content of isolated Trebouxia sp. Capital letters identify the fluorescence image; the lower-case letter indicates the corresponding bright-field images: A-a control; B-b algae treated with 200 μM c-PTIO. Each micrograph is representative of several images corresponding to independent samples. Magnification 1000×. Bar 20 μm
Table 1. NO end-products of the Trebouxia sp. photobiont isolated from Ramalina farinacea at different time points after the establishment of the algal suspension
Photosynthetic studies on isolated algae
To confirm that the visualized alterations in chlorophyll fluorescence were linked to alterations in the photosynthetic activity of the algae during NO deprivation, axenic cultures of Asterochloris erici, a well-characterized photobiont, were studied. The cells were cultured on cellulose-acetate discs, desiccated for 24 h, and rehydrated with 200 μM c-PTIO. Measurements were made in cells that had been maintained in culture conditions for 24 h. The significant decrease of Fv/Fm and ФPSII indicated that NO scavenging induces photo-inhibition of PSII (Figure 7). The degree of quinone A (QA) oxidation was determined as qP, which depends on the activation state of photosystem I (PSI) and the Calvin cycle [36]. After the dehydration/rehydration cycle, no differences were observed in qP, indicating that photoinhibition was produced before QA.
Figure 7. Effect of NO inhibition in Asterochloris erici photosynthetic parameters. Photosynthetic parameters of axenic cultures of Asterochloris erici desiccated for 24 h and then rehydrated with either deionized water or 200 μM c-PTIO. The algae were incubated under normal culture conditions for 24 h before chlorophyll a fluorescence was measured. Control algae were not desiccated but instead maintained under normal culture conditions. Fv/Fm, maximum photochemical efficiency of photosystem II (PSII); ФPSII, photochemical efficiency in light; qP, photochemical component of fluorescence relaxation. Different letters show significant differences between treatments. LSD test (p < 0.05), n = 3
The same treatments and measurements were carried out in whole thalli of R. farinacea but no alterations in photosynthesis at 24 h were observed (data not shown).
Discussion
This study investigated the role of NO during rehydration in Ramalina farinacea. The results showed that lichen NO plays an important role in the regulation of lipid peroxidation and photobiont photo-oxidative stress during rehydration.
NO is a well-studied critical signaling molecule involved in abiotic stress responses [14] and plant defence [13]. Our results demonstrated that, in addition to its utility for quantification methods, DAN is an excellent fluorescence microscopy probe for the histophysiological characterization of NO production in lichen.
The ability of ROS production to induce oxidative stress depends on the balance between cellular pro-oxidants and antioxidants, with an imbalance between the two resulting in oxidative damage. Thus, studies of ROS release using probes such as DCFH2 only determine the levels of pro-oxidant species but do not indicate the degree of oxidative stress. Instead, lipid peroxidation, measured as MDA, has long been used to characterize oxidative damage in cells and was the approach used in this study.
Our data showed that rehydration is accompanied by ROS and NO generation and thus confirmed the results of Weissman et al. [20]. The kinetics of ROS release is biphasic with an initial exponential phase (20-30 min) followed by a linear phase up to 1 h. The quantification of NO end-products showed that released NO reaches a maximum 1-2 h post-rehydration. Despite the presence of ROS, lipid peroxidation significantly decreased during the first hours following rehydration, reaching a minimum after 2 h, which coincided with the maximum levels of NO end-products.
Our microscopy studies revealed that the production of ROS and NO is closely related to lichen morphology: ROS was mainly associated with the hyphae of the cortex whereas NO was clearly localized to the medullar hyphae of the mycobiont. Confocal microscopy confirmed that the medulla is free of intracellular ROS, which were seen only in a few punctate zones around several large photobionts (Figure 1C). Since ROS are now recognized as key signaling molecules in yeast and in plants [14,15,37], these areas could constitute points of communication between the fungus and algae and are perhaps related to the mutual up-regulation of protective systems, as suggested by Kranner et al. [5]. Further investigations are needed to clarify this point.
NO scavenging during lichen rehydration resulted in increased ROS production and lipid peroxidation. Moreover, the initial exponential phase of free radical production is eliminated. This finding demonstrates that NO is involved in antioxidant defense and the regulation of lipid peroxidation especially during the first minutes after rehydration. In plants and in animals, NO is known to modulate the toxic potential of ROS and to limit lipid peroxidation, acting as a chain-breaking antioxidant to scavenge peroxyl radicals [12,16,38].
The incidence of the confocal laser on the algae of NO-deprived rehydrating thalli caused a rapid photo-oxidative burst and isolated photobionts showed evidence of oxidative destruction of chlorophyll even in the absence of the photo-stress caused by a confocal laser. Furthermore, NO-endproducts quantification supports the ability of Trebouxia photobionts to produce NO, eventually in important amounts (Table 1). Chlorophyll autofluorescence informs about the levels and integrity of this molecule. No appreciable changes in chlorophyll autofluorescence were seen during rehydration but the inhibition of NO in thalli hydrated for 24 h induced a reversible decrease in this parameter during 1 h. NO has been shown to ameliorate ROS toxicity in the chlorophycean alga Scenedesmus obliquus, probably by preventing the photo-inhibition that leads to photo-oxidation and pigment bleaching [39]. Our studies on the physiology of photosynthesis show that the inhibition of NO action altered the photosynthetic activity of the photobionts. These results suggest that NO is involved in PSII stabilization and could be related with the limited role of classical antioxidant systems during desiccation-rehydration cycles in Asterochloris (formerly Trebouxia) photobionts recently reported [7]. Several authors have demonstrated that, in higher plants, NO reversibly binds to PSII [40-44] and modulates electron transfer and quenching processes [45]. The fact that the same dose of c-PTIO than that used for photobionts did not alter photosynthetic activity in the photobionts of intact lichens suggests that the mycobiont is involved in stabilizing the photobiont's chlorophyll. Assays with higher doses of c-PTIO and specific inhibitors of fungal NO synthases are needed to confirm this possibility.
Conclusions
These data provide the first evidence of an important role for NO in oxidative stress regulation during the early stages of rehydration in the lichen Ramalina farinacea, including chlorophyll photostability of the trebouxioid photobionts (summarized in Figure 8). Our results also raise important questions about the evolutionary role of NO in the establishment of lichen symbiosis, due to its dual role as antioxidant and mediator in cell communication.
Figure 8. Schematic representation of the findings of the present work on the functional relation of nitric oxide (NO) with oxidative stress during rehydration of Ramalina farinacea in the context of current knowledge. Rehydration induces the functional reconstitution of electron chains, the most relevant being chloroplast photosynthesis and mitochondrial oxidative phosphorilation. During the process of reconstitution, membrane molecular architecture is not optimal and an elevated electron leaking from electron chains occurs. Electron leaking causes a burst of intracellular ROS. Nitric oxide is released mainly from mycobiont medular hyphae (NO production by photobionts has not been confirmed in the lichen but is likely). A decrease in lipid peroxidation of lichen thalli coincides with the peak of NO-endproductos. NO participates in chlorophyll photoprotection and stabilization during rehydration
Future research should be directed at functional (NO donors, NO synthase inhibitors, exposure to SO2, Cu+/2+, etc.), ultrastructural (sites of NO synthesis, immunohistochemistry), and cell communication (co-culture of isolated symbionts, NO donors, c-PTIO) studies of NO, with the aim of clarifying the role of this multifaceted molecule.
Abbreviations
a.u.: arbitrary units; BHT: 2,6-di-tert-buthyl-4-methylphenol; c-PTIO, carboxy-PTIO: 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, potassium salt; DAN: 2,3-diaminonaphthalene; DCF: 2',7'- dichlorofluorescin; DCFH2: 2',7'- dichlorodihydrofluorescein; DCFH2-DA: 2',7'- dichlorodihydrofluorescein diacetate; MDA: malondialdehyde; NAT: 1-H-naphthotriazole; ROS:reactive oxygen species; TEP: 1,1,2,2-tetraethoxypropane
Authors' contributions
EB and MC conceived Objectives and designed the study and general design of the work. FG and EB collected and identified R. farinacea thalli. Microscopy and image handling were performed by FG-B and J R-A. FG designed and carried out photobionts isolation and physiology of photosynthesis experiments. Studies on lipid peroxidation and NO-endproducts quantification were made by AEP. MC and FG wrote the paper and EB made final considerations. All authors read and approved the final manuscript.
Acknowledgements
This project was funded by the Spanish Ministry of Education and Science [project numbers CGL2006-12917-C02-0 and CGL2009-13429-C02-01], project Prometeo 2008/174 of the Generalitat Valenciana and the project AECID PCI/A/024755/09 of the Spanish Ministry of Foreign Affaires.
We are grateful to F. Gasulla, J. Gimeno-Romeu, E. Barreno, (ICBIBE, University of Valencia) and A. Guéra (Plant Biology, University of Alcalá) for communicating unpublished data, to Dr. R. Catalá (CIB, Madrid), Dr. P. D'Ocón (UVEG, Valencia) and Dr. J. Medina (INIA, Madrid) for critical revision of the manuscript, and J.L. Rodríguez Gil for MDA protocol optimization.
English revision was done by Wendy Ran.
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Research article
Evidence for somatic gene conversion and deletion in bipolar disorder, Crohn's disease, coronary artery disease, hypertension, rheumatoid arthritis, type-1 diabetes, and type-2 diabetes
Kenneth A Ross
Author affiliations
Department of Computer Science, Columbia University, New York, NY 10027, USA
Citation and License
BMC Medicine 2011, 9:12 doi:10.1186/1741-7015-9-12
The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1741-7015/9/12
Received:15 October 2010
Accepted:3 February 2011
Published:3 February 2011
© 2011 Ross; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background
During gene conversion, genetic information is transferred unidirectionally between highly homologous but non-allelic regions of DNA. While germ-line gene conversion has been implicated in the pathogenesis of some diseases, somatic gene conversion has remained technically difficult to investigate on a large scale.
Methods
A novel analysis technique is proposed for detecting the signature of somatic gene conversion from SNP microarray data. The Wellcome Trust Case Control Consortium has gathered SNP microarray data for two control populations and cohorts for bipolar disorder (BD), cardiovascular disease (CAD), Crohn's disease (CD), hypertension (HT), rheumatoid arthritis (RA), type-1 diabetes (T1D) and type-2 diabetes (T2D). Using the new analysis technique, the seven disease cohorts are analyzed to identify cohort-specific SNPs at which conversion is predicted. The quality of the predictions is assessed by identifying known disease associations for genes in the homologous duplicons, and comparing the frequency of such associations with background rates.
Results
Of 28 disease/locus pairs meeting stringent conditions, 22 show various degrees of disease association, compared with only 8 of 70 in a mock study designed to measure the background association rate (P < 10-9). Additional candidate genes are identified using less stringent filtering conditions. In some cases, somatic deletions appear likely. RA has a distinctive pattern of events relative to other diseases. Similarities in patterns are apparent between BD and HT.
Conclusions
The associations derived represent the first evidence that somatic gene conversion could be a significant causative factor in each of the seven diseases. The specific genes provide potential insights about disease mechanisms, and are strong candidates for further study. Please see Commentary: http://www.biomedcentral.com/1741-7015/9/13/abstract webcite.
Background
Gene conversion is a process in which genetic information is transferred unidirectionally between highly homologous but non-allelic regions of DNA [1]. The genome contains many pairs of homologous regions, reflecting frequent gene duplication during evolution. Gene conversion is usually triggered by a double strand break (DSB), which can occur during meiosis or mitosis [1]. The DSB is repaired using the homologous sequence as the template. In mammalian cells, the sister chromatid is the most frequent conversion substrate [2], typically leading to perfect repair of a DSB. Gene conversion from other sequence, however, can lead to DNA changes. Gene conversion has recently been implicated in a number of diseases, as a source of both inherited and de-novo germ-line mutation [1]. It has been hypothesized that somatic gene conversion is relatively frequent but has escaped attention due to the technical difficulty of measurement [1].
An informative example of gene conversion is the IDS gene, located on the X chromosome. Mutations in IDS cause Hunter syndrome. There is a pseudogene IDS2 located 20 kb from IDS in an inverted orientation relative to IDS, with 88% overall homology to IDS [3]. 20% of Hunter syndrome mutations involve structural rearrangements induced by the interaction of the two nonallelic homologous regions [3,4]. The rearrangements appear to be independent events, indicating a recurrent mutation rather than common ancestry. Observed rearrangements include deletions, inversions, and gene conversion events [3,4]. Among the regions exhibiting gene conversion, a complex pattern of alternating sequence fragments from each of the duplicons is apparent. The IDS2 pseudogene is missing several IDS exons, but exhibits homology with IDS on each side of this 'gap'. Some of the deletion events observed in the IDS gene appear to represent conversion of IDS sequence by IDS2 in the vicinity of this gap, leading to the elimination of those exons [4]. A one kilobase recombinational hotspot has been identified for the IDS/IDS2 events; this hotspot exhibits 98% identity compared with the 88% overall identity of the duplicons [3]. Lagerstedt et al. [3] suggest that recombination is initiated in this high-identity region, and spreads through branch migration until a region of sufficient sequence divergence is reached. Lagerstedt et al. propose a model in which gene conversion leads to changes in both duplicons, and in which mismatched base pairs in the heteroduplex DNA may be corrected to generate additional conversion [3]. Figure 1 illustrates this model. These observations lead to two important conclusions. First, when looking for evidence of gene conversion, one should examine all duplicons for a given sequence. Second, one should examine the entire contiguous high-homology sequence in those duplicons, and not limit the analysis to the immediate neighborhood of a particular locus.
Figure 1. A model of gene conversion between duplicons. Two homologous but non-allelic sequences are shown, with homology indicated by a common green color. After a double strand break in the original sequence, the template sequence is used to form a heteroduplex DNA structure with the original sequence during the process of repair. A possible repair outcome is shown, illustrating changes to both the template and original sequences far from the location of the break, as well as changes and deletion in the original sequence in the vicinity of the break.
Somatic gene conversion can have multiple kinds of effects. Most obviously, conversion of a coding sequence by a non-identical homologous sequence may lead to a dysfunctional gene product, or an immunogenic novel amino acid sequence. Conversion of a regulatory, promoter, suppressor, or enhancer sequence may alter gene expression, either up or down. Since converted sequence usually retains the methylation status of the source sequence [5], conversion may result in either the methylation of previously unmethylated promoter sequence, suppressing gene expression, or in the demethylation of previously methylated sequence, enabling gene expression where it was not previously expressed. Gene conversion may also be correlated with other effects of nonallelic homologous pairing. Crossover and conversion occur in the same hot spot regions, and gene conversion appears to be preferred over crossover when interacting regions are short [6]. Non-allelic crossover may lead to insertions, deletions and/or inversions. Homologous pairing within a short region of DNA could create DNA loop structures that alter transcription patterns [7]. Conversion could potentially occur during DNA/RNA pairing [8,9]. Any of the effects mentioned above could have a major impact on cell function, and provide plausible causative mechanisms for disease.
I propose to examine somatic gene conversion in the context of disease using single nucleotide polymorphism (SNP) microarray data. Because conversion tracts are short, linkage disequilibrium (LD) between a gene conversion locus and nearby SNP markers is likely to be weak or nonexistent [10]. As a result, it becomes necessary to analyze single-SNP markers without expecting to see correlated patterns in nearby markers as one would expect in a traditional disease association study.
The Wellcome Trust Case Control Consortium (WTCCC) data set was obtained using an Affymetrix 500 K platform [11]. Genotyping was performed on two large British control populations (58C, NBS), in addition to disjoint populations for bipolar disorder (BD), Crohn's disease (CD), coronary artery disease (CAD), hypertension (HT), rheumatoid arthritis (RA), type-1 diabetes (T1D) and type-2 diabetes (T2D). The WTCCC data has been extensively analyzed using a traditional genomewide association study [11]. This previous analysis required the presence of three concordant SNP markers in order to identify a disease-associated haplotype. Such an analysis is likely to miss gene conversion events because of the weak LD. Further, by focusing the analysis at the called-genotype level, such an analysis is insensitive to somatic changes to the genome.
DNA samples in the WTCCC study are obtained from lymphocytes. One might be concerned that an analysis of somatic mutation in lymphocytes may not be informative about somatic mutation in other tissues more closely associated with the diseases in the WTCCC study. Fortunately, there is some evidence that a phenomenon related to gene conversion known as sister chromatid exchange (SCE) is informative about disease when measured in lymphocytes. SCE involves crossover between homologous sister chromatids mediated by the homologous recombination pathway [12], and has been interpreted as indicating general genome instability and/or a response to DNA damage [13]. SCE is elevated in lymphocytes of individuals with CD [14], CAD [15], T1D [16], and T2D [17], but not RA [18], although in some cases the elevation may be related to treatment rather than disease [19]. SCE is also elevated in multiple sclerosis [20], systemic lupus erythematosus [21], several cancers [14], and in individuals with viral infections [22].
Since SCE analysis using lymphocytes (rather than tissues directly affected by the disease) is informative, one might expect lymphocytes to also show disease-associated gene conversion behavior. Because blood cells are widely circulating, they are likely to encounter agents of double strand breakage such as viruses, and therefore exhibit gene conversion if conversion is occurring anywhere in the body. Further, a disease may be associated with damage to a particular tissue, for example by autoimmune processes, and the destroyed tissue is unavailable for analysis. Other cell types such as lymphocytes might therefore serve as useful proxies for damaged tissues. If the mechanisms of in-vivo gene conversion are sequence-specific rather than tissue-specific, then lymphocytes would exhibit the same conversion experienced by the damaged tissue, without eliciting the destruction response.
To identify somatic changes in a population I propose a novel data analysis technique. The technique takes advantage of the fact that a sample contains DNA from many cells of a single individual. If a significant proportion of those cells have undergone gene conversion at a locus, then the resulting change in the genotype of those cells should be measurable as a perturbation in the intensity for the two allele probes at that locus. An SNP with a distribution of perturbations specific to a disease population serves as a marker for a potential disease-associated locus. More details about how such perturbations are measured, and why such perturbations would have a signature different from other sources of variation such as paralogous sequence variants, can be found in the Methods section below.
Once a set of SNPs showing the signature of gene conversion is identified in a disease population, it would be desirable to validate those associations using an independent source of information that links the disease to those SNPs significantly more closely than to randomly chosen SNPs. As noted above, one needs to consider not just the SNP locus itself, but all regions with homology to the duplicon containing the SNP. The most direct form of association between a region and a disease is to find a gene in the region that is known to be associated with the disease, or that participates in a critical pathway known to be relevant for the disease. Additional evidence might include data showing that the gene is expressed in the relevant tissue with function related to disease pathogenesis. Most regions of high homology contain at most a few genes, and so the analysis can be relatively specific. One could also look for adjacent genes for which the duplicon could plausibly contain an upstream enhancer locus. I use 30 kb as a threshold for this type of adjacency.
When duplicons are nearby on the same chromosome, the intermediate region between them is an additional region of interest. Improper recombination between such regions could lead to inversions, insertions, or deletions of the intermediate sequence. In some situations, somatic deletion of a genomic region can generate patterns similar to those that would be generated by gene conversion. Deletion might be suspected when the duplicons occur in an aligned fashion nearby on the same chromosome, a configuration that could lead to misaligned recombination.
In the presence of an agent that induces genetic damage, a cell may respond by inducing the homology-directed repair pathway [23]. If this pathway is induced in each of many cells in response to the same agent, the same homology-biased mutations may happen in a variety of tissues. Mutations in stem cells will persist in lineages descending from those cells.
The damage-initiating agent may act locally or globally. A local agent, such as a virus that damages DNA in a position-specific manner, could induce gene conversion selectively in the region surrounding the target sequence. A global agent, such as a deficient or inactivated DNA repair pathway [24], would lead to DNA damage in a broad (but not necessarily random) fashion, inducing generalized gene conversion at many loci. Local gene conversion will be identifiable as a perturbation in the disease population that is absent in the control population and other disease populations. Perturbations due to global gene conversion may be present, to a lesser degree, in other populations whose diseases are caused by global agents. The perturbations should presumably be absent in the control population and in populations for diseases caused exclusively by local agents.
Increased SCE exchange rates are likely to be correlated with a global causative agent. Based on the SCE data for five of the seven studied diseases [14-18], one might hypothesize that RA is caused by a local agent, while CD, CAD, T1D, and T2D are caused by global agents. This hypothesis will be evaluated in the following analysis.
Methods
Raw signal intensity and genotype calling data were obtained from the WTCCC in an anonymized form, and the analysis of the data was approved by a Columbia Institutional Review Board. Each disease cohort contained approximately 2,000 individuals, while the two control cohorts each contained approximately 1,500 individuals. The Affymetrix platform supports 500,568 SNP loci, of which 459,653 passed the WTCCC quality control procedures [11].
For a SNP locus with an A/B polymorphism, the microarray generates a pair of intensity values IA and IB. Each intensity value is the average intensity over a small number of oligonucleotide probes containing the allele together with some flanking sequence. The (IA, IB) point typically falls within one of three clusters corresponding to the three genotypes AA, AB, and BB.
Consider now an individual with an AA genotype. Suppose that 20% of the sampled cells of this individual have undergone gene conversion in which one of the A alleles has been converted into a B allele by a homologous sequence, while the flanking sequence has remained unchanged. The left example of Figure 2 shows this kind of conversion. (Conversion of both A alleles would be rare, and is ignored.) This individual will display an overall (IA , IB) intensity pair that is 20% of the way from the AA cluster to the AB cluster. In another individual with a heterozygous AB genotype, a 20% conversion rate at the same locus would yield an overall (IA , IB) intensity pair that is 10% of the way from the AB cluster to the BB cluster, since only the conversion of the A allele will cause a change in probe intensities. In an individual with a BB genotype, no change would be observed.
Figure 2. Effects of gene conversion on probe intensity signals. A microarray has two probes for a SNP, each 25 bp long (top). An individual with an AA homozygous genotype at the SNP locus is shown. Two examples of gene conversion are illustrated. The left example considers the case when the duplicon contains sequence that exactly matches the B probe. The right example considers the case when the duplicon contains sequence that does not match either probe.
Because there is experimental variation in intensity measurements, it may be difficult to determine whether a small perturbation in a single measurement represents gene conversion or merely noise. However, it is possible to study the distribution of perturbations for a population at a locus. If a population has a significant spread of intensities between clusters, when control populations do not, then one can hypothesize that gene conversion at that locus is happening in a population-specific manner. See the cluster plot for RA in Figure 3 for an example. If the population is a disease cohort, then the locus may be associated with the disease phenotype.
Figure 3. Cluster plots for SNP rs4988327 in the WTCCC data. Note the high spread for RA, and the resulting increase in no-calls (orange) relative to calls (green).
Returning to the example above, consider the complementary situation in which the flanking sequence near an SNP probe has been converted. Whether or not the SNP locus is changed, the converted sequence will no longer match either probe sequence. The right example of Figure 2 shows this kind of conversion. If many cells in an individual are converted in this fashion, a reduced signal from this sequence will be measured by both probes of the microarray. For a locus at which this effect is associated with the disease phenotype, all clusters will shift radially towards the origin in the cluster plots for the disease population.
Calling algorithms attempt to identify the boundaries of clusters corresponding to the AA, AB and BB genotypes. For example, the Chiamo algorithm [11], considers all populations simultaneously, and estimates cluster boundaries in a way that allows for some population-dependent differences. The intensity distributions vary from SNP to SNP, and so clustering is performed separately for each SNP.
Based on the analysis above, gene conversion for a particular population should be accompanied by either (a) an increase in the spread of the two-dimensional intensity distribution relative to the control population, or (b) a translation of the clusters towards the origin, relative to the control population. In case (a), there should be an increase in the number of points that are either between clusters, or on the fringe of a cluster. In case (b), there should be a decrease in the distance between clusters, leading to an increase in the number of points whose cluster assignment is ambiguous. Either way, there will be an increase in the number of no-calls generated by the calling algorithm, relative to the control populations. This is one 'signature' of gene conversion that I will try to identify.
The Chiamo calling algorithm has been applied to the WTCCC data, and it is possible to use those calls to help recognize the signature of gene conversion. Chiamo generates a confidence score for a call; the authors of the Chiamo algorithm recommend that when this score is below 0.9, the genotype should be considered a 'no-call.' When clusters are more dispersed, their peripheries can begin to overlap with each other. In such a situation, the Chiamo algorithm will have less certainty about points falling in the intermediate regions. Chiamo will define cluster boundaries more tightly, resulting in an increase in the no-call rate for intermediate points [11]. An example of this phenomenon is given in Figure 3, where the orange points (that are particularly frequent in RA at this locus) are no-calls.
An increase in no-calls between two clusters can lead to a biased allele distribution in the called genotypes. For example, if there are many no-calls between the AA and AB clusters, then the A allele will be underrepresented among the subpopulation whose genotypes are called with high confidence. This bias is another possible signature for gene conversion. (See Additional file 1 for an extended discussion of no-calls.) Note that there may be cases of gene conversion that do not show this signature because the non-called points do not change the observed allele frequencies.
Additional file 1. Supporting text and tables. Detailed information about the identified SNPs, copy number variation, SNP interactions, and the mock study.
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To identify gene conversion events, I take three complementary approaches. The first approach that I call the 'stringent' filter is designed to optimize precision, that is, to minimize the number of false positives while possibly missing some true positives. The second approach is designed to provide better recall, that is, to include more true positives at the risk of also including false positives. This second approach is called the 'relaxed' filter. The third approach, termed the 'no-call-only' filter, looks only for extreme no-call rates, since some gene conversion loci may not exhibit changes in called allele frequencies.
For the stringent filter, called SNPs with high no-call rates in a population relative to the union of the two control populations are initially selected. A chi-squared statistic is calculated for each SNP based on a 2 × 2 chi-squared test comparing calls/no-calls for both the disease population and the control population. Only SNPs with an increase in the no-call rate in the disease population and a chi-squared statistic corresponding to P < 5 × 10-5 in a one-sided test are retained by this initial selection.
A further selection is applied to test for a bias in the genotype distribution in the disease population relative to controls. Bias is assessed in one of two ways; an SNP that displays bias according to either of these tests is retained. Only SNPs in which the control population has at least ten individuals for each of the AA, AB and BB genotypes are considered. First, the three genotype frequencies in the disease population are compared with the corresponding frequencies in the control population using a 3 × 2 chi-squared test to determine the likelihood that they have a common distribution. Only SNPs with a chi-squared statistic corresponding to P < 5 × 10-4 in a two-sided test are retained. Second, the three genotype frequencies in the disease population and control population are separately assessed for departure from Hardy-Weinberg Equilibrium using a conventional 3 × 2 chi-squared test. Only SNPs with a chi-squared statistic corresponding to P < 5 × 10-4 in a two-sided test in the disease population and a chi-squared statistic corresponding to P > 0.01 in the control population are retained.
Gene conversion appears to require at least 300 base pairs of homology in humans [1]. Among known gene conversion loci, the smallest degree of identity between the homologous regions is 88% [1]. One should therefore not expect newly discovered loci to have identity much below 88%. I will thus use 85% identity as a lower bound for the stringent filter.
The candidate SNPs were evaluated for homologous flanking sequence elsewhere in the genome. The UCSC database of segmental duplications [25] was used to identify genomewide duplications with at least 1,000 base pairs of homology (after elimination of low-complexity repeats) and at least 90% identity. Additionally, each SNP that met the other stringent filter conditions was subjected to manual analysis using the BLAST network service at NCBI to identify duplications that may not meet the thresholds of the segmental duplication database, but that may still be relevant for gene conversion. (I used the Megablast algorithm with default parameters. When a duplicon contains several almost-contiguous segments, the identity of the duplicon is the identity reported by BLAST for the segment containing the region that maps to the SNP under consideration.) The three filters are summarized in Table 1. The relaxed and no-call filters use different homology criteria from the stringent test so that the segmental duplication database can be used to automate the analysis. Because the segmental duplication database excludes regions with low complexity repeats, some SNPs in regions with more than 90% homology (for example, rs9378249) are not in the segmental duplication database.
Table 1. Summary of the three data filters.
The analysis does not consider SNPs on the Y chromosome. For the X chromosome, the analysis is limited to the female subpopulation within each cohort. As a result, some statistical power is lost, particularly for cohorts such as CAD that have a relatively small number of female members.
Cluster plots for all SNPs mentioned in the text can be found in Additional file 2.
Additional file 2. Zip archive containing cluster plots for all SNPs mentioned in the main text. The 58C and NBS populations are approximatel 1,500 while the other populations are each about 2,000.
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Sources of variation
Copy number variations at an SNP locus mean that in addition to the conventional AA, AB, and BB genotypes, there may be additional genotypes such as AAB and B. Each of these alternative genotypes would have its own cluster in the cluster plot, which can be examined for signs of more than three clusters. Each SNP was also assessed for known copy-number variation using the Database of Genomic Variants [26], since copy-number variants could also cause changes in no-call frequencies and genotype distributions that may be related to disease. (See Additional file 1 for further discussion of copy number variation.) Note that somatic deletion would generate genotypes like B in some cells, but since most cells retain the normal copy number, the effect will be a small perturbation in the cluster plot rather than a separate cluster. Germ-line mutations would not give the same perturbation patterns as somatic conversion. For a germ-line mutation that changed one allele to another, the individual would appear as part of another cluster in the corresponding cluster plot. If a germ-line mutation deleted or duplicated an allele, then the individual would appear as part of a cluster with a nonstandard copy number. If this deletion/duplication was common, then the cluster plot would show features typical of CNV loci, such as the presence of more than three clusters.
A paralogous sequence variant occurs when the homologous sequence to the mapped SNP sequence possesses a polymorphism. Suppose an SNP has probes for alleles A and B. If the paralogous sequence also has an A/B polymorphism, then the cluster plot will have five clusters, corresponding to AAAA, AAAB, AABB, ABBB, and BBBB. If the paralogous sequence has an A/C polymorphism, then the probes will not detect the signal from the C allele, and there will be clusters for AA, AB, BB, AAA, AAB, ABB, AAAA, AAAB, AABB. In either case, the cluster plot will differ significantly from what is expected under a gene conversion hypothesis.
Some polymorphisms on the microarray platform may have been misidentified, with the true polymorphism being in paralogous sequence with no polymorphism at the mapped SNP locus. As long as the paralogous sequence is part of a larger region of homology with the mapped SNP locus, the outcome of the gene conversion analysis will be unchanged by such phenomena because both duplicons are examined.
A foundational somatic mutation could occur during early development, leading to a lineage of cells within the individual carrying the mutation. This kind of mutation will not be identified by the present analysis unless the blood cells being genotyped come from more than one such lineage. Even then, the relevance of a foundational mutation to disease would be unclear because the mutation would also have to have been in a lineage ancestral to the diseased tissue.
Results
Putative gene conversion events detected using the stringent filter
31 instances of putative gene conversion with duplicon identity of at least 85% were identified using the stringent filter, covering 23 distinct SNPs. This data is summarized in Tables 2, 3 and 4; additional information about the associations can be found in Table S1 in Additional file 1. The SNPs in Table 4 fall within the MHC region and are identified by the stringent filter for T1D. Since T1D has significant associations at the haplotype level in the MHC region [11], it is difficult to separate a conversion signal from the broader association signal for these SNPs. The same is true for RA [11], but no MHC SNPs were identified for RA using the stringent filter.
Table 2. SNPs identified for various cohorts using the stringent filter (Part 1).
Table 3. SNPs identified for various cohorts using the stringent filter (Part 2).
Table 4. SNPs identified in the MHC region for T1D using the stringent filter.
In all 28 of the 28 instances in Tables 2 and 3, the change in allele frequency is consistent with what would be predicted by a gene conversion hypothesis (see Additional file 1). Additional SNPs that met the stringent filter conditions except that identity between duplicons was 71%-83% are discussed in Additional file 1.
The strength of the evidence for a putative SNP/disease association is determined by consulting the published literature in search of a known association. The strength of the evidence is summarized using the scale of Table 5, where a higher number corresponds roughly to stronger evidence. The score for a SNP is the maximum score for any gene in any duplicon associated with the SNP; genes for which a duplicon occurs 30 kb or less upstream of the gene are included. Note that the score for an SNP does not give any weight to genes occurring between neighboring homologous regions (except for the 30 kb-upstream genes mentioned above). The evidence score therefore ignores the possible deletion and/or duplication of genes in the intervening sequence. The code for the strength of the evidence is given in parentheses in the heading for each SNP.
Table 5. Numeric codes describing the strength of evidence for an association of a gene with a disease.
To assess the significance of the set of identified regions, the duplicons for the SNPs identified by the stringent test (which should have few false positives) are assessed for association with the corresponding disease. The code for the strength of the evidence is given in parentheses in the heading.
rs4471699 in CD (6)
Of the characterized genes in the various duplicons, SULT1A3 has the most obvious connection to the CD phenotype involving inflammation of the small and/or large intestine. SULT1A3 is highly expressed in the small intestine [27] where it specifically sulfates dopamine and is important for the metabolism of several neurotransmitters [28]. SULT1A3 shows reduced expression in the colons of CD patients [29]. (The related genes SULT1A1 and SULT1A2, which are also located in a segmentally duplicated region of chromosome 16, have reduced expression in CD [30].) Eisenhofer et al. [28] suggest that the production of dopamine sulfate in the intestine 'reflects an enzymatic "gut-blood" barrier for detoxifying dietary biogenic amines.' Dysfunction of this pathway could lead to toxicity in the small and large intestines.
The UQCRC2 gene is a part of the mitochondrial respiratory complex III. Apolipoprotein E4 binds to UQCRC2, and overexpression of a fragment of this protein impairs the function of complex III [31]. Mitochondrial dysfunction has been associated with CD in several case reports, including one with dysfunction in complex III [32].
Strikingly, the duplicon containing rs4471699 and the closest matching duplicon have recently been shown to be endpoints of a region deleted in the germ-line in certain cases of autism, and duplicated in others [33,34]. Among the common features of autism are gastrointestinal abnormalities [35]. Mitochondrial dysfunction also occurs with increased frequency in autism [36,37].
rs669980 in RA (5)
CBWD1 (and by inference also CBWD2) has 25% protein identity with the cobW gene of P. dentrificans that is thought to be involved in vitamin B12 processing [38], and possibly cobalt chelation [39,40]. Vitamin B12-binding proteins are found in the synovium of RA patients [41,42]. Low serum vitamin B12 levels are noted in a significant percentage of RA patients [43]. Methyl B12 appears to suppress cytokine production in T lymphocytes [44], which may be relevant to RA. Improper vitamin B12 processing can lead to elevated plasma homocysteine levels, which has been observed in multiple RA cohorts [45].
Dysregulation of cobalt chelation could also have secondary mutagenic effects, since cobalt is genotoxic [46].
rs10502407 in T2D (6), CAD (6)
CIDEA has known associations to obesity, insulin resistance, and T2D [47-49], which are also risk factors for CAD [50]. The duplicon is located upstream of CIDEA in a potential enhancer locus.
rs12134625 in CAD (3)
The FUSIP1 gene specifically represses splicing during mitosis [51,52] and in cells subject to heat shock [53]. Cells lacking FUSIP1 are defective in recovery after heat shock [53]. Splice repression after heat shock prevents the possible accumulation of inaccurately spliced mRNAs, until the heat-damaged splicing apparatus is restored to normal [53]. FUSIP1-null mice display multiple cardiac defects during embryonic development, due to improper processing of pre-mRNA encoding cardiac triadin [54]. Somatic defects in FUSIP1 that lead to mis-spliced triadin transcripts could be a pathogenic mechanism in CAD.
rs9551988 in CAD (3), BD (3), HT (3)
PSPC1 has sequence-specific RNA-binding domains, and localizes to paraspeckles [55]. While the function of paraspeckles is not fully understood, Prasanth et al. [56] describe how paraspeckles store CTN-RNA, which is cleaved under conditions of stress and released for immediate translation into protein. Prasanth et al. argue that this mechanism allows the cell to provide a rapid stress response, rather than having to wait for RNA transcription [56]. The released mRNA encodes SLC7A2, also known as CAT2, a cationic amino acid transporter involved in L-arginine transport, a necessary step in nitric oxide (NO) synthesis [56,57]. Insulin directly effects vascular endothelium and smooth muscle via nitric oxide release [58,59]. The pathway for insulin-induced NO synthesis involves L-arginine transport and the SLC7A2 gene [58,60,61]. The physiological implications of a dysregulation of insulin in obesity, CAD, and HT are well known [58,59]. A dysregulation of SLC7A2 function could have similar effects. In preeclampsia (HT and proteinuria in pregnancy) the L-arginine NO system of circulating leukocytes appears dysregulated [62]. The L-arginine NO pathway appears to be involved in the pathogenesis of BD [63,64].
rs935019 in HT (4)
The two duplicons are immediately adjacent and aligned within the GYPC gene. Such an arrangement provides an opportunity for improper recombination due to misalignment. Indeed, deletion variants of the GYPC gene have been attributed to unequal crossover at these duplicons [65]. One of these deletions frequently occurs spontaneously in E. coli during cloning [65], suggesting that spontaneous somatic deletions are also likely.
The GYPC gene codes for the GPC and GPD proteins, which regulate the shape and mechanical properties of red blood cells [66]. While there is no direct evidence linking GYPC to HT, the tissue-specificity and function of GYPC make such a link plausible.
rs12227938 in HT (3)
The HERG gene encodes pore-forming alpha-subunit protein important for repolarizing K+ current in the heart [67]. The ALG10B gene (also known as KCR1) modulates HERG, reducing the sensitivity of cardiac cells to arrhythmic disturbance [68,69]. ALG10B suppresses heart rhythm and regulates cardiac automaticity [70]. Polymorphisms on ALG10B are associated with the risk of acquired long QT syndrome, a cardiac rhythm disturbance [71]. Somatic defects in ALG10B would have direct relevance to HT.
SNP A-1797773 in T2D (4)
VPS35 is part of the retromer protein complex, which has a variety of sorting-related functions [72]. Mutant VPS35 is associated with improper insulin secretion [73].
rs12381130 in T1D (0)
This particular duplicon has homology with many other regions. Interestingly, on chromosomes 3, 4, 8, and 11 there are pairs of homologous duplicons about 4 Mb apart. Gene conversion at rs12381130 could be a marker of more general conversion and/or improper recombination at these locations, potentially leading to somatic deletions, duplications or inversions of the sequence between duplicon pairs on a chromosome.
rs11060028 in CD (6)
GLT1D1 appears to be a glycosyltransferase, but relatively little is known about its specific function. The chromosome 10 duplicon is 16 kb upstream of ABCC2 in a possible enhancer locus. ABCC2 is expressed on the apical membrane in the jejunum, ileum and colon [74]. It is an efflux transporter, responsible for extruding toxic substances from the cell [29,74]. ABCC2 expression is reduced in CD, in both the ileum and colon [29].
rs3805006 in T1D (4)
rs3805006 is located within an intron of ITPR1, and 7 kb upstream of the noncoding RNA gene EGO [75]. ITPR1, together with the related receptors ITPR2 and ITPR3, regulate calcium release within the insulin secretion pathway in pancreatic beta cells [76]. The ITPR3 gene was associated with T1D in a Swedish population [77], although see [78,79].
rs9378249 in BD (0) and HT (0)
This SNP falls within the MHC region on chromosome 6. There is no general association of the MHC region with either BD or HT in the WTCCC data [11], although the region has recently been implicated in schizophrenia [80].
In the cluster plots for rs9378249, the no-calls for BD, HT, and T1D are located in the middle of the heterozygote cluster. This kind of clustering pattern strongly suggests variation between populations in the magnitude of the intensity measurements. Intensity variations could be a result of either somatic gene conversion or somatic deletion in certain populations, assuming in both cases that the control populations have higher intensity than the affected populations.
A diagram of the homology between the two duplicons is given in Figure 4. From this diagram, it becomes apparent that conversion of the lower region by the upper region could eliminate the DHFRP2 sequence entirely.
Figure 4. The structure of the homology between the HLA-B and HLA-C containing duplicons on chromosome 6. Genes and pseudogenes are shown in blue. Corresponding homologous regions are shown in matching shades of green, together with the degree of homology according to the segmental duplication track of the UCSC browser (the two rightmost segments) or Blast (the leftmost segment). The pink region is about 91% homologous to the DHFR region on chromosome 5.
Relatively low raw intensity levels at a locus would be expected if there were a significant number of deletions at that locus in somatic cells. Low intensity at rs7761068, which resides in the putatively deleted region and is the closest SNP to DHFRP2 in the microarray data set, could be interpreted as an indicator of more frequent somatic deletion of the region containing DHFRP2.
To determine a threshold for low/high intensity at rs7761068, the two control populations were pooled and the three genotype clusters were analyzed separately. For the first homozygous cluster, which is close to the y-axis in the cluster plot, the median y-intensity is 1.227. For the second homozygous cluster, which is close to the x-axis in the cluster plot, the median x-intensity is 1.493. For the heterozygous cluster, the median (x + y)-intensity is 1.888. Based on these numbers, an individual is defined to have low intensity at rs7761068 if the x, y, and x + y values are all lower than the corresponding thresholds; otherwise the individual is said to have high intensity at rs7761068. Each of the populations was then partitioned into low and high intensity fractions.
The results shown in Figure 5 strongly suggest that there is increased deletion in all disease populations besides RA. (A 2 × 2 chi-squared test comparing each population with the combined controls yields P = 0.02 for CD, and P < 10-13 for the other five populations.) rs9378249 displays an intensity distribution with features similar to rs7761068 shown in Figure 5, suggesting that deletion due to conversion and/or deletion of the green regions is more likely to be responsible than interactions between the pink region containing DHFRP2 and the region containing DHFR.
Figure 5. Proportion of each of the nine populations having low measured intensity at rs7761068. The intensity thresholds were chosen so that 50% of the combined control population would have low intensity.
DHFRP2 is a pseudogene with homology to DHFR. DHFR codes for dihydrofolate reductase, an enzyme required for the synthesis of thymine nucleotides. Impaired T synthesis causes misincorporation of uracil into DNA, leading to various kinds of DNA damage [81]. While DHFRP2 is noncoding, its mRNA could interact with DHFR mRNA via an antisense regulatory mechanism [82]. The DHFR gene locus shows evidence of both sense and antisense transcription [83], consistent with a role for antisense regulation. (Since interactions of DHFRP2 and DHFR have not been demonstrated, the evidence level of this hypothesis is zero.)
In BD patients, folate sensitive fragile sites are expressed more often than in controls [84]. Polymorphisms in the MTHFD1 gene, which encodes several folate enzymes, are associated with BD [85]. Polymorphisms in the MTHFR gene, which encodes 5,10-methylenetetrahydrofolate reductase, have been associated with HT [86] and BD [87]. High homocysteine levels, which are often associated with folate deficiency, are associated with hypertension [88], and folate supplementation appears to decrease the risk of developing HT [89].
rs841245 in HT (5)
PPFIBP1 encodes the liprin-beta-1 gene, which is highly expressed in the heart [90]. Liprin-beta-1 interacts specifically with the S100A4/Mts1 protein in vivo [91]. The S100A4/Mts1 protein is more highly expressed in individuals with HT, and appears to cause changes in vasculature [92-95].
rs12070036 in BD (5)
ZNF678 has unknown function. It has diverged significantly from all other known zinc-finger proteins [96], and is associated with human variation in height [97].
The chromosome 12 duplicon at 7.2 Mb is located 3.3 kb upstream of PEX5 in a potential promoter region. PEX5 is a gene responsible for recognizing PTS proteins in the peroxisome [98]. Defects in PEX5 cause one of several peroxisome biogenesis disorders, accompanied by reduced plasmalogen biosynthesis in the brain [99,100]. Plasmalogen is a lipid that is abundant in myelin, and peroxisome dysfunction leads to demyelination and axon degeneration in the central nervous system [101]. Somatic mutations in a PEX5 promoter could lead to situations in which some neurons are myelin-deficient, causing aberrant signaling. Demyelination has been previously suggested as a pathogenic mechanism in BD [102], and an association between BD and multiple sclerosis (a demyelinating disease) have been observed [103,104]. Valproate treatment for BD appears to change the behavior of the peroxisome in neurons [105].
rs4988327 in RA (6)
The scatter plot for this SNP is shown in Figure 3.
LRP5 is a member of the canonical WNT5a signaling pathway that is initiated by IL6 in rheumatoid synovial fibroblasts [106]. LRP5 is also associated with bone mineral density and with susceptibility to osteoarthritis [107,108].
rs11010908 in T2D (0)
While there are no characterized genes in the duplicons, two of the duplicons are adjacent, spanning a 370 kb region that includes the genes ANKRD26, YME1L1, MASTL, and ACBD5. ANKRD26-knockout mice develop hyperphagia-induced obesity and insulin resistance [109], as might be expected for a gene associated with T2D.
rs295470 in CAD (5)
The function of ACTG1 appears to be the maintenance of the actin cytoskeleton [110]. A muscle-specific ACTG1-knockout leads to progressive myopathy [111]. Conversely, injection of a human ACTG1 construct (but not constructs based on ACTC1 or ACTG2) into adult rat cardiomyocytes caused a cessation of beating, suggesting a dominant negative effect of overexpression of ACTG1 [112]. ACTG1 appears to play an important role in the structure and normal function of striated muscle [111,113].
RBP2 cDNA is down-regulated by low density lipoprotein, which may be relevant to CAD [114]. RBP2 participates in the uptake and/or metabolism of vitamin A, which is converted to retinol. Low plasma retinol is associated with coronary events [115].
rs2122231 in BD (0) and HT (0)
rs2122231 is located within a region of human ERV9 retroviral sequence. Gene conversion between this sequence and other ERV9 sequence could change ERV9 expression behavior. Variation in ERV9 expression has been associated with psychiatric disorders, including BD and schizophrenia [116,117].
ERV9 long terminal repeat (LTR) sequence also appears in the promoter of the beta globin gene [118]. Disruptions of ERV9 expression could affect beta globin transcription, providing a plausible link to HT. There are many ERV9 LTR sequences in the human genome; in the absence of evidence that this particular region is responsible for ERV9 expression, the evidence level for these associations is 0.
SNP_A-1948953 in HT (3) and BD (3)
LNX proteins including LNX1 interact with members of the Notch signaling pathway that could affect the formation of neuronal cell shape and synaptic connections in the brain [119]. LNX1 interacts with CAST in neurons, and CAST is associated with neurotransmitter release [120]. These properties of LNX1 may be relevant for BD.
LNX1 binds with CXADR, the coxsackievirus and adenovirus receptor [121]. Coxsackievirus seroprevalence has been associated with HT [122].
Interestingly, LNX1 RNA is a much closer match to the SNP_A-1948953 duplicon than the LNX1 DNA; there are gaps in homology that coincide with the LNX1 introns.
rs9839841 in CD (4)
The duplicon for this SNP is on the Y-chromosome, suggesting that gene conversion should be observed only in males. The rs9839841 SNP is a C/T polymorphism on chromosome 3. The corresponding Y-chromosome locus has a 35 bp flanking sequence that is identical to the chromosome 3 sequence containing the T allele. As a result, the microarray will show a base intensity for the T allele that is higher in males than in females. One should thus interpret the scatter plots and clustering results with caution, as they may be influenced by the relative frequency of each gender in the population. In support of a true CD association at this locus for males, Figure 6 shows a scatter plot limited to males for the CD, 58C and NBS populations. The CD population shows a higher spread despite having approximately the same number of data points as each control population.
Figure 6. Cluster plot for males at the rs9839841 locus. The populations are CD (788 males), 58C (752 males), and NBS (720 males). Note the higher spread of the data points in CD.
RFTN1 modulates T-cell signals, particularly Th17, and influences the severity of autoimmune responses [123]. RFTN1 is also needed for B-cell receptor signal transduction [124]. CD and some other chronic inflammatory diseases are mediated by Th17 cells [125,126].
rs4850057 in T2D (6) and BD (4)
UNC13B expression is reduced in pancreatic beta cells of rat models of T2D [127]. Conversely, overexpression of UNC13B amplifies insulin exocytosis [127]. These results are directly relevant to T2D in which insulin exocytosis is dysregulated [128,129].
UNC13B also modulates neurotransmitter release in neurons [130-132], a pathway relevant for BD.
The HLA region in T1D
It is difficult to separate a conversion signal from the broader association signal for T1D in the MHC region; the MHC region on chromosome 6 has extensive association with T1D [11]. Recent high resolution studies have identified an association signal at the HLA-B locus (but not the HLA-C locus) that is independent of the MHC class-II loci [79]. HLA-C has been linked with T1D when considered in combination with KIR genes that are expressed in natural killer cells [133,134].
There are many plausible ways that disruption of an immunity-related gene could modulate T1D pathogenesis. Gene conversion provides additional candidate hypotheses. For example, gene conversion in the duplicons associated with rs389600 could lead to disruption of HLA-G expression. HLA-G expression is immunoprotective in pancreatic islets [135]. An association between the HLA-G region and T1D has previously been observed [136].
Significance of stringent test associations
The ways that the identified genes appear to be relevant to the corresponding disease are diverse. This diversity makes it difficult to formally quantify the significance of the noted associations. In particular, it might be that any sample of genes from duplicated regions leads to many associations with disease pathways if the literature is examined to sufficient depth. To eliminate this possibility, and to quantify the degree of 'background' association one would expect, I conducted a mock association study.
In the mock study, I identified ten SNPs for each disease. The SNPs were chosen to reside on known segmental duplications from the segmental duplication database. A chi-squared statistic comparing the distributions of AA/AB/BB genotypes in controls and in the disease samples was computed, and the ten SNPs that minimized this statistic were chosen. (The selected SNPs for a disease sample are therefore those whose genotype distributions are closest to the controls.) For each disease I searched for disease associations using the literature in the same way that associations were sought for SNPs selected by the various filters. The details are presented in Tables S15 and S16 in Additional file 1.
The hypothesis being tested is that the associations of the stringent test and the mock test differ in the degree of association to the corresponding disease. The rate at which known evidence was found in the stringent test and the mock study is summarized in Table 6. SNPs in the MHC region for T1D were excluded. A Fisher's exact test of the difference between the stringent filter and mock study at evidence level three gives P < 10-9. Even if one limits the stringent test results to SNPs belonging to duplicons in the segmental duplication database, a Fisher's exact test at evidence level three gives P < 10-8. There are consistent disease associations for 22 of the 28 identified instances, and one can reject the null hypothesis that the observed associations are random.
Table 6. Comparison of the stringent and mock tests.
A permutation test
Another way to assess the significance of the stringent test associations is via a permutation test. By switching the labels of cases and controls with probability 0.5 and applying the stringent test conditions, one can test the null hypothesis that the distribution among cases relative to controls is the same as the distribution of controls relative to cases.
In order to perform this test without manually checking for homology, I limit the analysis to associations in regions of at least 90% homology identified by the segmental duplication database. SNPs in the MHC region for T1D are excluded. With those limitations, there are 16 SNP/disease pairs satisfying the original stringent test. Switching the labels of cases and controls for each disease and SNP yields five qualifying SNP/disease pairs.
Based on this information, it is possible to approximate the permutation test distribution as a binomial distribution with N = 21 and a probability of 0.5. The probability p that one would observe at least 16 associations under such a distribution is 0.013, allowing us to reject the null hypothesis.
The relaxed filter
Seventeen stringent-filter SNPs with homology sufficient to satisfy the segmental duplication database constraints are also returned by the relaxed filter. 65 additional instances covering 50 distinct SNPs survive the relaxed filter. Four of these SNPs are among those identified (for other diseases) using the stringent filters. Four additional SNPs are distinct from those identified by the stringent test, but reside in the same duplicons as SNPs from the stringent test. This data is summarized in Table 7. P values for these associations are given in Tables S2 and S3 in Additional file 1.
Table 7. Additional SNPs identified using the relaxed filter.
By design, the gene associations identified solely by the relaxed filter may include false positives. Nevertheless, several of these associations appear to be plausible for the disease(s), and are promising candidates for further study.
The region containing rs10502407 in chromosome 18 has known associations with bipolar disorder. GNAL, and possibly other genes in this region, are subject to epigenetic regulation, and constitute potential susceptibility genes for BD and schizophrenia [137].
rs3858741 is identified as a gene conversion locus for BD, CD and HT and rs9551988 is associated with T2D. These two SNPs are within the same duplicon. The discussion of rs9551988 for the stringent filter analysis covers the BD, HT, and T2D associations. The NO pathway also appears to be important for CD [138,139].
ALG10B is associated with HT in the stringent filter. The association with CAD in the relaxed filter can also be attributed to elongated QT intervals, as can the association with T1D [140]. ALG10B also appears to modulate K+ current in neurons [141], making the link to BD plausible. rs11053044 is identified as a gene conversion locus in T2D; rs11053044 falls within the ALG10 duplicon. Elongated QT intervals are also observed in T2D [142]. Variants of the pore-forming alpha-subunit potassium channel gene KCNQ1 are associated with reduced insulin secretion and T2D [143], and with forms of the long QT-syndrome [143]. VPS35 is associated with BD and CD. VPS35 appears to regulate Wnt signaling [144]. Wnt signaling is important for the proper structure of the absorptive epithelium of the small intestine [145], a plausible link with CD. The Wnt pathway is also associated with BD [146].
The SNP rs9624808 is identified in T1D by the relaxed test; rs9624808 is in same duplicon as rs4988327. LRP5 has been identified as a susceptibility locus for T1D [147,148].
The SNP rs1291361 associates HTR7 and HEBP1 with BD. HTR7 is a serotonin receptor that mediates impulsive behavior [149], and appears to have variants associated with schizophrenia [150]. HEBP1 appears to function in the brain's response to oxidative stress [151].
PARP4, associated with T2D, is a DNA repair molecule involved in nick sensing [152].
ROCK2, associated with CAD, HT, RA, and T1D, is involved in various functions including actin cytoskeleton organization, and abnormal activation of the ROCK pathway has been associated with CAD and HT [153].
DHFR, associated with T1D, converts dihydrofolate into tetrahydrofolate, a necessary step for the de-novo synthesis of purines. See Figure 4 and the discussion of rs9378249, which is also associated with T1D by the stringent filter.
XCL1 and XCL2 are associated with RA. XCL1 is produced by T cells in RA [154]. XCL1 and XCL2 regulate the movement of cells expressing XCR1 [155], which is upregulated in synovial fluid in RA [156]. The UGT1A molecules, associated with BD, are responsible for metabolizing and/or eliminating a variety of chemicals, including mutagens and toxins [157].
CYP3A4, associated with RA, is involved in vitamin D metabolism [158].
PDSS1 is associated with CAD and BD by the relaxed filter. A germ-line mutation in PDSS1 was identified in two siblings with cardiac disease and mental retardation associated with coenzyme Q10 deficiency [159].
The no-call-only filter
Seventeen stringent-filter associations meet the no-call-only filter condition on the p value; see the pn column of Table S1 in Additional file 1. (Ten of these also satisfy the homology requirements of the no-call-only filter.) Eight relaxed-filter associations meet the no-call-only filter condition; see the pn column of Tables S2 and S3 in Additional file 1. Table 8 shows the remaining 50 associations covering 37 distinct SNPs. One of these SNPs (rs4471699) is among those identified (for other diseases) using the stringent filter. Nine of these SNPs are among those identified (for other diseases) using the relaxed filter. This data is summarized in Table 8.
Table 8. Additional SNPs identified using the no-call-only filter.
Beyond the SNPs already identified by the relaxed filter, the following no-call-only filter associations appear to be promising candidates for future study.
SULT1A3 in BD and T2D. Impaired sulfation has been linked with various neurological diseases [28,160]. Sulfoconjugation of monoamines via SULT1A3 occurs within the brain, and could represent an important detoxification pathway [28,161]. SULT1A3 is important for the degradation of dopamine in neurons [162], and dopamine dysregulation has been linked with both BD [163] and T2D [164].
TRIM48 and TRIM53 in BD, CD, and T2D. TRIM proteins such as TRIM48 are thought to function during the cellular response to viral infection [165].
CENPI in BD, CAD, HT, T1D, and T2D. CENPI is located on the X chromosome, and is essential for proper segregation during mitosis [166]. Disruption of CENPI results in daughter cells having extra/missing chromosomes [166].
HCCA2 (also known as MOB2) in RA and T1D. HCCA2 appears to be important for proper segregation during mitosis [167,168].
MYPT2 in HT. MYPT2 is expressed in the heart and skeletal muscle where it dephosphorylates myosin and is involved in muscle contraction [169,170]. Note that the matching duplicon is on the Y chromosome, meaning that somatic gene conversion could only happen in males.
GPC5 in RA. GPC5 expression appears to be reduced in arthritis [171] and GPC5 is located within a quantitative trait locus for arthritis [172]. A SNP within GPC5 appears to be significant for parovirus-induced arthritis [173]. Polymorphisms in GPC5 also appear to be associated with the response of multiple-sclerosis patients to interferon beta therapy [174], and GPC5 appears to be a risk factor in multiple sclerosis [175].
HSD17B7 in HT. HSD17B7 catalyzes the conversion of estrone to estradiol [176], and also is involved in cholesterol biosynthesis [177]. Estradiol treatment lowers blood pressure in hypertension [178-181]. Disruption of HSD17B7 could lower endogenous estradiol concentrations leading to an increase in blood pressure.
DPY19L2 in BD. In C. elegans, the DPY19 gene is required to properly polarize and orient migrating neuroblasts during development [182].
ITGB2 in RA. The ITGB2 gene encodes the CD18 adhesion molecule present on several kinds of immune cells. CD18 expression is upregulated in macrophages and T-cells in the peripheral blood and synovial fluid of RA patients [183,184].
Cluster plot artifacts
The 58C DNA samples were obtained from cell lines, while the other samples (including the NBS control sample) were obtained directly from blood cells [11]. Genomewide, the samples were statistically similar [11]. Nevertheless, it is conceivable that certain SNPs are systematically affected by the procedures used to establish cell lines. A systematic bias that reduces the no-call rate at a SNP in the 58C population could make other populations appear to have high no-call rates at the SNP relative to the combined controls. A significant difference between the 58C and NBS populations in cluster positions for a SNP could be an indicator of such a bias. At the same time, one cannot exclude the possibility that the reasons for this bias may themselves be related to gene conversion. For example, a cell that has undergone a conversion-induced mutation at a locus may not be viable as a cell-line founder, meaning that only cells with unmutated sequence at that locus will be present in the cell-line samples.
A small number of individuals in the WTCCC data generated outlying low-intensity points at multiple loci in the CAD/RA/NBS cohorts, a probable artifact of different procedures for those cohorts [11]. High no-call rates can also occur at a locus with copy number variation, where there are typically more than three clusters. I therefore visually examined all cluster plots for SNPs identified by the various filters, looking for clear examples of any of these three patterns.
The results are summarized in Table 9. For the stringent filter rows labeled with a 58C disparity, the no-call rate for 58C is less than one third of that for NBS. Four of the seven stringent filter SNPs (rs12070036, rs12381130, SNP_A-1797773, rs9257223) have significantly higher no-call rates than the NBS population alone (P < 0.005 for a one-sided chi-squared test). The remaining three SNPs have no-call rates that are not significantly difierent from the NBS population (P > 0.05). The P value for the stringent filter comparison with the mock study remains below 10-8 at evidence level three even if all stringent filter SNPs in Table 9 are excluded.
Table 9. SNPs with anomalous cluster plots.
The SNP rs7761068 was considered in Figure 5 for the analysis of rs9378249. The proportion of low-intensity individuals at rs7761068 does not segregate with the RA, CAD and NBS populations, and the 58C and NBS populations have similar intensity distributions, suggesting that the observed effect at rs7761068 is not artifactual.
Since each cohort has a different proportion of males, a duplicon on a sex chromosome could skew the cluster plot results in a population specific way. Such skew is clear for rs9839841, where a duplicon is on the Y chromosome, and where 94% of the no-calls in CD are for males. Measurements of the male proportion of no-calls for all of the other stringent filter SNPs were close to the proportions in the population as a whole (data not shown). This observation excludes the possibility that a probe sequence for these SNPs is absent from the reference human genome yet occurs frequently in the population on a sex chromosome.
Linkage
In the present study, concordant observations at several adjacent SNPs were not expected [10], and the analysis did not require such observations. Looking at the 28 SNPs identified by the stringent filter in Tables 2 and 3 retrospectively, one can look for evidence of linkage in the form of a significantly increased no-call rate at SNPs adjacent to the target SNP. Evidence of linkage at the 28 loci, within the SNP resolution available on the microarray platform, is summarized in Table 10.
Table 10. Linkage between stringent-filter SNPs and adjacent SNPs.
These linkage results demonstrate that strong linkage is unusual, and that when it occurs, linkage is typically limited to one neighboring SNP. These results also suggest that linkage is more common in BD and HT than in other conditions.
Somatic deletion
While the filters discussed previously are designed to identify gene conversion, it is possible that they also capture cases of somatic deletion. Somatic deletion at a SNP locus would be indistinguishable from somatic conversion within the flanking sequence of the SNP. Looking at the stringent filter results, approximately half of the loci have pairs of duplicons within a few megabases of each other on the same chromosome. This pattern could lead to deletions through gene conversion, improper recombination, or due to removal of sequence fragments forming hairpin-like structures [185]. Somatic duplication is also possible. For rs12381130 and rs11010908, there is no disease-related gene within any of the duplicons, while disease-related genes do occur between duplicons. (The LRP5 gene resides on the chromosome 11 interval for rs12381130, and the ANKRD26 gene resides on the chromosome 10 interval for rs11010908.) For rs9378249, the data suggest that there is a somatic deletion of the DHFRP2 pseudogene.
There is another kind of deletion that could give rise to results that might appear like gene conversion. Consider a SNP locus in which there exists a duplicon having 100% sequence identity in the flanking sequence. This duplicon would add to the signal of one of the alleles at the SNP locus.(Cross-hybridization with less that 100% identity is possible, but is ignored here.) Assuming the duplicon is not polymorphic, this additive signal would be consistent across individuals. The positions of the clusters would be different from a situation without such a duplicon, but AA/AB/BB clusters would still be able to be differentiated from one another.
Imagine a disease associated phenomenon in which there is increased deletion of the duplicon (but not the SNP region) due to improper recombination. In such a case, there would be a bias towards a loss of signal for the allele that is present in the non-polymorphic duplicon. This is the opposite bias to what one expects from gene conversion of the SNP region by its duplicon (although as discussed in Additional file 1, for conversion of major to minor alleles, such a bias is still possible).
To investigate this possibility, I re-examined the results of the stringent filter to identify cases where there is (a) 100% identity of the duplicon within the SNP's flanking sequence, and (b) a change in the allele distribution away from the allele in the duplicon. There is one such SNP, namely rs9551988, that accounts for three of the five observations (Table S1) where the allele frequency changes away from the allele in the non-polymorphic duplicon. Given the additional information that the duplicons for rs9551988 are 500 kb away from each other on the same chromosome and in the same orientation, it is reasonable to infer that deletion is the likely explanation for the results observed at this locus.
Now imagine a disease associated phenomenon in which there is increased deletion of the SNP region (but not the non-polymorphic duplicon) due to improper recombination. In such a case, there would be a bias towards a relative loss of signal for the allele that is not present in the non-polymorphic duplicon. This is the same bias that one expects from gene conversion of the SNP region by its duplicon. I therefore re-examined the results of the stringent filter to identify cases where there is (a) 100% identity of the duplicon within the SNP's flanking sequence, and (b) a change in the allele distribution towards the allele in the duplicon. There are four such cases, namely rs669980, rs935019, SNP_A-1797773, and rs9839841. Of these, only rs935019 represents a case with nearby aligned duplicons on the same chromosome. For rs935019, variation in copy number has been observed in cloning experiments [65], suggesting that deletion is the most likely explanation for this locus.
An additional example was observed during the examination of SNPs using BLAST to determine whether they reside in a region with homology elsewhere in the genome. rs2812 met the stringent filter conditions for CAD except that it did not reside in a duplicated region. Nevertheless, a 400 bp duplicon occurred both upstream and downstream of rs2812, together spanning a 2 kb region including the SNP. rs2812 is located within the PECAM1 gene, which has previously been associated with CAD [186-188]. Out of approximately 250 SNPs that were examined in this way, rs2812 was the only one for which this kind of duplication pattern was observed. Nevertheless, the present study was not designed to identify such patterns, and additional longer-range (or inter-chromosomal) duplication that increases the likelihood of sequence deletion may exist.
Known de-novo non-allelic conversion sites
Five pairs of genes have been identified as loci of de-novo germ-line gene conversion between non-allelic regions, leading to a disease phenotype [1]; see Table 11. If these conversion events are frequent enough to be noticed even in the germline, then such loci may be likely to be sites of relatively frequent somatic conversion. I therefore examine SNPs located in duplicons related to these gene pairs to determine whether the cluster plots support this hypothesis.
Table 11. De-Novo conversion events in disease [1].
I consider all SNPs appearing in one of the two duplicons shared by the two genes. Coverage is limited by the resolution of the microarray. In fact, no SNPs are available for the CYP21A1P/CYP21A2 genes. For the SBDSP/SBDS pair, there are four almost-contiguous segmental duplications in the segmental duplication database, spanning just over 500 kb. I consider all SNPs in all of the four duplicons. I visually inspected the cluster plots for the SNPs in the corresponding duplicons. The target pattern is one in which for every population (including controls) there is a substantial number of points between clusters. The results of the visual cluster plot analysis are summarized in Table 12. The visual analysis is supported by the WTCCC quality control procedures: for seven of the nine identified SNPs (all except rs6578592 and rs1465306) the SNP was excluded for quality control reasons such as departure from HWE in the control population. (One additional SNP, rs1880278, was also excluded for quality control reasons but did not show features predicted for gene conversion.)
Table 12. Possible conversion in duplicons for genes previously observed to have undergone germ-line conversion.
Given the small sample size and sparse coverage of the duplicons, the results of Table 12 are suggestive, but far from definitive.
Disease-specific patterns
Based on the SCE data, RA was predicted to be a local disease. Four SNPs that are associated with RA (rs4988327, rs10768666, rs4236384, rs9976299) have cluster plots in which RA alone has an increased number of no-calls. When other disease populations have correlated behavior, the RA population sometimes appears to remain close to the control population, as exemplified in Figure 5. In contrast, no other disease population has an associated SNP for which that population alone has an increased no-call rate.
These results are broadly consistent with a view of RA as a local disease, and of the remaining diseases as global diseases. The distinction is not clear-cut, however, since there are RA-associated SNPs with no-call behavior that is similar across multiple diseases.
An alternative interpretation of the distinctness of RA is based on the observation that lymphocytes may be the initiators of RA pathogenesis. Since lymphocytes are the cells being genotyped, lymphocyte-specific autoimmune dynamics could amplify the signal attributable to pathogenic mutations. For example, a mutation in a T cell that leads to cell activation and replication would substantially increase the population of cells exhibiting the mutation. Of the four SNPs showing RA-specific spread in the cluster plots, rs9976299 is notable for being within the ITGB2 gene which encodes the CD18 adhesion molecule. CD18 expression is upregulated in macrophages and T-cells in the peripheral blood and synovial fluid of RA patients [183,184].
BD and HT co-occur at four different stringent-filter SNPs. Three of these SNPs display similar linkage patterns with neighboring SNPs for both BD and HT. These factors suggest that BD and HT may have a common ultimate cause that is different from the other five diseases. A general similarity between HT and BD has previously been identified using a classification algorithm over the same WTCCC data set [189]. Individuals with BD have a more than twofold increased risk of HT [190].
Discussion
Based on prior data for loci such as IDS [3,4], disease related genes were sought in one of several duplicons, only one of which contains the identified SNP. For 8 out of the 28 stringent filter SNPs, the disease related gene is on a duplicon not containing the SNP, emphasizing the importance of examining all duplicons. Such genes would not be identified using a conventional association study.
Confounding factors could perturb cluster plots, potentially leading to false associations. Loci that did not meet the WTCCC quality control requirements have been excluded. The WTCCC reports a disparity between the NBS/RA/CAD cohorts and the other cohorts for some SNPs [11]; such disparities are rare among the SNPs meeting the filter conditions (Table 9). Additional quality control issues not identified by the WTCCC are possible. Nevertheless, it is hard to imagine how a quality control artifact could lead to population-specific effects that correlate with disease related genes.
Copy-number variation can be discounted as a general explanation for the observed phenomena, since none of the stringent test SNPs (and only one of the no-call SNPs) showed more than three clusters. Further, few of the stringent filter SNPs are within known CNV loci (Additional file 1). Even if copy number variation was the mechanism responsible for some of the present results, the results would still be interesting as novel cohort-specific associations.
The present paper provides support for the hypothesis that many complex diseases are caused in part by somatic mutation in regions with homology elsewhere in the genome. Diseases such as cancer often display gross karyotypic changes that could be due to improper recombination between nonallelic homologous regions in somatic tissue. Because detection of somatic mutations is technically much more demanding than that of germline mutations, somatic gene-conversion events in cancer have probably been underestimated [1].
Some puzzling epidemiological features of autoimmune diseases are consistent with a somatic mutation hypothesis. Association with viruses can be explained by the mutagenic actions of those viruses. Associations of autoimmune disease with higher latitudes has been hypothesized to relate to lower vitamin D levels [191]; vitamin D is associated with lower rates of double-strand breaks [192] and with protection from viral infections [193]. Complex inheritance patterns spanning multiple diseases would result from a common underlying genetic susceptibility based on sequence homology, combined with stochastic effects such as tissue-specific viral infection.
In order to be identified as a conversion region in this study, the region must contain a locus that is within the SNP repertoire of the microarray chip. A substantial amount of somatic gene conversion might affect loci with alleles that are fixed in the population. If so, alternative platforms will be needed to detect such conversion. It is likely that there is additional disease-specific somatic gene conversion that the present study has not detected even among the covered SNPs. Spread in the cluster plots might not be apparent if a particular disease-causing somatic mutation was rare enough that the perturbation was small relative to experimental variation.
On the other hand, common gene conversion events might preferentially include SNP loci. If a conversion event is common in somatic tissues, it may also be relatively common in the germ-line. If the germ-line event is not deleterious, a polymorphism could result. The consequences of somatic and germ-line changes are different, and a somatic mutation may cause disease where a germ-line change does not. For example, a somatic mutation may result in a novel protein that is immunogenic. Alternatively, some of the loci associated with a conversion event may be phenotypically neutral, and these may lead to polymorphisms as a result of partial conversion events in the germ-line.
The phenotype of a somatic mutation is likely to be very different from the phenotype of a germ-line mutation. Outside of cancer, there is very little data about phenotypes associated with somatic mutations. It is therefore dificult to correlate the observations of this paper with existing knowledge about somatic mutation. Correlations with genomewide studies of disease associated polymorphisms are possible in principle. However, given the methodologies used in those studies (for example, requiring multiple concordant SNPs [11]), it is not expected that correlations will be found given the absence of linkage disequilibrium for gene conversion [10].
It may well be that somatic gene conversion is, in some cases, a normal adaptive phenomenon. Such effects might be detectable using SNP microarrays by examining the intensity plots directly without employing a calling algorithm. The quality control protocols of SNP array studies typically exclude loci where the called allele frequencies depart from HWE in the control population, which would exclude loci for which somatic gene conversion was common. It may be worth re-examining such loci, particularly those in duplicated regions.
The present report suggests that somatic gene conversion is associated with mutations and genomic rearrangements that lead to disease. Working backwards, one could generate hypotheses for further study by identifying genomic regions with high degrees of homology that contain disease-relevant genes. For example, the BRCA1 gene that is involved in DNA stability and repair pathways [194] itself contains a segmental duplication that includes part of the gene and its promoter region [195,196]. Some BRCA1-related cancers appear to be caused by gene conversion events in individuals carrying one mutant BRCA1 allele [197]. Once BRCA1 function is compromised, gene conversion and rearrangement at other loci may become more frequent.
Gene conversion could be a cause of the disease phenotype, or it could alternatively be a side-effect of an underlying disease-causing genetic disorder with no direct bearing on the phenotype. The fact that disease associations are found for most of the stringent filter SNPs is strongly suggestive of a causative link in which the specific conversion events are the proximate causes of the phenotype.
I have used the output of the Chiamo algorithm without modification. Spread is inferred from a high number of no-call results at a locus. While this method of inferring spread appears to have been effective, more effective methods might be possible. Methods could measure suitably defined 'spread statistics' given allele intensity distributions for several populations.
The success of the analysis supports the hypothesis suggested by the sister chromatid exchange studies that DNA in lymphocytes undergoes similar transformations to DNA in tissues affected by disease. In principle, it may be possible to test for various somatic mutations using a blood sample. Specialized microarrays could be developed to detect specific sequences resulting from common somatic mutations.
Several important questions remain. The present study does not allow the quantification of risk associated with any particular gene conversion locus. Even the identification of which individuals have substantial conversion at a locus is approximate. Locus-specific experimental studies of conversion frequencies in health and disease are needed.
The present study also does not quantify the degree of conversion necessary to cause disease. In lymphocytes, for example, mutations in a very small number of cells could cause disease if those cells undergo clonal expansion. In other tissues, many cells might need to be mutated before tissue function is compromised. Stem cell mutations (which may be relatively common due to frequent mitosis) could lead to a regular inflow of mutated cells.
Disease associations with a number of specific genes have been suggested by the present work. Changes at these loci in somatic tissues may represent the proximate cause of disease. Nevertheless, the ultimate cause of disease is the factor that causes the DNA damage. Environmental factors are likely to play a significant role. The association of the folate-dependent thymine nucleotide synthesis pathway with several diseases, together with an increase in the frequency of SCEs under methotrexate treatment [198], also suggests another kind of ultimate cause in which impaired DNA synthesis leads to homology-driven repair [199].
Conclusions
That somatic gene conversion may occur frequently has been previously suggested, but progress has been hampered by the technical difficulty of measuring somatic gene conversion on a large scale [1]. The present study is the first to use genome-scale SNP data to infer somatic gene conversion loci in specific populations. For more than 75% of the loci, genes within the locus associate with the corresponding disease in a manner consistent with known gene/disease associations.
Any single association identified in this report should be considered tentative, and subject to experimental confirmation. Nevertheless, taken together, the associations provide compelling evidence that somatic gene conversion and/or somatic deletion at particular loci influence each of the seven studied diseases. The techniques developed are not specific to the WTCCC data, and could be applied to other data sets to identify putative gene conversion for other diseases.
Abbreviations
BD: bipolar disorder; bp: base pair; CAD: coronary artery disease; DSB: double strand break; HT: hypertension; kb: kilobases; RA: rheumatoid arthritis; SCE: sister chromatid exchange; SNP: single nucleotide polymorphism; T1D: type-1 diabetes; T2D: type-2 diabetes; WTCCC: Wellcome Trust Case Control Consortium.
Competing interests
The author declares that he has no competing interests
Acknowledgements
This work was funded by the NIH under award U54-CA121852. The NIH played no role in the study design, data collection/analysis, or in the decision to submit the manuscript for publication. This study makes use, with permission, of data generated by the Wellcome Trust Case Control Consortium. A full list of the investigators who contributed to the generation of the data is available from http://www.wtccc.org.uk. Funding for the WTCCC project was provided by the Wellcome Trust under award 076113. Access to the data was approved by an Institutional Review Board at Columbia University. The author thanks Itsik Pe'er and Martin Lindquist for helpful discussions.
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Pre-publication history
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1741-7015/9/12/prepub
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And what did I see?: why has devised hpricot, a permissive HTML parser with tree-crawling functions. It's written in C, so it's fast. Just another great piece of software named after apricots.
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You are here: Home / News / EEA invites submissions of evidence on global trends to support forthcoming assessments
EEA invites submissions of evidence on global trends to support forthcoming assessments
Published : Jul 07, 2009 Last modified : Apr 13, 2011 07:17 PM
Understanding the state of Europe's environment and its future prospects is impossible without an appreciation of the situation and trends outside the continent. Many environmental issues are inherently transboundary and are influenced by numerous other international forces, including social, technological, economic and political interaction. At the same time, many global socio-economic drivers operate over decades, necessitating a long-term perspective.
For these reasons, forthcoming EEA assessments, including the next European State of the Environment and Outlook Report (SOER), to be released at the close of 2010, will address long-term global interlinkages. One section of SOER 2010 will comprise an exploratory assessment of long-term global mega-trends, driving forces and uncertainties that will shape Europe's environment and policies over coming decades.
To support this novel assessment, EEA invites interested scientific and research communities and organisations, NGOs and the private sector to submit evidence on:
• key global mega-trends;
• their drivers and certainties and uncertainties;
• how these mega-trends might interact; and
• what environmental consequences they might imply for Europe.
If you think you may have relevant evidence, please follow this link: Call for evidence.
Any information used in the SOER or other EEA assessments will be referenced appropriately. Please note, however, that the submission of evidence is voluntary, not subject to any compensation and for the exclusive purpose of informing EEA analysis.
While the EEA is entitled to make unrestricted use of the submissions (unless explicit confidentiality restrictions are imposed), it is under no obligation to do so. Parties will not be informed unless their evidence is being used in an EEA assessment.
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European Environment Agency (EEA)
Kongens Nytorv 6
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Denmark
Phone: +45 3336 7100
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Carbon
Carbon
Ultrapure carbon as graphite. Original size in cm: 1 x 3. Source: images-of-elements.com
This article has been reviewed by the following Topic Editors: Jay Gulledge, J. Emmett Duffy, Andy Jorgensen
Carbon is the sixth most abundant element in the universe and is unique due to its dominant role in the chemistry of life and in the human economy. It is a nonmetallic element having the symbol C, the atomic number 6, an atomic weight of 12.01115, and a melting point about 360ºC. There are four known allotropes of carbon: amorphous, graphite, diamond, and fullerene. A new fifth allotrope of carbon was recently produced, a spongy solid called a magnetic carbon “nanofoam” that is extremely lightweight and attracted to magnets.
Previous Element: Boron
Next Element: Nitrogen
6
C
12.011
Physical Properties
Color black
Phase at Room Temp. solid
Density (g/cm3) 2.266
Hardness (Mohs) 0.8
Melting Point (K) 3823.2
Boiling Point (K) ---
Heat of Fusion (kJ/mol) ---
Heat of Vaporization (kJ/mol) ---
Heat of Atomization (kJ/mol) 717
Thermal Conductivity (J/m sec K) 1.59
Electrical Conductivity (1/mohm cm) 0.727
Source Coal, Petroleum, Natural gas
Atomic Properties
Electron Configuration [He]2s22p2
Number of Isotopes 3
Electron Affinity (kJ/mol) 121.85
First Ionization Energy (kJ/mol) 1086.4
Second Ionization Energy (kJ/mol) 2352.6
Third Ionization Energy (kJ/mol) 4620.4
Electronegativity 2.55
Polarizability (Å3) 1.8
Atomic Weight 12.011
Atomic Volume (cm3/mol) 5.3
Ionic Radius2- (pm) ---
Ionic Radius1- (pm) ---
Atomic Radius (pm) 77.2
Ionic Radius1+ (pm) ---
Ionic Radius2+ (pm) ---
Ionic Radius3+ (pm) ---
Common Oxidation Numbers -4, +4
Other Oxid. Numbers -3, -2, -1, +1, +2, +3
Abundance
In Earth's Crust (mg/kg) 2.00×102
In Earth's Ocean (mg/L) 2.8×101
In Human Body (%) 22.85%
Regulatory / Health
CAS Number 7440-44-0 synthetic
7782-42-5 natural
OSHA Permissible Exposure Limit (PEL) TWA: 15 mppcf
OSHA PEL Vacated 1989 TWA: 2.5 mg/m3
NIOSH Recommended Exposure Limit TWA: 2.5 mg/m3
IDLH: 1250 mg/m3
Sources:
Mineral Information Institute
Jefferson Accelerator Laboratory
History
The name derives from the Latin carbo, for "charcoal". It was known in prehistoric times in the form of charcoal and soot. In the year 1797, the English chemist Smithson Tennant proved that diamond is pure carbon. It is found in abundance in the sun, stars, comets, and atmospheres of most planets. Carbon in the form of microscopic diamonds is found in some meteorites.
Natural diamonds are found in kimberlite of ancient volcanic "pipes," found in South Africa, Arkansas, and elsewhere. Diamonds are now also being recovered from the ocean floor off the Cape of Good Hope. About 30% of all industrial diamonds used in the U.S. are now made synthetically.
The energy of the sun and stars can be attributed at least in part to the well-known carbon-nitrogen cycle.
Uses
Due to carbon’s unusual chemical property of being able to bond with itself and a wide variety of other elements, it forms over 10 million known compounds. Carbon is present as carbon dioxide in the atmosphere and dissolved in all natural waters. It is a component of rocks as carbonates of calcium (limestone), magnesium and iron.
The fossil fuels (coal, crude oil, natural gas, oils sands, and shale oils) are chiefly hydrocarbons. Carbon is the active element of photosynthesis and the key structural component of all living matter. The isotope carobon-12 is used as the basis for atomic weights. Carbon-14, a radioactive isotope with a half-life of 5,730 years, is used to date such materials as wood and archaeological specimens. In 1960, W.F. Libby was awarded the Nobel Prize in Chemistry for developing the carbon dating method.
Organic chemistry, a major subfield of chemistry, is the study of carbon and its compounds. Because carbon dioxide is a principal greenhouse gas, the global carbon cycle has become a focus of scientific inquiry in relation to global warming, and the management of carbon dioxide emissions from the combustion of fossil fuels is a central technological, economic, and political concern; furthermore, imbalances to the carbon cycle due to deforestation, overgrazing, peatland exploitation and other land cover changes are thought to be significantly implicated in climate change. Methane, CH4, is another important carbon compound that is also a significant greenhouse gas.
Forms
Carbon atom.
Carbon is found free in nature in three allotropic forms: amorphous, graphite, and diamond. A fourth form, known as "white" carbon, is now thought to exist. Ceraphite is one of the softest known materials while diamond is one of the hardest.
Graphite exists in two forms: alpha and beta. These have identical physical properties, except for their crystal structure. Naturally occurring graphites are reported to contain as much as 30% of the rhombohedral (beta) form, whereas synthetic materials contain only the alpha form. The hexagonal alpha type can be converted to the beta by mechanical treatment, and the beta form reverts to the alpha on heating it above 1000°C.
In 1969 a new allotropic form of carbon was produced during the sublimation of pyrolytic graphite at low pressures. Under free-vaporization conditions above ~ 2550K, "white" carbon forms as small transparent crystals on the edges of the planes of graphite. The interplanar spacings of "white" carbon are identical to those of carbon form noted in the graphite gneiss from the Ries (meteroritic) Crater of Germany. "White" carbon is transparent birefringent material.
Carbon found in organic molecules—molecules that contain carbon atoms bonded to hydrogen atoms and to other carbon atoms—is called organic carbon. Carbon is the most abundant element found in organisms. For this reason, carbon is considered the fundamental building block of all life. Plants acquire carbon from the atmosphere through photosynthesis. Using inorganic carbon in the form of carbon dioxide (CO2) from the atmosphere and energy from sunlight, plants convert CO2 to organic carbon as they produce stems, leaves, and roots. Carbon may also be converted from inorganic to organic forms using chemical energy in the absence of light by chemoautotrophs. Heterotrophs—organisms such as animals, fungi, and many types of bacteria that cannot synthesize their own food from carbon dioxide—obtain their carbon from organic compounds.
Compounds
In combination, carbon is found as carbon dioxide (CO2) in the atmosphere of the Earth and dissolved in all natural waters. It is a component of great rock masses in the form of carbonates of calcium (limestone), magnesium, and iron. Coal, petroleum, and natural gas are chiefly hydrocarbons.
Carbon is unique among the elements in the vast number and variety of compounds it can form. With hydrogen, oxygen, nitrogen, and other elements, it forms a very large number of compounds, carbon atom often being linked to carbon atom. There are over ten million known carbon compounds, many thousands of which are vital to organic and life processes.
Without carbon, the basis for life on Earth would not be possible. While it has been thought that silicon might take the place of carbon in forming a host of similar compounds, it is now known that stable compounds with very long chains of silicon atoms cannot be formed. The atmosphere of Mars contains 96.2% CO2. Some of the most important molecular compounds of carbon are carbon dioxide (CO2), carbon monoxide (CO), carbon disulfide (CS2), chloroform (CHCl3), carbon tetrachloride (CCl4), methane (CH4), ethylene (C2H4), acetylene (C2H2), benzene (C6H6), acetic acid (CH3COOH), and their derivatives.
Isotopes
Carbon has many isotopes, but just three are stable enough to exist in detectable amounts in nature. Carbon-12, a stable (non-radioactive) isotope, comprises nearly 99% of all carbon on Earth. In 1961 the International Union of Pure and Applied Chemistry adopted the isotope carbon-12 as the basis for atomic weights. Carbon-13, also a stable isotope, is the next most abundant, comprising slightly more than 1% of all carbon on Earth. Carbon-14 is the most abundant radioactive isotope of carbon at 1 part per trillion. It has a half life of 5730 years and has been widely used to date such materials as wood, archaeological specimens, etc, through radiocarbon dating. All other isotopes of carbon are highly unstable and extremely rare.
Carbon Cycle
Carbon is conveyed among features of the lithosphere, biosphere, atmosphere and oceans; in addition it is transformed to different molecular forms as well as physical forms. The composite of all these transformations is termed the carbon cycle. The most important forms of carbon in the Earth's atmosphere are carbon dioxide, carbon monoxide, methane and black carbon; these forms are variously important as plant metabolite (carbon dioxide, black carbon); toxic agent (carbon monoxide, black carbon) and radiative forcing agent (methane, carbon dioxide, black carbon).
Carbon is stored on in the following major dynamic sinks: (a) as organic molecules in living and dead organisms found in the biosphere; (b) in gas and particulate form in the atmosphere; (c) as organic matter in soils; (d) in the lithosphere as fossil fuels and sedimentary rock deposits such as limestone, dolomite and chalk; and (e) in the oceans as dissolved atmospheric carbon dioxide and as calcium carbonate shells in marine organisms; and as methane clathrates, deep frozen methane formations under circumpolar seabeds.
Carbon moves from the atmosphere to the biosphere chiefly by the process of photosynthesis; conversely carbon moves from the biosphere to the atmosphere by several processes, including: burning of organic matter; methane emission from ruminants; deforestation, with some carbon lost to the atmosphere via decay, soil carbon disturbance loss or forest product use; and decay of organic matter, with a fraction of decayed matter being released to the atmosphere.
Other sizable sinks of carbon are the oceans themselves, seabed carbonates, biota and decaying matter present in soils, methane clathrates. These are very large carbon sinks, even compared to the atmosphere; furthermore, the status of research on their size and dynamics is embryonic, such that important new perspectives are likely to materialize on these sinks over the next decade.
Biological Implications
Carbon is a critical element to all life. It is one of the six bulk elements and is the second-most common element in the human body. By mass it is the most abundant constituent of all the major molecules that organisms are formed from, including nucleic acids (e.g., DNA), proteins, carbohyrdrates, and lipids. As a result, living organisms are intimately involved in the carbon cycle. . Some carbon compounds such as carbon monoxide (CO) or the cyanide ion CN- pose health and mortality risks to most fauna including humans.
Further Reading
• D.R.Lide, ed, (2005). CRC Handbook of Chemistry and Physics (86th ed.). Boca Raton (FL): CRC Press. ISBN 0-8493-0486-5.
• A.J.A.Janse (2007). Global Rough Diamond Production Since 1870. Gems and Gemology (GIA) XLIII (Summer 2007): 98–119.
• D.Gorman, A.Drewry, Y.L.Huang & C.Sames (2003). The clinical toxicology of carbon monoxide. Toxicology 187 (1): 25–38.
• The Chemistry of Carbon, New York University.
• Understanding the Global Carbon Cycle, The Woods Hole Research Center.
• P.Falkowski, R.J.Scholes, E.Boyle, J.Canadell, D.Canfield, J.Elser, N.Gruber, K.Hibbard et al. (2000).The Global Carbon Cycle: A Test of Our Knowledge of Earth as a System. Science 290 (5490): 291–296
• T.M.Smith, W.P.Cramer, R.K.Dixon, R.Leemans, R.P.Neilson, A.M.Solomon, A. M. (1993). The global terrestrial carbon cycle. Water, Air, & Soil Pollution 70: 19–37.
• Joel S.Levine, Tommy R.Augustsson, Murali Natarajan (1982). The prebiological paleoatmosphere: stability and composition. Origins of Life and Evolution of Biospheres 12 (3): 245–259
Citation
Cutler J. Cleveland (Lead Author);C Michael Hogan (Contributing Author);Jay Gulledge, J. Emmett Duffy, Andy Jorgensen (Topic Editor) "Carbon". In: Encyclopedia of Earth. Eds. Cutler J. Cleveland (Washington, D.C.: Environmental Information Coalition, National Council for Science and the Environment). [First published in the Encyclopedia of Earth November 18, 2008; Last revised Date October 3, 2011; Retrieved May 18, 2013 <http://www.eoearth.org/article/Carbon>
The Author
Cutler J. Cleveland is Professor of Earth and Environment at Boston University, where he also is on the faculty of the Center for Energy and Environmental Studies. Professor Cleveland is Editor-in-Chief of the Encyclopedia of Energy (Elsevier, 2004), winner of an American Library Association award, the Dictionary of Energy (Elsevier, 2005), Handbook of Energy (Elsevier, forthcoming), and is the Founding Editor-in-Chief of the Encyclopedia of Earth. He is the recipient of the Adelma ... (Full Bio)
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Ecoregions of Bulgaria
Ecoregions of Bulgaria
Oil cape, Bulgaria. Source: Wikimedia Commons
This article has been reviewed by the following Topic Editor: Peter Saundry
Bulgaria has sixecoregions that occur entirely or partly within its borders:
1. Balkan mixed forests covers most of Bulgaria
2. East European forest steppe
3. Pontic steppe
4. Euxine-Colchic deciduous forests (only a very samll part of Bulgaria is included in this ecoregion)
5. Rodope montane mixed forests
6. Aegean and Western Turkey sclerophyllous and mixed forests extends into Bulgaria in the far south east corner of the country
Balkan mixed forests
The Balkan mixed forests ecoregion covers much of Bulgaria and bordering countries, excluding the Rodope Mountains. The vegetation of this ecoregion, especially that of the forests and grasslands, is Central European in character.
The diversity of flora and fauna is relatively high compared to the rest of Europe and there are a high number of endemic plant species. Mixed oak forests are characteristic, with Quercus frainetto as the dominant tree species. Oak forests are interspersed with pine, silver fir (Abies alba) and Norway spruce (Picea abies) forests, woodland-pastures, shiblyak and grasslands. High valleys and sheltered slopes feature forests dominated by beech (Fagus sylvatica) and hornbeam (Carpinus orientalis and C. betulus). The region’s herpetofauna is among the most diverse in Europe.
The ecoregion has a good network of protected areas; however, the changing political climate threatens them with fragmentation
East European forest steppe
Pontic steppe
Euxine-Colchic deciduous forests
Extending along the southern Black Sea coast, the vegetation in the Euxine-Colchic deciduous forests ecoregion ranges from temperate rainforest to coastal bottomland forests, peatlands and coastal sand dunes. Primarily is Turkey, the ecoregion extends acros the border to the southeastern tip of Bulgaria. The old-growth forests are home to one of the largest brown bear populations in Europe, and the migratory populations of many waterfowl, passerines, and raptors fly through the eastern end of the region. The draining of wetland habitat for agriculture, logging, and poaching are among the greatest threats to the flora and fauna.
Rodope montane mixed forests
Rodope Mountain, Greece Photograph by © WWF-Canon/Michel Gunther The Rodope Montane Mixed Forests Ecoregion is composed of the Balkan (Stara Planina) and Rhodope Massifs in the central Balkan Peninsula.
Central European in character, mixed deciduous forests (Fagus sylvatica, Carpinus orientalis, C. betula, Quercus spp.) grow on mountain slopes while the higher elevations are dominated by conifers (Abies alba, Picea albies, Pinus nigra).
On the highest peaks, forests are replaced by heaths and alpine grasslands. It is estimated that the flora of the region includes about 3,000 vascular plant species. Many are endemics from the Pleistocene glaciation, as the region served as a refuge for species that never re-established to the north.
The position of the ecoregion at the crossroads of several floristic elements (European, Alpine, and Mediterranean) also enhances floral diversity . Several of Europe’s threatened fauna species are found here such as otter (Lutra lutra), pine marten (Martes martes), imperial eagle (Aquila heliaca), cinereous vulture (Aegypius monachus), and ferruginous duck (Aytha nyroca).
Although there is a good network of protected areas, the ecoregion faces many threats from the changing political climate, expanding agriculture, and increasing tourism.
Aegean and Western Turkey sclerophyllous and mixed forests
Situated in parts of Turkey, Greece, (and entending into a small part of the southwestern most corner of Bulgaria) and on some of the the Aegean Sea islands, the Aegean and Western Turkey sclerophyllous and mixed forests ecoregion enjoys a Mediterranean climate and encompasses islands, coastal areas and some inland plains. It still supports a few areas of pine forest, and hosts rare and endemic species such as oriental sweetgum and cretan palm. The endangered loggerhead turtle nests here, and fox, wolf and wild boar are included among its mammal populations. Many resident and migratory birds are found here, including threatened species such as the pygmy cormorant, Dalmatian pelican, white-headed duck, and the lesser kestrel. The dense human populations that have inhabited this ancient area of civilization have severely degraded most of the original habitat, beginning in the earlier Holocene, and accelerating in more modern times with the recent human population explosion. .
See also:
Context
Ecoregions are areas that:
[1] share a large majority of their species and ecological dynamics;
[2] share similar environmental conditions; and,
[3] interact ecologically in ways that are critical for their long-term persistence.
Scientists at the World Wildlife Fund (WWF), have established a classification system that divides the world in 867 terrestrial ecoregions, 426 freshwater ecoregions and 229 marine ecoregions that reflect the distribution of a broad range of fauna and flora across the entire planet.
Citation
World Wildlife Fund (Lead Author);Peter Saundry (Topic Editor) "Ecoregions of Bulgaria". In: Encyclopedia of Earth. Eds. Cutler J. Cleveland (Washington, D.C.: Environmental Information Coalition, National Council for Science and the Environment). [First published in the Encyclopedia of Earth July 21, 2012; Last revised Date September 27, 2012; Retrieved May 18, 2013 <http://www.eoearth.org/articles/view/178214/Greece/>
The Author
Known worldwide by its panda logo, World Wildlife Fund (WWF) leads international efforts to protect endangered species and their habitats. Now in its fifth decade, WWF works in more than 100 countries around the globe to conserve the diversity of life on Earth. With nearly 1.2 million members in the U.S. and another 4 million worldwide, WWF is the world's largest privately financed conservation organization. WWF directs its conservation efforts toward three global goals: 1) saving endangered ... (Full Bio)
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From Forensics Wiki
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Follow-up Workshop for Digital Forensics Educators
This is a short note to invite you back to the Follow-up Workshop for Digital Forensics Educators at Erie Community College, Tuesday, August 16, 2011 from 9-4. If you hadn’t participated last year…you’re still invited.
Dr. Garfinkel has requested that we have a more interactive workshop with participants sharing their methodologies and course work with the group. This would facilitate more sharing amongst colleagues and give him a better focus regarding the forensics corpora course integration.
We would like to start lining up presenters so that the program can start taking shape. The format for sharing can be as simple as a short talk about your program or PPTs or group activities. Please respond with what you would like to do. I look forward to seeing you in August. Regards, Donna
Registration Link (http://kuroski.net/Forensics2011/login.php)
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Go4Expert Member
8Apr2008,22:11 #21
Its wonderful how such an apparently trivial problem of finding prime numbers is turning into such an animated discussion on various algorithmic techniques - each with its own benefits & pitfalls!
Wikipedia's page on the prime number is as exhaustive as its page on human evolution! It just goes to show how fascinating Mathematics is that it throws a challenge in something as apparently simple as finding primes.
It also demonstrates the importance of algorithmic design & analysis for effective computing - even the biggest super-computer on the planet cannot achieve its true computational potential if the algorithm involves excessive looping or is inefficient!
Also, a special thanks to Niraj (friendsofniraj), who has given an extremely succint explanation of how the Square Root logic works - there was no need for me to read a single word on Wiki about the algorithm (although I did to satiate my curiosity - and might I say, it is very thoroughly dissected in Wikipedia!) And also the keen eye of Shabbir noticed the redundancy in the calculation of Square Root through the inner-loop!
All in all - a very fascinating discussion!
Regards,
Rajiv
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Hi to all! Nice to be here
Newbie Member
24Jan2008,08:38 #1
Just found this forum lately while doing some shift for LAMP architecture. I am an Oracle DBA / Developer who wants to see and feel the new rush on opensource tools..
Go4Expert Founder
24Jan2008,09:19 #2
Hi and to the forum
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Array problems to use the Koch L-system rule.
Light Poster
25Jul2011,18:34 #1
Hi,
I have a problem with recusion for my project. This is what it must do:
when my funtion is executed once it should output a char 'F' then when it recurs once again, every F ---> F+F-F-F+F so that when it recurs 3 times the output becomes
F+F-F-F+F + F+F-F-F+F - F+F-F-F+F - F+F-F-F+F + F+F-F-F+F
help.
Light Poster
25Jul2011,20:04 #2
Maybe I should use an array of strings instead of characters. . .
What do you suggest?
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About this Journal Submit a Manuscript Table of Contents
Anatomy Research International
Volume 2012 (2012), Article ID 837626, 9 pages
doi:10.1155/2012/837626
Review Article
Origin and Regenerative Potential of Vertebrate Mechanoreceptor-Associated Stem Cells
1Department of Cell Biology, University of Bielefeld, Universitätsstraße 25, 33501 Bielefeld, Germany
2Department of Molecular Neurobiology, University of Bielefeld, Universitätsstraße 25, 33501 Bielefeld, Germany
Received 9 July 2012; Accepted 4 September 2012
Academic Editor: David Yew
Copyright © 2012 Darius Widera et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Meissner corpuscles and Merkel cell neurite complexes are highly specialized mechanoreceptors present in the hairy and glabrous skin, as well as in different types of mucosa. Several reports suggest that after injury, such as after nerve crush, freeze injury, or dissection of the nerve, they are able to regenerate, particularly including reinnervation and repopulation of the mechanoreceptors by Schwann cells. However, little is known about mammalian cells responsible for these regenerative processes. Here we review cellular origin of this plasticity in the light of newly described adult neural crest-derived stem cell populations. We also discuss further potential multipotent stem cell populations with the ability to regenerate disrupted innervation and to functionally recover the mechanoreceptors. These capabilities are discussed as in context to cellularly reprogrammed Schwann cells and tissue resident adult mesenchymal stem cells.
1. Introduction
Meissner corpuscles (MCs, also called tactile corpuscles) were first described in 1852 by the German physiologists Rudolf Wagner and Georg Meissner [1]. These are encapsulated, rapidly adapting mechanoreceptors responsible for sensing light touch on the skin. Recently, due to their immunocytochemical properties, it has been proposed that MC may also act as nociceptors [2]. They can be found within the dermis, beneath the basal layer of skin regions sensitive to light touch. Within the murine, rat and human palatal mucosa, MCs are located centrally within palatal ridges (rugae palatinae) and are often accompanied by Merkel cell-neurites [3] (see Figure 1(a)). Remarkably, an anterior-posterior gradient of Nestin-expressing cells within the rat palate could be identified (see Figure 2). In particular, numerous Nestin-positive MCs can be observed in the lamina propria of hard palate, whereas nearly no MCs are present in the soft palate. In humans, the number of MCs gradually decreases with age [4].
Figure 1: Anatomical localization of Meissner corpuscles (MCs) and Merkel cell-neurite complexes (MCN) within rodent hard palate. (a) MCs are located centrally within palatal rugae in the lamina propria, whereas MCN can be found within the basal layer. (b) Nestin expression within rat palatal MCs and adjacent to MCN. Cryosections of rat hard palate were stained with mouse anti-Nestin antibody (clone Rat401) followed by incubation with secondary Alexa555-coupled anti-mouse detection antibody. Confocal analysis revealed strong immunoreactivity in numerous cells within MCs and adjacent to MCN.
Figure 2: Anterior-posterior gradient of cells Nestin expressing within the rat palate. Cryosections of rat palate were stained with mouse anti-Nestin antibody (clone Rat401) followed by Alexa 555 coupled detection antibody and nuclear counterstaining using Sytox Green. Confocal analysis revealed the presence of numerous Nestin-positive MCs and Merkel cell-neurite complexes within the hard palate (anterior pole), whereas within the soft palate (posterior pole) only few Merkel cell neurites complexes and nearly no MCs were detected.
A further type of highly specialized mechanoreceptors is the Merkel cell-neurite complexes (Merkel nerve endings), which are, in contrast to Meissner corpuscles, not encapsulated and seem to act as slowly adapting mechanoreceptors responsible for sustained sensing of mechanical pressure. In mammals, they are widely distributed and can be found in the basal layer of the palatal/oral mucosa (see Figure 1(a)), as well as in hairy and glabrous skin. Merkel cells were first described in 1875 by Friedrich Siegmund Merkel [5] and were originally termed “Tastzellen” (German: touch cells) (reviewed in [6]).
Remarkably, after injury, such as experimental nerve crash or freeze injury, MCs and Merkel cells seem to harbor a limited capacity to regenerate [79]. However, the exact cellular and developmental origin of plastic cells within MCs and Merkel cell-neurites responsible for this limited plasticity remains unclear. Recently, we demonstrated high expression of neural crest and general stem cell markers as well as pluripotency-associated transcripts within rat palatal mucosa. In addition, immunocytochemical analysis revealed high expression of Nestin within the MCs and adjacent to Merkel cell-neurite complexes, suggesting the presence of stem cells or other cells with progenitor properties within these mechanoreceptors [3].
In the following, we review the cellular composition of Meissner corpuscles and Merkel cell-neurite complexes. We focus on their developmental ancestry, marker expression, and the potential origin of multipotent stem cells within these highly specialized mechanoreceptors, factors which might explain their regenerative potential.
2. Cellular Composition of Meissner Corpuscles and Merkel Cell-Neurite Complexes
At the cellular level, MCs consist of a coiled arrangement of endings from up to six myelinated axons that terminate between layers of very thin, flattened, specialized Schwann cells called lamellar cells (see schematic diagram in Figure 4) [1013]. MCs are connected with thin axons and contain the myelinated axons as well as several unmyelinated nerve fibers [10, 14].
MCs contain several fibroblastoid cells, generating the connective tissue capsule. This capsule is distinguishable from the surrounding tissue, since this extracellular matrix is mainly of fibrillar connective tissue consisting of fibrillary proteins like collagens and fibronectins. Importantly, lamellar cells itself are embedded within the so-called basal lamina consisting among others of laminin and collagen type IV (reviewed in [15]). Notably, the extracellular matrix ECM, including the basal lamina, plays an essential role in connection, support, and growth regulation of cells in vivo and may therefore have major impact on cells responsible for regenerative processes. This may be due to direct influence on differentiation and proliferation or due to guidance of cells migrating into lesioned MCs.
However, although the acellular components and the cellular composition of MCs have been studied intensely using morphological and immunocytochemical analyses, the existence of further cell types like adult neural crest-derived stem cells or tissue resident mesenchymal stem cells has not been previously reported.
Merkel cell-neurite complexes consist of large, oval Merkel cells (Merkel-Ranvier Cells), which are in synapse-like contact with flat terminal endings of myelinated nerve fibers. In addition to their postulated function as receptor cells essential for tactile discrimination, Merkel cells may also exhibit an endocrine function, as they contain dense core granules comprising various neuropeptides [1618]. In addition, due to the close spatial proximity of Merkel cells to associated myelinated nerve fibres, several myelinating Schwann cells are known to be located adjacent to Merkel cell-neurite complexes.
3. Expression Pattern
Within MCs, neuronal cells can be identified based on their characteristic expression of neuronal markers including neurofilaments, -III-tubulin, synaptophysin, protein gene product 9.5 (PGP 9.5) [19], and neuron-specific enolase (NSE) [3, 13, 20, 21]. In addition, MCs expressed a panel of specific ion channels (reviewed in [22]). Nonneuronal cells of MC show robust expression of S100, Nestin (Figure 1(b)), Vimentin as well as the epidermal growth factor receptor (EGFR) [3, 20, 21, 23]. Several studies reported strong immunoreactivity for the neurotrophin receptors TrkA, TrkB, and p75 NTR [20, 21, 24]. Human MCs are negative for cytokeratins and GFAP, as described by Vega and colleagues [20]. Very recently, Meissner corpuscles have been described to express acid-sensing ion channels 2 (ASIC2) [25].
Merkel cells are characterized by the expression of cytokeratins CK8, CK18, CK19, and CK20 (reviewed in [6]) and the absence of CK4 and CK13 [26]. CK20 is of note as the most specific marker of Merkel cells; its expression shows no overlap with other cell types. Moreover, Merkel cells show expression of p75NTR, whereas no expression of Vimentin or GFAP can be detected by immunocytochemistry [27, 28]. Although we detected Nestin-positive cells in close proximity to Merkel cell-neurite complexes (Figure 1(b)), there are no direct evidences that Merkel cells itself are positive for this intermediate filament [3]. Indeed, a study by Eispert suggested that Nestin is absent in human Merkel cells of different anatomical origin [29]. Merkel cells are known to express a variety of neuronal markers like NSE, Synaptophysin, and PGP 9.5, whereas Neurofilament is absent [26, 28, 3033]. Additionally, it was reported that Neurotrophin-3 (NT-3) is required for the development of Merkel cells [34]. Recently, it has been reported that CK20-positive Merkel cells within human skin express Sox2—a pluripotency-associated transcription factor which is crucial for embryonic development as well as embryonic and adult neural crest-derived stem cell populations [35].
4. Developmental Origin of Meissner Corpuscles and Merkel Cell-Neurite Complexes
It has been suggested that in addition to the Schwann cell-related cells of the MC, the nerve fibres within NC are also of neural crest origin [36]. In this study, the authors used lineage tracing in Wnt1Cre/RetfCFP mice and demonstrated expression of CFP in neuronal cells, thus elucidating their neural crest ancestry. However, the discussion surrounding the developmental origin of Merkel cells is controversial. In the avian system, it has been suggested that Merkel cells also arise from the neural crest [37]. To address the question of the ontogenic origin of mammalian Merkel cells, Maya Sieber-Blums and colleagues used Wnt1Cre/Rosa26R reporter mice in which cells of neural crest lineage can be clearly traced back via expression of -galactosidase (-gal) [38]. In this study, CK8-immunopositive Merkel cells showed clear coexpression of -gal, suggesting their neural-crest origin. In addition, electron microscopy after peroxidase immunostaining revealed presence of -gal and Merkel cell typical dense core granules. A contrary model for the developmental origin of mammalian cells was proposed in 2009 by deletion of Atoh1 in either epidermal (Krt14Cre ) or neural crest deleter mice (Wnt1Cre ). In this approach, the authors selectively deleted Atoh1, which is necessary for the survival of Merkel cells, in epidermal, Krt14-expressing cells or in Wnt1-positive progeny of the neural crest. Interestingly, epidermal deletion of Atoh1 resulted in loss of Merkel cells in the skin, whereas neural crest-specific deletion showed no significant effects [39]. As the debate surrounding these findings continues, the final assessment of the developmental origin of Merkel cells requires further experimental analysis and as the exact nature of this origin remains elusive.
5. Regenerative Potential of MCs and Merkel Cell-Neurite Complexes
In the last two decades of the twentieth century, several reports provided evidences for regenerative capacity of MCs and Merkel cell-neurite complexes [79, 4042]. Through transection of the nerve innervating Meissner corpuscles in mice, Chizuka Ide observed that after an initial degeneration, at least some corpuscles start to regenerate, including re-innervation 30 days after the section and proper morphological appearance 4 months after the surgery [40]. After freezing injury, complete disintegration of cells within the corpuscles occurs after 25 days. In an advanced experimental procedure involving freezing damage, it was observed that the regenerated axons demonstrated extensive branching and morphologically specialized axonal terminals as well as the occurrence of newly formed lamellar cells [41]. Here, the author postulated that Schwann cells migrated into the site of the lesion and differentiated into lamellar cells, although the ancestry of the regenerated axon was not investigated further. Studies by Zelena and colleagues revealed that the regeneration potential of Meissner corpuscles after nerve crush and experimental freeze injury is higher in older animals than in young rats [8, 9].
In addition to MCs, Merkel cells as well seem to regenerate after injury. In an intriguing initial study, reinnervation and an increase of the Merkel cell number after experimental nerve crush were observed 40–100 days after the surgery [7, 43]. Importantly, the authors were able to show that the newly formed Merkel cells appeared physiologically normal and demonstrated typical histological features via electron microscopy and toluidine blue staining.
Although these reports clearly suggested that mechanoreceptors such as MCs and Merkel cell-neurite complexes can regenerate at least to a certain degree, the exact cellular origin of such unexpected plasticity remains unclear. Thus, the existence of so far unknown plastic progenitors or stem cells responsible for the regeneration process seems likely and is further discussed here.
6. Potential Stem Cell/Progenitor Populations within MCs and Merkel Cell-Neurite Complexes
6.1. Mammalian Adult Neural Crest-Derived Stem Cells
In recent years, several studies revealed the persistence of neural crest-derived stem cells in adult mammals [3, 4451].
In general, such neural crest-derived stem cells (NCSCs) are pluripotent in the early embryonic development (able to differentiate into cells of all germ layers) and become more restricted after migration from their niche between the ectoderm and the neural tube. A limited number of NCSCs that persist in the adult are able to generate multiple cell progenies in vitro and in vivo and aretherefore, multipotent stem cells (reviewed in [52]). In particular, such cells have been described to efficiently differentiate into neuronal and glial cells, osteogenic cell types, adipocytes, and chondrocytes, as well as into melanocytes and muscle cells. Such adult NCSCs express in vitro and in vivo high levels of Nestin, which is an intermediate filament originally described in Schwann cells and important for the self-renewal of neural stem cells [53, 54]. Adult NCSCs show expression of Vimentin, Sox2, and, depending on the cultivation method, the neurotrophin receptor p75NTR. As observed in adult human NCSCs isolated from respiratory mucosa, such cells also express TrkA (Hauser et al., unpublished observation). In addition, their expression pattern further includes Sox9, Sox10, Klf4, c-Myc, and Oct4 (see [52] for the full list). When cultivated under serum-free conditions in vitro, NCSCs form self-adherent clusters similar to neural stem cell-derived neurospheres.
In 2009, we reported Nestin-expressing NCSCs adjacent to MCs and Merkel cell-neurite complexes within the palatal ridges (palatal rugae/rugae palatinae) of adult rats [3]. Due to their anatomical origin within the palate, we termed these palatal neural crest-derived stem cells (pNCSCs). In their niche, the palatal ridges of hard palate, pNCSCs demonstrated high expression of Nestin, as demonstrated by immunocytochemical analysis and RT-PCR. In particular, Nestin immunoreactivity was detected in the periphery, centre, and on the top of Meissner corpuscles, as well as in close proximity to Merkel cell-neurite complexes within the basal layer of the palatal mucosa. We were further able to successfully isolate and expand rat and human palatal NCSCs in vitro as free-floating neurosphere cultures [3, 21] (see also Figure 3). Such cultivated pNCSCs were positive for a set of stem cell markers including Nestin, p75NTR, Sox9, Notch1, Slug and Snail in addition to Sox2, Klf4, Oct4, and c-Myc. Using appropriate differentiation protocols we demonstrated that pNCSCs were not only able to differentiate into GFAP-expressing glial cells, but also into -III-tubulin, NF- and Map2-positive neuronal cells. Due to such tremendous plasticity, tissue-resistant NCSCs are strong candidates for the observed in vivo regeneration of MCs and Merkel cell-neurite complexes after dissection and consequent degeneration. However, at least in case of regeneration after freeze injury, in which all cells within the mechanoreceptors including adjacent cells perish, there must be a further source of plastic cells capable of migration to the site of the lesion. These may be other NCSCs attracted by injury signals (e.g., chemokines and growth factors) from greater spatial distances or different cell types like cellularly reprogrammed Schwann cells or their progenitors (see the following).
Figure 3: Cultivated palatal neural crest-derived stem cells form self-adherent neurospheres and express the intermediate filament Nestin. Human palatal NCSCs were isolated according to protocol described in [3], fixed using 4% paraformaldehyde and stained using primary antibody against human Nestin and Alexa 555-coupled secondary antibody. DNA was stained using SYTOX green (bar 20 m).
Figure 4: Putative multipotent stem cell/progenitor populations within Meissner corpuscles. After lesion several cell populations may participate to the regeneration process. Firstly, multipotent adult neural crest-derived stem cells NCSCs may directly contribute to the regeneration process. Secondly, lamellar cells and other Schwann cells may be cellularly reprogrammed and re-enter the cell cycle after lesion. This process may be accompanied by migration of Schwann cells and their progenitors from the periphery to the site of the lesion. Finally, tissue resident fibroblast-like MSCs may also play an important role in recovery of mechanoreceptors.
6.2. Schwann Cell Progenitors and Dedifferentiated Schwann Cells
Schwann cells are directly related to the neural crest and share several markers with NCSCs (e.g., Vimentin, Nestin, or p75NTR, reviewed in [52]). Schwann cell progenitors (SCPs) are considered as late NCSCs that have established contact with axons [55]. It has been proposed that Schwann cell progenitors and Schwann cells have an extraordinarily unstable phenotype as differentiated Schwann cells can be switched back into more primitive cell types by injury signals or by appropriate cultivation methods [56, 57]. Consequently, these data suggest that Schwann cells and their immature ancestors—Schwann cell progenitors—may act as stem cells. In accordance with this hypothesis, Schwann cells have been described to perform differentiation into melanocytes after the lesion of the adult sciatic nerve [58, 59]. After injury, Schwann cells can reenter the cell cycle and dedifferentiate [56, 60]. Dupin and colleagues demonstrated differentiation of Schwann cells into myofibroblasts, indicating stemness characteristics of such cells in vitro [57].
Recently, we demonstrated successful cellular reprogramming of adult myelinating Schwann cells into an immature multipotent NCSC phenotype [21]. After isolation and expansion of Schwann cells under culture conditions mimicking an in vivo injury, we observed significantly elevated expression levels of p75, c-Myc, Sox2, Klf4, Oct4, Sox9, and Slug. Importantly, we were also able to differentiate such cultivated adult Schwann cells into ectodermal and mesodermal progeny. Such cellular reprogramming into immature neural crest-like phenotype also seems to occur in vivo in response to injury. This has been impressively demonstrated in a Wnt Cre/lox-EGFP mouse model [61]. After injury, mature Schwann cells residing at the nerve roots dedifferentiate into proliferating p75NTR-positive immature Schwann cells, which migrate into the lesion site.
It might be assumed that a similar injury-induced reprogramming mechanism could switch mechanoreceptor-associated Schwann cells, such as lamellar cells of MCs, into more primitive phenotype. Since mechanoreceptor-associated, subcutaneous Schwann cells are permanently exposed to mild mechanical stress, a cellular turnover and a latent cellular plasticity may be a hallmark of those cells. Indeed, in our study we detected proliferating, Ki67-positive cells not only in the basal cell layer of the palatal mucosa, but also in the center of MCs [21]. The expression of p75NTR is usually observed in immature, plastic Schwann cell progenitors. However, we and others reported that lamellar cells within MCs show strong p75NTR-immunoreactivity in addition to the well-described expression of S100 [20, 21]. Within palatal MCs, S100-immunoreactivity was detected in 100% of the cells in the investigated region, whereas not all cells expressed p75NTR (~50%) [21]. In addition, we observed a high degree of co-expression between Nestin and p75NTR (nearly 100%). Such co-expression of Nestin and p75NTR is a typical marker of immature cells such as early Schwann cell progenitors. However, the most likely source of multipotent neural crest-related stem cells within palatal MCs may be myelinating Schwann cells and not Schwann cell progenitors. Although SCPs are defined as neural crest stem cells which have established contact to nerve fibers, myelinization seems to be crucial for Nestin expression as Nestin has always been observed within myelinating Schwann cells and not in Schwann cells directly in contact with unmyelinated axons [21].
6.3. Tissue Resident Mesenchymal Stem Cells as Further Putative Stem Cell Type Responsible for Regeneration
A further adult stem cell type is represented by mesenchymal stem cells. Beside their initially described niche within the mammalian bone marrow, the presence of adult MSCs has also been confirmed in almost all tissues including muscle, fat, and the dermis of diverse types of mucosa [62]. Similar to adult NCSCs, MSCs are able to generate osteogenic, adipogenic, and chondrogenic progeny, although their neuronal differentiation potential is a subject of an ongoing scientific debate [6365]. At least in vitro, MSCs seem to show the ability to differentiate into neuron-like and Schwann cell-like phenotypes [66, 67] (reviewed in [65]). Concerning their morphological and immunocytochemical properties, as well as the expression profile, MSCs show high degree of similarity to fibroblasts, making them nearly indistinguishable in vivo [6871]. Importantly, in addition to typical MSC markers such as CD105, CD73 and CD90, MSCs express also Nestin and Vimentin [64, 72, 73].
As discussed previously, MCs contain not only glial cell, such as lamellar cells, but also fibroblast-like cells which, in the classical view, generate the connective encapsulation of the corpuscle. Remarkably, we observed Nestin immunoreactivity not only in lamellar cells of MCs, but also adjacent to the connective encapsulation of the corpuscles [3, 21]. Therefore, it cannot be excluded that MCs and adjacency of Merkel cell-neurite complexes contain both at least some tissue resident MSCs.
Taking into consideration that MSCs may be able to differentiate into multiple progenies, also this cell type can address the question of mechanoreceptor regeneration.
In this context, it is noteworthy that in our studies, we focused on cranial mucosa, a tissue where also tissue-resident MSCs are clearly of neural crest origin [74]. Thus, such cranial neural crest-derived MSCs may reveal a higher degree of plasticity than their trunk counterparts, which are not of neural crest ancestry [74].
7. Conclusions
As summarized previously, there are different multipotent cell populations in anatomical adjacency of mechanoreceptors which may be responsible for regenerative processes after lesion (see Figure 4). Firstly, due to their extraordinarily high plasticity, NCSCs may directly contribute to the regeneration process in vivo. A second plastic cell population is represented by cellularly reprogrammed Schwann cells, which reenter the cell cycle and dedifferentiate into more primitive phenotypes after lesion in their endogenous niche. Tissue resident MSCs may also play an important role in recovery after degeneration of mechanoreceptors. Additionally, a cooperative manner action, including more than one cell type, cannot be excluded at this time.
A definitive identification of the cellular base of regeneration remains a future challenge, the elucidation of which may help to develop effective treatments for acute injuries and age-related diseases accompanied by loss of mechanoreceptors, such as peripheral neuropathies. In vitro cultivation of autologous, mechanoreceptor-associated stem cells may be the basis of new therapeutic strategies.
Conflict of Interests
The authors declare no potential conflict of interests.
Acknowledgments
Experimental work described herein that was performed in our laboratory was supported by a grant of University of Bielefeld to D. Widera, grants of the German Research Council (DFG) to C. Kaltschmidt, and a grant of the German Ministry of Research and Education (BMBF) to B. Kaltschmidt. The authors thank Johannes FW Greiner and Kyle J. Lauersen for critical reading. They acknowledge support for the Article Processing Charge by the Deutsche Forschungsgemeinschaft and the Open Access Publication Funds of Bielefeld University Library.
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Sensors 2006, 6(10), 1224-1233; doi:10.3390/s6101224
Article
Flow-through Bulk Optode for Spectrophotometric Determination of Thiocyanate and Its Application to Water and Saliva Analysis
Department of Analytical Chemistry, Faculty of Chemistry, University of Murcia, 30071 Murcia, Spain
* Author to whom correspondence should be addressed.
Received: 23 June 2006 / Accepted: 3 October 2006 / Published: 9 October 2006
(This article belongs to the Special Issue Sensors in Flow Analysis)
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Abstract: A flow-through spectrophotometric bulk optode for the flow-injectiondetermination of thiocyanate is described. As active constituents, the optode incorporatesthe lipophilized pH indicator 5-octadecanoyloxy-2-(4-nitrophenylazo)phenol andmethyltridodecyl ammonium chloride, dissolved in a plasticized poly(vinyl)chloridemembrane entrapped in a cellulose support. The optode is applied, in conjunction with theflow injection technique, to the determination of thiocyanate at pH 7.5 (TRIS/H2SO4). Thesensor is readily regenerated with a 10-2 M NaOH carrier solution. The analyticalcharacteristics of this optode with respect to thiocyanate response time, dynamicmeasurement range, reproducibility and selectivity are discussed. The proposed FI methodis applied to the determination of thiocyanate in waters from different sources and in humansaliva samples in order to distinguish between smokers and non-smokers.
Keywords: Flow-through optode; spectrophotometry; thiocyanate; water; saliva.
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MDPI and ACS Style
García, S., Mª; Ortuño, J.A.; Sánchez-Pedreño, C.; Albero, I., Mª; Fernández, J., Mª Flow-through Bulk Optode for Spectrophotometric Determination of Thiocyanate and Its Application to Water and Saliva Analysis. Sensors 2006, 6, 1224-1233.
AMA Style
García S, Mª, Ortuño JA, Sánchez-Pedreño C, Albero I, Mª, Fernández J, Mª. Flow-through Bulk Optode for Spectrophotometric Determination of Thiocyanate and Its Application to Water and Saliva Analysis. Sensors. 2006; 6(10):1224-1233.
Chicago/Turabian Style
García, Soledad, Mª; Ortuño, Joaquín A.; Sánchez-Pedreño, Concepción; Albero, Isabel, Mª; Fernández, José, Mª. 2006. "Flow-through Bulk Optode for Spectrophotometric Determination of Thiocyanate and Its Application to Water and Saliva Analysis." Sensors 6, no. 10: 1224-1233.
Sensors EISSN 1424-8220 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
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Nano Express
Highly sensitive hydrogen sensor based on graphite-InP or graphite-GaN Schottky barrier with electrophoretically deposited Pd nanoparticles
Karel Zdansky
Author Affiliations
Institute of Photonics and Electronics, Academy of Sciences of the Czech Republic, Chaberska 57, 18251 Prague 8, Czech Republic
Nanoscale Research Letters 2011, 6:490 doi:10.1186/1556-276X-6-490
Published: 10 August 2011
Abstract
Depositions on surfaces of semiconductor wafers of InP and GaN were performed from isooctane colloid solutions of palladium (Pd) nanoparticles (NPs) in AOT reverse micelles. Pd NPs in evaporated colloid and in layers deposited electrophoretically were monitored by SEM. Diodes were prepared by making Schottky contacts with colloidal graphite on semiconductor surfaces previously deposited with Pd NPs and ohmic contacts on blank surfaces. Forward and reverse current-voltage characteristics of the diodes showed high rectification ratio and high Schottky barrier heights, giving evidence of very small Fermi level pinning. A large increase of current was observed after exposing diodes to flow of gas blend hydrogen in nitrogen. Current change ratio about 700,000 with 0.1% hydrogen blend was achieved, which is more than two orders-of-magnitude improvement over the best result reported previously. Hydrogen detection limit of the diodes was estimated at 1 ppm H2/N2. The diodes, besides this extremely high sensitivity, have been temporally stable and of inexpensive production. Relatively more expensive GaN diodes have potential for functionality at high temperatures.
Keywords:
hydrogen sensor; metal nanoparticles; electrophoresis; Schottky barrier; InP; GaN
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Very High Activity
Contributors : Robert Lemke
Analyzed 8 days ago based on code collected 8 days ago.
Recent Kudos...
... for TYPO3 CMS given by:
Christian Jul Jensen
robregonm
Christoph Koehler
Project Commits
Ohloh did not measure any commits by this contributor.
Project Languages
Ohloh did not measure any lines of code written by this contributor.
Copyright © 2013 Black Duck Software, Inc. and its contributors, Some Rights Reserved. Unless otherwise marked, this work is licensed under a Creative Commons Attribution 3.0 Unported License . Ohloh ® and the Ohloh logo are trademarks of Black Duck Software, Inc. in the United States and/or other jurisdictions. All other trademarks are the property of their respective holders.
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OpenWetWare:How to join
From OpenWetWare
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Revision as of 23:58, 20 April 2007
Use this page to sign up for an OpenWetWare account. Accounts are only needed for editing pages; all pages are viewable by anyone.
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Personal tools
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“The Master,” “Amour” top London Film Critics’ Circle noms
PanARMENIAN.Net - Michael Haneke’s Amour and Paul Thomas Anderson’s The Master each received seven nominations for the London Film Critics’ Circle Awards, Deadline said.
Amour was nominated in the best film, director, screenwriter, actor, actress and supporting actress categories along with a nod as best foreign language film.
The Master also was mentioned in the best film, director, screenwriter, actor and supporting actress races as well as supporting actor.
Skyfall is the most heavily nominated British film with five nods inlcuding two for Judi Dench as best supporting actress and British actress of the year; the latter shared with her role in The Best Exotic Marigold Hotel. Life Of Pi, Argo, Lincoln, Les Misérables and UK indie Sightseers are all nominated four times each.
The London Film Critics’ Circle will hold its 33rd awards ceremony on January 20.
Partner news
Top stories
The jewels were to be loaned to celebrities who have arrived on the French Riviera town for its famous annual film festival.
The list of the finalists also includes Hungary, Azerbaijan, Georgia, Romania, Norway, Iceland, Finland and others.
Set in the gritty blue-collar neighborhood of God’s Pocket, story follows a man stuck with a debt he can't pay.
"Catching Fire" follows Katniss and fellow Hunger Games victor Peeta as they embark on a "Victor's Tour" throughout 12 districts of Panem.
Partner news
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This work is licensed under a Creative Commons Attribution-ShareAlike 3.0 United States License.
An XML version of this text is available for download, with the additional restriction that you offer Perseus any modifications you make. Perseus provides credit for all accepted changes, storing new additions in a versioning system.
hide References (19 total)
load Vocabulary Tool
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Citation URN: urn:cts:latinLit:phi0474.phi037.perseus-lat1:3.43
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ab-dicō āvī, ātus, āre,
to disown, disavow, reject: ubi plus mali quam boni reperio, id totum abdico atque eicio: abdicari Philippum patrem, Cu.—With se and abl, to give up an office before the legal term expires, resign, abdicate (cf. depono, to lay down an office at the expiration of the term): dictaturā se abdicat, Cs.: se consulatu: respondit aedilitate se abdicaturum, L.—Once absol. (of consuls), to abdicate, resign, C.—With acc: abdicato magistratu, S.: causa non abdicandae dictaturae, L.
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[31]
There also is the Cainochorion, (New Castle,) as it is called, a fortified and precipitous rock, distant from Cabeira less than 200 stadia. On its summit is a spring, which throws up abundance of water, and at its foot a river, and a deep ravine. The ridge of rocks on which it stands is of very great height, so that it cannot be taken by siege. It is enclosed with an excellent wall, except the part where it has been demolished by the Romans. The whole country around is so covered with wood, so mountainous, and destitute of water, that an enemy cannot encamp within the distance of 120 stadia. There Mithridates had deposited his most valuable effects, which are now in the Capitol, as offerings dedicated by Pompey.
Pythodoris is in possession of all this country; (for it is contiguous to that of the barbarians, which she holds as a conquered country;) she also holds the Zelitis and the Megalopolitis. After Pompey had raised Cabeira to the rank of a city, and called it Diospolis, Pythodoris improved it still more, changed its name to Sebaste, (or Augusta,) and considers it a royal city.
She has also the temple of Men surnamed of Pharnaces, at Ameria, a village city, inhabited by a large body of sacred menials, and having annexed to it a sacred territory, the produce of which is always enjoyed by the priest. The kings held this temple in such exceeding veneration, that this was the Royal oath, ‘by the fortune of the king, and by Mēn of Pharnaces.’ This is also the temple of the moon, like that among the Albani, and those in Phrygia, namely the temple of Mēn in a place of the same name, the temple of Ascæus at Antioch in Pisidia, and another in the territory of Antioch.
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Google Pages Host Trojan Horse Scam
Jun 20, 2006 • 7:10 am | (0) by | Filed Under Other Google Topics
A WebmasterWorld thread discusses the news that Google Pages was used to host a trojan horse that can infect computers. The trojan horse was "designed to steal bank details relating to certain financial institutions." It did not do too much damage, from what I hear. Also, this is not a big deal - dozens of similar services (Yahoo!'s Geocities, etc.) have the same issues, I believe.
Forum discussion at WebmasterWorld.
Previous story: Checking Declined Yahoo! Search Ad Listings Reasons in UK
blog comments powered by Disqus
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Person:Lou Henry (2)
• F. Charles Henry (add)
• M. Florence Weed (add)
1. Lou Henry1874 - 1944
m. 10 Feb 1899
Facts and Events
Name Lou Henry
Gender Female
Birth[1] 29 Mar 1874 Waterloo, Black Hawk, Iowa, United States
Marriage 10 Feb 1899 Monterey, Monterey, California, United Statesto Herbert Clark Hoover
Death[1] 7 Jan 1944 New York City, New York, United States
the text in this section is copied from an article in Wikipedia
Lou Henry Hoover (March 29, 1874 – January 7, 1944) was the wife of President of the United States Herbert Hoover and served as First Lady from 1929 to 1933.
Marrying her engineer husband in 1899, she traveled widely with him, including to Shanghai, China, and became a cultivated scholar and linguist. A proficient Chinese speaker, she is the only First Lady to have spoken an Asian language. She oversaw construction of the presidential retreat at Rapidan Camp in Madison County, Virginia. She was the first First Lady to make regular, nationwide radio broadcasts to the American public.
Lou Henry grew up in Iowa and California. She enrolled at Stanford as its only female geology major in 1894. There she met future president Herbert Hoover. They married after her graduation and she accompanied him to China where he worked as a civil engineer.
This page uses content from the English Wikipedia. The original content was at Lou Henry Hoover. The list of authors can be seen in the page history. As with WeRelate, the content of Wikipedia is available under the Creative Commons Attribution/Share-Alike License.
References
1. 1.0 1.1 Lou Hoover, in National First Ladies Library, [1].
First Ladies of the United States
Martha Dandridge Washington · Abigail Smith Adams · Martha Jefferson Randolph · Dolley Payne Madison · Elizabeth Kortright Monroe · Louisa Johnson Adams · Emily Donelson · Sarah Yorke Jackson · Angelica Singleton Van Buren · Anna Symes Harrison · Jane Irwin Harrison · Letitia Christian Tyler · Priscilla Cooper Tyler · Julia Gardiner Tyler · Sarah Childress Polk · Margaret Smith Taylor · Abigail Powers Fillmore · Jane Appleton Pierce · Harriet Lane · Mary Todd Lincoln · Eliza McCardle Johnson · Julia Dent Grant · Lucy Webb Hayes · Lucretia Randolph Garfield · Mary Arthur McElroy · Rose Cleveland · Frances Folsom Cleveland · Caroline Scott Harrison · Mary Harrison McKee · Frances Folsom Cleveland · Ida Saxton McKinley · Edith Carow Roosevelt · Helen Herron Taft · Ellen Axson Wilson · Edith Bolling Galt Wilson · Florence Kling Harding · Grace Goodhue Coolidge · Lou Henry Hoover · Eleanor Roosevelt · Bess Wallace Truman · Mamie Eisenhower · Jacqueline Kennedy · Lady Bird Johnson · Pat Ryan Nixon · Betty Bloomer Warren Ford · Rosalynn Carter · Nancy Davis Reagan · Barbara Pierce Bush · Hillary Rodham Clinton · Laura Bush · Michelle Robinson Obama
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Place:Woerden, Zuid-Holland, Netherlands
Watchers
NameWoerden
TypeGemeente
Coordinates52.08605°N 4.88394°E
Located inZuid-Holland, Netherlands (1814 - 1989)
Also located inUtrecht, Netherlands (1989 - )
Contained Places
Voormalige gemeente
Barwoutswaarder ( 1964 - )
the text in this section is copied from an article in Wikipedia
Woerden is a municipality and a city in the central Netherlands. Due to its central location between Amsterdam, Rotterdam, The Hague and Utrecht, and the fact that it has rail and road connections to those cities, it is a popular town for commuters who work in those cities.
source: Getty Thesaurus of Geographic Names
source: Family History Library Catalog
General Info
• Woerden is a municipality (gemeente) in the Netherlands
• Between 1814-1989, Woerden was located in Zuid-Holland. After 1989, Woerden has been located in Utrecht, Netherlands.
• Because of the 1900 rule, the name continues to reflect its placement in Zuid-Holland.
Research Tips
This page uses content from the English Wikipedia. The original content was at Woerden. The list of authors can be seen in the page history. As with WeRelate, the content of Wikipedia is available under the Creative Commons Attribution/Share-Alike License.
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Ginseng
Jump to: navigation, search
Ginseng
Panax quinquefolius foliage and fruit
Panax quinquefolius foliage and fruit
Scientific classification
Kingdom: Plantae
Division: Magnoliophyta
Class: Magnoliopsida
Order: Apiales
Family: Araliaceae
Subfamily: Aralioideae
Genus: Panax
L.
Species
Subgenus Panax</br>
Section Panax
Series Notoginseng
Panax notoginseng
Series Panax
Panax bipinnatifidus
Panax ginseng
Panax japonicus
Panax quinquefolius
Panax vietnamensis
Panax wangianus
Panax zingiberensis
Section Pseudoginseng
Panax pseudoginseng
Panax stipuleanatus
Subgenus Trifolius</br>
Panax trifolius
Ginseng refers to species within Panax, a genus of 11 species of slow-growing perennial plants with fleshy roots, in the family Araliaceae. They grow in the Northern Hemisphere in eastern Asia (mostly northern China, Korea, and eastern Siberia), typically in cooler climates; Panax vietnamensis, discovered in Vietnam, is the southernmost ginseng found. This article focuses on the Series Panax ginsengs, which are the adaptogenic herbs, principally Panax ginseng and Panax quinquefolius. Ginseng is characterized by the presence of ginsenosides.
Siberian ginseng (Eleutherococcus senticosus) is not a ginseng at all. It is another adaptogen, but a different species named "Siberian ginseng" as a marketing ploy; instead of a fleshy root, it has a woody root; instead of ginsenosides, eleutherosides are present, (see below).
Etymology
The English word ginseng derives from the Chinese term rénshēn (simplified: ; traditional: ), literally "man root" (referring to the root's characteristic forked shape, resembling the legs of a man). The difference between rénshēn and "ginseng" is explained by the fact that the English pronunciation derives from a Japanese reading of these Chinese characters. However, the current Japanese word for these characters 人参 (ninjin) means carrot, and ginseng is referred to in Japanese as 朝鮮人参 (chosen ninjin). The Korean name is 고려인삼 高麗人参 (goryo insam).
The botanical name Panax means "all-heal" in Greek, and was applied to this genus because Linnaeus was aware of its wide use in Chinese medicine.
Traditional uses
Both American and Panax (Asian) ginseng rhizomes are taken orally as adaptogens, aphrodisiacs, nourishing stimulants, and in the treatment of type II diabetes, including sexual dysfunction in men. The rhizome is most often available in dried form, either in whole or sliced form. Ginseng leaf, although not as highly prized, is sometimes also used; as with the rhizome it is most often available in dried form.
This ingredient may also be found in some popular Energy Drinks: usually the "tea" varieties or Functional Foods. Usually ginseng is in subclinical doses and it does not have measurable medicinal effects. It can be found in cosmetic preparations as well, with similar lack of effect. It is considered a wasteful use of important herbs by herbalists.
Ginseng root can be double steamed with chicken meat as a soup. (See samgyetang.)
Modern science and ginseng
As with herbalism in general, ginseng's medical efficacy remains controversial. It has been difficult to verify the medicinal benefits of ginseng using modern science, as there are contradictory results from different studies, possibly due to the wide variety and quality of ginseng used in studies. Another issue is the profit potential of corporate research since ginseng cannot be patented.[original research?] As a result, high-quality studies of the effects of ginseng are rare. Incidentally, one of the better studies involving ginseng actually uses a proprietary ginseng extract. [1]
Ginseng is promoted as an adaptogen (a product that increases the body's resistance to stress), one which can to a certain extent be supported with reference to its anticarcinogenic and antioxidant properties,[2] although animal experiments to determine whether longevity and health were increased in the presence of stress gave negative results.[3]
A comparative, randomized and double-blind study at the National Autonomous University of Mexico does indicate it to be "a promising dietary supplement" when assessed for an increase in quality of life [4].
Panax ginseng appear to inhibit some characteristics associated with cancer in animal models; nevertheless, this effect is unclear in humans.[5]
There are references in the literature, including seemingly authoritative compendiums that appear to show interactions with ginseng. Herbalist Jonathan Treasure of the United States National Institute of Mental Health traces the growth of misinformation on an alleged adverse herb-drug interaction between the monoamine oxidase inhibitor phenelzine and Asian ginseng (Panax ginseng C.A. Meyer). This originally was mentioned in a 1985 editorial by Shader and Greenblatt in the Journal of Clinical Psychopharmacology. Shader and Greenblatt devoted a couple of lines to the case of 64 year-old woman who took an undisclosed dose for an undisclosed time of a dietary supplement product called “Natrol High” while concurrently taking phenelzine 60 mg qd. She experienced symptoms of “insomnia, headache, and tremulousness”. Treasure contacted Natrol by email and discovered within ten minutes that there was no Panax ginseng in the formula, but instead eleutherococcus which was then called by the popular name "Siberian ginseng" and it was given in a subclinical dosage mixed with a variety of other herbs. The purported interaction effects are well-known side effects of phenelzine alone, which had been given in a high dosage and are not at all suggestive of eleutherococcus. However this misinformed article with a misidentified herb has been picked up in literature searches, megastudies and is now documented by conventional medical authorities such as Stockley’s , and is repeated in several botanical monographs e.g. World Health Organization (WHO 1999).[6][7][8]
Ginseng and Reproductive Activity
A 2002 study by the Southern Illinois University School of Medicine (published in the annals of the New York Academy of Sciences) found that in laboratory animals, both Asian and American forms of ginseng enhance libido and copulatory performance. These effects of ginseng may not be due to changes in hormone secretion, but to direct effects of ginseng, or its ginsenoside components, on the central nervous system and gonadal tissues[9]In males, ginsenosides can facilitate penile erection.[10] This is consistent with traditional Chinese medicine and Native American medicinal uses of ginseng.
Side effects
One of Panax ginseng's most common side-effects is the inability to sleep.[11] Other side-effects include nausea, diarrhea, euphoria, headaches, epistaxis, high blood pressure, low blood pressure, mastalgia, and vaginal bleeding.[12]
Overdose
The common adaptogen ginsengs (Panax ginseng and Panax quinquefolia) are generally considered to be relatively safe even in large amounts.
Panax ginseng is not recommended within Chinese Medicine to be administered along with anti-infective herbs unless a person is quite debilitated, because of the fear that the pathogen will be tonified. Herbalists in China believed this and according to Xu Dachun in his brief essay on ginseng (1757 A.D., during the Qing Dynasty): "if one administers Ginseng of a purely supplementing nature, then one will merely supplement the evil influences and help them settle down. In minor cases, the evil influences will, as a result of such mistaken therapy, never leave the body again. In serious cases, death is inevitable."[13]
Common classification
File:Ginseng in Korea.jpg
Ginseng roots in a market in Seoul, 2003
Panax quinquefolius American ginseng (root)
Ginseng that is produced in the United States and Canada is particularly prized in Chinese societies, and many ginseng packages are prominently colored red, white, and blue.
According to Traditional Chinese/Korean Medicine, American Ginseng promotes Yin energy, cleans excess Yang in the body, calms the body. The reason it has been claimed that American ginseng promotes Yin (shadow, cold, negative, female) while East Asian ginseng promotes Yang (sunshine, hot, positive, male) is that, according to traditional Korean medicine, things living in cold places are strong in Yang and vice versa, so that the two are balanced. Chinese/Korean ginseng grows in northeast China and Korea, the coldest area known to many Koreans in traditional times. Thus, ginseng from there is supposed to be very Yang. Originally, American ginseng was imported into China via subtropical Guangzhou, the seaport next to Hong Kong, so Chinese doctors believed that American ginseng must be good for Yin, because it came from a hot area. However they did not know that American ginseng can only grow in temperate regions. Nonetheless the root is legitimately classified as more Yin because it generates fluids.[14]
The two main components of ginseng are in different proportions in the Asian and American varieties, and may well be the cause the excitatory versus tonic natures.[4]
The ginseng is sliced and a few slices are simmered in hot water to make a decoction.
Most North American ginseng is produced in the Canadian provinces of Ontario and British Columbia and the American state of Wisconsin, according to Agri-food Canada. P. quinquefolius is now also grown in northern China.
A randomized, double-blind study shows that an extract of American ginseng reduces influenza cases in the elderly when compared to placebo.[1]
Panax ginseng Asian ginseng (root)
According to Traditional Chinese/Korean Medicine Panax Ginseng promotes Yang energy, improves circulation, increases blood supply, revitalizes and aids recovery from weakness after illness, stimulates the body. Panax Ginseng is available in two forms:
The form called white ginseng is grown for four to six years, and then peeled and dried to reduce the water content to 12% or less. White Ginseng is air dried in the sun and may contain less of the therapeutic constituents. It is thought by some that enzymes contained in the root break down these constituents in the process of drying. Drying in the sun bleaches the root to a yellowish-white color.
The form called red ginseng is harvested after six years, is not peeled and is steam-cured, thereby giving them a glossy reddish-brown coloring. Steaming the root is thought to change its biochemical composition and also to prevent the breakdown of the active ingredients. The roots are then dried.
Red ginseng
Red ginseng (Korean=홍삼, simplified Chinese: ; traditional Chinese: ), is Panax ginseng that has been heated, either through steaming or sun-drying. It is frequently marinated in an herbal brew which results in the root becoming extremely brittle. This version of ginseng is traditionally associated with stimulating sexual function and increasing energy. Red ginseng is always produced from cultivated roots, usually from either China or South Korea.
In 2002, a preliminary double-blind, crossover study of Korean red ginseng's effects on impotence reported that it can be an effective alternative for treating male erectile dysfunction.[15]
A study shows that Red ginseng reduces the relapse of gastric cancer versus control[16]
A study of ginseng's effects on rats show that while both White ginseng and Red ginseng reduce the incidence of cancer, the effects appear to be greater with Red ginseng.[17]
Falcarinol, a seventeen-carbon diyne fatty alcohol was isolated from carrot and red ginseng, shown to have potent anticancer properties on primary mammary epithelial (breast cancer) cells.[18] Other acetylenic fatty alcohols in ginseng (panaxacol, panaxydol, panaxytriol) have antibiotic properties.[19]
Wild ginseng
Wild ginseng is ginseng that has not been planted and cultivated domestically, rather it is that which grows naturally and is harvested from wherever it is found to be growing. It is considered to be superior to field farmed ginseng by various authorities, and it has been shown to contain higher levels of ginsenoside. Wild ginseng is relatively rare and even increasingly endangered, due in large part to high demand for the product in recent years, which has led to the wild plants being sought out and harvested faster than new ones can grow (it requires years for a ginseng root to reach maturity). Wild ginseng can be either Asian or American and can be processed to be red ginseng.
There are woods grown American ginseng programs in Maine, Tennessee, Virginia and North Carolina. [20][21] and United Plant Savers has been encouraging the woods planting of ginseng both to restore natural habitats and to remove pressure from any remaining wild ginseng, and they offer both advice and sources of rootlets. Woods grown plants have comparable value to wild grown ginseng of similar age.
Ginseng alternatives
These mostly adaptogenic plants are sometimes referred to as ginsengs, but they are either from a different family or genus. Only Jiaogulan actually contains ginsenosides, although ginsenosides alone do not determine the effectiveness of ginseng. Since each of these plants have different uses, one should research their properties before using. Descriptions and differentiation can be found in David Winston and Steven Maimes book Adaptogens[22]
Other plants which are referred to as ginsengs may not be adaptogens (although notoginseng is in the Panax family):
References
1. 1.0 1.1 McElhaney JE et al (2004). "A placebo-controlled trial of a proprietary extract of North American ginseng (CVT-E002) to prevent acute respiratory illness in institutionalized older adults". J Am Geriatr Soc 52 (1): 13–19. PMID 14687309.
2. Davydov M, Krikorian AD. (October 2000). "Eleutherococcus senticosus (Rupr. & Maxim.) Maxim. (Araliaceae) as an adaptogen: a closer look.". Journal of Ethnopharmacology 72 (3): 345-393. PMID 6685799.
3. Lewis WH, Zenger VE, Lynch RG. (August 1983). "No adaptogen response of mice to ginseng and Eleutherococcus infusions.". Journal of Ethnopharmacology 8 (2): 209-214. PMID 6685799.
4. Caso Marasco A, Vargas Ruiz R, Salas Villagomez A, Begona Infante C. (1996). "Double-blind study of a multivitamin complex supplemented with ginseng extract.". Drugs Exp Clin Res. 22 (6): 323–329. PMID 9034759.
5. Shin HR, Kim JY, Yun TK, Morgan G, Vainio H (2000). "The cancer-preventive potential of Panax ginseng: a review of human and experimental evidence". Cancer Causes Control 11 (6): 565–576. PMID 10880039.
6. [1] Treasure, Jonathan. Medline & The Mainstream Manufacture of Misinformation 2006
7. Stockley, IH (2002), Stockley's Drug Interactions. 6th ed. London: Pharmaceutical Press.
8. WHO (1999), "Radix Ginseng", in,WHO Monographs on Selected Medicinal Plants, Geneva: World Health Organization, 168-182.
9. Hong B; Ji YH; Hong JH; Nam KY; Ahn TYA double-blind crossover study evaluating the efficacy of korean red ginseng in patients with erectile dysfunction: a preliminary report. J Urol. 2002; 168(5):2070-3 (ISSN: 0022-5347)Department of Urology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea
10. de Andrade E; de Mesquita AA; Claro Jde A; de Andrade PM; Ortiz V; Paranhos M; Srougi MStudy of the efficacy of Korean Red Ginseng in the treatment of erectile dysfunction. Sector of Sexual Medicine, Division of Urological Clinic of Sao Paulo University, Sao Paulo, Brazil.
11. http://www.umass.edu/cnshp/faq.html
12. http://www.aafp.org/afp/20031015/1539.html
13. http://www.itmonline.org/arts/ginseng.htm
14. Chinese Herbal Medicine: Materia Medica, Third Edition by Dan Bensky, Steven Clavey, Erich Stoger, and Andrew Gamble 2004
15. Hong B, Ji YH, Hong JH, Nam KY, Ahn TY. (2002). "A double-blind crossover study evaluating the efficacy of Korean red ginseng in patients with erectile dysfunction: a preliminary report". Journal of Urology 168 (5): 20–21. PMID 12394711.
16. Suh SO, Kroh M, Kim NR, Joh YG, Cho MY. (2002). "Effects of red ginseng upon postoperative immunity and survival in patients with stage III gastric cancer.". American Journal of Chinese Medicine. 30 (4): 483–94. PMID 12568276.
17. Yun TK, Lee YS, Lee YH, Kim SI, Yun HY (2001). "Anticarcinogenic effect of Panax ginseng C.A. Meyer and identification of active compounds.". Journal of Korean Medical Science 16 (S): 6–18. PMID 11748383.
18. [2]
19. [3]
20. http://www.state.tn.us/environment/na/ginseng.shtml
21. http://www.ces.ncsu.edu/depts/hort/hil/hil-127.html
22. Winston, David & Maimes, Steven. “Adaptogens: Herbs for Strength, Stamina, and Stress Relief,” Healing Arts Press, 2007
See also
External links
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Posterior gluteal line
Jump to: navigation, search
Bone: Posterior gluteal line
Right hip bone. External surface. (Posterior gluteal line is red arch near top, labeled at center left.)
Latin linea glutea posterior
Gray's subject #57 232
Dorlands
/ Elsevier
l_10/12496152
The posterior gluteal line (superior curved line), the shortest of the three gluteal lines, begins at the crest, about 5 cm. in front of its posterior extremity; it is at first distinctly marked, but as it passes downward to the upper part of the greater sciatic notch, where it ends, it becomes less distinct, and is often altogether lost.
Behind this line is a narrow semilunar surface, the upper part of which is rough and gives origin to a portion of the Gluteus maximus; the lower part is smooth and has no muscular fibers attached to it.
This article was originally based on an entry from a public domain edition of Gray's Anatomy. As such, some of the information contained herein may be outdated. Please edit the article if this is the case, and feel free to remove this notice when it is no longer relevant.
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Australian Bureau of Statistics
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6202.0 - Labour Force, Australia, Preliminary, Feb 1998
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Summary results of the monthly Labour Force Survey containing estimates of employed and unemployed persons classified by sex, full-time/part-time status, states and territories and some age groups; and persons not in the labour force.
6202.0 was published as Labour Force, Australia, Preliminary until March 2003. As the publication had provided final summary data for a number of years to that point, the misleading qualification preliminary was removed from the April 2003 issue onwards.
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Australia's 13th Census of Population and Housing was conducted on Tuesday 6 August 1996. Data from the 1996 Census is available through the following products.
Basic Community Profile for Australia
Detailed tables of key characteristics for people, families and dwellings.
Basic Community Profile for Other Areas
Detailed tables of key characteristics for people, families and dwellings for states, Statistical Divisions, Statistical Subdivisions and Statistical Local Areas.
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A summary of findings and selected characteristics for Australia and each state and territory.
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Census characteristics for Urban Centres and Localities across Australia, ranking the top 200 based on person, family and dwelling characteristics.
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Household, family and labour force characteristics for Australia and each state and territory.
Selected Social and Housing Characteristics
A range of social and housing statistics produced from the 1996 Census for Australia, the states and territories, and their regions.
Socio-Economic Indexes for Areas (SEIFA96)
Summary measures derived from 1996 Census data. SEIFA includes five indexes to allow ranking of regions and areas, providing a method of determining the level of social and economic well-being in each region.
1996 Household Sample File (HSF)
A comprehensive Confidentialised Unit Record File (CURF) of 1996 Census variables, containing a 1% random sample of private dwellings and associated family and person records, and a 1% random sample of persons from non-private dwellings.
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Commentary and data on a number of key social indicators from the 1996 Census, with a focus on regional distributions and comparisons. Topics include cultural diversity, living arrangements, education, work and housing.
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Tell me more ×
Answers OnStartups is a question and answer site for entrepreneurs looking to start or run a new business. It's 100% free, no registration required.
When it comes to enterprise software, would you say there is a difference between a hosted solution and an SaaS solution? Or are they the same thing?
If there are differences and you offered a "hosted" solution would you still use a "subscription" based pricing model?
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6 Answers
up vote 4 down vote accepted
Hosted and SaaS are different: - Hosted means the software you access is not on your own infrastructure, it is "hosted" by someone else. That means that you could have bought a bunch of software and have them hosted. This is not SaaS. - SaaS is software that you pay as you go (as a service) and there lies the difference.
Now let's bring a bit of confusion: A SaaS application is usually hosted.. But a Hosted solution is not necessarily a SaaS solution.
To answer you question about subscription, SaaS is "pay as you go", so it is subscription based (otherwise the provider wouldn't be able to stop the "service" if you stop paying). But a hosted solution will have a subscription on the hosting part, not necessarily on the software you access (and that is hosted).
Let me know if you need more clarity.
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An hosted solution is usually what offer SaaS providers.
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A "Hosted" solution is when a third-party is used to house IT infrastructure, be it hardware-only (customer owns, loads, and runs the apps), software-only (1 or 2 separate 3rd parties may be involved, one provides the remote cloud (aka data center) and one provides the remote application management (usually one 3rd party hires the other, but some companies have remote management of apps that are located at an internal location).
SaaS is software-based, butit too can appear in various ways, inlcuded hosted SaaS. Take a video SaaS vendor (stores IP video remotely for others). It can look like a software-only hosting option where the vendor runs the video app,but customer finds colocation space to host it. Or the SaaS vendor can also own the data center, and sell the app by the computing slice charging you both for the hardwar cpu cycles used and the number of users on the app or the number of computing cycles that the app takes. So SaaS can be billed for software-only, or both hardware and software.
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These are both slippery terms, and there's definitely overlap.
SaaS generally implies that the provider has a system somewhere in the cloud, and that as a customer I pay for software features and utilisation rather than for the platform hosting as such.
Hosted solutions are often software that you could install for yourself, and as an option you can access the service through the cloud. It's commmon - but not universal - to find that payment for hosted services mirrors the underlying software's pricing structures plus structures common in 'raw' hosting.
But the difference is really more a matter of convention than anything. I've certainly seen companies who seem totally SaaS to me position their services as 'hosted' in order to connect better with enterprises who like 'cloud,' know what 'hosting' is but are not decided about SaaS. And I've seen common software packaged up with hosting and called SaaS to differentiate from commodity hosts.
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Definitions vary all over the place, so your results may vary. This works for me.
Hosting - running and maintaining a computer system on someone's behalf.
SaaS - Software-as-a-Service is a model of software deployment whereby a provider licenses an application to customers for use as a service on demand. One example of SaaS is the Salesforce.com CRM application.
IaaS - Infrastructure-as-a-Service is the delivery of computer infrastructure (typically a platform virtualization environment) as a service. Rather than purchasing servers, software, data center space or network equipment, clients instead buy those resources as a fully outsourced service. One such example of this is the Amazon web services.
PaaS - Platform-as a-Service is the delivery of a computing platform and solution stack as a service. It facilitates the deployment of applications without the cost and complexity of buying and managing the underlying hardware and software layers. PaaS provides the facilities required to support the complete lifecycle of building and delivering web applications and services. An example of this would the GoogleApps or Heroku.
So, whether its SaaS, IaaS, Paas - its all "hosted" - that is, running somewhere other than on your hardware, and you pay for it (of course, there are hybrid solutions that incorporate your hardware + their hardware, but let's keep it simple for now.)
How you pay - per use, monthly, quarterly, annually - is a condition defined by the provider and not a deciding factor on what type of provider it is.
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Most software vendors that offer hosted solutions will install their software on a server and network that they manage for you. In the enterprise, a company may want to start with a hosted solution and then bring it in-house as their company and infrastructure grows (We're hiring a network admin and buying servers for other purposes, might as well bring the app in-house and save money and get a better connection.). Some companies may prefer this option instead of purely SAAS with no chance bringing the data in-house.
You can buy an annual contract for the software and then pay monthly for the additional hosting. Number of users is generally tied into the cost.
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Answers OnStartups is a question and answer site for entrepreneurs looking to start or run a new business. It's 100% free, no registration required.
I seem to struggle at one thing, being all over the place. I believe I have good entrepreneurial mindset and wondering if I am doing it all wrong. My aim is to quit the corporate job and run my own show.
So far I have started 2 companies and currently running them at the same time. Here is the scenario.
One is a groupon clone website
• very time consuming, service driven
• high need for funding in terms of growth
A niche publishing company
• require good content strategy
• less time consuming
• doesn't require funding. i can fund mostly from my pocket.
I am passionate about these two as I think there's a gap in the market.
Do you think having two companies, running them while working full-time is a good strategy?
Should I concentrate on one and drop the other? Which one?
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closed as not constructive by Karlson, Christian, littleadv, Gary E Mar 12 at 19:00
As it currently stands, this question is not a good fit for our Q&A format. We expect answers to be supported by facts, references, or specific expertise, but this question will likely solicit debate, arguments, polling, or extended discussion. If you feel that this question can be improved and possibly reopened, see the FAQ for guidance.
1 Answer
You are potentially working on generating a disaster.
I would sit down an check on these things:
1. How much time I actually spend working at my full time job?
2. How much time I actually spend working at company 1?
3. How much time I actually spend working at company 2?
4. How much money does company 1 bring in now? How much is the potential return from Company 1?
5. How much money does company 2 bring in now? How much is the potential return from Company 2?
When you actually figure this out you will get a clearer picture of what you should do next. As it stands you simply trying to substitute your your judgement with the collective judgement of this forum. That's no way to run your business or run your life. On top of this depending how many hours a day you spend running things you will want to consider that at some point you might start working for the hospital bills. Yes sometimes we all think that we are 10 feet tall an bulletproof until that comes back to bite you.
So sit down, figure out what you are spending and what you are earning and this will make your decision clearer.
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Tell me more ×
Answers OnStartups is a question and answer site for entrepreneurs looking to start or run a new business. It's 100% free, no registration required.
A few months ago, I was asked to join a startup for $85k/year. All the company really had was a pretty big angel investor and one guy with the idea (wealthy father and son with the idea). Before they brought me on, they had spent some money on marketing the hype behind the idea itself, built a pre-registration and that was it. When they brought me on I built the very first MVP that works well. After 3-4 months they awarded me 2% of the company's shares and said I have an opportunity to earn more as long as I grow with the company. Building the whole software up until now, makes me believe that I am entitled to much more than 2%, but i really don't know how this works. Do I ask for more upfront? or is this a type of thing where I get some sort of 5 year plan to earn up to 20% of the company. The CEO of the company is assuring me that I can own up to 20% of it as long as I stay with the company/work hard etc. I just don't want to spend 12 hours a day on it if i'm not going to have a bigger stake in it. What are things I should watch out for, and how much equity should I be looking to get in this situation? Keep in mind that the angel investor still is able to expand for us by hiring more programmers, hiring internal help or anything we really need to get to the next level.
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5 Answers
Sadly for you it doesn't work like this. These guys have the vision and the money, so all they need is the capability to deliver which they can get from any decent developer. Yes, you built it, but just like when a property developer gets builders in to build that fabulous house, it isn't the builder that takes a big slice of the profits. You just pay the builder the going rate for a builder.
Saying that, I think they've been very generous giving you 2% a year on top of the $85k. I wouldn't rock the boat; think about how easily they could go out and hire another developer.
If they are talking about giving you even more, upto 20%, then wow - this is much more than you should ever expect as a developer on the payroll. You're onto a good thing - that is - if the product/website/idea actually takes off and is worth anything. Focus on that.
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If you are getting $85k/year (which is a very good remuneration), then why are you expecting equity?
If it was my company and I was paying you $85K/year with no connection to the success of company, I wouldn't give any equity at all.
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Don't be upset after seeing the previous three answers. While they are all great points, you may have other choice.
As Joel mentioned in another related question, Forming a new software startup, how do I allocate ownership fairly?, equity is mainly about risk-taking and vesting. (You may also find other great points there)
It's nothing wrong to get a salary being a developer. It's also nothing wrong to wear the hat of entrepreneur to seek for equity as one of the primary members of the startup, when the founder(s) appreciate that.
Now back to your question, the boss said you could get 20% as long as you stay and work hard etc. That's a pie in the air. It's hard to judge if you work hard or not, it's hard to make them happy every year to award you a slice of that pie by promise.
May I suggest, if you are serious about your future equity and confident at the vision, you need to take risk, more specifically, by vesting. A choice is to negotiate a plan to convert a rather part of your salary into equity timely, instead of extra rewarding from the boss only when he is happy.
If both agreed, you need a paper to write them down, or better a contract.
By taking risk you gain a fairer position against boss and investors. The boss will have more confidence on your motivation, and you will only be happy to code 12 hours per day, though not recommended by doctor.
Please note this is only a suggestion probing another possibility. There is risk. Please consider is thoroughly by yourself before taking action.
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I wouldn't say you deserve more than that. They are paying you $85k a year to develop their site. You are a developer and therefore you get paid to do that kind of thing. It's the service you are offering. I would be happy that you got offered equity!
Good luck :-)
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Entrepreneurship isn't really about the "know-how", it's about the "know-what".
You had the know-how all along. In fact, most people with a programming background could have started just about any of the big software companies that once were just startups. The key skill isn't knowing HOW to program, it's knowing WHAT to program (and also what NOT to program). At the same time, as a programmer myself, I understand your point of view: two identically working programs can be built in very different ways. You're putting all your energy into writing quality code and you're thinking about it pretty much all day. That is a necessary but non-sufficient condition for startup success.
In your case, I think a 2% equity grant was a generous contribution; not to mention that you're also getting a salary. Your programming skill on its own isn't worth much (around 80K per year); it's the skill of vision that makes or breaks a startup and that also makes your programming skills worth more.
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From NAS-Central Buffalo - The Linkstation Wiki
Revision as of 13:07, 9 March 2008 by Mindbender (Talk | contribs)
Jump to: navigation, search
9.3.08: Howto: Overclock the Kurobox Pro/Linkstation Pro/Linkstation Live
Original source for the v2-hardware was this japanese article. thx to dommer we now have instructions for the v1 hardware. (Comparison v1 vs v2)
3.3.08: Pictures & Info from the LS-HGL
26.2.08: Forums & Downloadsection moved
Mindbender
3.2.08: Please participate in this vote
Mindbender
28.1.08: Attempt to summarize all opinions
Mindbender
24.1.08: Noteworthy post from the WHAT IS GOING ON?!?!?-Thread
Read it. Mindbender
19.1.08: Please read WHAT IS GOING ON?!?!? and post your opinion. Mindbender
15.1.08: The cs05q3armel-optware-feed was reported to be working on the Linkstation EZ aka LS-LGL by mobster in IRC!
You need to follow the LS-Pro bootstrapping steps. Mindbender
13.1.08: The cs05q3armel-optware-feed is working on the Linkstation Pro DUO!
I enhanced the bootstrap instructions so you just need to read them and follow them step by step. Mindbender
12.1.08: info + pics from the Linkstation Pro DUO
very similar to the rest of the arm9-boxes, acp_commander works. Mindbender
12.1.08: Reorganized Wiki Sidebar, added 2 new forum sections for the LS EX(LS-LGL) and the LS Pro DUO. Mindbender
5.1.08: the buffalo wiki from nas-central.org was moved to buffalo.nas-central.org and a general, vendor independent wiki was placed at nas-central.org
Take a look at the new subwikis of nas-central.org (by clicking on the links in the "nas-central-links"-box on the left in the navigation-bar) and the new blog! Mindbender
2.1.08: Happy new year! wiki & forum maintainance scheduled on Saturday 5th Jan 07, 5 pm UTC
expected length 2 hours Mindbender
26.12.07: Missing sources in the user-dirs and upload-folder in the downloadsection
Anyone with access to the downloadsection please help, thanks. Mindbender
25.12.07: Merry christmas from the nas-central.org team :)
19.11.07: Status of Development - 19.11.07
19.11.07: LS Pro/LS Live v1 Boot Modes
Note the 2 different melodies. There are reports that this does not work on the v2 hardware. (Someone with serial please check what happens when the reset button is pressed on the v2)
12.11.07: Disassemble_the_TSPROv2/TSLIVE
Here you see some info regarding JTAG/serial/PCIe x1 on these boxes:
8.11.07: Migration of terastation.org wiki content to nas-central.org
Help transfering over the content of the terastation.org wiki to this one here!
Wiki page for coordination
7.11.07: Updated 2007 Gentoo Image available for PPC Linkstations/Kuroboxes
Release Thread
15.10.07: Optware bootstrap-files available for LS Pro/LS Live and Tera Pro v2/Tera Live
Currently there are no kernel modules inside...which will change via a seperate feed. But there are loads of packages!
11.9.07: Also notice the new page about the global overview over all NAS-hacking-Communities.
I hope it helps NAS-Users in general to find to their NAS-Community the first time more easily.
11.9.07: While we are fixing hundreds of links review the history of LinkStationwiki.net. Also checkout the history of LinkStationwiki.org if you want to know how everything started.
10.9.07: We are nas-central.org now!
8.9.07: Howto: Resizing the system partition with parted magic live cd
8.9.07: The Server has been installed at osuosl.
The plans are to have everything moved within a week (in case nothing unexpected happens).
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Wikia
SRD:Improved Multiweapon Fighting
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Improved Multiweapon Fighting [General]Edit
PrerequisitesEdit
Dex 15, three or more arms, Multiweapon Fighting, base attack bonus +9.
BenefitEdit
In addition to the single extra attack a creature gets with each extra weapon from Multiweapon Fighting, it gets a second attack with each extra weapon, albeit at a –5 penalty.
NormalEdit
With only Multiweapon Fighting, a creater can only get a single attack with each extra weapon.
SpecialEdit
This feat replaces the Improved Two-Weapon Fighting feat for creatures with more than two arms.
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14th Regiment, Kansas CavalryEdit This Page
From FamilySearch Wiki
United States U.S. Military Kansas Kansas Military Kansas in the Civil War 14th Regiment, Kansas Cavalry
Contents
Brief History
The 14th Regiment, Kansas Cavalry was organized at Fort Scott and Leavenworth April, 1863, as a Battalion of 4 Companies for escort to General Blunt. The regiment organized at Fort Scott, December, 1863. It mustered out June 25, 1865.[1]
For more information on the history of this unit, see:
Companies in this Regiment with the Counties of Origin
Men often enlisted in a company recruited in the counties where they lived though not always. After many battles, companies might be combined because so many men were killed or wounded. However if you are unsure which company your ancestor was in, try the company recruited in his county first. Men from Brown and Shawnee counties enlisted in this regiment in companies A, D, H and K.
The Civil War Soldiers and Sailors database lists 1,760 men on its roster for this unit. Roster.
Other Sources
• Beginning United States Civil War Research gives steps for finding information about a Civil War soldier or sailor. It covers the major records that should be used. Additional records are described in 'Kansas in the Civil War' and 'United States Civil War, 1861 to 1865' (see below).
• National Park Service, The Civil War Soldiers and Sailors System, is searchable by soldier's name and state. It contains basic facts about soldiers on both sides of the Civil War, a list of regiments, descriptions of significant battles, sources of the information, and suggestions for where to find additional information.
• Kansas in the Civil War describes many Confederate and Union sources, specifically for Kansas, and how to find them.. These include compiled service records, pension records, rosters, cemetery records, Internet databases, published books, etc.
• United States Civil War, 1861 to 1865 describes and explains United States and Confederate States records, rather than state records, and how to find them. These include veterans’ censuses, compiled service records, pension records, rosters, cemetery records, Internet databases, published books, etc.
References
1. National Park Service, The Civil War Soldiers and Sailors System, (accessed 14 December 2010).
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Changes related to "Maryland, Naturalization Indexes (FamilySearch Historical Records)"
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NOTE: If you are a developer, please use a private wiki based on foswiki/trunk on a daily base ...or use trunk.foswiki.org to view this page for some minimal testing.
Use Item9693 for docu changes for 1.2 and 2.0.
Item10355: Fatwilly theme looks unfinished
Priority: CurrentState: AppliesTo: Component: WaitingFor:
Enhancement Closed Extension PatternSkin
Some tweaking needs to be done:
• Add icons to topic actions at the top
• Remove underlines from topic action bar at bottom
• Sidebar:
• Top of side bar sticks over top bar
• Add gradient bg for headers in sidebar
• Remove colored web color line
-- ArthurClemens - 15 Feb 2011
Also popup.gif is missing from the MANIFEST file
-- GeorgeClark - 07 Mar 2011
The FatWilly theme reverted the pre fix in Item2433. Verbatim blocks need overflow:auto
-- GeorgeClark - 08 Mar 2011
I fixed the right bar overlapping issue and the underline links in the topic actions bar.
-- ArthurClemens - 09 Mar 2011
To do: the search box is empty, no hint that it is seach. this is only on f.o.
-- ArthurClemens - 10 Mar 2011
Personal sidebar does not need a bottom border.
-- ArthurClemens - 10 Mar 2011
Very bad: the login screen has everything centered. Quick fix applied on f.o.
-- ArthurClemens - 11 Mar 2011
Topic revision: r23 - 16 Apr 2011, KennethLavrsen
The copyright of the content on this website is held by the contributing authors, except where stated elsewhere. see CopyrightStatement.
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GlobalVoices in Learn more »
Special topic archive · 22 posts
This initiative is a companion piece to Communication and Human Development: The Freedom Connection?. Global Voices has been commissioned by the Canadian International Development Research Centre (IDRC) to write these stories on the future of ICTs and development. Learn more
Latest stories about The Future of ICT for Development
29 April 2010
East Timor: Connecting Civil Society
Providing internet access to civil society has been a key priority of the few information and communication technology (ICT) initiatives that exist in East Timor so far.
14 December 2009
ICT4D: Past mistakes, future wisdom
What makes an ICT4D project fizzle out? What are the common mistakes that donors, planners and implementers make when trying to run an ICT4D project? Practitioners discuss in a public Twitter chat.
7 December 2009
ICT4D for Women: Opportunities and Risks
Mobile phones present opportunities for development as well as risks for further abuse and marginalization of women. Gender awareness is crucial when it comes to using ICT for development.
3 December 2009
M-banking: Going where no bank has gone before
Millions and millions of low-income, unbanked people stand to benefit (and maybe prosper?) from the development of mobile financial services in the next years, but there are several technological, logistical, and security challenges that must be addressed first.
26 November 2009
India's tryst with e-health: A healthier future for its rural millions?
About 700km away from Bangalore, across a couple of remote villages in the Bidar district, a quiet revolution has been going on. No, not a political one, but a remarkable pilot project in telemedicine.
24 November 2009
Can ICTs aid small-scale farmers?
The world's small-scale farmers grow a large amount of food and provide many important jobs in rural areas. However, they do their work at great economic and environmental risk. How can ICTs make the jobs and lives easier for the world's farmers?
17 November 2009
Impact of ICT on Indigenous Cultures: Rejuvenation or Colonization?
Can ICT truly preserve and protect distinct identities and culture? The cultural debate surrounding deployment of ICT in the field of indigenous/ knowledge and culture simply refuses to die down.
9 November 2009
Uganda & Kenya: In Search of e-Governance
Good governance has been linked to gains in economic and human development. Governments have begun using technologies to offer more citizen services, expand transparency and make information more accessible. We look at how Kenya and Uganda use ICTs to create better governance.
3 November 2009
The future of ICT4D: How soon is now?
In the final of three posts on the future of ICTs for development, we examine a few projects that could change the way people leverage technology in rural areas.
27 October 2009
Disaster Management and the role of ICTs
In a first post of the series, we explore the role of ICTs in Disaster Management and the paradigm shift in Disaster Management strategies that came about post the aftermath of the Indian Ocean Tsunami in 2004.
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GlobalVoices in Learn more »
A Comparison of China's and America's Richest People
This post also available in:
Español · Comparación de los más ricos de China y Estados Unidos
Swahili · Matajiri Wakubwa China na Marekani Walinganishwa
Liz Carter from the Tea Leaf Nation translated an info-graphic by CN politics [zh], which compares the character of China's and America's richest people.
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An unofficial blog that watches Google's attempts to move your operating system online.
Send your tips to gostips@gmail.com.
September 19, 2006
Captions on Google Video
Google Video tries to promote captions and features a small list of videos that have captions. Although adding video captions was available in the video status section, I didn't see any video with captions until today.
It's really strange that Google supports only SubViewer (*.SUB) and SubRip (*.SRT) formats, instead of focusing on professional formats used in television, for example. Most people who upload their videos won't take the time to create subtitles, as this requires a software and it's not very easy to do.
Some speech recognition combined with an automated translation software would be really useful in this area. Or at least a collaborative captioning system, similar to the way volunteers translate Google interface.
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wiki:Help/Action/SelectConnectedWays
Version 4 (modified by skyper, 2 years ago) (diff)
small changes
This page is incomplete: The icon image link is missing
Languages:
Plugins -> UtilsPlugin2 -> All Connected ways
Keyboard shortcut: Crtl+Shift-E
Select all connected ways.
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[Date Prev][Date Next][Thread Prev][Thread Next][Date Index][Thread Index]
Re: [libreoffice-marketing] Re: Media Contacts for TDF
Hi,
thanks everyone for your great feedback! I've incorporated this now on the TDF, challenge and conference website.
Remember to use these changed section for new press releases, too. :)
Florian
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References:
[libreoffice-marketing] Media Contacts for TDFItalo Vignoli <italo.vignoli@gmail.com>
[libreoffice-marketing] Re: Media Contacts for TDFFlorian Effenberger <floeff@documentfoundation.org>
Re: [libreoffice-marketing] Re: Media Contacts for TDFItalo Vignoli <italo.vignoli@gmail.com>
Re: [libreoffice-marketing] Re: Media Contacts for TDF"C. Olofson" <c.olofson@gmail.com>
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Shops Pandan Mewah is on Mewah 8/1, J; is on Mewah 2/11, J; is near Mewah 2/7, J; is near Mewah 2/5, J; is near Mewah 4, J; is near Mewah 3/2, J; is near Mewah 2/9, J; is near Mewah 2/1, J; Shops Pandan Mewah is geographically located at latitude(3.1274 degrees) 3° 7' 38" North of the Equator and longitude (101.7658 degrees) 101° 45' 56" East of the Prime Meridian on the Map of Kuala Lumpur.
The locations related to Shops Pandan Mewah are represented by the shortest path a radio wave would travel and may not be nearest by road. For example, Shops Pandan Mewah is located 161 metres from Mosque Pandan Mewah. Shops Pandan Mewah is located 195 metres from Chin Lin Auto Workshop. Shops Pandan Mewah is located 233 metres from Hospital Ampang. Shops Pandan Mewah is located 254 metres from Hata Square. Shops Pandan Mewah is located 260 metres from Pandan Mewah Industrial Park.
Featured Places Of Interest Located NearbyHospital Ampang is located 0.2 Kilometres away from Shops Pandan Mewah. Hospital Ampang - 1 Photo(s) Featured.
De Palma Hotel 3.6km, Maluri Hotel 4km, Flamingo Hotel 4.1km, are places to stay (hotel, service apartment, inn) located near Shops Pandan Mewah.
Hata Square 0.3km, Pandan Mewah Industrial Park 0.3km, Shops Tasik Tambahan 4 14 0.4km, are places to shop (shopping mall, shop houses) located near Shops Pandan Mewah.
Flamingo Bowl 4.1km, Yayasan Seni Art Gallery 4.5km, Cultural Craft Complex 5.9km, are places of interest (attraction) located near Shops Pandan Mewah.
SMK Pandan Mewah 0.5km, SJK Ampang Campuran 0.6km, SJK Taman Tasik 0.7km, are places of learning (school, college, university) located near Shops Pandan Mewah.
Field Pandan Mewah 0.3km, Park Tambahan 4 13 0.5km, Jelawat 11 Park 0.5km, are parks, playgrounds, open fields or commons located near Shops Pandan Mewah.
Shops Pandan Mewah
Mosque Pandan Mewah
Chin Lin Auto Workshop
Hospital Ampang
Hata Square
Pandan Mewah Industrial Park
Field Pandan Mewah
Mcdonalds Drive Thru
Pandan Court
Pandan Mewah Heights Condo
Shops Tasik Tambahan 4 14
Xi Yin Furniture Store
Sri Mayang Apartment
Park Tambahan 4 13
SMK Pandan Mewah
Loji Kumbahan
Projet Pandan Mewah
Jelawat 11 Park
Field Taman Putra
Click here to zoom in
Where do you want to go?
Location Information
Latitude °
Longitude °
PlaceName
Category
Shops Pandan Mewah
Esso Pandan Mewah is about 0.6 km away.
Pandan Indah Industrial Park is about 0.6 km away.
Moon Tie Quaa Temple is about 0.6 km away.
Park Putra 6 is about 0.6 km away.
SJK Ampang Campuran is about 0.6 km away.
Surau Taman Sri Raya is about 0.6 km away.
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Dynamic Range
Info
Search:
Alternate term for density range. See Density Range.
The term dynamic range is also used to refer to the measure of the sensitivity of any optical device. See Scanning.
All text and images are licensed under a Creative Commons License
permitting sharing and adaptation with attribution. (See Copyrights for details.)
PrintWiki – the Free Encyclopedia of Print
About PrintWiki Policies Hosted by WhatTheyThink
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Let us not forget that the greatest composers were also the greatest thieves. They stole from everyone and everywhere. Casals, Pablo
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Pau Carlos Salvador Casals i Defill (December 29, 1876 October 22, 1973), commonly known as Pablo Casals, was a virtuoso Catalan cello player (and later conductor). He made many recordings throughout his career, of solo, chamber, and orchestral music, also as conductor, but Casals is best remembered for the recording of Bach's Cello Suites he made from 1936 to 1939.
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Art, like Nature, has her monsters, things of bestial shape and with hideous voices. Wilde, Oscar
Excerpt from The Picture of Dorian Gray · This quote is about art · Search on Google Books to find all references and sources for this quotation.
A bit about Wilde, Oscar ...
Oscar Fingal O'Flahertie Wills Wilde (October 16, 1854 November 30, 1900) was an Anglo-Irish playwright, novelist, poet, and short story writer. One of the most successful playwrights of late Victorian London, and one of the greatest celebrities of his day, known for his barbed and clever wit, he suffered a dramatic downfall and was imprisoned after being convicted in a famous trial of "gross indecency" for homosexual acts.
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Victory and defeat are each of the same price. Jefferson, Thomas
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Youth troubles over eternity, age grasps at a day and is satisfied to have even the day. Gilmore, Dame Mary
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It's easy! Just pick the product you like and click-through to buy it from trusted partners of Quotations Book. We hope you like these personalized gifts as much as we do.
Make and then buy your OWN fantastic personalized gift from this quote
The secret to humor is surprise. Aristotle
Make a fabulous personalised bracelet or other form of jewellery with this quote
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212 - The Extra Degree
The one extra degree makes the difference. This simple analogy reflects the ultimate definition of excellence. Because it's the one extra degree of effort, in business and life, that can separate the good from the great. This powerful book by S.L. Parker and Mac Anderson gives great examples, great quotes and great stories to illustrate the 212° concept. A warning - once you read it, it will be hard to forget. Your company will have a target for everything you do ... 212°
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Quotes about medicine
These are quotes tagged with "medicine". You can also search for quotes containing the word medicine.
"They do certainly give very strange, and newfangled, names to diseases."
Plato on medicine
3 fans of this quote
"He who lives by medical prescriptions lives miserably."
Proverb on medicine
"Quackery has no friend like gullibility."
Proverb on medicine
"Desperate maladies require desperate remedies."
Proverb, French on medicine
"It is medicine, not scenery, for which a sick man must go searching."
Seneca on medicine
"By medicine life may be prolonged, yet death will seize the doctor too."
Shakespeare, William on medicine
3 fans of this quote
"Some remedies are worse than the disease."
Syrus, Publilius on medicine
"In medicine sins of commission are mortal, sins of omission are venial."
Tronchin, Theodore on medicine
"Sleep is better than medicine."
Unknown, Source on medicine
"The art of medicine consists of amusing the patient while nature cures the disease."
Voltaire on medicine
9 fans of this quote
"She saw she had fallen into the hands of one of those doctors who have strayed too far from apparent in the direction of the soul."
West, Rebecca on medicine
"Do not forget: in medicine, there are more important things than life and death: dollars and cents. "
Kocher, Gerhard on medicine
"Medicine knows no limits, especially not its own."
Kocher, Gerhard on medicine
"From year to year, it is more obvious: the goal of medicine is not health but the further extension of the health system."
Kocher, Gerhard on medicine
"You can't treat a car like a patient. A car needs love. "
Kocher, Gerhard on medicine
"Mankind has survived all catastrophes. It will also survive modern medicine. "
Kocher, Gerhard on medicine
"Many of us will die for science without knowing it. "
Kocher, Gerhard on medicine
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Quotes about the end
These are quotes tagged with "the end". You can also search for quotes containing the word the end.
"the world doesn't end with a bang, but a whimper"
Unknown on the end
"in my end is my beginning."
Unknown on the end
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31 January 2013
Cats That Look Like Famous Paintings
Joseph Ducreux - Yawning Self-Portrait
Rembrandt - Self Portrait
Edgar Degas - Two Dancers
John William Waterhouse - Echo and Narcissus
cats
Frederic Leighton - June Flame
Gerard Hoet - Young Man Playing the Flute
Tizian - Venus of Urbino
Ford Madox Brown - Romeo and Juliet
Guido Reni - Repentance of St Peter
paintings
Sir Joshua Reynolds - Princess Sophia Matilda
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Robotic Surgery More Accurate
Posted 11 Feb 2006 at 16:28 UTC by Rog-a-matic
A new study from Imperial College in London shows that robot assisted knee surgery is significantly more accurate yet slightly slower than conventional surgery. There were no additional side effects and in most cases recovery was faster. Other reports suggest similar success with robotic assisted surgery. The real question is: will AI programmers be able to make improvements in bedside manors? That should be an easier task than the surgery part :)
See more of the latest robot news!
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Help Wikitravel grow by contributing to an article! Learn how.
Wikitravel:TourBusStop
From Wikitravel
Revision as of 07:14, 20 May 2009 by Tatatabot (Talk | contribs)
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This is the Wikitravel TourBus stop.
Wikitravel is a project to create a free, complete, up-to-date and reliable world-wide travel guide. So far we have 26,275 destination guides and other articles written and edited by Wikitravellers from around the globe.
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Is the starting point for those who want to help.
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Difference between revisions of "Youngstown"
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(Get out)
(added listing Cafe Cimmento)
Line 48: Line 48:
+
+
*<eat name="Cafe Cimmento" alt="" address="" directions="" phone="" url="" hours="" price="" lat="" long="">In the Downtown</eat>
*<eat name="Rosetta Stone Cafe" alt="" address="" directions="" phone="" url="" hours="" price="" lat="" long="">Great new restaurant in the Downtown</eat>
*<eat name="Rosetta Stone Cafe" alt="" address="" directions="" phone="" url="" hours="" price="" lat="" long="">Great new restaurant in the Downtown</eat>
*<eat name="Handel's Ice Cream" alt="" address="" directions="" phone="" url="" hours="" price="" lat="" long="">Voted one of America's best ice cream parlors</eat>
*<eat name="Handel's Ice Cream" alt="" address="" directions="" phone="" url="" hours="" price="" lat="" long="">Voted one of America's best ice cream parlors</eat>
Revision as of 03:39, 5 May 2008
Youngstown is a mid-sized city in Northeast Ohio and is the County Seat of Mahoning County.
Contents
Get in
By plane
• Youngstown-Warren Regional Airport, YNG. The airport is a conveniently located off Routes 11 and 82 in Vienna, OH. Its an hour away from Akron-Cleveland and Pittsburgh.
By car
Youngstown is served by the following interstate highways:
• I-76 serving Akron and Youngstown and connecting to beyond Pennsylvania to the east.
• I-80 is the Ohio Turnpike (a toll road) that runs across the northern part of the state, serving Cleveland, Akron and Youngstown (where I-80 and I-76 criss cross).
I-680 provides access to downtown areas and outer suburbs
Get around
WRTA provides the inner city and suburbs with bus transportation.
See
• Stambaugh Auditorium. Former home of The Youngstown Symphony.
• Chevrolet Centre. Home of the Steelhounds, Thunder, and site of many major concerts
• Powers Auditorium. Built in 1931 by the Warner Brothers. Home to The Youngstown Symphony.
• Youngstown Playhouse. America's Oldest ongoing community theater
• Oakland Center for the Arts. Great local theater productions
• Butler Institute of American Art. First museum of American Art.
Do
Sports
Outdoors
• Mill Creek Metroparks. Located in the heart of Youngstown, OH. Mill Creek Park encompasses approximately 2,600 acres, 20 miles of drives, and 15 miles of foot trails, and a rare collection of gardens, streams, lakes, woodlands, meadows and wildlife for all to enjoy. Visitors will find a wide range of recreational opportunities such as hiking, biking, picnicking, boating, Par-3, or championship golf, tennis, sand volleyball and more. Many of the facilities can be found at the James L. Wick, Jr. Recreation Area. Universally accessible trails, fishing pier, playground and picnic areas are located throughout the Park. Cabins and pavilions for group events are available for rental year-round. Mill Creek Park is the largest metropolitan park in the country, more than three times larger than Central Park in New York City!
Buy
• Southern Park Mall. Boardman
• Eastwood Mall. Located in Niles
Eat
• Cafe Cimmento. In the Downtown
• Rosetta Stone Cafe. Great new restaurant in the Downtown
• Handel's Ice Cream. Voted one of America's best ice cream parlors
• Fifth Season
• Springfield Grille
• Bean Counter Cafe in Downtown
• Scarsellas
• MVR
• Golden Dawn
• Oscars
Drink
• Downtown Draught House. Tons of beer selections. West Federal Plaza - Downtown
• Core. Federal Plaza in the Downtown
• The Cell. Nightclub near YSU
• Imbibe Martini Bar, in the Downtown.
• Irish Bob's, South Ave.
• Micky's, Market St. in the Uptown.
• Shenanigans, Market St. in the Uptown.
• University Pizzeria and Italian Eatery, Lincoln Ave. (Youngstown State University campus)
Sleep
• Best Western Meander Inn, 870 N Canfield Niles Road, +1 330 544-2378 (fax: +1 330 544-7926), [1].
• Fairfield Inn & Suites Youngstown Austintown, 801 North Canfield Niles Road, +1 330 505-2173 (fax: +1 330 505-3629), [2].
Get out
Golf
The Youngstown Region was ranked #4 in the country for golf by Golf Digest.
Mill Creek Golf Course - West Golf Drive - Youngstown
Pine Lakes Golf Club - 6233 Liberty Street - Hubbard
Reserve Run Golf Course - 625 E. Western Reserve Rd. - Youngstown
Kennsington Golf Club - 4171 Westford Place - Youngstown
This article is an outline and needs more content. It has a template, but there is not enough information present. Please plunge forward and help it grow!
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Australian Bureau of Statistics
Celebrating the International Year of Statistics 2013
ABS Home > Statistics > By Catalogue Number
3236.0.55.003 - Household and Family Projections, Australia: Projected Families -- Electronic Delivery, 2001 to 2026
Latest ISSUE Released at 11:30 AM (CANBERRA TIME) 25/06/2004
Page tools: Print Page Print All RSS Search this Product
• Data Cube (I-Note)
The text below was previously contained in a preview file on the details tab. It has been moved to be more accessible.
Classifications
At 30 June30 June 2001-2026
Projection seriesI, II, III
Part of stateCapital city/balance of state
Family typeCouple family with children
Couple family without children
One-parent family, male parent
One-parent family, female parent
Other family
Using this Dataset with SuperTABLE
When creating your own table from this SuperTABLE you must select at least one value from each of the following fields:
• At 30 June,
• Projection series,
• Part of state, and
• Family type
Failure to do this will result in incorrect data in your table.
Table for validation purposes:
PROJECTED FAMILIES BY FAMILY TYPE, AUSTRALIA, SERIES II
Family type
At 30 June 2001
At 30 June 2026
Couple family with children
2,491,540
2,610,263
Couple family without children
1,917,612
3,108,107
One-parent family, male parent
139,760
202,691
One-parent family, female parent
698,409
989,569
Other family
98,650
111,219
Total
5,345,971
7,021,849
© Commonwealth of Australia 2013
Unless otherwise noted, content on this website is licensed under a Creative Commons Attribution 2.5 Australia Licence together with any terms, conditions and exclusions as set out in the website Copyright notice. For permission to do anything beyond the scope of this licence and copyright terms contact us.
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Australian Bureau of Statistics
Celebrating the International Year of Statistics 2013
ABS Home > Statistics > By Release Date
3302.0.55.001 - Life Tables, Australia, 2003 to 2005
Previous ISSUE Released at 11:30 AM (CANBERRA TIME) 30/11/2006
Page tools: Print Page Print All RSS Search this Product
• About this Release
This product contains Australian life tables for males and females for the reference period. A life table is a statistical model used to represent mortality of a population. In it's simplest form, a life table is generated from age-specific death rates and the resulting values are used to measure mortality, survivorship and life expectancy.
© Commonwealth of Australia 2013
Unless otherwise noted, content on this website is licensed under a Creative Commons Attribution 2.5 Australia Licence together with any terms, conditions and exclusions as set out in the website Copyright notice. For permission to do anything beyond the scope of this licence and copyright terms contact us.
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"warc_url": "http://www.biomedcentral.com/1471-2148/7/S1/S2/abstract"
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This article is part of the supplement: First International Conference on Phylogenomics
Software
SCaFoS: a tool for Selection, Concatenation and Fusion of Sequences for phylogenomics
Béatrice Roure, Naiara Rodriguez-Ezpeleta and Hervé Philippe*
Author Affiliations
Canadian Institute for Advanced Research, Centre Robert Cedergren, Département de biochimie, Université de Montréal, Montréal, Québec H3C3J7, Canada
For all author emails, please log on.
BMC Evolutionary Biology 2007, 7(Suppl 1):S2 doi:10.1186/1471-2148-7-S1-S2
Published: 8 February 2007
Abstract
Background
Phylogenetic analyses based on datasets rich in both genes and species (phylogenomics) are becoming a standard approach to resolve evolutionary questions. However, several difficulties are associated with the assembly of large datasets, such as multiple copies of a gene per species (paralogous or xenologous genes), lack of some genes for a given species, or partial sequences. The use of undetected paralogous or xenologous genes in phylogenetic inference can lead to inaccurate results, and the use of partial sequences to a lack of resolution. A tool that selects sequences, species, and genes, while dealing with these issues, is needed in a phylogenomics context.
Results
Here, we present SCaFoS, a tool that quickly assembles phylogenomic datasets containing maximal phylogenetic information while adjusting the amount of missing data in the selection of species, sequences and genes. Starting from individual sequence alignments, and using monophyletic groups defined by the user, SCaFoS creates chimeras with partial sequences, or selects, among multiple sequences, the orthologous and/or slowest evolving sequences. Once sequences representing each predefined monophyletic group have been selected, SCaFos retains genes according to the user's allowed level of missing data and generates files for super-matrix and super-tree analyses in several formats compatible with standard phylogenetic inference software. Because no clear-cut criteria exist for the sequence selection, a semi-automatic mode is available to accommodate user's expertise.
Conclusion
SCaFos is able to deal with datasets of hundreds of species and genes, both at the amino acid or nucleotide level. It has a graphical interface and can be integrated in an automatic workflow. Moreover, SCaFoS is the first tool that integrates user's knowledge to select orthologous sequences, creates chimerical sequences to reduce missing data and selects genes according to their level of missing data. Finally, applying SCaFoS to different datasets, we show that the judicious selection of genes, species and sequences reduces tree reconstruction artefacts, especially if the dataset includes fast evolving species.
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The Scheme of Rizhao City Brand
Qingjun Wu
Abstract
Nowadays, the competition of cities essentially is a competition for city brands. In fact, it is necessary for each city to implement a city brand strategy on their developing road.Rizhao , a seaside city in China, has many fine resources such as the coastal deep harbor, the seashore sand beach and the University City ,which, however, still are separated rather than combined together.By developing the nationwide theme, the following article associates resources fully in an attempt to build up the Rizhao city brand.
Full Text: PDF
This work is licensed under a Creative Commons Attribution 3.0 License.
International Business Research ISSN 1913-9004 (Print), ISSN 1913-9012 (Online)
Copyright © Canadian Center of Science and Education
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< Kids Say The Damnedest Things
Fierce HTML Character Entities >
Interesting Google Searches: "hacker's guide" turns up various pieces of hardware and software that have hacker's guides. Gems include The noweb Hacker's Guide, A Hacker's Guide to Ncurses Internals, and the justifiably-patronizing Manager FAQ ("Your manager probably doesn't have the same appreciation for meta-humor, recursion, and obscure technical puns that you do.").
Filed under:
[Main] [Edit]
Unless otherwise noted, all content licensed by Leonard Richardson
under a Creative Commons License.
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Rate This Article
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Materials flow of mercury in the economies of the United States and the world
Materials flow of mercury in the economies of the United States and the world
This article has been reviewed by the following Topic Editor: Cutler J. Cleveland
Introduction
Materials flow studies provide insights into the dynamics that affect flow, that quantity of a specific material moving from one medium and (or) location to another, in this case, mercury. These studies permit decision-makers to leverage knowledge of materials flow into more efficient management with respect to social goals. For example, policy might be directed toward minimizing environmental impact by adjusting some aspect of a particular material’s flow. Materials flow studies address the life cycle of materials from extraction, through processing, manufacturing, use, re-use, and disposition. Materials flow studies characterize not only the movement of materials (including losses to the environment), but also the stocks: a stock (inventories or products in use) is where a specified material resides, relatively unaltered, for a period of time.
Mercury was selected for study because of its demonstrated toxic effects on the environment and its potential for impact on human health. Mercury is widely dispersed throughout air, soils, and water. It is mobile within the environment, so any policy-oriented solution or technological advancement that limits the amount going into the environment could yield benefits.
Figure 1: Generic materias flow diagram
A materials flow study of mercury in the United States was last published by the U.S. Bureau of Mines in 1994 and contained data through 1990. This study updates the information through 1996. This year was selected because the most complete set of data was available, and estimations and assumptions were thus held to a minimum. In that brief span of time between 1990 and 1996, major changes, precipitated mainly through government policy, have occurred within the mercury lifecycle. One purpose of this report is to document those changes; another is to identify trends in mercury production and usage for the future. More recent data from 1998 confirm that these trends have continued.
Mercury and its compounds have a long history of human use. It has been found in Egyptian tombs dated back to 1500 BC. Cinnabar, a mercury-sulfide mineral, was used as a red pigment in early Egypt and China. Spiritualists associated mercury metal with mystic qualities, and alchemists tried to transform it into gold. It was used for centuries as a curative for syphilis. Modern uses for mercury include electrical switches, thermometers, dental amalgams, lighting (mercury vapor and fluorescent lamps), flow meters, batteries, fungicides, electrochemistry, catalysis, explosives, gold recovery, and bactericides.
Mercury is the only metal that is liquid at room temperature (20°C). Mercury is a good electrical conductor and is highly resistant to corrosion. It has a high charge density to weight ratio, which makes mercury batteries preferable for space missions. Mercury is easily separated from its parent minerals through the application of heat, enhancing its ability to be recovered in a pure state. Mercury has the highest solubility in water of any metal, and easily vaporizes into the air; these two properties make it very mobile in the environment.
Mercury vapor can be carried over great distances in the atmosphere, and be deposited into lakes and streams. Under anaerobic (oxygen-deficient) conditions, deposited mercury undergoes biochemical change to become methylmercury. Methylmercury can enter and proceed through the food chain, bio-accumulating in fish tissue to levels that can endanger populations of animals and humans that feed farther up the chain. Government advisories, which warn consumers about mercury-contaminated fish, have become more frequent throughout both the industrialized and the developing world. These concerns have been the main impetus for greater regulatory control of mercury.
Although natural sources of mercury exist in the environment, such as mineral deposits, hot springs, and volcanoes, increased amounts of mercury have entered into the biosphere from anthropogenic (human-derived) sources. Some of the more significant anthropogenic mercury-emission sources include municipal and medical waste incineration, coal combustion, manufacturing process leaks, and the leaching of solid wastes in landfills.
In the past, management and regulatory responses to the growing mercury problem have generally been constrained by a lack of information on sources, method of transport, chemical interaction with the environment, and biological significance of mercury in the environment. However, significant research advances during the past decade have allowed scientists to identify, measure, and examine the important biogeochemical processes that determine the fate and biological availability of mercury in the environment. A detailed discussion of these issues is outside the scope of this study, but the following references may be helpful: U.S. EPA, “Mercury Study Report to Congress,” 1997; “Mercury as a global pollutant,” in Water, Air, and Soil Pollution, v. 80, 1995; “Mercury pollution – Integration and synthesis,” edited by Carl J. Watras and John W. Huckabee, 1994; and “Mercury as an environmental pollutant,” in Water, Air, and Soil Pollution, v. 56, 1991.
A primary requirement for any materials flow analysis is substantiated data. Where U.S. data were unavailable because they were either not collected or not reported, certain calculations were made based on estimations and assumptions. Flow splits (numerical fractions into which a single flow from one stock distributes to two or more different stocks) were estimated if data were unavailable. In addition, approximations were made for byproduct mercury production from gold mines, mercury incorporated in lighting fixtures, and disposal splits between recycling and land filling. As mercury in the environment is an international issue, global interregional mercury flows were estimated for 1990 and 1996. Unless specifically noted, the figures in this study were produced using U.S. Geological Survey’s data (Mineral Commodity Summaries, 1997–1998, and Minerals Yearbook, 1995–1997). The details concerning the quantification of various textual and graphical parameters mentioned are contained in the Appendix.
Mercury flow analysis
Domestic
Figure 2: Components of U.S. apparent supply of mercury (1970–1998).
U.S. mercury apparent supply[1] is shown in figure 2 for the period 1970 to 1998. These data suggest three different time periods, characterized by different market dynamics. From 1970 to 1986, U.S. primary mercury mine production and net imports contributed significant amounts to the mercury market. Net imports, during this first period, grew strongly from 33 percent of apparent supply in 1970 to 83 percent in 1974, and then decreased slowly to 58 percent in 1978. Thereafter, net imports plunged to 13 percent of apparent supply in 1980, remaining steady until 1984, when they advanced to 42 percent. The market share of U.S. primary mercury mine production was inversely correlated to net imports, indicating direct substitution of one for the other. Throughout this period, U.S. secondary mercury production from scrap supplied between 4 and 26 percent of apparent supply.
Figure 3: U.S. industrial reported consumption of mercury (1970–1997).
The second distinct period spanned 1986–1992; it was characterized by a rapid decrease in U.S. apparent mercury supply, caused by legislation to eliminate mercury in batteries (54 percent of demand for mercury in 1984, and 2 percent in 1992) (fig. 3). Also contributing to the reduction in apparent mercury supply was the elimination of mercury in paint as a fungicide (16 percent in 1989, and 0 percent in 1992). U.S. stockpile releases continued through this period, and secondary production showed little change. From 1989 through 1992, the United States exported mercury, most likely from industry stocks held to manufacture batteries and paint additives, but also from large U.S. Government stockpile releases (1991 and 1992). Mine production of primary mercury in the United States ceased in 1991.
The third distinct historical period, from 1993 to 1998, was one of adjustment to current conditions where apparent mercury supply had bottomed out. This period is characterized by increases to consumer and producer stocks, increasing net imports, no primary mine production, and greatly expanded secondary mercury production, supported by favorable (State-level) legislation mandating mercury recycling.
Figure 4: U.S. apparent supply and reported consumption of mercury (1970–1998).
The term “reported consumption” has a long history and was used in the past by the U.S. Bureau of Mines (USBM) and is currently used by the U.S. Geological Survey (USGS). Reported consumption is collected data from surveyed respondents and represents the mercury metal purchased from producers (at market prices, at time of purchase) by nonproducers. Distribution of U.S. mercury reported consumption among industrial sectors for the period 1970 through 1997 is shown in figure 3. This illustration demonstrates the details of the distribution of mercury among market sectors, and shows the major impact of curtailment of the use of mercury for batteries and paints, illustrating the discussion of figure 2.
Figure 4 compares apparent mercury supply with reported consumption. If stock changes were added to apparent supply, the result would track more closely with reported consumption.
In figure 4, when apparent supply exceeds the reported consumption line, inventories of producers and consumers are increased that year, and where the apparent supply fails to reach the reported consumption line, inventories are depleted. Between 1984 and 1989, mercury apparent supply never achieved the full reported consumption level; indicating continual consumer and producer inventory declines [2]. During 1991 and 1992, when net imports were negative (exports are greater than imports) and reported consumption was leveling off from the steep decline experienced between 1983 and 1991, the United States was actually depleting its total mercury stocks.
Emissions to the environment
In 1996, the burning of fossil fuels emitted 76 t of mercury into the atmosphere, as shown in figure 5[3]. Almost 87 percent or 66 t originated from the burning of coal. The single largest point source of anthropogenic mercury emissions is coal-fueled utility boilers used for electrical generation. Recovery of mercury presents a problem because it is present in coal in very small quantities, but the enormous amount of coal burned produces a large overall contribution. Oil and gas combustion, mainly in business and residential boilers and furnaces for space heating, emitted 10 t of mercury into the air. The concentration of mercury in oil and gas is even less than in coal. Oil and gas burners are widely dispersed, small, and the stacks are generally uncontrolled.
Figure 5: Domestic flow of mercury in 1996, in metric tons.
Various types of waste combustors and incinerators [4] emitted 4 t of mercury into the atmosphere in 1996. Two principal contributors were municipal waste combustors (27 t) and medical waste incinerators (15 t). Mercury additions to both municipal and medical wastes have been reduced, mainly by eliminating the use of mercury-containing batteries by mandate, and by the use of a new class of electronic medical instrumentation to replace those that formerly required mercury, for example, medical thermometers and blood pressure gauges. Hazardous waste (solid and liquid) was burned both in hazardous waste combustors and cement kilns. These sources emitted 11 t of mercury into the atmosphere in 1996.
Several factors are at work to reduce the total mercury emission levels from these sources. These factors include mandated stack emission controls, similar to those for municipal and medical waste combustors; and closure of hazardous waste combustors that had been justified by subsidies to the combustors for co-generated electricity. Finally, although hazardous waste can be utilized in cement kilns, its use limits production, because the fuel value of the waste is variable and its use requires more control. During periods of high capacity usage, cement kilns run on conventional fuels.
A total of 144 t of mercury entered the U.S. environment from all anthropogenic sources in 1996. This is 35 percent of the total mercury entering otherwise useful applications (417 t). A significant amount of mercury, about 13.9 t, entered the environment from spills, breakage, and other leaks as mercury was used. Comparison of figures 5 and 6 indicates that mercury mine closures in the early 1990’s were responsible for a significant reduction of mercury releases to the environment (78 t) from the milling and roasting of mercury ores. Domestic mercury releases to the environment in 1996 decreased by 97 t over 1990 levels, that is, 144 t (1996) versus 241 t (1990). Mercury releases from incineration decreased by 47 percent (100 to 53 t) from 1990 to 1996. This reduction took place because of fewer mercury-containing products entering waste streams and more efficient stack emission controls on incinerators. Mercury disposed of in landfills, excluding soil amendments [5], in 1996 (295 t) was 61 percent less than in 1990 (755 t).
Sources—1996
As shown in figure 5, U.S. mercury sources in 1996 included secondary production (446 t), by-product from gold mines (65 t), and mercury metal imports (340 t). These sources contributed 851 t, exceeding metal exports of 45 t, and reported consumption 372 t by a total of 434 t. The 434 t represent an increase of consumer stocks of 125 t, and an increase of producer stocks of 309 t. Secondary production of mercury, by itself, was greater than reported mercury industrial usage in the United States in 1996.
Figure 6: Domestic flow of mercury in 1990, in metric tons (Jasinski, 1994): natural and incidental releases, and mined, recycled, and imported metal supplied to domestic and exported markets. W, withheld.
In 1996, no government stockpile sales or chlor-alkali mercury-cell closures took place, so no mercury entered the economy from those particular sources. Although there is little reason for government stockpile sales in the near term, future market disruptions can be expected as occasional mercury-cell closures occur within the chlor-alkali industry.
The chlor-alkali industry uses mercury-containing electrolysis cells as one technology to produce chlorine from chloride salts of sodium or potassium. Approximately 3,000 t of mercury resides in this mercury cell stock. Mercury cell chlorine plants are slowly being phased out in favor of non-mercury technologies.
Sources—1990 versus 1996
In 1996, secondary production (446 t) was more than four times the level of secondary production in 1990 (mercury recovered from old scrap, fig. 6). Although no domestic, primary mine production of mercury occurred in 1996, 448 t was produced from U.S. mercury mines in 1990. The change from primary mine production to increased secondary production over the 6-year period is important because it eliminated a major source of mercury to the environment, approximately 72 t from milling and roasting, probably reflecting increased recovery efforts due to legislation. Currently, much of the recycling occurs in States that encourage and support recycling (see table 1).
From 1990 to 1996, there was a total trade turnaround of 591 t, indicating a growing dependence on foreign sources for the current mercury needs of the United States. Production of mercury, as a by-product of gold mining operations, decreased from 114 t in 1990 to 65 t in 1996, a 43 percent decrease. Imports of mercury rose sharply from 15 t in 1990 to 340 t in 1996. On the other hand, mercury exports decreased dramatically from 311 t in 1990 to 45 t in 1996.
Compared to 1990, when the U.S. Government stockpile released 245 t of mercury to the market, 1996 saw no such sales. Sales were suspended in 1994 pending the release of the Mercury Study Report to Congress, and have not resumed as of this writing (1999).
Disposition—1996
Figure 7: Domestic product flow of mercury through end uses in 1996, in metric tons.
Figure 7[6] illustrates that in 1996, 372 t of mercury flowed into private stocks. Private stocks are mercury residences that are nongovernmental stockpiles. They are controlled by producers of mercury metal, manufacturers of products containing mercury, users of mercury in other production processes (such as chloralkali plants), retail consumers, wholesale distributors, and scrap brokers. Private stocks include inventories (for example, ores and scrap, work-in-progress inventories, inventories for sale) and products in use (such as dental fillings, switches, fluorescent lamps). In 1996, 794 t flowed out of private stocks, of which 56 percent went into secondary production to be recycled and returned to useful applications. The balance, 44 percent, either was disposed into landfills (295 t), or was lost to the air from incineration processes (53 t). The total unrecovered mercury (lost during incineration or to landfills) of 348 t represents a private stock-wasting rate of 5 percent in 1996.
The majority of the private mercury stock was split between (1) chlor-alkali plants (45 percent), at 14 locations, and (2) wiring devices and switches (39 percent), widely dispersed in virtually every facet of the economy. Should these mercury-cell chloralkali plants close, most of the associated mercury stocks could be easily recovered. On the other hand, recovery of the mercury held in electrical devices would be much more problematic, although in some States, such as Minnesota, companies like Honeywell offer a free recycling program for mercury-containing thermostats.
The chlor-alkali industry used 136 t of mercury in 1996, almost triple the mercury usage in the next category, wiring devices and switches (49 t). Except for the chlor-alkali plants, and wiring devices and switches, which together make up 84 percent of private mercury stocks, all other private stocks had larger outflows of mercury in 1996 than inflows.
At the beginning of 1996, private stocks totaled 6,800 t of mercury, exceeding all U.S. Government mercury stockpiles of 4,600 t. Together, these private sector and government stocks (11,400 t) represent approximately a 27-year supply of mercury at the 417 t level of industrial and exported demand in 1996.
Disposition—1990 versus 1996
Figure 8: Domestic flow of mercury in 1990, in metric tons (Jasinski, 1994): products manufactured, in use, and obsolete.
A nearly 50 percent reduction in total mercury flows to industry occurred between 1990 and 1996. The mercury flows to industry went from 711 t in 1990 to 372 t in 1996. Comparing figure 7 with figure 8, mercury flowing into all specified industrial sectors in 1996 was lower than 1990 levels: dental 30 percent; laboratory 38 percent; measurement and control devices 62 percent; wiring devices and switches 30 percent; lighting 66 percent; batteries 100 percent; and chlor-alkali plants 45 percent.
Excluding the chlor-alkali industry’s private mercury stocks, which were not estimated in the 1990 report, the sum of all other private stocks decreased from 4,300 t in 1990 to 3,800 t in 1996, a compound annual stock reduction rate of slightly more than 2 percent.
In 1990, the paint sector was still adding mercury to waterbased paints, mainly as a fungicide. In 1996, this sector does not appear because mercury-containing paints were banned from the market by legislation in 1992. In 1990, mercury-containing dry cell batteries used 105 t of mercury; in 1996, virtually no mercury went to dry cell batteries because of both legislation and technological advances.
Case Study—Chlor-Alkali
Since 1989, the use of mercury for the production of chlorine and caustic soda (37 percent of total mercury consumption in 1996) has been the largest component of U.S. mercury consumption. Mercury is used in electrolytic cells (mercury cells) to decompose chloride compounds. During this process, small amounts of mercury are emitted to the air, water, and land as sludge and as wastewater. A detailed description and a flow diagram of this process are included in Information Circular 9412. Because mercury cells (a mature technology) account for a major part of total industrial usage of mercury, a more detailed look at the flow of mercury within this process is warranted.
Figure 9: 1996 mercury flow in the mercury cell process of the U.S. chlor-alkali industry, in metric tons.
The chlor-alkali industry employs three classes of stocks (inventories) that are shown in figure 9 [7]. They include new purchases or make-up (averaging about 150 t per year during the 1990’s) that are held in warehouses to be used to restore any losses from the process; an average mercury inventory (134 t) passing through the recycling processes within the plants; and an average mercury inventory cycling through mercury cells (2,770 t).
Figure 10: 1990 mercury flow in the mercury cell process of the U.S. chlor-alkali industry, in metric tons; n.a., not applicable.
In 1996, Toxic Release Inventory data (1997) indicated[8] that the chlor-alkali industry released approximately 8.0 t of mercury directly to the environment (7.6 t to air, 0.2 t to land, 0.2 t to water), 7 t to off-site recyclers, and 19 t to landfills. Less than 1 t is associated with the caustic product that leaves the plant, most likely distributed to paper mills. Subtracting these known losses of 35 t from the given 1996 purchase of 136 t of mercury by the industry leaves a total of unaccounted mercury in 1996 of 101 t. In 1999, the “missing” mercury continues to be the subject of intense scrutiny by the industry and the Environmental Protection Agency (EPA).
Comparing figure 9 (1996) with figure 10 (1990), reveals several trends: there were four more mercury cell chlor-alkali plants operating in 1990 than in 1996, and the incremental mercury inventory of the chlor-alkali industry to support those plants was 544 t. Mercury purchases by the chlor-alkali industry in 1990 amounted to 247 t, which were 111 t more than in 1996 (136 t). The industry landfilled 41 t less mercury in 1996 (19 t) than in 1990 (60 t), a 68 percent decrease. Releases and losses embodied in caustic product remained about the same. In 1990, the private stocks held by the chlor-alkali industry were approximately 3,600 t. These stocks had been reduced in 1996 to 3,050 t. This difference is an overall decrease of 15 percent, or about 2.5 percent per year. This inventory reduction for mercury cells most likely flowed out of the United States as exports, and is part of the negative trade balance in 1991 and 1992 (fig. 2).
Figure 11: U.S. mercury reported consumption, production, price, and legislation (1970–1997).
The chlor-alkali industry has been closing some mercury cells and tightening mercury flow controls on the remaining operational cells. No new mercury cell plants are being constructed. New, more efficient, and less costly technologies have been available for a long time, and they are being installed where new chlorine capacity is needed. However, some very efficient, large-capacity mercury cell operations still exist and will remain operational into the foreseeable future.
Legislation
Figure 11 illustrates time series for mercury reported consumption, production, and price alongside dates of regulatory and control legislation. Even though numerous regulations are in place, Federal and local governments are implementing new actions to further reduce mercury contamination of the environment from all anthropogenic sources and to limit the use and disposal of mercury. Recently established regulatory actions could reduce mercury emissions from municipal and medical waste about 90 percent by the year 2000, when proposed rules become effective. Table 1 lists legislation and programs that have affected mercury flows in the 1990’s.
Table 1: Legislation and programs affecting mercury.
Year
Authority
Summary
1992 EPABanned the land disposal of high mercury content wastes that are generated from the electrolytic production of chlorine and caustic soda (effective 5/8/92).
1992 New Jersey Restricted the sale and disposal of batteries containing mercury.
Banned the sale of products that have cadmium, mercury, or other toxic materials in packaging after 1/1/93.
1992 California and Minnesota Placed restrictions on the disposal of fluorescent light tubes.
1993 EPACanceled the registrations for the last two mercury-containing fungicides approved for use in the United States at the request of the manufacturer. Cancellation became effective 11/1/93.
1993 Florida
Arkansas, Minnesota, and New Jersey
Approved emissions regulations for resource recovery plants to limit stack emissions of mercury.
Limits the release of mercury to the environment from the disposal of batteries.
Banned the sale and distribution of mercuric-oxide button cell batteries.
Phase out the amount of mercury permitted in alkaline batteries.
1994 U.S. Food and Drug Administration
Minnesota
Congress
Set level of 1 part per million in fish as the safe maximum limit for human consumption. Several States, primarily in the Northeast, issued warnings against eating freshwater fish because of elevated levels of mercury.
Prohibited the disposal of thermostats and other mercury-containing devices unless the mercury was removed.
Suspended sales from the National Defense Stockpile because of questions raised by the EPA as to the potential environmental problems associated with the release of mercury effective 7/94.
1995 33 States Issued freshwater fish consumption advisories because of elevated levels of
mercury contamination.
1996 Public Law 104-142
U.S. Coast Guard
The Mercury-Containing and Rechargeable Battery Management Act of 1996 was made law on May 13, 1996. Title I prohibited the sale of regulated batteries after May 1997 without a label indicating recyclability or proper disposal. Title II phases out the use of alkaline-manganese and zinc-carbon batteries containing intentionally added mercury and button cell mercuric-oxide batteries.
Signed an agreement with the Georgia Environmental Protection Division to remove from Georgia’s waterways discarded zinc-air batteries containing mercury.
1997 EPA, U.S. Department of Justice, U.S. Attorney for Arizona
EPA
EPA
Settled lawsuit brought by Defenders of Wildlife. The suit was concerned with mercury pollution of certain Arizona waterways.
Released its 1996 summary of State-issued warnings to the public to avoid or limit eating fish from certain water bodies (Environmental Protection Agency, 1997b).
Issued its report on mercury (Environmental Protection Agency, 1997c) fulfilling the requirements of section 112(n)(1)(B) of the Clean Air Act as amended in 1990.
Outlook
The following are examples of current actions and efforts to curtail the use of mercury in any nonessential and (or) substitutable application:
• The Chlorine Institute (Report to EPA, 1998) has committed to a 50 percent reduction of mercury used in the chlor-alkali industry by 2005. This will initially occur through tighter controls over mercury cycling within mercury-cell plants, and eventually, the closing of these plants will shift large private stocks into the market supply line.
• Under the Clean Air Act, EPA has established mercury emissions limits (1) for municipal waste combustors, which should result in a 90 percent decline from 1990 levels by 2000, and (2) for medical waste incinerators, which should result in a 95 percent decline from 1990 levels by 2002.
• The EPA and the American Hospital Association agreed to establish several goals regarding waste management, one of which would eliminate specific mercury-containing waste by the year 2005.
• The EPA’s Mercury Study Report to Congress predicted that high deposition rates of anthropogenic mercury (from both global and domestic sources) will occur in the Great Lakes Region. The major factors contributing to this phenomenon are proximity to sources and local climate. The increasing concern regarding mercury contamination within the Great Lakes Basin was the impetus for an international agreement between the Governments of Canada and the United States. The agreement sets a goal to significantly reduce the human use and release of mercury from anthropogenic sources in the Great Lakes Basin by 2006.
International
Mercury presents a global issue because emissions from identifiable point sources, wherever they are located, find their way into water and air for transport across national borders. Legislation and regulation have been created in many countries to address the mercury issue and are responsible for dramatic decreases in mercury use, and consequently in the available supply of mercury-containing products. As a current, net importer of mercury, the United States must consider the importance of the international flow of mercury, because all of the producing mercury mines are foreign; 86 percent of the mercury cells of the worldwide chlor-alkali industry is outside of the United States; there is a large global trade in mercury (2,037 t in 1990 and 1,395 t in 1996); and environmental regulations are not uniform or similarly enforced from country to country.
Emissions to the environment
There are several specific international environmental questions regarding mercury. Because People’s Republic of China (PRC) is the largest combustor of mercury-containing coal as well as being the largest importer of mercury in the world, what are the internal Chinese flows of mercury and their associated emissions? What are the impacts of the unchecked use of mercury by artisanal miners in Brazil, Ghana, Venezuela, Philippines, and other countries? Are the mercury-emission control levels adequate in foreign chlor-alkali plants? What are the environmental consequences of mercury production in Tajikistan, Kyrgyzstan, and the Ukraine, considering the uncertainties surrounding the poor level of environmental controls within the former Soviet Union (FSU)? Although these questions cannot be answered here, the following anecdotal evidence addresses the concerns.
By Countries
Brazil: The Kayapo Indian Area is situated in the south of the State of Para, Brazil. A study by Antonio Barbosa, a chemist from the University of Brasilia, has confirmed that newborn Kayapo children suffer from high levels of mercury contamination— although not to a sufficiently high degree to yield classic mercury poisoning symptoms. The study showed that mercury levels in Kayapo women drop significantly during pregnancy as the mercury is transferred from the mother and accumulates in the fetus. For this reason, newborn children have higher levels of mercury concentration than their mothers.
Germany: As reported by Drozdiak (1996), the Rhine River was considered the “sewer of Europe” for decades. Originating in the Alps, the continent’s busiest waterway absorbed pesticides from the Swiss chemical factories, potassium salts from Alsatian mines, and heavy metals from German industry. By 1970, mercury and cadmium concentrations had reached very high levels. However, in 1995, French biologists found that salmon and sea trout had returned to the upper Rhine for the first time in 50 years. Lead, mercury, and dioxin levels have been cut by 70 percent since 1986 when an international commission was created to clean up the river.
Japan: According to Takeuchi (1960), effluent containing mercury from an acetaldehyde manufacturing plant was discharged into the small bay of Minamata, Japan. This discharge continued from the years before 1953, when Minamata disease began to occur, to September 1958. A total of 121 cases of Minamata disease were identified in adults, children, and fetuses. About half of the adults, one-third of the children, and about one-eighth of the fetal victims died. Characteristically, the children and adults had eaten a great amount of fish and shellfish that contained a considerable amount of mercury. From 1 ppm to 50 ppm were detected in some organs on a wet weight basis. In fetal cases, all of the mothers had eaten large amounts of seafood and river fish. This provided evidence that alkyl mercury penetrates the placental barrier in humans.
In 1959, when the causative agent of the disease was found to be organic mercury, the mud of Minamata Bay was correspondingly found to contain an extremely large amount of mercury. The maximum concentration (133 ppm to 2,010 ppm) was found near the drainage channels from the chemical plants. This Japanese experience served to focus world attention on mercury emitted to the environment.
New Guineaz: Morgan (1995) reported that the gold workings at Porgera, New Guinea, have been operational since 1990. In that time they have yielded more than 6 million ounces of gold, dumping about 40,000 cubic meters of treated tailings into the Porgera River each day. In tests conducted by Phillip Sherman, University of Tasmania, Australia, mercury concentration in the river water was 64 times pre-mining levels.
Russia: In January 1995, the Arkhangelsk Pulp and Paper Combine of Novodvinsk, Russia, emitted as much as 16 t of mercury compounds into the Svernaya Dvina River (as reported in The Environmental Database [TED Case 245, 1997]). The silt beds of the Svernaya Dvina River were found to contain high levels of mercury salts. Contamination levels were more than 600 times allowable concentration limits. Although the contamination was quickly taken away by high water levels and a strong current to the White Sea (considered to be critical Arctic habitat), the pulp and paper combine continues to emit mercuric substances to the river. The plant is the principal employer in the town, and its water treatment plant serves as the water treatment for the community as well as the plant, so it would be very difficult to shut down for necessary improvements.
Tajikistan: In Tajikistan, the Shing-Mangianskaya mountain range contains many antimony-mercury, gold-sulfides, and gold-rare metal mines. Wastes from these mines as well as natural background materials from these mines have contributed large quantities of mercury and other metals to the Zeravshan River. In the Iskanderkul-Yagnobsky region of that range, which largely specializes in mercury-antimony mining, two areas are notably polluted with mercury, antimony, arsenic, lead, and possibly thallium. The largest area consists of the Jijikrut mine and the Anzob Processing Facility situated in the middle stretch of the Yagnob River. This mine and its processing facility have been operational for more than 30 years. During this period, several million metric tons of mercury-antimony mining waste have been accumulated and occasionally washed into the river. In the second area, around the Konchoch-Skal mine, several hundred thousand metric tons of mercury-antimony waste have accumulated, and considerable amounts have washed into the Konchoch River.
By Processes
Artisanal Gold: Artisanal small-scale gold mining of placer deposits occurs mostly in developing countries. Examples include Brazil, Venezuela, Colombia, Guyana, and Suriname, which border the Guyana Shield in South America; the Philippines and New Guinea in Oceania; and Nicaragua in Central America.
In Brazil, the amount of mercury entering the environment was estimated at about 200 t/yr [Trade and Environment Database (TED) case 132]. As described in TED case 132, gold recovery is performed by removing sediments from river bottoms and adjacent areas and feeding them through a number of mercury-coated sieves. The mercury amalgamates with the gold in the sediments, separating the gold from the rest of the material. Considerable amounts of mercury remain in the gold-depleted soil, and much of this finds its way into the rivers. The gold-mercury amalgam is then retorted. Heat drives off the mercury, leaving the gold product. While most of the mercury condenses and is recovered, some of this mercury is emitted to the air, where it resides for a time before being deposited on nearby land or water surfaces through precipitation. Mercury deposited on land ultimately reaches streams and rivers through runoff. Roughly 1.0 kilogram of mercury enters the environment for every kilogram of gold produced by artisans. Another estimate according to research by Veloso de Araujo (1995), in the Alta Floresta area, State of Mato Grosso, Brazil, was that a typical month’s gold production of 230 kilograms (kg) emitted 240 kg of mercury to the atmosphere as elemental mercury vapor, and 60 kg of mercury into rivers.
Coal Combustion: On a worldwide basis, coal is the most widely used primary fuel, accounting for approximately 37 percent of total fuel used for electricity production. The amount and percentage of global mercury contributions originating from the burning of fossil fuels such as coal are unknown. If the rest of the world parallels the United States with respect to mercury emissions, then coal burning may be the single largest anthropogenic source of mercury to the atmosphere. Although all countries recognize that burning coal can degrade the environment, not all have pursued or are actively implementing methods to significantly reduce emissions.
Canada, most European countries, and Japan are widely recognized as having strict regulations limiting emissions from coal-fired plants. Although these regulations are not specifically targeted at reductions in mercury emissions at this time, some of the emission control technologies currently employed prevent nearly one-half of the mercury contained in coal from being emitted to the atmosphere. As stricter regulations are implemented and as mercury is targeted, mercury contributions to the environment from these sources should decrease.
Some countries, such as PRC, India, Russia, and other countries of the FSU, are taking some measures to reduce emissions from coal burning, but they have not been very effective. Russia, other countries of the FSU, and some other countries in Eastern Europe have actually decreased coal burning, in part because of depressed economies, but also because of substituting natural gas for coal in some utility plants. As industrializing countries increase coal burning, but allow pollution controls to lag far behind, emissions to the environment, including mercury, will increase. PRC uses coal to produce nearly 80 percent of its electrical energy. Chinese coal, in general, contains about the same amount of mercury as U.S. coals; however, preliminary studies of coals burned in other countries indicate that mercury can be as much as 10 times higher. At current growth rates, PRC will continue to be the largest coal-burning nation in the world.
A broad estimate of mercury emissions from coal burning could be made. A global study would have to consider average mercury contents and destination of coal shipments, identify significant coal combusting facilities, and identify recovery technologies used. A detailed estimate of global mercury contributions from coal combustion requires collection and analysis of data from individual coal-fired utility plants and industrial boilers throughout the world. The USGS is planning to undertake a study that will collect information and estimate quantities and composition of air pollution produced through coal combustion in the United States. An examination of mercury emissions will be included in that study.
Oil and Natural Gas Combustion: On a worldwide basis, gas and oil are the second and third most widely used primary fuels, accounting for approximately 16 percent and 10 percent of total fuel used for electricity production, respectively. The top five consumers of oil for generating electrical power include the United States, Japan, Russia, PRC, and Germany. Studies have shown that the oil refining process also releases mercury. Oil burning is a contributor to mercury emissions on a global basis, but much less than coal.
Burning natural gas to generate electricity is not a significant mercury emissions factor on a worldwide basis. In the United States, the mercury emissions rate from burning natural gas is relatively low, contributing approximately 0.1 percent of total U.S. mercury emissions. The net effect of the substitution of natural gas for coal to produce electricity should result in lowering atmospheric loading of mercury. As in the case of coal and oil, data are insufficient to provide an estimate of global mercury emissions from natural gas-fueled plants.
Although the mercury content of natural gas is generally believed to be comparatively low, there are exceptions. Approximately 6–8 t of mercury is recovered annually from North Sea gases processed in the Netherlands. High levels of mercury are also known to exist in natural gas burned in Germany, but the mercury is not recovered. These two examples illustrate the need for further research into the small-scale mercury content of natural gas and emissions from its combustion.
Sources
Presently, the world-class, producing primary mercury mines are located in Algeria, PRC, Kyrgyzstan, and Spain. (Unless noted, the production numbers in this section are from the Gobi Report, 1998 (see Appendix, p. 26).) Before the worldwide collapse of mercury markets in the early 1990’s, Italy, Mexico, Slovakia, Slovenia, and Turkey were all active minor producers. Although none of these countries are presently producing mercury from primary mines, each retains significant reserves.
The Western European region was the world’s largest mercury supplier in terms of net trade in 1996 (1,141 t). Spain, the largest producer within this area, provided 92 percent of this region’s total output. The FSU, principally the nations of Kyrgyzstan, Tajikistan, Russia, and Ukraine, was the world’s second largest supplier of mined mercury (785 t in 1996). Northeast Asia (PRC, Japan, and Korea) has widely scattered but extensive mercury reserves. In 1996, 508 t of mercury was produced from mines in the PRC. Algeria produced 347 t of mercury from its mines in 1996; this accounted for all of Africa’s production.
Some mercury is produced as a by-product of gold production, for example, in Mexico. Mercury is also produced as a byproduct of zinc production. Finland started by-product mercury production from zinc operations in 1971, and produced 88 t in 1996 as reported in the USGS Minerals Yearbook, 1998.
Figure 12: 1990/1996 international net mercury trade flow, in metric tons.
Disposition
Approximately 40 percent of the mercury produced in 1996 was used in the world’s chlor-alkali industry (1,344 t). Western Europe, with the world’s largest reservoir of mercury-cell chloralkali capacity, consumed 631 t of mercury just for that purpose in 1996. North America, Eastern Europe, and India/Pakistan were also significant users of mercury (136, 184, and 133 t, respectively) for chlorine production. North America, Western Europe, and northeast Asia were the principal economies using mercury (a total of 860 t, 81 percent of global manufacturing) for the production of mercury-containing goods.
The international use of mercury for chlor-alkali production decreased for the period 1990–1996 by 33 percent, and for mercury-containing products by 42 percent. Estimated mercury use by small-scale gold miners in Brazil decreased from 200 t in 1990 to 100 t in 1996. Mercury use by gold miners in other developing countries is probably significant based on anecdotal evidence, but is not quantifiable at this time. Stock changes in the world were extensive both in 1990 and 1996 (25 percent of their respective years’ production). Besides the United States, Western Europe seems to be the only region that is actively reducing mercury stocks. On the other hand, PRC has apparently been adding mercury to its stockpiles in levels far in excess of their own needs.
Figure 13: Global mercury flow by use, 1990 versus 1996, in metric tons.
Trade, 1990 versus 1996
Major net mercury exporters in 1996 included Western Europe, the FSU, and Africa. Major net mercury importers in 1996 included Asia, Eastern Europe, and South America. These net trade flows to and from various regions of the world are displayed in figure 12, which also illustrates the 32 percent decrease in worldwide mercury trade from 1990 to 1996.
Figure 13 presents a slightly more detailed picture of global mercury demand. Global production of mercury in 1996 (3,337 t) decreased by 2,019 t (38 percent) over 1990 levels (5,356 t).
Case study—Chlor-Alkali
Table 2 reports data from the Chemical Marketing Association, Inc. (1999) showing 1992 and 1997 world chlorine production capacities.[9] The following observations can be made:
1. Most of the growth in chlorine capacity is occurring in Asia, including the subcontinent of India.
2. Sixty-five percent of mercury cell capacity is located in Europe.
3. Mercury cells as a percentage of total capacity have remained the same or decreased everywhere (only exception is northeastern PRC), that is, decommissioning is occurring and most new capacity is not mercury cell.
Lindley (1997) reported major improvements in reducing mercury emissions from mercury cell processes. However, the main emission route is still to air. From 1977 to 1995, the European chlor-alkali industry reported a drop in mercury emissions from 220 t to 18 t, a 92 percent decrease.
Table 2: World chlorine capacity from chlor-alkali plants, in thousands of metric tons.
Total
World
North
America
South
America
West
Europe
East
Europe
FSU Africa Middle
East
India
Pakistan
N.E.
Asia
S.E.
Asia
All Cells
1992 45,5394
100%
13,575
30%
1,696
4%
11,223
25%
1,896
4%
3,773
8%
535
8%
800
2%
1,523
3%
9,706
21%
667
1%
199749,437
100%
14,686
30%
1,787
4%
10,640
22%
1,791
4%
3,676
7%
584
1%
1,294
3%
2,135
4%
11,794
24%
1,050
2%
CAGR 1.721.59
1.05
(1.06)
(1.13)
(0.59)
1.77
10.10
6.99
3.94
9.50
Hg-Cell
199212,625
100%
2,016
16%
460
4%
6,984
55%
1,437
11%
248
2%
295
2%
263
2%
898
7%
0
--
5
nil
1997 11,640
100%
1,809
16%
424
4%
6,445
55%
1,174
10%
248
2%
222
2%
276
2%
916
8%
50
nil
5
nil
CAGR*(1.61) (2.14) (1.62) (1.59)(3.94) 0 (5.53)
0.97
0.40
nil
0
%Hg-Cell
199228 15
27
62
76
7
55
33
59
0
1
199724 15
24
61
66
7
38
21
43
nil
nil
*CAGR=Compound Annual Growth Rate. Numbers in parentheses are negative.
Data provided by CMAI through personal communication.
Legislative Approaches
Netherlands: Maxson and Vonkeman (1996) stated that the Dutch, to encourage recycling, have banned the disposal of mercury-containing wastes, and closed the borders to their export since January 1996. Mercury will only be allowed in products whose life cycles can be controlled.
The Dutch government has implemented strict measures to reduce mercury emissions from industry. The Dutch consider that the health risk to the general population of mercury in the air, food, and water is now negligible. The aquatic environment is not well enough understood to fully appreciate the risks, although it is generally agreed that predators (both birds and mammals) that feed on fish and (or) other aquatic organisms undergo some risk. Mercury is still a problem in Dutch soils, and especially in dredged harbor sediments.
Despite the low estimated human risk from mercury, the Dutch government has called for reductions in mercury emissions to soil, water, and air of 80, 70, and 50 percent, respectively, by 2000 relative to 1985 emissions. Actual reductions in emissions of 40, 70, and 65 percent are expected. The ability to meet the target for water emissions is somewhat uncertain, while the target for emissions to soil will clearly be missed, implying that accumulation of mercury in the soil continues. More stringent targets will be set for 2010, and additional regulatory measures are under consideration.
Sweden and Denmark: Both Sweden and Denmark have already taken steps to ban the use of mercury in nonessential applications, as reviewed by Maxson and Vonkeman (1996). Furthermore, both countries have committed themselves to closing their borders to the transfer of mercury-bearing wastes. They have therefore had to address questions similar to those being addressed by the Netherlands. The key difference is that both Denmark and Sweden have far less mercury circulating in the environment than the Netherlands, and they do not have such significant secondary sources of mercury. Therefore, neither country expects to have to deal with mercury surpluses as large as those the Netherlands will have to deal with.
Officially, Sweden considers mercury, as an environmental pollutant, to be a global problem requiring an international approach. The main strategy for risk reduction in Sweden is to phase out all uses of mercury. Formal government legislation with regard to products was enacted early in 1991. The importation, manufacture, and sale in Sweden of the following products were prohibited:
• as of 1 January 1992, mercury in glass thermometers;
• as of 1 January 1993, other mercury-containing thermometers, measuring instruments, and electrical devices including level switches, thermostats, relays and circuit breakers;
• as of 1 January 1995, mercury-containing pressure switches and electrical contacts for the continuous transfer of current.
The most recent Environmental Government Bill (1993/94:163), which also has the support of Parliament, proposes that all remaining products and uses of mercury, with a few exceptions, should be phased out by the year 2000 or sooner. One notable exception is the continued use of mercury in the chlor-alkali industry, which is permitted until 2010.
Early in the evolution of Danish mercury controls, the Danes had thought that controlling industrial emissions of mercury would be sufficient to reduce human exposure to acceptable levels. It has gradually become clear that this is not sufficient. Products containing mercury are still being produced, and give rise to diffuse mercury pollution during use, and to mercury-containing waste after disposal.
In general, the official Danish position has now developed to the point that exposure of humans to mercury should be kept to an absolute minimum. This can be achieved only by minimizing the use of mercury for all purposes. The long-term goal of the Danish EPA is to bring all uses of mercury to an end.
Through subsidies promoted by the Danish Government for recycling and cleaner technologies, financial support is available for research, development, and dissemination of information promoting substitution and recycling of heavy metals. Projects have been completed concerning the substitution of mercury in products, as well as improved collection aimed at recycling of specific industrial products containing mercury.
One of the key objectives of The Cleaner Technology Action Plan 1993–97 is to support the development of environmentally safer products. Concerted efforts are being made by industry, research institutions, and others to develop and test, in particular, dental filling materials that do not contain mercury. An evaluation has been carried out for the possibilities of beginning or improving existing arrangements for the collection, recycling, and (or) proper disposal of used products containing mercury, especially electronic equipment and construction/demolition wastes. As part of an ongoing project concerning the recycling of fluorescent light tubes, the possibility of recapturing mercury vapor has been investigated, in order to avoid mercury emissions and potential occupational health problems.
Japan: Because of several well-publicized industry-related disasters involving pollution by heavy metals, dating as far back as the 1950’s, the Japanese public and industry are particularly sensitized to such issues. It is generally not considered necessary in Japan to pass legislation aimed at the potential hazards of products. Whenever sufficient consensus develops that might otherwise lead to legislated restrictions, industry “voluntarily” regulates itself to respond to the problem. Target restrictions are set by a consensus between government and industry, and industry is then free to take the measures it considers most appropriate to meet those restrictions. In the case of limit values for pollutants, it is common practice for the Japan Environment Agency (JEA) and industry to agree on a provisional value for the first 5 years, which is then reviewed at the end of that period.
Norway: Maxson and Vonkeman (1996) commented that the official position of Norway is that mercury is one of the heavy metals whose effects on the environment and on human health are most severe. Risk reduction measures should therefore be based on a concern for both health and for the environment. Despite decreases in Norwegian discharges, concentrations continue to increase in the soil and aquatic systems, leading to restrictions on the consumption of certain fish and shellfish.
Brazil: The gold mining situation in Brazil (representative of similar activity in some other developing countries) is important for several reasons (Maxson and Vonkeman, 1996). First, Brazil is one of the largest present markets for mercury, used extensively in the unsophisticated gold mining operations characteristic of the interior regions of the country. Second, together with similar operations in other developing countries, gold mining is the largest single source of mercury pollution to the environment (especially surface waters) in the world. In the last 20 years, an estimated 1,200 t of mercury has been emitted to the Brazilian environment due to artisanal gold mining activities! Third, the health effects due to this pollution are already visible among workers and residents in these areas. Fourth, these mining operations, and the use of mercury, have proven nearly impossible for national or regional authorities to control.
Russia, Ukraine, Kyrgyzstan: Following the disintegration of the FSU, substantial stocks of mercury came onto the international market, according to Maxson and Vonkeman (1996). In desperate need of foreign currency, certain republics offered this mercury at prices as low as 25 percent of the average world price in 1990, according to The Economist in a 1991 report. The European Commission took measures to prevent FSU mercury from being “dumped” on the European market, but the general effect was nevertheless a depression of world prices, which continues to this day.
FSU mines present a curious phenomenon. They are government controlled, but they are likely to be under the influence of one or two key individuals. They are not likely to be subsidized, but they are capable of producing mercury at very low cost. Finally, they are likely to sell mercury when certain organizations or individuals need quick cash, rather than when market prices might suggest they should sell. In effect, like state-owned mercury mines in other countries, FSU sales may be little influenced by market conditions. However, FSU sales may have significant influence on world market prices.
Conclusions
Environmental concerns, prompted by incidents such as the mercury-caused deaths and injuries at Minamata, Japan, have produced many rules, regulations, and mandates that, over the years, have greatly reduced worldwide mercury production, use, and emissions to the environment.
In the United States, there was no mercury mine production in 1996, whereas 448 t was produced in 1990. Mercury mine closures, in the early 1990’s, were responsible for a significant reduction of mercury to the environment from the milling and roasting of mercury ores. In 1996, secondary production was more than four times the level of secondary production in 1990. The change from primary mine production to secondary production over the 6-year period is important because it not only eliminated a major source of mercury to the environment, namely 72 t from milling and roasting, but it also reflected an increased awareness for recycling. Much of the recycling occurs in States with mercury recycling mandates, and some of it is subsidized. Recycling of domestic mercury scrap in 1990 occurred at the rate of 130 t per year. In 1996, the rate was 446 t, a 243 percent increase.
Domestic mercury use decreased 48 percent from 1990 to 1996. In 1996, mercury flowing into all specified industrial uses was less than 1990 levels: dental use was down 30 percent, laboratory use down 44 percent, measurement and control devices down 62 percent, wiring devices and switches down 30 percent, lighting down 66 percent, chlor-alkali down 45 percent, and mercury use in batteries and paints down 100 percent. In 1990, the paint industry added mercury to water-based paints, mainly as a fungicide. In 1996 this use has disappeared. Mercury-containing paints were banned from the market by legislation in 1992.
In 1990, mercury-containing dry cell batteries used 105 t of mercury; in 1996, virtually no mercury went to dry cell batteries. In 1990, the U.S. government stockpiles released 245 t of mercury to the market. In 1996, there were no sales. Sales were suspended in 1994 pending the release of the Mercury Study Report to Congress (1997b), and have not resumed as of this writing (1999). Mercury imports rose sharply from 15 t in 1990 to 340 t in 1996. On the other hand, mercury exports decreased dramatically from 311 t in 1990 to 45 t in 1996. This is a total trade turnaround of 591 t, indicating a growing dependence on foreign supply for the remaining mercury needs of the country.
Mercury emissions to the environment in 1996 decreased by 97 t over 1990 levels. Mercury losses from incineration processes decreased by 47 percent between 1990 and 1996. This reduction was a function of less mercury-containing products entering waste streams as well as stack emission controls on incinerators. Mercury disposed in landfills, excluding soil amendments, in 1996 was 61 percent less than in 1990.
With regard to the international production and flow of mercury: all producing mercury mines are foreign; 86 percent of the mercury cell sector of the worldwide chlor-alkali industry is outside of the United States; there is a large trade (2,037 t in 1990 and 1,395 t in 1996) in mercury; and environmental regulations are not internationally uniform.
The Western European region was the world’s largest mercury supplier in 1996. Spain, the largest producer within this area, provided 92 percent of this region’s total output. The FSU, principally the nations of Kyrgyzstan, Tajikistan, Russia, and Ukraine, was the world’s second largest source of mined mercury (785 t in 1996). PRC has widely scattered, but extensive mercury reserves and produced 508 t of mercury in 1996. The nation of Algeria produced 347 t mercury from its mines in 1996, which accounted for all of Africa’s production. Global production of mercury in 1996 decreased by 2,019 t over 1990 levels.
Approximately 40 percent of the mercury produced in 1996 was used in the world’s chlor-alkali industry. Western Europe was, by far, the world’s largest reservoir of mercury-cell chlor-alkali capacity, and used 631 t mercury in 1996. North America, Eastern Europe, and India/Pakistan were also significant users of mercury (154, 184, and 133 t respectively) for chlorine production. North America, Western Europe, and northeast Asia were the principal economies using mercury for the production of mercury-containing goods. Major net mercury exporters, in 1996, included Western Europe, the FSU, and Africa. Major net mercury importers, in 1996, included Asia, Eastern Europe, and South America.
PRC is the largest consumer of mercury-containing coal and the largest importer of mercury in the world. In the future, those interested in mercury in the environment will want to know what the flows and their associated emissions are in PRC. Although estimated mercury use by artisanal gold miners in Brazil decreased 50 percent from 1990 to 1996, the impact of the continued use of mercury by artisanal gold miners in countries throughout the world is an important international environmental concern. Additionally, the consequences of mercury mining and production in Tajikistan, Kyrgyzstan, and the Ukraine are of interest because of the uncertainty of environmental control within the states of the FSU.
Appendix
Introduction
This Appendix sets forth the methodology for the calculations of mercury flow and stocks represented in figures 2–13. Figures 2, 3, 4, and 11 present a historical perspective on domestic mercury sources and use over the period 1970 to 1997. In the shorter span between 1990 and 1996, mercury production and use changed dramatically. Many changes were mandated by legislation passed because of concerns about mercury and its impact on the environment. Figures 5–10, 12, and 13 compare domestic mercury flow for 1990 and 1996. Much of the methodology used for the study of mercury flows in 1990 has been retained, but several important changes have been made in the development of the 1996 values. This Appendix explains them in detail. Several elements constrained the development of these data: they had to be consistent among themselves; estimates, where possible, had to agree with authoritative sources; and some rationale was required for estimates where data were not available. In the data tables, column and row data may not add to totals due to independent rounding. The authors welcome suggestions for estimate improvement that are based on better data or more pertinent experience.
Perspective figures
Figure 2: Components of U.S. mercury apparent supply (1970–1998), was developed from time series data provided by the commodity specialists of the USGS. Domestic apparent supply for each year in the range is shown by a bar having four segments, including net imports, mine production (including byproduct), secondary production from scrap, and U.S. Government stockpile releases. (See footnote 2, p. 3.)
Figure 3: U.S. industrial reported consumption of mercury (1970–1997), shows how mercury consumption was distributed by sector for the period. Most of the data used to generate figure 3 were available. However, values for the laboratory sector had to be estimated for 2 years (1995, 1996), where an extrapolation yielded a rate of decline of 2 t per year that was extended from the previous 5 years of data.
Pharmaceutical and agricultural usage was reported for the first few years of the period, but not at the end of the series; thus, the values, where reported, were added into the “Other” category, and no delineation for those categories was made.
The breakout of lighting, wiring devices and switches, and batteries did not appear in the series until 1978. Previous to that time, only the total was reported. To show these three categories from 1970 to 1978, the average of the values for lighting and wiring devices and switches for the years 1978, 1979, and 1980 was used for each year from 1970 through 1977, and the values for batteries for those years were taken to be the difference between the sum of the estimates for lighting and wiring devices and switches and the total reported value for the three.
Figure 4: U.S. apparent supply and reported consumption (1970 – 98), shows how consumer and producer mercury stock changes have been distributed over time. Numbers for most of the supply items were available, but net imports of mercury had to be estimated for the period 1978 to 1988, during which U.S. exports of mercury were not published. To make this estimate, we assumed a straight-line appreciation of exports from the value of 33 t in 1977 to 221 t in 1989, and subtracted the extrapolated values from imports, which were known for this period.
One artifact of the linear growth assumption, especially if the growth was really a step function with a long period of low export levels, is the failure of apparent supply to close with reported consumption, as indicated in figure 4. The reported consumption line lies above the apparent supply line for most of the period in question. This could lead to a misinterpretation of the period as one of generally decreasing producer/consumer stocks.
Figure 11: U.S. reported consumption, production, price, and legislation (1970–1997), juxtaposes discrete legislation passage dates with the time series data for primary mercury production, reported consumption of mercury, and world mercury price normalized to 1997 dollars.
Comparative figures
Figure 5 versus figure 6
In the left third of figure 6 are estimated mercury emissions to the environment from natural sources, fuel combustion, and kiln/smelter activities. To improve the 1996 estimate, the more rigorous estimates made by the EPA in its Mercury Study Report to Congress (1997b) were used. The EPA’s estimates were for 1995, and these data have been incorporated into figure 5 as the element titled “Emissions from.” Furthermore, the item in figure 6, “Mercury released from natural emissions,” was not duplicated in figure 5 because the update (fig. 5) is targeted towards the anthropogenic mercury flow system. “Emissions from” represents actual mercury releases to the environment and excludes a large body of mercury that is retained in landfills from previous mercury disposal activities. The EPA estimated a loss rate from landfills based on effluent gas data, and that small contribution is recorded in figure 5.
Table 3: Mercury and gold production, 1991-1992, in metric tons.
Item 1991
1992
Mercury
58 60
Gold
290 320
Ration-Mercury:Gold
0.200.19
The middle third of figure 6 and the middle third of figure 5 are both representations of supply, but different in many important ways. The figure 6 categories “Mercury mine production” and its feeders “Milling and roasting” and “Mercury contained in ore” were dropped from figure 5 because domestic primary mercury mining has been completely replaced by secondary production from scrap. The concept of estimating mercury contributed/ released to the environment from production activities was retained, but the EPA’s estimates are reported. The net result of both changes is a rather large decrease (78 to 0.4 t) in mercury contribution to the environment from domestic mercury production activities.
The figure 6 item “Mercury recovered from old scrap” and the figure 5 item “Secondary production” are identical. Note the four-fold increase in supply from this item. State mandates for mercury recycling are largely responsible for this major supply change. The figure 6 item “Recovered at gold mining operations” corresponds to the update’s “By-product from gold mining.” The original estimate was based on actual survey data. For this update, an estimate of 65 t was reported based on the following observations. In 1991 and 1992, although no primary domestic mercury mine production took place, Mineral Commodity Summaries (MCS) data were reported for both mercury and gold production as shown in table 3.
In 1996, gold production was reported by MCS at 326 t. Multiplying by the ratio (0.2), the estimate for mercury is 65 t. Kenney and others (1995), using 1994 data, reported that about 30 gold and silver mining/recovery operations collectively recovered about 73 t of by-product mercury using retorts. Whether any of this mercury actually was available to the market rather than being added to producer stocks was not determined.
The terms “Industry stocks (1/1/90)” and “Industry stocks (12/31/90)”, which appear in the middle and right third of figure 6, have been combined into one item, “Net change consumer stocks” (fig. 5). The word “consumer” is used because the MCS footnotes this item as “Consumer stocks only.” The convention of subtracting start-of-year from end-of-year stocks is used. This item could have been presented in the “Sources” column in figure 5, where the increasing stocks would have been represented by negative flows, but we preferred to keep all flows positive. Therefore, an increase in stocks will be a positive flow in the “Destinations”
column.
In the middle sector of figure 6, an item labeled “Unaccounted mercury” is reported as withheld (W). In figure 5, a new item, “Net change producer stocks,” was created to distinguish it from consumer stocks, which are directly reported. The methodology for calculating “Net change producer stocks” is as follows: first, the “Destinations” column quantities of “Metal exports” (45 t), “Net changes consumer stocks” (125 t), and “Industrial usage” (372 t), all of which are reported, are totaled (542 t); next, the “Sources” column quantities of “Secondary production” (446 t), “By-product from gold mining” (65 t), “Metal imports” (340 t), “Net change in government stocks” (0 t), and “Net change in mercury cells” (0 t) are totaled (851 t); finally, the reported “Destinations” total of 542 t is subtracted from the “Sources” total of 851 t and the difference of 309 t is ascribed to “Net change producer stocks.” This amount of mercury is an increase in producer stocks for 1996. Secondary production has completely replaced primary production, and it is uncertain whether either secondary producers, or by-product mercury producers, actually sell all of their production. Therefore, it seemed reasonable to create the producer stock category.
Table 4: U.S. mercury imports, 1996
Origint Value (1000$)
$/t
Canada 137 791 5,770
Kyrgyzstan 33 266 8,060
Russia 79 302 3,820
Spain 68 327 4,810
Other 23 92 4,000
Total 340 1,778
But why would producers increase stocks by an amount equivalent to nearly the total of industrial usage? Table 4 shows the only mercury imported at full market price to be 33 t from Kyrgyzstan. The remaining imports of 307 t, an amount approximately the same as the 309 t attributed to producer stocks, came into the United States at less than market price. One conclusion is that the producers were willing, in 1996, to buy mercury at below market prices and hold it for future sales.
“Released from National Defense & DOE stockpiles” from figure 6, and “Net change in government stocks” from figure 5 are essentially the same in concept, but the new title seemed more descriptive of the scenario where the government could actually buy mercury (unlikely, but possible), as well as sell it.
A stock in the “Sources” column labeled “Net change in mercury cells” was added to figure 5. The mercury cell process used in production of chlorine in chlor-alkali plants retains over 3,000 t of mercury. These types of plants, while viable, have been slowly closing. Japan has recycled virtually all of the mercury in its now-closed mercury cells, and for a period of time was a large mercury exporter. Although there was no reported change in the amount of mercury in domestic mercury cells in 1996, the potential for change in the future can be accommodated with this new category.
In figure 6, all the outputs go to a box titled “Total U.S. Supply.” his box has been deleted from figure 5, but the total 1996 low amount (851 t) is shown prior to being split. This amount 851 t) represents mercury metal that will be used for products “Industrial usage”), inventory changes (“Net change producer stocks” and “Net change consumer stocks”) and exports (“Metal exports”).
Finally, figure 5 shows an arrow representing mercury flow from “Industrial usages” going to “Total addition to the environment.” This estimate (13.9 t) was taken directly from the EPA Mercury Study Report to Congress.
Figure 7 versus figure 8
Generally, figure 7 corresponds to figure 8 in concept. Note that mercury embedded in exports and imports of products containing mercury was not included in any of the sectors’ analysis. With the exceptions of the wiring devices and switches sector, which is known to be considerable and growing, and the electric light sector, where imports and exports are approximately equal, the remaining sectors’ imports and exports are negligible.
Table 5: Mercury usage in the dental sector (1986–1996), in metric tons.
Dental sector: It was determined that 90 percent of current year mercury used for dental applications was used in teeth, 8 percent was lost in the dental office in the first year, and 2 percent was lost within 10 years. Furthermore, Harris (1998) has reported the average life of a mercury amalgam filling to be 5–8 years. Seven years was assumed for the calculation. The updated estimates for mercury flow through the dental sector are illustrated in table 5.
The amount of mercury added to product stocks in 1996 was reported as 31 t (fig. 7). The mercury retained in products included 7 years’ worth of mercury in teeth (233 t) plus the mercury retained in the dental offices, which is eventually lost within 10 years. It is assumed that about 1/10 of the 10-year mercury total is lost every year. The mercury in Row H represents the 10-year office contribution (3 t) that is retained in this sector in 1996. This brings the total 1996 stock to 236 t. The mercury leaving the sector includes all of the mercury from teeth in the eighth year[10] (48 t), the mercury lost from dental offices within the first year (2.6 t), and the fraction of mercury that dissipates in 1 year from the mercury that is retained in dental offices for 10 years, the sum of Row F (0.8 t) for a total of 51 t. The 51 t of mercury that exited the dental sector in 1996 split into 46 t recycled, and by difference, 5 t into dissipative (incineration and landfill) loss. This split is based on the assumption that 90 percent of mercury generated by the dental sector is recycled, mostly as spent fillings that are being replaced yearly, or new mercury collected within dental offices from amalgam scrap.
Table 6: Mercury usage in the laboratory sector (1990–1996), in metric tons.
Laboratory sector: Because mercury usage for 1996 was withheld, the estimate for 1996 was based on the consistent annual decrease of 2 t between 1990 and 1994. Therefore, this sector usage was estimated at 20 t in 1996.
The EPA reported that mercury is used in laboratories in instruments, and as reagents and catalysts. Without specific data on the distribution of mercury to these three subsectors, we assumed that 1/3 of the annual input goes to each subsector. The life within each subsector was based on the EPA’s estimate of a 5-year life for instruments, reagent 1 year, and catalyst 2 years. Applying these assumptions allowed us to generate table 6, which provides the mercury flows through this sector.
The amount of mercury going into laboratory product applications in 1996 is estimated to be 20 t. The mercury retained in the laboratory sector includes 5 years’ worth of mercury in instruments (43 t) plus 1 year’s worth of mercury in reagents (7 t), plus 2 years’ worth of mercury in catalysts (15 t), a total of 65 t. Mercury exiting the laboratory sector in 1996 includes the sixth year of mercury in instruments (11 t), the second year of mercury in reagents (8 t), plus the third year of mercury in catalysts (9 t), a total of 28 t. The 28 t of mercury that leaves the laboratory sector in 1996 splits into a 25 t flow into recycling, and, by difference, a 3 t flow into dissipative (incineration and landfill) loss (fig. 7). This split is based on the assumption that 90 percent of mercury generated by the laboratory sector is recycled, mostly as spent instruments and catalysts.
Table 7: Mercury usage in the measurement and control devices sector (1990–1996), in metric tons.
Measurement/control devices sector: Table 7 contains mercury usage provided by the USGS for measurement and control devices for the last 7 years. In 1996, 41 t of mercury went into product applications. In addition to these base data, the EPA reports that the major portion of mercury production in this sector is thermometers, which have an estimated average life of 5 years.
Table 8: Mercury usage in the wiring devices and switches sector (1963–1996), in metric tons.
The mercury retained in products includes 5 years’ worth of mercury used for this sector (331 t). Mercury exiting this sector in 1996 was 108 t, which was 1990’s mercury use. The 108 t of mercury that left the measurement/control devices sector in 1996 was split in half, 54 t each flowed into recycling and into dissipative (incineration and landfill) loss. The 50 percent assumption, based on recycling, is arbitrary, yet seems reasonable given the fact that these devices are widely spread throughout society.
Wiring devices/switches sector: Neglecting mercury imports for wiring devices and switches, which could be substantial in this sector, the amount of mercury going into product applications in 1996 was reported in the USGS Minerals Yearbook as 49 t. The EPA’s Mercury Study Report to Congress noted that electrical switches containing mercury were not manufactured prior to the 1960’s. This study also reported that 10 percent of electrical switches were discarded after 10 years, 40 percent after 30 years, and 50 percent after 50 years, and that thermostats had approximately a 20-year life. The results of incorporating this information into the estimates are shown in table 8.
The mercury retained in product is the sum of the numbers in column D (2,670 t), which was determined by subtracting the 1 percent per year wasting rate from the reported yearly usage. Mercury exiting this sector in 1996 is the sum in column C (32 t) that splits into two equally divided streams of 16 t each. Again, this split is based on the assumption that 50 percent of mercury generated by this sector will be recycled.
Electric light sector: The electric light sector includes both fluorescent lamps and high-intensity discharge lamps (HID). The EPA reported[11] the following: the average mercury content of each fluorescent lamp unit has decreased from 46 to 23 milligrams over the period 1990–1995; the 1992 split between fluorescent and HID lamps is 96:4 (confirmed by Kenney and others (1995), for 1996 production); HID lamps average about 62 milligrams mercury per unit; and the average life of both fluorescent and HID lamps is 4 years. Table 9 shows the decrease in mercury content, per fluorescent lamp, by year.
Table 9: Mercury content in a fluorescent lamp (1990-1996), in milligrams.
Year
19901991 19911993
1994 1995
1996
Hg 46 3834
30
27
23
1
Table 10 illustrates the methodology for calculating the mercury flow through the electric light sector. Lamp production and sales were extracted from Department of Commerce data, and the mercury usage from the USGS Minerals Yearbook was based on lamp wholesaler survey estimates. Disregarding mercury imports and exports[12] (equal in 1996) of fluorescent and HID lamps, the amount of mercury going into product applications in 1996 was estimated to be 11 t. The mercury retained in products included 4 years’ accumulation of mercury usage for this sector (64 t) and the mercury exiting this sector in 1996 equals the total mercury usage calculated from 1991 (23 t).
Table 10: Mercury usage in the electric light sector (1991-1996).
Year
Lamp
Production
(millions)
Fluorescent
fraction
(96%)
Hg per unit
(milligrams)
Hg usage (t)
HID fraction
(4%)
Hg per unit
(milligrams)
Hg usage (t) Total Hg
usage
(t)
1996 550528
19
10
22
621.4
11
1995 550 528 2312
22
62 1.4 13
1994 550 528 27 14 22 621.4 15
1993 550 528 30 16 22 621.4 17
1992 550 528 34 1822
621.4 19
1991 550 528 42 22 22 62 1.4 23
The stream split was based on the lamp-recycling rate reported by Kenney and others (1995). They determined that in 1995, the United States had capacity (24 hour operation, 300 days per year) to recycle 60 percent of lamp production. However, only about 1/5 of capacity was being utilized. This calculates out to approximately 3 t. Therefore, the electric light sector in 1996 split into a 3 t flow into recycling, and a 20 t flow into dissipative (incineration and landfill) loss.
Batteries sector: Although mercury batteries were no longer being produced in 1996, some of the mercury-containing batteries from prior years’ production were still in the system. Despite the fact that in-service batteries retain their utility for as long as 2 years, Kenney and others (1995) reported that the mean retention time for batteries in households was 4 years. The 4-year life estimate was used to calculate the mercury in product inventory based on usage listed in table 11.
Table 11: Mercury usage in the battery sector (1991–1996), in metric tons.
The mercury retained in product includes 4 years’ worth of mercury used for this sector (32 t). Mercury exiting this sector in 1996 includes the fifth year of sector mercury use (18 t). The split into recycling (2 t) and dissipative loss (16 t) is based on the assumption that 10 percent of mercury generated by this sector is recycled. The 10 percent assumption is arbitrarily based on the fact that batteries are widely spread throughout society, and the level of adherence to recycling mercury mandates is high, to keep these materials out of municipal solid waste.
Chlor-alkali sector: Mercury usage in the chlor-alkali industry has decreased due to the decreasing proportion of mercury cells making up chlorine production capacity and tighter mercury recycling in the mercury cell subsector. The mercury flowing through the chlor-alkali sector is shown in table 12.
Table 12:Mercury usage in the chlor-alkali sector (1991–1996), in metric tons.
The amount of mercury going into this process was 136 t in 1996. The mercury retained in the process, actually within the working mercury cells, is estimated to be 2,770 t. Additionally, the industry’s on-site recycling activity contains an inventory of 134 t, and a make-up inventory of purchased mercury of 150 t. The sum of these three inventories represents the industry’s total working mercury inventory of 3,050 t. The mercury leaving this sector is estimated from EPA Toxic Release Inventory (TRI) data and is discussed in the next paragraph.
A figure of 136 t of mercury leaving the sector in 1996 is based on the fact that 136 t of mercury was purchased in 1996: the system being essentially in equilibrium requires purchases to make up for system losses. Toxic Release Inventory data informs that 19 t flows into landfills, 7 t flows into off-site recycling, and 8 t is lost to fugitive and stack emissions. One can estimate, based on plant data, that about 1 t leaves the plant associated with impurities in the caustic product. This leaves 101 t of outflows from the chlor-alkali industry as unaccounted. For development of figure 7, the only concern was with the mercury leaving the system for off-site recycling and for land filling. This total is 26 t.
Other Sector: Table 13 demonstrates how end-use mercury reporting has changed over the years, as percent of total usage in the various sectors. The following assumptions about the flows through this category
Table 13: Reported mercury end-use categories (1970, 1980, 1996), in percent.
[<<, much less than; W, withheld; NR, not reported]
Category 1970
1980
1996
Agriculture 4 WNR
Amalgamation <<1 NR NR
Catalysts 4 NR NR
Dental preparations <<1 NR 8
Electrical apparatus
23
NR
NR
Lightspart of electrical
apparatus
W
8
Wiring devices/switchespart of electrical
apparatus
513
Batteriespart of electrical
apparatus
38
0
Other electrical
part of electrical
apparatus
W
NR
Chlor-Alkali30
24
37
Laboratory1
<<1
W
Measurment/control devices
4
3
11
Paint
21
22
NR
Pulp/paper
<<1
NR NR
Pharmaceuticals <<1
NR NR
Other 117
23
were made. A product life of 3 years and a 10 percent recycling rate for this category were chosen based on assumptions made for prior sectors. The mercury flowing through the “Other” sector is shown in table 14.
Table 14: Mercury usage in the “Other “sector (1992–1996), in metric tons.
The amount of mercury going into product inventories in 1996 is estimated to be 84 t. The mercury retained in product includes 3 years’ worth of mercury used for this sector (349 t). Mercury exiting this sector in 1996 includes the fourth year of sector mercury use (112 t). The 112 t of mercury that leaves the sector in 1996 splits into a 11 t flow into recycling (10 percent), and, by difference, a 101 t flow into dissipative (incineration and landfill) loss.
Unaccounted sector: The purpose for this category is to make the “Outflows” sum to the totals in the “Disposition” column of figure 7. It is an artifact of the methodology used. With regard to “Secondary production,” 64 percent is “Unaccounted.” With regard to “Landfill” and “Incineration loss,” 33 percent is “Unaccounted.” Further research is suggested to reduce the relative size of the contribution from the “Unaccounted” section.
Obsolete product disposition estimates: The “Disposition” column in figure 7 has three categories: Secondary production” to represent recycled material; “Incineration loss” to represent the fraction of mercury that is released to the environment from municipal waste combustors (MWC), medical waste incinerators (MWI), hazardous waste combustors (HWC), cement kilns, and sewage waste combustors; and “Land or landfill,” which includes mercury contained in items that are applied to land as soil amendment or that are directly landfilled, and collected mercury from incineration activities.
The subcategory “Secondary production” is the connection between figure 5 and figure 7. The 446 t of mercury from figure 5 establishes the limit in figure 7 for the sum of recycled materials from the industry sectors.
The EPA, in its Mercury Study Report to Congress, estimated mercury incineration losses as follows: 26.9 t from MWC, 14.6 t from MWI, 6.4 t from HWC, 4.4 t from cement kilns, and 1 t from sewage sludge incineration, a total of 53.3 t.
The EPA reported mercury collection efficiencies for various control devices as follows: flue gas desulfurization 30.85 percent; spray dryers 25.59 percent; fabric filters 28.47 percent; electrostatic precipitators 23.98 percent. It was assumed that 27 percent applied overall.
The total mercury going into all types of incinerators would be:
Mercuryinput = 53.3/(1–0.27) = 73 t (1)
Approximately 12 percent of generated municipal solid waste (MSW) goes into MWC; the remainder is directly landfilled. Correspondingly, about 95 percent of medical waste goes into MWI, 95 percent of hazardous waste goes into HWC, or cement kilns, and 26 percent of sewage sludge is incinerated, 36 percent is used as soil amendment, and 38 percent is directly landfilled.
The total mercury going into municipal waste combustors in 1996 would be:
Mercuryinput to MWC = 26.9/(1–0.27) = 37 t (2)
The total mercury in MSW would be:
MercuryMSW = 37/0.12 = 308 t (3)
The mercury from MSW going to the landfill would be:
MercuryLandfill = 308–37 = 271 t (4)
The mercury from the MWC going to the landfill would be:
MercuryTo Landfill From MWC = 37 * 0.27 = 10 t (5)
The total mercury from MSW, the amount going directly, and the amount from MWC, going to the landfill would be (271 + 10) = 281 t. Similarly, the total mercury from MW going to the landfill is 7 t, the total mercury from HW going to the landfill is 4 t, and the total mercury from sewage sludge incineration is 1 t. The total mercury input to landfills from these sources would be 295 t (fig. 7).
Figure 9 versus figure10
In 1996, the industry purchased 136 t of mercury to service a make-up mercury inventory, estimated from the average of 3 years (1995, 1996, and 1997) of annual purchases to be about 150 t. In addition to the make-up inventory, the industry retains about 134 t of mercury in its on-site recycling activities and about 2,770 t in the mercury cells themselves. The sum of these three inventories represents the industry’s total working mercury inventory of 3,050 t, as shown in figure 7.
The mercury leaving the industry is estimated from EPA Toxic Release Inventory data as follows: 19 t flow into landfills, 7 t flow into off-site recycling, and 8 t lost to fugitive and stack emissions.
Some mercury is known to leave the system attached to the caustic product, which is mainly used at paper mills. This is about 1/2 t per year (rounded to 1 t in fig. 9). This leaves 101 t of outflow from the chlor-alkali industry as unaccounted; this number is incorporated into the 116 t which outflows from the “Unknown” sector listed under the “Product/process stocks 1/1/96” in figure 7.
The estimate of the amount of mercury contained, and continuously recycled, through mercury cells in chlor-alkali plants was derived as follows: Art Dungen (Chlorine Institute, written commun., December 11, 1998), reported that 14 mercury-cell plants were operating in the United States in 1996. Within these plants, there were a total of 726 working mercury cells. A single mercury cell contains between 7,000 and 10,000 pounds of mercury. Given this information, it was assumed that the distribution of 7–10 thousand pound mercury cells was normal, such that the average of 8,500 pounds mercury per cell applied. The estimate for mercury in mercury cells was therefore:
Mercurytotal in Hg-cells = 8,500 lb/cell * 726 cells * 0.90718/2,000 = 2,800 t (6)
F. Anscombe (EPA, oral commun., December 11, 1999) reported that one mercury cell plant of which he had knowledge held 200,000 pounds of mercury in 24 cells. This calculates to 8,330 pounds mercury per cell. Using this number to replace the 8,500 in the calculation above yields a total of 2,740 t. Averaging the two estimates gives 2,770 t of mercury residing in mercury cells (fig. 9).
From figure 9, 150 t of make-up mercury, 134 t of mercury cycling within the recycle loop, and 2,770 t of mercury in cells were added to obtain the 3,050 t estimate of total mercury within the chlor-alkali segment of the industry.
Figure 10 shows flows in 1990. The chlor-alkali industry purchased 247 t of mercury to service a make-up mercury inventory, estimated from the average of 5 years (1988, 1989, 1990, 1991, and 1992) of annual purchases to be about 275 t. In addition to the make-up inventory, the industry retained about 153 t of mercury in its on-site recycling activities, and about 3,170 t in the mercury cells themselves. These estimates were made by taking the ratio of chlorine capacity for 1990 and 1996. The sum of these three inventories represents the industry’s total working mercury inventory of 3,600 t in 1990.
The mercury leaving the industry in 1990 is estimated from EPA Toxic Release Inventory data as 60 t flow into landfills, no flow into off-site recycling, and 9 t lost to fugitive and stack emissions.
Some mercury is known to leave the system attached to the caustic product, which is mainly used at paper mills. This is about 1/2 t per year (rounded to 1 t in fig. 10). This leaves 177 t of outflow from the chlor-alkali industry as unaccounted in 1990.
Figure 12 and figure 13
Table 15 contains the data for the production, use, and flow of mercury on a global basis and underlies the presentation in figures 12 and 13.
Table 15: Global mercury production, use, and flow 1990 and 1996, in metric tons. (n.a., not available)
For table 15, the listed “Regions” include the following countries: North America—Canada, Mexico, and United States; South America—Central and South America; West Europe—Belgium, Denmark, Finland, France, Germany, Greece, Ireland, Italy, Luxembourg, Norway, Portugal, Spain, Sweden, Switzerland, and United Kingdom; East Europe—Albania, Bulgaria, Czech Republic, Bosnia-Herzegovina, Federal Republic of Yugoslavia, Hungary, Macedonia, Poland, Romania, Slovakia, and Slovenia; FSU—Kyrgyzstan, Tajikistan, Russia, and Ukraine; Middle East—Iran, Iraq, Israel, Jordan, Saudi Arabia, Turkey, and United Arab Emirates; Africa—Algeria, Egypt, Gabon, Libya, Morocco, South Africa, and Tunisia; India and Pakistan; Northeast Asia—PRC, Japan, Korea, and Taiwan; Southeast Asia—Australia, Indonesia, Singapore, and Thailand.
Production
“Production” was taken from the Gobi Report (Gobi, 1998). Some discrepancies exist between the Gobi production data and the USGS data for the years in question. For example, total global mercury production for 1990 was 4,100 t according to USGS sources, and 5,356 t according to Gobi. Likewise, USGS reported total mercury production in 1996 as 2,795 t, versus 3,337 t by Gobi. As the Gobi Report provided the most complete set of trade flows, and considering that flow patterns were more important than flow precision, the Gobi production data were used for this part of the analysis.
Uses
This part of table 15 delineates four mercury uses: chloralkali production in mercury cells; manufactured products that contain mercury; artisanal gold mining; and stock changes. These parameters were estimated as shown.
Chlor-alkali production
Chemical Marketing Association Inc. (CMAI, 1999) provided a complete global listing of chlor-alkali plant capacity, broken out by production method, which allowed the isolation of each country’s mercury-cell chlorine capacity. Country estimates for mercury usage in chlor-alkali production were calculated by multiplying the mercury-cell chlorine capacity by the U.S. ratios as follows: (annual mercury purchases for mercury cells) / (mercury cell capacity).
The above ratio was different for 1990 and 1996, 0.1444 versus 0.0918 t mercury per thousand metric tons mercury cell chlorine capacity, respectively. The decrease in mercury usage rate was attributable to increased efficiencies in the operation of mercury-cell chlor-alkali plants in the United States, and tightened controls on system mercury losses, both of which are indicated by reduced purchases of mercury. We assumed that some of the developing countries had not accomplished the same increased mercury-cell efficiencies and loss controls as had the United States. Therefore, the 1990 ratio (less efficient than 1996 ratio and requiring higher use for same capacity) was utilized to estimate 1996 mercury use for those countries.
Manufactures (Products Containing Mercury)
Except for the United States, very little international sector (dental, instruments, lighting, and others) information was available. A reasonable estimate was feasible for the chlor-alkali industry from the existing data, but was not possible for other sectors. We decided to estimate the total amount of mercury that would likely be going into manufactured products for each country. The following assumptions were made: each country has a level of economic sophistication proportional to that of the United States; total chlorine production capacity (chlorine production being ubiquitous throughout the world) for each country is a good indicator of economic sophistication; and supporting data must be available for each country. Accordingly, annual ratios for the years 1990 and 1996 were calculated as follows: (U.S. mercury purchases for mercury-containing products) / (total U.S. chlorine production capacity).
Again, this ratio basis was different for 1990 and 1996, 0.0401 versus 0.0165 t mercury per thousand metric tons total chlorine capacity, respectively. The decrease in mercury usage rate was attributed to legislation that mandated reductions in mercury use in products, technological advances in lighting leading to reduced mercury use in that sector, and tightened controls on system mercury losses through recycling.
Following the same line of reasoning as in the previous chlor-alkali calculations, the assumption was made that developing countries had not passed the same mercury-conscious legislation as had the United States. For such countries, the 1990 ratio was applied to 1996 total chlorine production capacities for the purpose of estimating 1996 mercury use for the manufacture of mercury-containing products. Specifically, only Western Europe, Japan, and Australia were considered to be progressing economically, technologically, and legislatively along the same track as the United States.
Anecdotal information
The data in the “Manufactures” column, table 15, were not broken into individual sectors. The following information provides additional insights as to the state of each of these sectors.
Dentistry
Domestically, the use of mercury in dental applications has remained almost constant since 1980. Recently, concerns about the use of mercury-silver amalgams in women either pregnant or contemplating pregnancy has arisen because of evidence that the fetus preferentially takes mercury from the mother’s body. The use of mercury in dental amalgams (50 percent) is being seriously debated worldwide. As reported by Heavy Metal Bulletin (1996), the replacement of amalgam (with ceramics) has been suggested in Sweden, Finland, Denmark, and Norway. Canada, Germany, Austria, and Sweden have recommended legally non-binding restrictions for mercury amalgams. Japanese dentists are directly responsible for the safe disposal of mercury. Amalgam separators are legal requirements in Sweden, Norway, Germany, and Switzerland. In Denmark, mercury-containing product sales have been banned since 1994, with the exception of amalgam. Germany and Austria have laws to outlaw amalgam by the year 2000.
Batteries
Mercury-oxide batteries have been banned from commercial use in the United States and Europe. Demand for this product has been eliminated entirely in these areas. Although PRC has legislation on the books that will eliminate mercury-oxide battery production by the year 2002, the question of how many are being produced currently is unanswered. Whatever the number, the use is restricted to developing countries because such products cannot be exported to the United States or Europe. The substitutes for mercury-oxide batteries all contain traces of mercury, and battery-recycling programs have developed worldwide. Button batteries, primarily “button type” containing mercury, are still produced by Gold Peak (Hong Kong) and other companies. They are not used in consumer devices in United States. However, the Ever Ready amplifier battery, EP-675, may still be manufactured by the Ever Ready Company for use (overseas) in hearing aids.
Fungicides
Fungicides containing mercury are no longer produced in developed countries. Mercury-containing fungicides were previously added to latex paints. Agriculturally, mercury-containing fungicides were used to control brown mold in freshly sawn lumber and to combat Dutch elm disease and snow mold. Some golf courses still use mercury-containing fungicides. As with batteries, these uses have been controlled or prohibited by law in 28 The Materials Flow of Mercury in the Economies of the United States and the World developed countries. Again, no reliable information exists about production and use in developing countries, although there have been reports (Iraq) of local poisonings from grains treated with mercury-containing fungicides.
Fluorescent lamps
Fluorescent lamps contain about 200 parts per million mercury. From 1985 to 1995 the mercury content of fluorescent lamps has decreased by 35 percent. PRC is a major producer of fluorescent lamps; however, few data are available on Chinese lamp production, use, disposition, and trade. Chinese fluorescent lamps could conceivably be a vehicle for mercury flow into the United States.
Laboratory chemicals
Mercury used in laboratories in many cases finds its way into the municipal water treatment plant, where it goes straight through into receiving waters. With regard to the international use and disposal of laboratory chemicals, no data are available.
Electronic equipment
Electronic equipment such as thermostats and electrical switches can contain significant amounts of mercury. With regard to the international use and disposal of electrical equipment, no data are available.
The Danish Environmental Protection Agency (1994) reported that from 1977 to 1990, mercury use declined from 31 t to about 11.5 t (63 percent), closely tracking the U.S. experience. Although it is not reported in this way in other European countries, one may infer from European Union environmental legislation and the quality of annual mercury flow monitoring reports that Europe is reducing mercury use through product bans. Whether this is occurring in developing countries to the same degree is an open question.
Artisanal gold
One of the greatest environmental concerns is associated with artisan gold mining in the Amazon basin. The Center for Mineral Technology (CETEM, 1995), Brazil, estimated that 140 t of mercury were being used every year by artisanal gold miners in the Amazon region. In 1990, 50 percent of reported gold production was from artisanal miners. In 1996, only 20 percent of reported gold production was by artisanal gold miners. In table 15, the artisanal gold miner’s use of 200 t mercury in 1990 and that of 100 t mercury in 1996 were extrapolated from the CETEM estimate and reported artisanal gold production.
Stock changes
The parameter “Stock changes” is an artifact established to balance production, use, and flow (net imports). Information regarding stock changes on a country-by-country basis was limited. Any information available was used, and flow was estimated or adjusted. In some cases, it was estimated for a subject country as either 5 or 10 percent (based on economic sophistication) of the sum of estimated mercury use for chlor-alkali and manufactures for that particular country.
Net flows
“Net flows” was extracted directly from trade flow data contained in the Gobi Report. Net flow data were not available for all countries. For countries where net flow data were unavailable, an estimate was made to balance production and use. The assumption was made that global net flow summed to zero.
Notes
1. ^Apparent supply includes production (primary and secondary) + net imports + government stockpile releases
2. ^See discussion in Appendix, p. 19, regarding how this observation may not be consistent for the years 1978–1988.
3. ^Figure 5 is an update of figure 6, “Domestic flow of mercury in 1990” (Jasinski, p. 24). Both figures address supply, demand, and emissions to the environment. For a detailed explanation of figure 5 flow amounts and how they were derived, see pages 19– 20 in the Appendix. For the purposes of this analysis, it is assumed that all flow amounts are 100 percent mercury.
4. ^The terms “incinerator” and “combustor” have no technical differences and are incorporated in this report to follow historical usage.
5. ^Sewer treatment plant sludges are applied to soils as fertilizers. Such sludges contain nutrient minerals, but also traces of heavy metals, including mercury
6. ^Figure 7 is an update of figure 8, “Domestic flow of mercury in 1990 – continued” (Jasinski, 1994, p. 25). Both figures address U.S. industrial inflows and outflows. For a detailed explanation of figure 7 flow amounts and how they were derived, see pages 20–25 in the Appendix.
7. ^Figure 9 (1996) and figure 10 (1990) represent inflows and outflows in the domestic chlor-alkali industry. For a detailed explanation of how they were derived, see pages 25–26 in the Appendix.
8. ^All chlor-alkali plants operating mercury cells reported except for one minor plant in Vicksburg, Miss.
9. ^Although prior analyses were based on 1990 and 1996 data, only 1992 and 1997 data were available in this case.
10. ^As a rule, whenever there were no data about the fraction of mercury that each year contributes to sector outflow, that is, no recognizable distribution, we applied the convention that the next year’s sector usage (the first year beyond the average life of the product) was a fair representation of the outflow from the sector.
11. ^Incorporating National Electrical Manufacturer’s Association (NEMA) data.
12. ^ If this sector’s flow analysis is updated in the future, the analyst will want to obtain more data on imports. PRC, a major mercury producer, has also become one of the world’s largest producers and exporters of electric lamps.
Further reading
• Akelsson, Frank, Mercury balance for the Gothenberg municipality, accessed June 8, 1998.
• Baratov, Oleg, and Skochilov, Yuri, 1996. Geo-ecology and sustainable
development of Tajikistan: Ecostan News
, accessed July 9, 1998.
• Center for Mineral Technology (CETEM), 1995. Gold mining (Garimpo) and mercury emissions into the atmosphere: Socio-economic evaluation, in Activities report 1995, p. 11–12.
• Center for Renewable Energy and Sustainable Technology (CREST), Facts about mercury-containing thermostats, accessed May 13, 1998.
• Chemical Marketing Association, Inc. 1999. (CMAI), Chlorine, world capacity tables: Chemical Marketing Association, Inc., 11601 Katy Frwy, Number 22, Houston, Tex., 48 p.
• Chu, Paul, and Porcella, D.B., 1995. Mercury stack emissions from U.S. electric utility power plants, in Porcella, D.B., Huckabee, J.W., and Wheatley, B., eds., Mercury as a global pollutant: Water, Air, and Soil Pollution, v. 80, p. 134–144.
• Cimi–Indianist Missionary Council, 1998. archive/nl/9410/0267.html Kayapo children and mercury pollution, research by Antonio Barbosa, chemist, university of Brasilia, accessed July 28,1998.
• Danish Environmental Protection Agency, 1994. Heavy metals: Redegorelse fra Miljostyrelsen, No. 3, p. 27 -31.
• Drozdiak, William, 1996. The Rhine River is no longer the “sewer of Europe,” accessed August 18, 1998.
• Farid, L.H., Machaado, J.E.B., and Silva, O.A., 1991. Controle da Emmissao e Recuperacao de Mercurio em Rejritos de Garimpo, in Veiga, M.M., and Fernandes, F.R.C., eds., Pocone—Um Campo de Estudos do Impacto Ambiental do Garimpo: Rio de Janeiro, Brazil, CETEM/CNPq, p. 27–44.
• Gobi International, 1998. The gobi report on mercury, CD ROM: version 2.2. Harris, R.B., Ask the dentist: The Beacon, Virginia Beach, Va., accessed August 20, 1998.
• Heavy Metal Bulletin, 1996. Volume 3, Issue 3, Current amalgam status and restrictions world-wide, accessed May 29, 1998.
• Hedegard, Leif, Amalgam-related illness FAQ, Mercury sources, accessed May 21, 1998.
• Jasinski, S.M., 1994. The materials flow of mercury in the United States: U.S. Bureau of Mines Information Circular 9412, 28 p.
• Kenney, C.W., Queneau, P.B., and Hansen, Barry, 1995. State of the art in mercury recycling and Remediation of mercury-containing waste, in International Symposium on the Treatment and Minimization of Heavy Metal-Containing Waste, February 12–16, 1995, Las Vegas, Nev.: Metallurgical Society of the American Institute of Mining and Metallurgical Engineers, 27 p.
• Lindley, A.A., 1997. An economic and environmental analysis of the chloralkali productive process—Mercury cells and alternative technologies: Prepared for the European Commission, Directorate-General III, Industry, June 1997, 61 p.
• Lindqvist, Oliver, ed., 1991. Mercury as an environmental pollutant: Water, Air, and Soil Pollution, v. 56, 847 p.
• Masters, H.B., 1997. Mercury, metals and minerals annual review—1997: Mining Journal Ltd., p. 80.
• Maxson, P.A., and Vonkeman, G.H., 1996. Mercury stock management in the Netherlands, Background document prepared for the workshop, Mercury: ban it or bridle it?, 21 November 1996: The Hague, Netherlands, Institute for European Policy, Brussels, Belgium, December, 1996, 48 p.
• Mercury flow in Sweden, 1997. Swedish mercury consumption and deposits in 1996, accessed May 28, 1998.
• Mining Journal, 1997. World energy trends, East Europe/CIS slump: Mining Journal, v. 328, no. 8411, January 10, p. 24.
• Mishra, C.P., Wilburn, D.R., Hartos, D.G., Sheng-Fogg, C.D., and Bowyer, R.C., 1985, Mercury availability—Market economy countries: U.S. Bureau of Mines Information Circular 9038, 18 p.
• Morgan, C.J., 1995. Porgera pollution: Department of Anthropology & Archaeology, James Cook University, Australia, accessed August 18, 1998.
• National Electrical Manufacturers Association, 1996. The declining presence of mercury in batteries and municipal solid waste: National Electrical Manufacturers Association, Rosslyn, Va., 22 p.
• North Carolina Office of Waste Reduction and Recycling (NCOWRR), 1995. Guidance for used fluorescent lamp management, accessed June 16, 1998.
• Porcella, Don, Huckabee, John, and Wheatley, Brian, eds., 1995. Mercury as a global pollutant: Water, Air, and Soil Pollution, v. 80, 1336 p.
• Rachel’s Hazardous Waste News, 1990. Reporting research by Environmental Research Foundation, Annapolis, Md.: Rachel’s Hazardous Waste News, no. 198, p. 1.
• Roskill Information Services Ltd., 1990. The economics of mercury 1990, Seventh Edition: Roskill Information Services Ltd., section 2.10, p. 18.
• Takeuchi, Tadao, 1960. Biological reactions and pathological changes in human beings and animals caused by organic mercury contamination, in Hartung, R., and Dinman, B.D., eds., Environmental mercury contamination: Proceedings of the International Conference of Mercury, University of Michigan, 1973, 349 p.
• The Chlorine Institute, Inc., 1998. North American chlor-alkali industry plants and production data report—1997: The Chlorine Institute, Inc., Pamphlet 10, April 1998, 18 p.
• ———1998, Report to EPA, May 8, 1998: The Chlorine Institute Inc., 2001 L Street N.W., Washington, D.C., 20036-4919, 3 p.
• Toxic Release Inventory, 1997. Network].
• Trade and Environment Database Case 132, Brazil gold mining and the environment, post 1994, accessed August 18, 1998.
• Trade and Environment Database Case 245, The environmental database, mercury in Russia, January 1997, accessed July 9, 1998.
• U.S. Bureau of Mines, 1970–1995, Mineral commodity summaries, Mercury, pages vary.
• ———1970–1994, Minerals Yearbook, Mercury, pages vary.
• U.S. Department of Defense, 1998. Strategic and critical materials report to the Congress, October 1997–September 1998, 48 p.
• U.S. Department of Energy, 1998, International energy outlook 1998: EIA-0484 (98), p. 114.
• U.S. EPA, 1992. Chlor-alkali, Inorganic Chemical Industry, chapter 8 in Compilation of air pollution emission factors, Fifth Edition, Volume I: U.S. Environmental Protection Agency, p. 8.11-1–8.11-6.
• ———1997a, Locating and estimating air emissions from sources of mercury and mercury compounds: U.S. Environmental Protection Agency, Office of Air Quality Planning and Standards, December 1997, EPA-454/R-97-012, pagination varies.
• U.S. Geological Survey, 1997–1998. Mineral commodity summaries, Mercury, pages vary.
• ———1995–1997, Minerals Yearbook, Mercury, pages vary.
• USGS, 2005. The Materials Flow of Mercury in the Economies of the United States and the World. By John L. Sznopek and Thomas G. Goonan. U.S. Geological Survey Circular 1197.
• USGS, 2007. Minerals Information: Materials Flow Publications. U.S. Geological Survey.
• U.S. Geological Survey and U.S. Bureau of Mines, 1996. Mineral commodity summaries, Mercury, pages vary.
• Vanderbilt University Case Study, EcoDS case study: Mercury reduction in fluorescent light bulbs, accessed July 1, 1998.
• Veloso de Araujo, R.V., 1995. Gold mining (Garimpo) and mercury emissions into the atmosphere: Socio-Economic Evaluation, Project Report, Center for Mineral Technologies (CETEM), Activities, p. 11–12.
• Watras, C.J., and Huckabee, J.W., eds., 1994. Mercury pollution, integration and synthesis: Proceedings of International Conference on Mercury as a Global Pollutant, Monterey, Calif., June 1992, 727 p.
Citation
USGS (minerals information) (Lead Author);Cutler J. Cleveland (Topic Editor) "Materials flow of mercury in the economies of the United States and the world". In: Encyclopedia of Earth. Eds. Cutler J. Cleveland (Washington, D.C.: Environmental Information Coalition, National Council for Science and the Environment). [First published in the Encyclopedia of Earth August 23, 2007; Last revised Date August 23, 2007; Retrieved May 18, 2013 <http://www.eoearth.org/article/Materials_flow_of_mercury_in_the_economies_of_the_United_States_and_the_world>
The Author
The USGS Minerals Information Team’s mission is to collect, analyze, and disseminate information on the domestic and international supply of and demand for minerals and mineral materials essential to the U.S. economy and national security. Examples of mineral materials are cement and steel.The Team’s goal is to provide decision makers with the information required to ensure that the Nation has an adequate and dependable supply of minerals and materials to meet its defense and economi ... (Full Bio)
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User Profile
Name: Earth Track
Member Since: November 6th, 2006
Member Name: Earth.track
Biography:
Earth Track was founded in 1999 by Doug Koplow to more effectively integrate information on energy subsidies. Earth Track works to develop comprehensive and accurate information on government interventions in energy markets through direct research and by forging partnerships with organizations and individuals around the world. By developing this data, we aim to inform local, national, and international bodies about how the interaction of their many policies affect energy markets, environmental quality, trade, and fiscal health. Earth Track information ensures greater alignment between environmental goals and fiscal and regulatory policies.
Interventions come in a range of guises, including tax breaks, special taxes, below-market loans or insurance, loan guarantees, direct grants, regulatory exemptions, or subsidies associated with direct government provision of energy goods or services. They can act either as subsidies (artificially reducing the cost of certain commodities) or as taxes (artificially increasing the cost of certain commodities).
While global efforts are underway to curb climate change, restructure energy markets, and transition to cleaner energy sources, there is very little information on how existing policies impede the achievement of these goals. Without this information, both markets and governments make less informed decisions about what energy to buy and what new technologies to invest in. Earth Track's role is to:
• Consolidate and standardize information on government interventions in energy markets from hundreds of sources and data providers in countries around the world.
• Provide an unbiased source of information on these policies outside of the pressures and politics of international organizations.
• Present information on subsidies and complicated financial, accounting, and regulatory policies in a manner accessible to non-technical audiences.
• Present a holistic picture of the impact of government policies by energy type, type of policy, or geographic region.
• Quantify the value of existing subsidies and taxes whenever possible to allow evaluation of time trends, patterns across fuels and regions, and to serve as inputs to macro-economic models.
Website: Homepage
Disclaimer: The Earth Track is the original source for some content in the Encyclopedia of Earth. The Earth Track is listed as a content source on each article that uses such content. Topic editors and authors for the Encyclopedia of Earth may have edited this content or added new information. The use of information from the Earth Track should not be construed as support for or endorsement by that organization for any new information added by Encyclopedia of Earth personnel, or for any editing of the original content.
User Content
Title Role Type Website Date
Government grants Author Article Encyclopedia of Earth 2006-12-06 02:51:30
Government loan, loan guarantee, and insurance programs Author Article Encyclopedia of Earth 2006-12-06 02:51:55
Government research and development programs Author Article Encyclopedia of Earth 2006-12-06 02:51:08
Government-owned enterprises Author Article Encyclopedia of Earth 2008-12-12 21:48:07
Natural resource leasing in the United States Author Article Encyclopedia of Earth 2006-12-06 02:52:50
Subsidies and market interventions Author Article Encyclopedia of Earth 2013-02-21 14:37:29
Subsidies and the poor Author Article Encyclopedia of Earth 2006-12-06 02:53:18
Tax subsidies Author Article Encyclopedia of Earth 2008-07-30 15:05:13
Ten most distortionary energy subsidies Author Article Encyclopedia of Earth 2007-01-26 16:07:06
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Mount Vernon, New YorkEdit This Page
From FamilySearch Wiki
United States New York Westchester County City of Mount Vernon
Contents
Resources
Church Records
City Records
To locate additional published and transcribed records for Mount Vernon, New York check:
• Gordon L. Remington, New York Towns, Villages, and Cities: A Guide to Genealogical Sources (Boston: New England Historic Genealogical Society, 2002). American Ancestors online edition; At various libraries (WorldCat); FHL Book 974.7 D27r. Alphabetical list including date founded, if a town history exists, church and cemetery sources, and if a Civil War register (TCR) exists. The codes used under Church and Cemetery are defined in the link above the listing of towns, cities and villages.
Repositories
Archives, Libraries and Museums
Societies
City Clerk
City Clerk[1]
George Brown
City Hall
1 Roosevelt Square
Mount Vernon, NY 10550
Phone: 914-665-2348
Fax: 914-665-2496
City Historian
City Historian[2]
Dr. Larry Spruill
City Hall
1 Roosevelt Square
Mount Vernon, NY 10550
Phone: 914-665-2351
Vital Records
References
1. City of Mount Vernon New York at http://cmvny.com/departments/city-clerk/ (accessed 13 December 2011).
2. Elizabeth Petty Bentley, County Courthouse Book, 3rd ed. (Baltimore, Md.: Genealogical Pub., 2009), 497. At various libraries (WorldCat); FHL Book 973 D24bena 2009.
Places
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• This page was last modified on 6 February 2012, at 19:57.
• This page has been accessed 286 times.
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Linux
From Forensics Wiki
Revision as of 21:51, 31 August 2006 by Simsong (Talk | contribs)
Jump to: navigation, search
The wide variety of useful Linux utilities exist for desktop computers can also be used on Linux-based PDAs. These utilities can often be used as a part of the forensics investigation process.
Tools
dd
dd, or duplicate disk, is a Unix and Linux utility that allows the user to create a bitstream image of a disk or device. Once the Linux-based PDA is connected to another device and the dd utility is run, the mirror image can be uploaded onto memory cards or even an external desktop workstation connected via a network. Images created by dd are readable by forensics software tools such as EnCase and Forensic Toolkit. Since the device uses a Linux filesystem, the image may also be mounted and examined on a Linux workstation.
foremost
foremost is a Linux based program data for recovering deleted files and served as the basis for the more modern Scalpel. The program uses a configuration file to specify headers and footers to search for. Intended to be run on disk images, foremost can search through most any kind of data without worrying about the format.
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Bibliography: Pollution Free War
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Title: Pollution Free War
Author: Rob Young
Year: 1990
Type: SHORTFICTION
Storylen: shortfiction
Language: English
ISFDB Record Number: 1419725
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Copyright (c) 1995-2011 Al von Ruff.
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Molecules 2011, 16(6), 5054-5061; doi:10.3390/molecules16065054
Article
Cholesterol-Lowering Activity of the Major Polyphenols in Grape Seed
1 The Medical Food Research and Development Center, Department of Transfusion Medicine, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand 2 Program in Nutrition and Dietetics, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand 3 The Research Group of Herbal Medicine for Prevention and Therapeutic of Metabolic Diseases, Chulalongkorn University, Bangkok, Thailand
* Author to whom correspondence should be addressed.
Received: 8 April 2011; in revised form: 13 June 2011 / Accepted: 16 June 2011 / Published: 17 June 2011
(This article belongs to the Section Natural Products)
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Abstract: The major polyphenols in grape seed have been shown to have beneficial health effects in the prevention of dyslipidemia and cardiovascular diseases. In this present study, we investigated the cholesterol-lowering activity of three major polyphenolic compounds found in grape seed. The results showed that gallic acid, catechin, and epicatechin significantly inhibited pancreatic cholesterol esterase in a concentration-dependent manner. Moreover, they bound to taurocholic acid, taurodeoxycholic acid, and glycodeoxycholic acid at levels ranging from 38.6% to 28.2%. At the concentration of 0.2 mg/mL, gallic acid, catechin, and epicatechin reduced the formation of cholesterol micelles 27.26 ± 2.17%, 11.88 ± 0.75%, and 19.49 ± 3.71%, respectively. These findings clearly demonstrate that three major polyphenolic compounds present in a particular grape seed have cholesterol-lowering activity by inhibiting pancreatic cholesterol esterase, binding of bile acids, and reducing solubility of cholesterol in micelles which may result in delayed cholesterol absorption.
Keywords: polyphenols; grape seed; mechanism pancreatic cholesterol esterase; cholesterol micelles; bile acid
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MDPI and ACS Style
Ngamukote, S.; Mäkynen, K.; Thilawech, T.; Adisakwattana, S. Cholesterol-Lowering Activity of the Major Polyphenols in Grape Seed. Molecules 2011, 16, 5054-5061.
AMA Style
Ngamukote S, Mäkynen K, Thilawech T, Adisakwattana S. Cholesterol-Lowering Activity of the Major Polyphenols in Grape Seed. Molecules. 2011; 16(6):5054-5061.
Chicago/Turabian Style
Ngamukote, Sathaporn; Mäkynen, Kittana; Thilawech, Thavaree; Adisakwattana, Sirichai. 2011. "Cholesterol-Lowering Activity of the Major Polyphenols in Grape Seed." Molecules 16, no. 6: 5054-5061.
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Polymers 2010, 2(4), 418-439; doi:10.3390/polym2040418
Article
New Biocompatible Polyesters Derived from α-Amino Acids: Hydrolytic Degradation Behavior
Institute of Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Israel
* Author to whom correspondence should be addressed.
Received: 20 September 2010; in revised form: 2 October 2010 / Accepted: 8 October 2010 / Published: 13 October 2010
(This article belongs to the Special Issue Advanced Polymer Architectures)
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Abstract: New polymers were synthesized from α-hydroxy acids derived from the natural amino acids Ile, Leu, Phe, and Val, combined with lactic acid, glycolic acid and 6-hydroxyhexanoic acid by direct condensation. The toxicity was determined and the degradation process of these polyesters was investigated under physiological conditions by analyzing the composition of the degraded polymers and the oligomers cleaved in the buffer medium. The polymers were found to be non toxic to two cell lines. Polymers displayed a biphasic degradation behavior. In most cases, a linear relationship was found between the weight loss constant and the hydrophobicity of the polymers, Log P. Regarding the second stage of weight loss, it is apparent that polymers derived from α-hydroxy(L)isoleucine ((L)HOIle) and α-hydroxy(L)Valine ((L)HOVal) degraded much faster than those derived from α-hydroxy(L)leucine ((L)HOLeu) and α-hydroxy(L)phenylalanine ((L)HOPhe), probably due to different spatial orientation of the side chains. Copolymers of 6-hydroxyhexanoic acid displayed slow degradation rates as expected, whereas the degradation profile of copolymers of lactic acid was similar to the other homopolymers. These new polyesters may serve as potential biocompatible materials for medical applications.
Keywords: α-hydroxy acids; polyesters; toxicity; degradation; drug delivery
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MDPI and ACS Style
Cohen-Arazi, N.; Domb, A.J.; Katzhendler, J. New Biocompatible Polyesters Derived from α-Amino Acids: Hydrolytic Degradation Behavior. Polymers 2010, 2, 418-439.
AMA Style
Cohen-Arazi N, Domb AJ, Katzhendler J. New Biocompatible Polyesters Derived from α-Amino Acids: Hydrolytic Degradation Behavior. Polymers. 2010; 2(4):418-439.
Chicago/Turabian Style
Cohen-Arazi, Naomi; Domb, Abraham J.; Katzhendler, Jeoshua. 2010. "New Biocompatible Polyesters Derived from α-Amino Acids: Hydrolytic Degradation Behavior." Polymers 2, no. 4: 418-439.
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This article is part of the series Online First articles in Volume 6 (2011). These articles were published as Online First on SpringerLink in 2010. They should be cited with a 2011 publication year.
Nano Express
Anisotropic Confinement, Electronic Coupling and Strain Induced Effects Detected by Valence-Band Anisotropy in Self-Assembled Quantum Dots
L Villegas-Lelovsky1,2*, MD Teodoro1,3, V Lopez-Richard1, C Calseverino1,4, A Malachias4, E Marega3,5, BL Liang3,7, Yu I Mazur3, GE Marques1, C Trallero-Giner6 and GJ Salamo3
Author Affiliations
1 Departamento de Física, Universidade Federal de São Carlos, São Carlos, SP 13565-905, Brazil
2 Instituto de Física, Universidade Federal de Uberlândia, Uberlândia, Minas Gerais 38400-902, Brazil
3 Arkansas Institute for Nanoscale Materials Science and Engineering, University of Arkansas, Fayetteville, AR 72701, USA
4 Laboratório Nacional de Luz Síncrotron, Campinas, Brazil
5 Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, SP 13560-970, Brazil
6 Faculty of Physics, Havana University, 10400, Havana, Cuba
7 Department of Electrical Engineer, University of California, Los Angeles, CA 90095, USA
For all author emails, please log on.
Nanoscale Res Lett 2011, 6:56 doi:10.1007/s11671-010-9786-8
The electronic version of this article is the complete one and can be found online at: http://www.nanoscalereslett.com/content/6/1/56
Received:6 July 2010
Accepted:9 September 2010
Published:1 October 2010
© 2010 Villegas-Lelovsky et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
A method to determine the effects of the geometry and lateral ordering on the electronic properties of an array of one-dimensional self-assembled quantum dots is discussed. A model that takes into account the valence-band anisotropic effective masses and strain effects must be used to describe the behavior of the photoluminescence emission, proposed as a clean tool for the characterization of dot anisotropy and/or inter-dot coupling. Under special growth conditions, such as substrate temperature and Arsenic background, 1D chains of In0.4Ga0.6 As quantum dots were grown by molecular beam epitaxy. Grazing-incidence X-ray diffraction measurements directly evidence the strong strain anisotropy due to the formation of quantum dot chains, probed by polarization-resolved low-temperature photoluminescence. The results are in fair good agreement with the proposed model.
Keywords:
Molecular beam epitaxy; Self-assembled quantum dots; Inter-dot coupling; Anisotropic effects; Linear polarized photoluminescence emission; Grazing-incidence X-ray diffraction synchrotron; Optoelectronic
Introduction
Recent attention has been given to the study of coupled quantum dot (QD) arrays for their potential application in quantum information processing [1-3]. The self-assembling process and its control become essential concerns in the search for new proposals of optoelectronic and quantum computing devices. Also, the spinor states in quasi-zero dimensional systems and their electronics have become features of renewed interest [4-7]. High uniformity of size, shape and distribution control of dot arrays are required in many application proposals like detectors, low-threshold lasers and photonic crystals. The lack of control over the self-assembly process of formation of these QDs leads to inhomogeneous broadening in size and/or shape that may degrade the quality of a device application. Therefore, the need for probing size, shape and effective inter-dot coupling has become an important area of research in recent years [8-12].
The anisotropy observed in linearly polarized PL-emissions from self-assembled QDs has been studied in recent years, and several works have detected some correlation with the anisotropic shape of the QD array [13-16]. There is also an agreement about the complexity of valence-band effects in QDs as a relevant issue when dealing with optical response from transitions between these completely localized states [7,17,18].
In the present work, we addressed mechanisms of testing simultaneously one-dimensional (1D) lateral ordering of dots, inter-dot coupling and 2D anisotropy of self-assembled QDs from studies of grazing-incidence X-ray diffraction (GID) and polarized photoluminescence (PL) emissions under different excitation power. This work has been motivated by the plausibility of controlled self-assembling growth of 1D dot arrays (QD chains) [19] and their potential use for testing important quantum effects such as correlation of information and optical coupling between dots where the relevant aspects of effects associated with inter-dot coupling and QD shape, size and distribution deserve special attention. It is also discussed the interplay between shape and strain fields with the inter-dot correlation that is revealed in the GID measurements and PL-emission spectra from QD arrays. Two sets of samples are investigated: one shows chain-like 1D correlation between neighboring dots and the other exhibits a mostly random island distribution. Two different QD shape models are used in order to calculate and test the polarized optical emission spectra dependence with spatial dot correlation and local geometry. The experimental confirmation included in this work highlights and supports the importance of probing correlated distribution in QD arrays for the characterization and improving of the growth-controlled processes.
Theoretical Model
A multi-band k · p model based on the standard Kohn–Luttinger [20] and parabolic Hamiltonians to probe the electronic structure of holes and electrons, respectively, in dots grown along the [100] direction was developed. Due to strong valence-band admixture, such a procedure provides straightforward information on the relaxation of the inter-band optical transition selection rules, using lower computational efforts than in tight-binding calculation model, for example [13,14]. The built-in strain field distribution, which lead to the formation of self-assembled QD arrays, has been considered within the Bir–Pikus deformation potential model [21]. Uniform strain tensors are assumed, a model that neglects effects caused by variations at the QD interfaces [22,23]. This approximation works reasonably well for the study of ground-state properties of medium (~150 Å) and large (>250 Å) size dots.
The double quantum dots structures under investigation are schematically illustrated in Figure 1. According to realistic dimensions the dots are assumed to have semi-cylindrical shape with radius ρ, laterally separated by an inter-dot barrier of thickness d. Since the main focus is concentrated in the tunneling along the lateral direction , the confining potential is defined as V(ρ, z) = V(ρ) + V(z), where the infinite barrier model have been used, as represented in Figure 1b (Figure 1c), at top (left) and bottom (right) interfaces, whereas the finite barrier model at the internal interfaces have been adopted, as represented in Figure 1c, in order to account for inter-dot coupling effects.
Figure 1. (a) Schematic modeling of QD size and inter-dot coupling used in this study of self-assembled dots formed along the indicated crystalline directions. (b) Confinement model for random distribution dots in the (100) plane. (c) Confinement model for testing anisotropic size and plausible inter-dot electronic coupling.
For the GaInAs alloys in consideration, at the center of the Brillouin zone, the split-off band is energetically well separated from the topmost valence subbands. In the limit of decoupled split-off band, the four-band Hamiltonian provides a good description of low-lying hole states by considering the coupling between the heavy-hole (hh) (J = 3/2, jz = ±3/2) and the light-hole (lh) (J = 3/2, jz = ±1/2). In the effective-mass approximation, when spanned in this basis, the kinetic energy of the hole is described by the 4 × 4 Kohn–Luttinger Hamiltonian
(1)
where,
(2)
with the Luttinger parameters γi (i = 1, 2, 3), and the momentum operators .
The Hamiltonian of the hole in the quantum dot system is
(3)
where V(ρ) is an infinite barrier outside of the semi-cylindrical cross-section, and V(z) is a double quantum well potential with infinite high outside walls, whose finite barrier is due to the offset between the band edges in the well and barrier materials; ℋBP is the Bir–Pikus Hamiltonian [21].
By exploring cylindrical symmetry in the Kohn–Luttinger model, the wave function of a hole state can be written in the form
(4)
The indexes (j, n, m) label the quantization along the z-direction (j) and in-plane (n, m) quantum numbers, respectively, α denotes the spin-up (| ↑ >) and spin-down (| ↓ >) periodic Bloch function character, namely: |hh ↑⟩, |lh ↑⟩, |hh ↓⟩ or |lh ↓⟩ and, finally, are the weight coefficients in the basis set of envelope wavefunctions, Fj(z)fn,m(ρ, φ), at a position (ρ, φ) inside the dot. The solutions for the in-plane motion, fn,m(ρ, φ), are given by [24]
(5)
for semi-cylindrical confinement (Figure 1c). In these expressions, μn,m is the mth zero of the Bessel function of order n, Jn(x), whereas the form of function Fj(z) depends on the profile potential along z-direction between the dots. The depth of the quantum well is determined by the offset between the valence-band edges in the dot and the barrier materials. For the GaAs/In0.4Ga0.6As interface, the valence-band offset can be estimated as ΔEv = 214 meV. By analytically solving the Schrödinger equation for holes and regarding the mismatch between the Luttinger parameters in the GaAs/In0.4Ga0.6As interfaces, the transcendental equation is derived, which determines all subband energies (j) and the corresponding wavefunctions (see Appendix 1). The signal (±) in the Eq. 16 provides them, respectively, with symmetric or asymmetric character . Taking advantage of this fact, the Hilbert space for the hole wavefunctions Ψv(r) can be split into two orthogonal subspaces, labeled I and II, that are classified according to the parity of the quantum number j. As a result, the Hilbert subspace I(II) gathers spinor states with spin-up (spin-down) components having odd j-values (even j-values) that are coupled with states with spin-down (spin-up) and odd j-values (even j-values). Hence, the eigenvalue problem for the Hamiltonian in Eq. 3 can be solved independently for each class of states I and II. The hole state wavefunction (4) for a given subspace can then be written as
(6)
The hole states of the semi-cylindrical QDs system are calculated by exact diagonalization of the Hamiltonian ℋ, on a finite basis set expansion given by Eq. 6 using a standard numerical diagonalization technique. The matrix elements of the momentum operators and involved in the off-diagonal terms Eq. 2 of the Hamiltonian ℋKL are given in Appendix 2.
As shown schematically in Figure 1, effects associated with isotropic and anisotropic spatial confinements are simulated in the calculation by changing the lateral sizes, D011 and , in the (100) plane as well as the inter-dot distance (d). Two geometry cases will be studied: (i) Uncorrelated dots, which consider isotropic spatial confinement in the (100) plane, with , without inter-dot coupling. The spin quantization axis (z-axis) is chosen along direction [100] (Figure 1a) and 2D dot distribution is random; (ii) Correlated dots, which consider anisotropic spatial confinement () and include inter-dot coupling (Figure 1b) that leads to a chain-like 1D dot alignment. Here, the spin quantization axis (z-axis) must be set along the direction [25,26].
Figure 2. Oscillator strength contours for correlated dots with inter-dot distance d = 160 Å and strain order factor ε|| = –0.3%.
These two models were tested and compared in order to search for the main qualitative differences between optical emission probabilities for light polarized along and perpendicular to the z-axis, respectively. This modeling tests the different behavior of optical emissions associated fundamentally with the difference between heavy-hole (hh) and light-hole (lh) longitudinal and transversal ellipsoidal effective masses as well as the effects originated from the strain fields on these hole energy levels.
The oscillator strength for optical electric fields linearly polarized along the and parallel to [011] directions (see Figure 1) can be calculated as . For uncorrelated dot arrays, showing mostly random distribution (case (i)), they are given by
(7)
where P = ⟨s|px|x⟩ = ⟨s|py|y⟩ = ⟨s|pz|z⟩ is the isotropic conduction-valence-band momentum matrix element between functions at the Γ-point, is the overlap between jth electron and hole envelope functions along z-axis, and the factor 2 is due to double spin degeneracy.
All coefficients , shown in Eq. 7, are real when calculated for cylindrical uncorrelated dot array case, and using the expansion set in Eq. 3. This result leads to identical oscillator strengths and, consequently, equal PL intensities for both optical linear polarizations. More specifically,
(8)
according to Eq. 4, and this identity is independent of QD size. Besides, neither hydrostatic nor axial strain contributions would induce changes to Eq. 8 in this symmetric case (unless anisotropic strains are applied). Therefore, a distribution of cylindrical uncorrelated dots over the (100) plane would lead to identical linear PL-emission intensities polarized along and perpendicular to the z-axis.
In correlated arrays showing preferential dot diffusion, the compressive strain can be relaxed by forming 1D arrangement, as occurring for strain distribution in free-standing superlattices. In this case, the in-plane strain is defined by ε|| = εxx = εyy = (a - aw)/aw, where the lateral lattice constant (a) can be calculated as [27]
(9)
Here, Sα = (S11 + S12)α is the sum of elastic compliance constants, Lα (aα) is the width (bulk lattice constant) of the corresponding layers regions α = w (well) or b (barrier). In this way, a 3% strain can be relaxed to a value near 1%. Although shear strain contribution, which affects the separation between hh and lh subbands, becomes relaxed, the hydrostatic strain component leads to the effective reduction of the inter-dot potential barrier, which enhances the inter-dot coupling and tunneling. The envelope function spreading along the direction favors the confinement of a carrier with higher in-plane effective mass, which leads to the exchange of the ground-state character, since .
The effects associated with the anisotropic confinement, within the inter-dot coupled model and simulated by a semi-cylindrical dot shape (see Figure 1c), uses only the subset of the expansion functions in Eq. 5 that complies with null boundary conditions at the flat part of the semi-cylinder. The corresponding linear crossed polarized optical matrix elements, for this correlated dot array model (case (ii)), are given by
(10)
(11)
Here, the factor 2 occurs due to the summation over subbands j = 1,2 since these states are nearly degenerate for large inter-dot separation, d. It is clear that the identity in Eq. 8 has changed and no longer holds for all values of the inter-dot distance and QD sizes. We will be showing below that mass anisotropy of hole ground-state might be hold responsible for these anisotropic optical emission intensities once the dot confinement strength becomes relaxed in certain directions, whether by dot size anisotropy and/or by inter-dot coupling tuned by the strain fields.
First of all, let us analyze the effect of the spatial confinement in the case of a single dot with the semi-cylindrical shape, namely: the limiting case d → ∞ shown in Figure 1c. As the strength of the spatial confinement is relaxed along the direction by the QD size increase, the topmost valence band becomes occupied by a state with a stronger lh-character and reduced hh-contribution [4,28,29]. This effect is caused by the strong hole mass anisotropy, namely: while . It can be noted, from simple arguments, that hh- or lh-mass character of the valence-band ground-state can be interchanged by weakening the spatial confinement strength in the direction . Under weak confinement regime, the total energy determining the level position is mainly inversely proportional to the effective mass, as
(12)
and
(13)
where ⟨D[011]⟩ and denote mean confining lengths. Consequently, by tuning the confinement anisotropically, the condition Elh < Ehh can be attained due to the mass anisotropy of carriers. As a result, the corresponding envelope functions must be more extended in one direction than the other. Thus, the corresponding PL transitions allowed for certain light polarization can probe the anisotropic character of the Bloch functions that, in the multi-band calculations, are determined by the values of the expansion coefficients in Eq. 4. It is noted, from Eq. 10, that a state having small hh-character and, consequently, small values of coefficients and , produces smaller oscillator strength for optical transition polarized along the inter-dot coupling direction .
Figure 2 shows the oscillator strength values calculated for two coupled semi-cylindrical QDs with two values of the transverse diameter, D[011], as a function of the axial length, (see Figure 1c). Here, we have estimated the strain strength to hold with the uncorrelated dot array condition and confirm that the bigger the transverse size of dot array is (Figure 1b) the smaller must the strain order factor be. Furthermore, for compressive strain ε|| > ε, the crossing point can be shifted toward the dotted line. For dilation strain, with ε|| < ε, the crossing point is shifted away from the uncorrelated dot condition, and this condition can be attained in self-assembled QDs grown along the [100] direction. Certainly, shear strain field distribution is able to tune the equal oscillator strength condition for these mutually perpendicular polarized emissions in isolated anisotropic QDs.
Analogously to the exchange of ground-state character induced by anisotropic confinement and shear strain fields, this effect can be also produced by electronic coupling between nearest-neighboring QDs, an effect that leads to the enhancement of the effective value . The interchange of ground-state character is highly favored in coupled dots by increasing the inter-dot tunneling, as can be seen in Figure 3, which leads to the envelope function spreading along the coupling direction, . In order to show this effect, we have used the combination of dots with finite inter-dot separation, d. Note, in Figure 3, that coupled dots will show a left-shifted crossing point for equal oscillator strength, when compared to the uncorrelated dot case. As discussed before, this shift can be further modified by shear strain fields.
Figure 3. Oscillator strength contours fulfilling for correlated dots with inter-dot distances d = 160 Å (solid line) and 330 Å (dashed line) and strain order factors ε|| = –0.3% (red), ε|| = –0.4% (green) and ε|| = –0.5% (blue).
For the limiting cases (see Figure 4), and , the oscillator strengths for polarized emissions attain the conditions and , respectively, and these results are attributed to the anisotropy of hole effective masses. The crossing point where the polarized emissions have equal intensities can be shifted by the shear strain contribution to hh- and lh-energy level positions. In Figure 5, it can be observed that the crossing points are shifted to the right as the strain order factor and/or inter-dot distance are increased. Furthermore, two asymptotic limits > 400 Å and < 200 Å where the crossing points coincide were found out, respectively, for various strain strengths and for different inter-dot distances.
Figure 4. Calculated oscillator strengths for crossed linear optical polarizations along the directions (red line) and [011] (blue line) for two coupled QD's (Figure 1c) with semicylindrical shape and axis in the direction with (a) = 280 Å, d = 160 Å upon strain order of ε|| = - 0.3% (solid line) and -0.9% (dashed line). (b) = 350 Å, d = 330 Å and ε|| = - 0.1% (solid line), -0.2% (dashed line) and -0.3% (dashed-dotted line). The crossing point stands for isotropic optical emission.
Figure 5. Calculated oscillator strengths for crossed linear optical polarizations for a strained system of two coupled QDs with different inter-dot distances d = 160 Å (dashed line), 330 Å (dashed-dotted line) and infinite (solid line) corresponding to a single (isolated) QD. Here was taken a lateral size D[011] = 350 Å and a strain order factor ε|| = - 0.2%.
Experimental Confirmation of the Purposed Theory
Experiments that confirm this modeling were performed using In0.4Ga0.6As QDs grown by molecular beam epitaxy on semi-insulating (100)GaAs. The QDs were obtained using the Stranski–Krastanov growth mode. Two set of samples were prepared for the experiments: (A) QDs with strong anisotropy in shape along direction and with partial ordering along that; (B) QDs with weak or no anisotropy on the (100) surface and large separation in both in-plane directions. The shape and the distribution of QDs were controlled by the Arsenic background. The use of As2 or As4 background during the growth allows the control of group III element diffusion on GaAs (100) surfaces, providing choices for different dot samples with the same composition but different shapes and distribution. Details of growth mechanisms and the processes involved in diffusion controlling by the background Arsenic environment are described in Ref. [19].
Two sets of samples A were grown under As4 background. In one set, the layer of dots was left uncapped for morphology analysis, and in the other, the QDs were buried with GaAs for low-temperature PL analysis. The other two sets samples B were grown under the same conditions as the sets A, except that under As2 background. Surface morphologies of the two uncapped samples were performed by using atomic force microscopy (AFM), as shown in Figure 6, imaged by Nanoscope IV in the tapping mode and using a high-resolution Silicon tip. The (1 × 1) μm AFM images show the morphologies of the In0.4Ga0.6As uncapped dot samples. The mean dot size and the center-to-center distance along the direction of both sets are displayed in Table 1. The AFM pictures show clearly the effect of different Arsenic background both on dot formation and distribution. The predominantly anisotropic dot shape and distribution obtained along direction is for samples grown under As4 environment. Finally, these sets of samples enable us to use sample (B) as the reference for uncoupled QD arrays with mostly isotropic distribution on the (100) plane.
Figure 6. One layer AFM 1 × 1 μm image of In0.4Ga0.6As QDs in samples grown under different conditions. Sample A (left) shows 1D chain-like ordering along the direction. Sample B (right) shows mostly isotropic or randomized dot distribution in the (001) plane.
Table 1. Average QD parameters with dispersion obtained from a Gaussian fit of the AFM data
Grazing-incidence X-ray diffraction (GID) measurements were performed in both samples at the XRD2 beamline of the Brazilian Synchrotron Light Laboratory (LNLS), using a 4 + 2 axis diffractometer. The X-ray photon energy was fixed to 10 keV. Since both samples were capped by a GaAs 50 nm layer, the incident angle was fixed at 0.28°, slightly above the GaAs critical angle, maximizing the signal from the buried quantum dots. The diffracted signal was measured by integrating the exit angle from 0 to 1.2° [30].
Figure 7a and 7b show longitudinal θ - 2θ scans in the vicinity of the in-plane (022) and reflections for samples A and B, respectively. Such scans are sensitive to the strain relaxation inside the In0.4Ga0.6As QDs and GaAs surrounding lattice. For all scans, diffuse intensity is observed surrounding the narrow and intense GaAs Bragg peak, located at |H| = |K| = 2. For sample A, the longitudinal scan performed at the vicinity of the GaAs (022) reflection exhibits a much broader profile than the scan measured with the sample rotated by 90°, close to the reflection. Such a behavior indicates that a more effective strain relaxation for the islands may take place along the [022] direction, while a more strained lattice profile is found along the direction. The intensity distribution in both profiles of Figure 7a is almost symmetric with respect to the GaAs peak position, denoting the existence of compressively strained InGaAs inside the QDs, as well as on the GaAs matrix surrounding the QDs [31]. Similar diffraction profiles are observed in the longitudinal scans performed on sample B (Figure 7b). For this sample, the difference of widths of diffuse intensity on (022) and scans is not as pronounced as observed for sample A, indicating a less anisotropic relaxation.
Figure 7. Radial scans at the vicinity of the GaAs (022) (solid dots) and (open dots) reflections for sample A (a) and B (b). Lateral size from iso-strain regions in samples A (c) and B (d) obtained from the width of transversal scans.
In order to quantify the strain relaxation inside QDs in both samples, transversal scans were performed at several positions along the longitudinal profiles shown in Figure 1a and 1b. These scans (not shown here) are measured by fixing the θ - 2θ condition and varying the sample rotation angle θ solely. In momentum transfer space, the angular momentum transfer qa = (4π/λ)sin(2θ/2)sing(Δθ) is varied, where Δθ = (θ/2θ)/2. Such a procedure allows to obtain the average lateral size L of regions inside the QDs with constant strain status by evaluating the width Δqa of transversal scans, L = 2π/Δqa [30,32]. Values obtained for the local lateral size of iso-strain regions as a function of the in-plane strain status for samples A and B in the [022] and directions are shown in Figure 7c and 7d, respectively. For both samples, the lateral size of iso-strain regions along the QDs chain direction is larger than along the axis parallel to the chains. The ratio , which is a quantitative indicator of the anisotropic lattice relaxation inside QDs, is larger for sample A than for sample B, corroborating the qualitative information inferred from the widths of longitudinal scans.
Some considerations must be drawn before extending the analysis of the data shown in Figure 7c and 7d. For uncapped QDs, the relaxation of lattice parameter is monotonic from their base to their apex [32]. Capped QDs, in contrast, exhibit a non-monotonic gradient, with lateral and vertical strain variations. This condition generally implies in the existence of similar in-plane strain status on the island base and apex, both in contact with the GaAs matrix. It is therefore impossible to resolve vertically the position of iso-strain areas for the capped QDs with our GID measurements. Nevertheless, the lateral sizes observed represent a good approximation of the in-plane area of iso-strain regions projected on the substrate surface plane. Such approach allows for a visualization of the anisotropic strain relaxation. Since the diffraction signal observed at (|H|, |K|) > 2 is related to the existence of compressively strained GaAs surrounding the QDs [31], maps with the projected view of iso-strain areas were extracted from the experimental data by taking into account the L values for (|H|, |K|) < 2, which are directly related to compressively strained In0.4Ga0.6As from the islands (Tensile strained GaAs at the bottom and at the apex of the island also contribute to the diffracted intensity at |H| = |K| < 2. However, the total volume of material with local lattice parameter larger than aGaAs outside the island is considerably smaller than the amount of material contained inside the islands. For a discussion on tensile strained substrate material see [33]). These projection maps for QDs from samples A and B are shown in Figure 8a and 8b, respectively. The iso-strain projection areas were drawn following the condition that they are contained on curves delimited by
(14)
where x and y are the in-plane coordinates along the [011] and directions, respectively, considering the plane origin at the central QD position. Figure 8 shows the iso-strain areas for an in-plane region of approximately 1,100 Å × 700 Å, which contains 9 QDs for sample A and 4 QDs for sample B (see Table 1). The color scale in these maps refers to the in-plane strain with respect to bulk GaAs.
Figure 8. In-plane projection of iso-strain regions for a field of view with several islands for samples A (a) and B (b). The in-plane strain represented in the color scale is relative to the GaAs bulk lattice.
From Figure 8a, one clearly observes that iso-strain contour lines from one QD of sample A almost reach the neighbor QDs along the chain direction. An asymmetric ratio of 1.7 is found for the broader iso-strain contour lines of QDs in this sample, pointing out again to a more pronounced strain relaxation along the [011] axis. The physical presence of very close QDs along the chains may therefore induce a modulation of the strain field that allows for a gentle strain relaxation in the direction. In sample B (Figure 8b), the asymmetric shape of iso-strain regions is still observed, but with a ratio of 1.35. Although an elongation is observed along the direction, the QDs are too apart from each other and do not strongly influence the strain field of the neighbor QDs in this direction.
Since the GID measurements do not reveal directly the height above the substrate of each iso-strain region finite element method, simulations were performed using a commercial software package to provide complementary information on the strain configuration of capped islands. In our simulations, a three-dimensional box containing a single GaAs capped In0.4Ga0.6As island was created for each sample, with periodic contour conditions at all lateral edges in order to take into account the symmetry of QD chains and the possible interaction with the strain field from neighbor QDs. A 15 Å thick wetting layer of nominal concentration was inserted between the islands and the substrate, following Ref. [34]. The island profiles used in this simulation were extracted from the AFM measurements in uncapped islands (Figure 6) that resulted in the dimensions from Table 1. The nominal composition was kept, assuming thus a negligible deviation of island stoichiometry from the nominal values (Anomalous grazing-incidence diffraction measurements performed at the Ga - K edge do not point out to deviations (within an error bar of 7%) from the nominal In/Ga content inside QDs.). Such assumptions consider that islands do not undergo dramatic changes in morphology or composition under capping, which is a valid approximation for the growth temperatures used here and the reduced strain with respect to pure InAs islands [35]. Finally, a 500 Å-thick cap layer was added to the simulation, as represented in Figure 9a.
Two-dimensional cuts of the simulated data are shown in Figure 9b, d, and 9f for sample A and Figure 9c, e, and 9g for sample B. The selected cuts are schematically depicted at Figure 9a and were chosen to be at the island bottom (b) and (c), middle (d) and (e), and top (f) and (g). Since the representation used in Figure 8 cannot be directly correlated to the Cartesian in-plane strain components x and y, the maps of Figure 9b–g were drawn as a function of the axial (first) principal strain component. Such principal component analysis allows for the reduction of the dimensionality of the data set, providing a resulting representation with radial symmetry. The axial strain component is given by [36]
(15)
where εxy is the in-plane shear strain and εxx, εyy the normal in-plane strains. For all principal strain maps, the color scale represents the deviation of the local lattice parameter with respect to the bulk local lattice parameter. Therefore, higher principal strain values are found in positions where the In0.4Ga0.6As lattice of the islands is fully strained to the GaAs lattice constant. Finite values of the axial strain component are also observed in regions surrounding the islands, in which the GaAs local lattice is affected by the proximity to the island. Selected contour level edges were marked by dark lines in all maps as a guide to the eyes.
Figure 9. (a) Representation of the two-dimensional cuts shown in maps panels (b–g) performed on the finite element method simulations with periodic contour conditions at the substrate box edges. The color contours represent variations on the first axial principal strain, which allows a qualitative comparison with the GID data of Figure 8. Cuts on the bottom (b), middle (d) and top (f) of the average island of sample A show an elongated strain profile along the directions. Similar cuts for the average island of sample B are seen on (c), (e) and (g).
The maps generated by in-plane cuts in the simulation of QDs in sample A clearly exhibit elongated contours along the direction, most notably for the cuts at the island basis and middle. This indicates that for lower in-plane strain conditions, the lattice surrounding the islands behave as semi-continuous wires along the direction. In the QDs of sample B, an elongation of axial strain contour levels is also observed along the chain directions for all maps. However, the anisotropic effect is much more reduced with respect to the results obtained for sample A.
The effects of different dot size distributions and inter-dot coupling have been analyzed by low-temperature linear polarized PL measurements carried out on the samples A and B buried with GaAs. The samples were placed in a closed-cycle Helium cryostat (Janis—CCS-150) and excited using a 532-nm continuous wave YAG laser (Coherent Verdi V10—10 W). The PL signal was carried out by a monochromator (SpectraPro 2500i—0.5 m focal length) and detected by a liquid-nitrogen-cooled InGaAs photodiode detector array (Princeton Instruments—model 7498-0001). Figure 10a and 10b show the PL intensity at 10 K for samples A and B, respectively, where the emission spectra, for each sample type, collected with two linear polarizations, namely: along and along [011].
Figure 10. PL spectra for crossed linear polarizations, taken at T = 10 K with excitation wave length λ = 532 nm along and [011] directions for samples (a) A and (b) B. The degree of linear polarization: has been included in these panels. (c) PL peak position as a function of the excitation intensity.
In Figure 10a, one may see a polarization degree around 6%, as might be expected due to the elongation in the quantum dots profile revealed by the AFM images (Figure 6) and strain distribution (Figure 8). As highlighted in Figures 2 and 3, the oscillator strength grows for emissions linearly polarized along the larger dot size direction. This behavior is enhanced for inter-dot separation up to d ~ 160 Å. When d is further reduced, the inter-dot tunneling probability increases considerably, and this behavior is also enhanced. The PL intensity polarized along coupling direction is also enhanced in coupled QDs by the reduction of barrier heights due to hydrostatic strain of the order of 1%. Besides, the anisotropic PL-emissions from sample A, as shown in Figure 5a, can be qualitatively reproduced by the oscillator strengths, shown in Figure 3, calculated by using the nominal values for both samples. As seen in Figure 3, an effective increase in inter-dot tunneling (distance d ~ 160) Å would lead to the relaxation of the confinement along the direction. These effects would lead to a hole ground-state character exchange from predominant-hh to -lh, and to the intensity difference between these cross-polarized emissions, experimentally confirmed by Figure 10a.
For the isotropic case, PL-emissions occur when , a condition well-fulfilled for the cylindrical model of Figure 1b. By changing the dot shape and coupling along direction in (100) plane, the model shows that condition can be obtained for semi-cylindrical geometry only for a small combination of values that emulates uncoupled dot distribution in the (100) plane if strain effects are included into the Hamiltonian. According to the theoretical modeling, an isotropic dot distribution on the (100) plane (case (i)) accounts for isotropic crossed polarized PL-emissions, as shown in Figure 5b for sample B. However, according to Figure 10b, a small polarization degree is still present in symmetric QDs, associated with the elongation that remains, as revealed by the Figure 8b. Such feature might come from the anisotropic diffusion rate of Indium atoms during the growth, which presents a higher mobility than the Gallium atoms. Furthermore, the Indium diffusion coefficient is faster along the than along the [110] direction, and as a result, the quantum dots of sample B are not completely symmetric [37,38].
To confirm the results from X-ray measurements, Figure 10c displays the shift in peak position of the spectra as a function of the excitation intensity. Note, for sample A, a shift toward higher energies as the excitation intensity grows. Such a blue-shift for the elongated dots has been associated with the screening of the built-in electric field due to the presence of strain. On the other hand, for sample B no remarkable energy shift is observed showing that the strain is not so pronounced as in the previous case [39].
Conclusions
The control and simulation of size anisotropy and effective inter-dot tunneling effects, as described in this work, is an important issue to be addressed during the characterization of ordered sets of coupled dots. The strain fields, present during the growth process of these QDs have led to the appearance of anisotropic geometric shapes, mostly elongated along the preferential direction. These effects can be probed by polarized optical responses from different samples. In summary, we have shown that the shape, spatial distribution and the inter-dot coupling of InGaAs self-assembled QDs can be probed and characterized by using linearly polarized PL-emissions. Valence-band effects due to admixing between hole states and strong anisotropic effective masses have led to different PL intensities in samples with lateral QD ordering forming "chain-like" structures. The envelope function model used here to describe the polarized optical responses showed fairly good agreement with structural AFM and X-ray data and may be used to predict or characterize the strength of inter-dot coupling and/or anisotropic dot shape and distribution.
Appendix 1: Double Quantum Well Potential
After matching the wavefunctions fulfilling the hole-Shchrödinger equation at the interfaces using ∂z ln Fj(z|z=l = β∂z ln Fj(z)|z=l and ∂z ln Fj(z|z=l+d = β-1z ln Fj(z)|z=l+d, we are able to obtain the transcendental equation
(16)
where and and is the rate between the hole effective masses in the barrier and the well region, respectively, and , as well as l is the well width, d the inter-dot distance and V the barrier height. By carrying out numerical calculation, solutions of the Eq. 16 yields to the energy levels with the corresponding wavefunctions of the symmetric (+) and antisymmetric (-) hole states,
(17)
where
Appendix 2: Matrix Elements
The matrix elements of the momentum operators are necessary in order to build the Hamiltonian matrix form of the ℋKL. In polar coordinates the operators are written as,
Projecting on the wavefunctions (5) with t = (n, m) and t' = (n', m'), it is straightforward to show that
(18)
with
and
(19)
where are numbers ruled by
(20)
In the particular case where t = t' Eq. 20 can be reduced to
Also, it follows the relation
(21)
The other matrix elements for the high-order operators are evaluated numerically using Eqs. 18–21 and the matrix identity .
It is worth to show that the element matrix of the diagonal terms in Dhh(lh) accomplished
Taking into account the loss of translational invariance along the z direction by replacing the wave vector-component kz by the operator -iz it is therefore convenient to write the resulting expression for the element matrix in a symmetrized form
where index j stands for the piecewise wavefunctions (17). The resulting integrals in z-direction are solved numerically.
Acknowledgements
Authors acknowledge financial support from the agencies FAPESP and CNPq (GEM, VL-R), CONACYT/Mexico and FAPEMIG (LV-L) and LNLS-MCT (AM) and ICTP/Trieste (CT-G) and the National Science Foundation of the U.S. trough Grant DMR-0520550 (BLL, YuIM). LV-L thanks E. Gomez for technical assistance.
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CH391L/S13/BioBricksAndRegistry
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A tutorial on BioBrick™ assembly is available on the [[BioBricks construction tutorial]] page.
A tutorial on BioBrick™ assembly is available on the [[BioBricks construction tutorial]] page.
Specific assembly standards for different types of BioBricks can be found at the [http://partsregistry.org/Help:BioBrick_Assembly ''Help:BioBrick Assembly] page.
Specific assembly standards for different types of BioBricks can be found at the [http://partsregistry.org/Help:BioBrick_Assembly ''Help:BioBrick Assembly] page.
+
+
The BioBrick assembly standard was first introduced by Tom Knight in 2003, and he has updated it several times since. The latest draft, from 2008, is called BB2: [http://hdl.handle.net/1721.1/45139 Draft Standard for Biobrick BB-2 Biological Parts]. The standard is still required to be used in all iGEM competitions (as of 2013).
===Sharing BioBricks===
===Sharing BioBricks===
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However, BioBricks and the Registry are still in use today by many iGEM teams that don’t have access to the latest technology and equipment.
However, BioBricks and the Registry are still in use today by many iGEM teams that don’t have access to the latest technology and equipment.
+
+
Here is an informational video about synthetic biology, published in 2011, that references the registry: [http://youtu.be/rD5uNAMbDaQ "Synthetic Biology Explained" by James Hutson, Bridge8]
==References==
==References==
Revision as of 16:14, 4 February 2013
Contents
Overview of BioBricks and the Registry of Standard Biological Parts
Synthetic biology stands to reap the same benefits from standardization as those that came from standardization in mechanical engineering, like the defining of pitch, diameter, and form of screw threads in the middle of the 19th century.[1]
The Registry of Standard Biological Parts is a growing bank of genetic building blocks (promoters, DNA binding sites, protein-coding sequences, etc) that are built with the intention of being pieced together to create synthetic systems within organisms. The goal is to create a large functional group of parts (called BioBricks"™), categorized by type, so that new combinations can be built according to the engineering principles of "abstraction" and "standardization".
The principles of abstraction and standardization are what allow engineers to collect, refine, and repackage nature so it's easier to make new and reliable things.[2] Proponents of synthetic biology attest that these principles were never truly integrated into synthetic biology's precursor, genetic engineering. Thus, according to Tom Knight of MIT, who coined the term "synthetic biology", BioBricks and the Registry were created to provide biology with the same advantages similar to those which accompanied standardization in mechanical design - "the widespread ability to interchange parts, to assemble sub-components, to outsource assembly to others, and to rely extensively on previously manufactured components."[1]
BioBricks™ are trademarked in order to define and ensure that the standardized parts in the Registry remain free-for-use as part of a standard library or parts.
"Get Some, Give Some"
The registry is built on the idea of "get some, give some:" the Registry is a resource for users to find and integrate new parts into their systems, while they in turn provide the Registry with data regarding the effectiveness of obtained parts and new parts they have developed. In this way, the Registry continually grows and improves as a community resource.
BioBrick™ Assembly Standard
An actual circular diagram of a BioBrick, flanked by restriction sites and packaged within a plasmid "backbone".[1]
A BioBrick is a sequence of DNA with a predefined structure and function. This "payload" is held in a circular plasmid, which is an isolated, circular piece of DNA that can replicate in bacteria.
BioBricks™ are created with the intention of being easily joined and manipulated. In order for this to be possible, the BioBrick™ assembly standard requires the use of defined prefix and suffix sequences (flanking both sides of the BioBrick) that contain specific restriction endonuclease sites. These sites are called EcoRI, NotI and XbaI in the upstream, and SpeI, NotI, and PstI in the downstream. Naturally, the parts must also be engineered such that these sites are not present in the functional region of the sequence.[3]
Cutting the BioBrick at specific restriction sites (using restriction enzymes) is what gives a BioBrick its interlocking ends. The end of one BioBrick can then be connected, or ligated, together with the end of another BioBrick, allowing you to effectively string together BioBricks end to end to make devices, and then string devices together to make systems.
For example, to join together two BioBricks, you would first cut both plasmids with restriction enzymes, turning one into an "insert" by getting rid of the rest of the plasmid, and turning the other into a "vector" by opening a space in the plasmid in front of the BioBrick. You then mix together the insert and vector with a special enzyme called a "ligase" that can join together two broken pieces of DNA. Because A's always pair with T's and G's always pair with C's, the overhanging edges of single-stranded DNA that your restriction enzymes left behind will match up to make double stranded DNA. The result is a composite plasmid that contains two BioBricks, now side by side.[4]
It is important to note that this new larger composite part has the same restriction sites as the smaller parts it was originally made from. This is what is meant by preserving "key structural elements" that allow one component of any size to be easily connected to any other component.[1] Also note that the "scar" (point of ligation between BioBricks) doesn't match the restriction sites anymore, so the bond between BioBricks will hold through though subsequent rounds of splicing.
Composite components are always created this way, either by “prefixing” one component with another, or “postfixing” one component with another. In both cases, the result is a new, composite component, which can then be used in the same way, as either an insert or a vector, in more complex reactions.[1]
A tutorial on BioBrick™ assembly is available on the BioBricks construction tutorial page. Specific assembly standards for different types of BioBricks can be found at the Help:BioBrick Assembly page.
The BioBrick assembly standard was first introduced by Tom Knight in 2003, and he has updated it several times since. The latest draft, from 2008, is called BB2: Draft Standard for Biobrick BB-2 Biological Parts. The standard is still required to be used in all iGEM competitions (as of 2013).
Sharing BioBricks
Restriction enzyme cloning, or "subcloning", is a common way to share BioBrick parts.
Molecular parts are shared using one of several cloning techniques. One of these techniques is called restriction enzyme cloning, or "subcloning".
Restriction enzymes (or restriction endonucleases) are proteins that cut DNA at or near specific sites. These sites are recognized as a specific DNA sequence, and go by names such as EcoRI, XbaI, SpeI, PstI and NotI. Assuming the your gene of interest (YGOI for short) exists in a bacterial plasmid or vector (donor plasmid), the restriction enzymes are used to cut YGOI out of the donor plasmid and then cut the recipient plasmid at a specific location in a specific pattern, so that YGOI can then be "pasted" to that location in the recipient plasmid using a process called ligation.[5] [6]
History
The registry is an effort that was founded by Tom Knight of the Artificial Intelligence Lab at MIT in 2003. He coined the term "BioBrick" in his paper, "Idempotent Vector Design for Standard Assembly of Biobricks". Idempotent, a term borrowed from mathematics and computer science, in this context means that, during the assembly of complex biological components, the chemical reactions should not alter the key structural elements of the components.[1]
In the summer of 2004, the registry contained about 100 basic parts; today, this has expanded to over 700 available and 2000 defined parts.[7]
PoPS (Polymerase per Second)
MIT initially used a unit of measurement called TIPS (Transcription Initiations per Second) for measure rates of transcription at the ends of its parts; however, this was insufficient because there are places on the DNA (e.g. terminators) where transcription initiations are not taking place. PoPS is a relatively new unit developed during construction of standardized "ends" of DNA pieces that measures the inputs and outputs of BioBrick™ parts. PoPS measure the rate at which RNA polymerase moves past a point in the DNA, similar to measuring the current flow across a specific point in a wire. Devices that have an input and output in PoPS are composable - that is, they can be arbitrarily joined together to create complex devices and systems. Creation of devices allows us to characterize devices and eventually more complex systems, thus PoPS is important as a common signal carrier. PoPS differs from transcription rate in that it can also be measured at terminator sites; upstream, they are theoretically equivalent.
An example of a system from which PoPS is understandable is a PoPS based inverter, which takes in a PoPS signal and inverts it. A PoPS based inverter consists of a ribosome-binding site, repressor coding region, terminator and cognate promoter. A high PoPS input cause expression of the repressor, which then binds to the promoter and produces a low output signal. A low PoPS input means very little repressor expression, so the promoter is free to generate PoPS.
At the end of the day PoPS is a useful abstraction that we can use to think about transcription-based logic devices and characterize BioBrick™ parts; up to this point, there has been to way of measuring it in vivo.[8]
Using the Registry
Main Page
The main page of the registry has a welcome message, four main icons, a list of registry tools, and registry news. The areas of interest for the purposes of searching and finding parts and the iGEM competition are the main tabs and registry tools.
Icons
This tab opens up the Catalog of parts, devices, and systems for browsing and finding parts.
• Help
The help section of the page consists of FAQ's, an introduction to BioBrick standardized parts, instructions for various types of assembly, information about the Registry and its tools, a more in-depth look at designing systems using BioBricks©, information about the DNA Repositories, and help for users and groups.
• Users & groups
This icon links to the iGEM competition home page.
• DNA repositories
This icon opens up the DNA Part Repositories page, which contains a list of (and a convenient search feature for) the DNA for BioBricks available in plasmids in cells.
Registry Tools
Opens the search page for parts in the Registry.
• Add a part
Opens the page for adding basic parts, composite parts, and construction intermediates to the Registry.
Opens the page where parts can be requested for use by iGEM teams.
• Opens the instructions for preparing and sending DNA to the Registry.
• Allows you to use quick analysis of a single sequence or begin a more complex sequencing project in order to compare a sequence to the parts or a specific part in the Registry, combine several sequence readings to see if your part is correct, or save a sequence with the part's other information so future users can find it.
Catalog
The registry is focused primarily around the Catalog containing sorted and categorized entries. The Catalog is split into a hierarchy of parts, devices, and systems. Parts are the simplest entries in the catalog, basic building blocks for devices and systems. Devices consist of multiple parts pieced together to perform a particular function. Systems, the most complex of the three, are self-contained sequences that entirely specify all the parts encoding a device designed for a specific task.[9]
You can browse the parts and devices in the Catalog by:
• Type
• Function
• Chassis (the model in which the part works best)
• Assembly standard (each assembly standard is described in detail with the correct parts and methods included on the catalog page for that method)
• Contributor
• Other user-generated catalog pages
BioBrick™ Types
Screenshot of BioBrick types within the Registry catalog.
At a high level, the main types of BioBrick parts are "protein coding sequences", "promoters", and "primers". Protein coding sequences are like “recipes” that are read by a chemical called “polymerase” and then transcribed into RNA which is then translated into proteins like... bioluminescence, banana smell, and colors.
• Protein coding sequences are like “recipes” that are read by a chemical called “polymerase” and then transcribed into RNA which is then translated into proteins like... bioluminescence, banana smell, and colors.
• A promoter is a “switch” upstream of a coding sequence that controls when a protein actually gets made. In other words, it controls when the polymerase begins transcription into RNA. The frequency at which a protein is made can be measured, and the current standard of measurement is called "PoPS", or "polymerases per second".
• A primer allows you to select a specific region of DNA in order to make copies of it (called “amplification” or “cloning”).
BioBrick™ Names
The letters used in naming parts and their corresponding functions.
The names of parts in the Registry begin with BBa = BioBrick [version] alpha, followed by a letter indicating their function. These letters and their corresponding functions are displayed in the image to the right.
An example of a part is BBa_I721001. The easiest way to determine the function of this part is simply to take the name and enter it into the search bar in the top right corner of the page. I attempted searching multiple parts based on their name in the actual search page of the registry, however it could not find them. It is possible that this is due to them recently redoing their system, because the part has been in the registry since 2007 in this case. Decoding the name is fairly simple; the first part implies that it is a BioBrick part type alpha (I can't find any examples of beta parts in the system). The I, as shown in the chart to the right, is supposed to signify that it is from an IAP project from 2003 or 2004; however, this is not the case (explained below). The numbers are assigned to groups involved in the project; searching the part will pull up its page with that information. In this case, the part was contributed by Jeffrey Hoffman and his iGEM group from 2007 - this is a clear example of the mentions in the paper by Peccoud [10] that a large percentage of parts in the registry are mislabeled or inaccurate.
Finding a Part
The easiest way to find a basic part if you know the part name or number is to enter the information on the search page of the Registry. If you are searching for parts that serve a specific purpose, the quickest way is to browse the parts by type or function in the Catalog. Finding composite parts entails the same process as basic parts; to find composite parts that contain a specific basic part you can use the Superpart search section on the Registry search page.
This information and more can be found in greater detail on the registry's Help:Search page.
Ordering Parts
Once you've found a part you want to order, there are multiple ways of acquiring it from the registry. Before ordering, you should go to the part's main page on the registry and check if it's available. This information is in a box on the top right of the page, listing the DNA as available if the part can be requested. Once you've checked availability, it can be requested via email. An email should be sent to hq@igem.org containing your iGEM team or lab name, the name of the part, the plasmid backbone and resistance, and the source plate and well. This information and more is on the Registry's site on the Help:Requesting Parts page.
Addgene
Addgene is a nonprofit organization whose purpose is to create a plasmid repository that will ease sharing of plasmids between scientists. Plasmids can be found in the following categories: empty backbone, species of gene, popular plasmids, depositing scientist, special collections, expression system, consortiums, and vector type. Plasmids can be ordered directly from their website. Several useful tools are available on their website as well, including a sequence analysis program, vector database, and various protocols for operations involving plasmids.
Legacy
2013 was the ten-year anniversary of the creation of the BioBrick standard, the Registry of Standard Biological Parts, and the iGEM competition. Technology has advanced to the point that it is can be more practical to encode and manufacture a complex DNA sequence locally and directly, rather than order BioBricks and splice them together one at a time.
However, BioBricks and the Registry are still in use today by many iGEM teams that don’t have access to the latest technology and equipment.
Here is an informational video about synthetic biology, published in 2011, that references the registry: "Synthetic Biology Explained" by James Hutson, Bridge8
References
1. Knight, Tom. Idempotent vector design for standard assembly of biobricks. MASSACHUSETTS INST OF TECH CAMBRIDGE ARTIFICIAL INTELLIGENCE LAB, 2003. [Knight2003]
2. "Synthetic Biology Explained" <http://www.youtube.com/watch?v=rD5uNAMbDaQ> [SynBioExplained]
3. Canton B, Labno A, and Endy D. . pmid:18612302. PubMed HubMed [Canton2008]
4. http://agapakis.com/hssp/biobricks.html [agapakis]
5. Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular Cloning: A laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 1.63-1.70. [CloningColdSpringHarbor]
6. http://homepage.univie.ac.at/nikos.pinotsis/webPP/genetoprotein/cloning_strategy/clo_rest-enzymes.html [CloningNikosPinotsis]
7. http://partsregistry.org/Help:About_the_Registry [iGEMRegistry]
8. "PoPS." OpenWetWare, . 4 Oct 2007, 16:12 UTC. 8 Feb 2012, 21:11 <http://openwetware.org/index.php?title=PoPS&oldid=156219>. [PoPS]
9. Peccoud J, Blauvelt MF, Cai Y, Cooper KL, Crasta O, DeLalla EC, Evans C, Folkerts O, Lyons BM, Mane SP, Shelton R, Sweede MA, and Waldon SA. . pmid:18628824. PubMed HubMed [Peccaud2008]
All Medline abstracts: PubMed HubMed
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Community news
From OpenWetWare
Revision as of 21:46, 23 March 2006 by Reshma P. Shetty (Talk | contribs)
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News (edit)
Get Started
Start editing!
BE.109
OWW in the classroom.
John Wilbanks
Seminar Series.
What's new?
What's changing NOW!
3/23 The Main Page has undergone major revisions.
3/11 Community Portal has been updated!
2/17 Check out the new OWW Calendar.
2/17 Now running MediaWiki software version 1.5.6.
2/08 Welcome BE.109 & BE.180 students joining OWW!
Add events to the Calendar. Archive community news.
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Lidstrom:Oligo Orders
From OpenWetWare
Revision as of 14:40, 6 March 2012 by Janet B. Matsen (Talk | contribs)
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Invitrogen
• Most people order from Invitrogen. Ask Melissa for the excel sheet with which to order, and return it to her filled in.
• They ship for free, and have a good price.
IDT
• IDT is the synthetic biology industry standard.
• Janet and Amanda use IDT for larger oligo orders because it is cheap, we can enter orders ourselves, and they are delivered the next day if you order by 3pm. (Technically next-day delivery is not a rule, but it is true 99.9% of the time.)
• They charge $0.14/bp and ship to the lab for free for orders over $50. Conveniently when you check out they quote $0.35/bp so it is not hard to meet the $50 minimum; the price is adjusted to $0.14/bp after you finish checking out.
• Don't bother with same-day oligo orders. It just costs a lot extra to have a guarantee that your oligos will arrive ~10:30am the next day when you order by 1pm.
• Many labs have primers shipped to the biochemistry store on campus for free. We would need a second account to do this, and don't currently have one. So if your order is not free to ship, we use Invitrogen.
• Click "ship order when complete" not "ship items as available" because then you will lose your free shipping privileges.
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Protocols/Template
From OpenWetWare
(Difference between revisions)
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(PBMC Antigen Stimulation Assay)
(Isolation of Mononuclear Cells)
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=='''Procedure'''==
=='''Procedure'''==
-
==Isolation of Mononuclear Cells==
* This is a sterile procedure and all steps should be performed in a hood.
* This is a sterile procedure and all steps should be performed in a hood.
# Turn on the hood. Bring Ficoll and PBS to room temperature in the hood.
# Turn on the hood. Bring Ficoll and PBS to room temperature in the hood.
Revision as of 17:59, 15 October 2009
This page is a template and should not be edited.
Click here, copy the source, and paste it into your page.
Interested in posting a protocol on OpenWetWare? Here is a template to help you do so.
Click the view source tab and copy everything below this line. Paste it into your new protocol page. Then replace the text in this page with your own protocol. Feel free to add or delete sections as appropriate.
Contents
Overview
This is a protocol designed for T-regulatory assay for Mouse allergen (Mus m1) study.
Materials
List reagents, supplies and equipment necessary to perform the protocol here. For those materials which have their own OWW pages, link to that page. Alternatively, links to the suppliers' page on that material are also appropriate.
• supply 1 (i.e. tubes of a certain size? spreaders?)
• reagent 1
• X μL reagent 2
• component A (reagent 2 is made up of multiple components)
• component B
• equipment 1
• equipment 2
Procedure
• This is a sterile procedure and all steps should be performed in a hood.
1. Turn on the hood. Bring Ficoll and PBS to room temperature in the hood.
2. Obtain whole blood specimens collected in sodium heparin (green top) collection tubes and record subjects information, i.e. ID#, date collected, date received.
3. If performing Basophil Activation Assay on this sample, set aside 3mL of whole blood before the next step.
4. Dilute the remaining blood at 1:1 with PBS in a 50mL conical tube.
5. Place 15mL of Ficoll in a 50mL conical tube. Overlay with up to 30mL of diluted blood, gently add the blood on the ficoll solution to avoid of blood with ficoll solution.
6. Centrifuge @ 500g for 30minutes @ room temperature (slow acceleration, deceleration off to ensure no disruption of the density gradient).
7. Using a sterile transfer pipette, aspirate the buffy coat (peripheral blood mononuclear cells [PBMCs]) into a new 50mL conical tube (avoid aspirating the ficoll). Add PBS into the 50mL tube to bring the sample to a minimum of 2X the initial volume. Invert up and down gently to mix.
8. Centrifuge @ 500g for 20minutes @ room temperature (maximum acceleration and deceleration).
9. Aspirate and discard the supernatant. Resuspend the cell pellet first by tapping the tube until no clumps are visible, then adding 1mL of PBS. Set aside a 10μl aliquot of cells for counting as follows: Add 90μl of PBS into the 10μl of cells.
10. Add PBS to the cells in the 50mL tube for a total volume of 20mL, and centrifuge @ 300g for 15 minutes @ room temperature(maximum acceleration and deceleration).
11. In order to determine the volume to use for resuspending the PBMCs after the wash, the total number of cells in the sample must be determined. Combine the 100μL aliquot of cells in PBS set aside above with 100μL of 0.2% Trypan solution (if using the automated counter) or 0.4% Trypan solution (if manual counting). Mix well with pipette.
12. Carefully, introduce 10μL of the stained cells into the notch of a hemocytometer and record cell counts using a hand-held counter. Count the number of cells under a microscope or place 20μL of the stained cells onto a disposable slide and count using the automated counter.
13. After centrifugation is completed, aspirate and discard the supernatant. Resuspend the cell pellet by tapping the tube until no clumps are visible. Suspend PBMCs with PBS @ 10 million cells/mL in a 15mL conical tube.
PBMC Antigen Stimulation Assay
*This is a sterile procedure and all steps should be performed in a hood.
1. Remove stimulants from the freezer and thaw.
2. Label 24 well plate with specimen ID and date (this is for the 7 days cell culture). Label each well with the appropriate stimulant condition, ordered by priority (for cases where there are insufficient cells to test all stimulants).
1. Musm1 (allergen): @ 200μg/mL purified Musm1 protein in Aim-V.
2. AIM-V + IL-2 (negative control): AIM-V + IL-2 medium alone.
3. Beads (positive control): 1μg/mL anti-Cd3/Cd28 beads.
4. Tetanus: 200μg/mL tetanus in AIM-V.
3. Add an equal volume (1:1 dilution) of freshly prepared 10μM CFSE (in PBS) to the tube of cells. To make 1.5mL of PBS + CFSE (2x solutin): add 3μL of stock CFSE (5mM) into 1.5mL of PBS.
4. Incubate in 37deg;C water bath for 10 minutes.
5. After incubation, wash the CFSE stained cells in 10mL of AIM-V @ 300g for 10 minutes. Aspirate supernatant after centrifugation.
6. Resuspend CFSE stained cells in AIM-V medium @ 4 million cells/mL. For plating, each well should contain 2 milliion cells/mL.
• Begin the stimulation process by preparing AIM-V medium + a 2X solution of IL-2 by adding 2μL of IL-2 per mL of medium in a 15mL conical tube. For 5 stimulant conditions, you will need atleast 2.5mL of AIM-V + IL-2. Vortex gently.
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
1. List troubleshooting tips here.
2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
3. Anecdotal observations that might be of use to others can also be posted here.
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
References
Relevant papers and books
1. Goldbeter A and Koshland DE Jr. . pmid:6947258. PubMed HubMed [Goldbeter-PNAS-1981]
2. JACOB F and MONOD J. . pmid:13718526. PubMed HubMed [Jacob-JMB-1961]
3. Mark Ptashne. A genetic switch. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 2004. isbn:0879697164. [Ptashne-Genetic-Switch]
All Medline abstracts: PubMed HubMed
Contact
• Who has experience with this protocol?
or instead, discuss this protocol.
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Talk:CH391L/S13/Ancestral Sequence Reconstruction
From OpenWetWare
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Contents
Methods
• Gabriel Wu 16:59, 18 February 2013 (EST): How does Pauling's proposal for ancestral gene construction relate to the actual discovery of DNA structure?
• Benjamin Gilman 13:21, 21 February 2013 (EST): The Pauling and Zuckerkandl paper came out when the only protein sequence information we had came from limited peptide sequencing methods like Edman degradation (N-terminal sequencing). You might add something about the shift to using DNA or RNA sequences to infer protein sequence once techniques like Maxam-Gilbert and Sanger sequencing showed up in the '70s.
• Jeffrey E. Barrick 21:26, 24 February 2013 (EST):The idea, if not the algorithms, were probably fairly close to what goes on today.
• Kevin Baldridge 17:00, 18 February 2013 (EST):How do the methods here compare with those used for phylogenetic placement based on ribosomal RNA sequences?
• Jeffrey E. Barrick 21:26, 24 February 2013 (EST):Pretty much the same in theory.
• Aurko Dasgupta 00:36, 25 February 2013 (EST): The techniques used to estimate the ancestral sequences are essentially the same. However, most protein sequences are nowhere near as conserved as ribosomal sequences.
Ancestral Organism Resurrection?
• Gabriel Wu 17:06, 18 February 2013 (EST): What's the oldest intact DNA? What's the oldest speciesn being sequenced today?
• Catherine I. Mortensen 15:05, 21 February 2013 (EST):Has anyone heard of H. Salinarum?
• Benjamin Gilman 16:47, 21 February 2013 (EST): I know there's some debate about it, but it's possible that at least one sample of an ancient H. Salinarum ancestor is >100 million years old. A lot of people were skeptical because the genome didn't seem that different from those of current halophilic archaea, but at least some regions didn't match anything we know of now. I wonder if it's reasonable to assume that once they're adapted to high salt, halophiles might evolve more slowly because they face less competition.
• Thomas Wall 21:47, 21 February 2013 (EST): This wiki entry is a good start if you are interested on the subjects http://en.wikipedia.org/wiki/Ancient_DNA
• Thomas Wall 21:52, 21 February 2013 (EST): Here they found the half life of DNA in fossil samples, 521 years, http://rspb.royalsocietypublishing.org/content/279/1748/4724
• Gabriel Wu 17:08, 18 February 2013 (EST): Separate topic on sequencing wooly mammoths and neanderthals?
• Jeffrey E. Barrick 18:30, 20 February 2013 (EST):We could call that topic Ancestral organism resurrection. It could also talk about synthesizing the 1918 Spanish flu. Here's a review that might help with that topic.
• Thomas Wall 21:57, 21 February 2013 (EST): I always thought the coolest and somewhat plausible project for an animal was the Tasmanian Tiger (Thylacine), A museum was attempting to do it in Australia. That project was closed down but many more seemed to have sprung up. A recent paper about such things from a UT proff http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0002240
Caveats and Limitations
• Kevin Baldridge 17:10, 18 February 2013 (EST):On the topic of general to specific evolution, is there any consideration for hypermodified amino acids? Maybe the ancient proteins had post-translational modifications in the proteins that adjusted the specificity, but we don't know about it from the genetic sequence for the protein.
• Aurko Dasgupta 23:16, 23 February 2013 (EST): I have yet to find any specifics on how post-translational modification could affect final protein specificity. That said, I think it's an extremely pertinent point, and will keep looking for something that answers this.
• Siddharth Das 19:24, 19 February 2012(EST): With even the most powerful statistics tools and rigorous mathematics, how useful is this technology in terms of evolutionary studies, since it seems the genes themselves are inaccurate representations (varying mutation rate for the past million year, etc)? Furthermore, is it safe to assume that the genetic code was as conserved as it is today? There are exceptions even now; for example, in the fungus Candida, CUG codes for serine instead of leucine. On a different note, the wiki is very well organized.
• Jeffrey E. Barrick 21:28, 24 February 2013 (EST): There's a nice study where they show that the methods used to reconstruct the ancestral sequences (whether parsimony or maximum likelihood) tend to overestimate the thermal stability of the reconstructed proteins because they favor "consensus" proteins. Link
• Alvaro E. Rodriguez M. 21:39, 21 February 2013 (EST): One thing that seems to be missing in most topics currently is technical limitation to approaches like this. i.e. Do you need 2 sequences of a gene/protein or 10 to make it work.
Content and Formatting
• Gabriel Wu 17:06, 18 February 2013 (EST): Can you include a little more detail in your figures? At least give some detail about the methods used to determine time and explain the acronyms (e.g. PNCA, GNCA, etc).
• Alvaro E. Rodriguez M. 21:36, 21 February 2013 (EST): Adding to Gabriel's comment I already did some editing, but it would be better if you either define the acronyms and then use them or keep using the corresponding terminology.
• Gabriel Wu 16:59, 18 February 2013 (EST): Remove the cost and methods of gene synthesis (or just reference the dna assembly section we've already gone over). Expand the codon optimization section (unless this fits better in somewhere else).
• Jeffrey E. Barrick 17:16, 18 February 2013 (EST):Add a picture of the fluorescent proteins (take one from Matz Lab website?
• Alvaro E. Rodriguez M. 21:36, 21 February 2013 (EST):Expanding on this, a visual on how the process is carried (a pipeline image) would be more useful for the reader.
• Jeffrey E. Barrick 18:37, 20 February 2013 (EST):Please add the titles of papers in the bibliography on your own as is done in other topics. You might also send feedback to OWW to tell them the bibio extension is broken to see if someday they will fix it.
Reconstruction of co-evolution
• Gabriel Wu 17:20, 18 February 2013 (EST): From Andre: Discussion of Red Queen hypothesis and how ancestral gene reconstruction can show us evolution of interactions between host and virus proteins.
• Jeffrey E. Barrick 18:30, 20 February 2013 (EST):If it's specific proteins I'd put it in this topic. If it's whole viruses, I'd kick to it a whole-organism topic.
iGEM team connection
• Neil R Gottel 16:31, 21 February 2013 (EST):This seems to be a tricky topic to find a link to iGEM, since I'm not finding any teams who have incorporated this into their projects. Therefore, we should do it. It also has an obvious tie-in to BEACON (evolution of proteins!), so that makes it doubly worth pursuing. Since these analyses tend to give higher-temp versions of proteins, we could potentially use this as a starting point for making heat-stable versions of various biotech/industrial enzymes, then tweaking/evolving to increase efficiency. The actual goal is to make some sort of new protocol that others can follow, and therefore cite when we publish in Nature. Or Science, I'm not picky.
• Thomas Wall 23:02, 21 February 201 3 (EST): I think this might be a good idea, generate some buzz
• Aurko Dasgupta 23:02, 23 February 2013 (EST): This is definitely something I'd support. The only thing is that we don't seem to have anyone with any background in sequence recontruction type stuff do we? If some grad student on campus works with this kind of thing, we should try to see if they'd be interested in advising anything done on a project involving the development of a functional reconstructed protein.
• Jeffrey E. Barrick 20:59, 24 February 2013 (EST):What would be an interesting protein family tree to reconstruct? I feel like one would want to explore more than thermostability (esp. since there are some caveats to the reconstruction procedure where it may be making more consensus proteins than ancestral proteins).
• Benjamin Gilman 16:51, 21 February 2013 (EST): Is "RetroBioBricks" too hard to say?
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‘ [60] rudeness, the roysterers who swarmed there, besides the damning oaths they belched out against each other, looked very sourly upon us, as if they grudged us the horses which we rode and the clothes we wore.’ They had proceeded but a little distance, when they were overtaken by some half dozen drunken rough-riding cavaliers, of the Wildrake stamp, in full pursuit after the beautiful Quakeress. One of them impudently attempted to pull her upon his horse before him, but was held at bay by Ellwood, who seems, on this occasion, to have relied somewhat upon his ‘stick,’ in defending his fair charge. Calling up Gulielma's servant, he bade him ride on one side of his mistress, while he guarded her on the other. ‘But he,’ says Ellwood, ‘not thinking it perhaps decent to ride so near his mistress, left room enough for another to ride between.’ In dashed the drunken retainer, and Gulielma was once more in peril. It was clearly no time for exhortations and expostulations, ‘so,’ says Ellwood, ‘I chopped in upon him, by a nimble turn, and kept him at bay. I told him I had hitherto spared him, but wished him not to provoke me further. This I spoke in such a tone as bespoke an high resentment of the abuse put upon us, and withal pressed him so hard with my horse that I suffered him not to come up again to Guli.’ By this time, it became evident to the companions of the ruffianly assailant that the young Quaker was in earnest, and they hastened to interfere. ‘For they,’ says Ellwood, ‘seeing the contest rise so high, and probably fearing it would rise higher, not knowing where it might stop, came ’
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25
A cube is a solid figure contained by six equal squares.
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Mike Tyson’s Punch-Out, The Book
An unofficial 240-page encyclopedia.
Fall Preview 2012
Our take on the best in art, movies, books, and music for the last Fall before that apocalypse thing.
Been Down So Long It Looks Like Up To Me
Justin Blyth has a new zine out through Arkitip
Skulls
Illustrations by István Orosz for the book Ship of Fools.
via, laughing squid
“Bookstores for Gazers”
NY’s art book stores get some due shine.
Morning Dose of the Heart of Coppola
“A mix of Orson Welles’ reading of Heart of Darkness, Apocalypse Now and the documentary Hearts of Darkness: A Filmmaker’s Apocalypse.”
Lunchtime Laughter
PERIODS. EAST OF EDEN feat. Penn Badgley
Gary Carter, Inventor of the F-Bomb
Editors for Merriam-Webster traced the word back to a 1988 Newsday article in which the Mets legendary catcher talked about trying to clean-up his language. The word is one of the new additions to the 2012 dictionary along with sexting, game changer, energy drink, gastropub, and 20 others.
via, AP
Todd James – Yield To Temptation Limited Edition
While the regular edition of this great little book won’t out until the Fall, there are 35 customized copies with a drawing by Todd James that you can buy now. And you should take that opportunity, because drawings by Todd James are not that easily obtained on the regular.
Available through PictureBox
Colorful Resistance
Erik Parker has a new monograph out through Skira Rizzoli that takes an image heavy look through his art career thus far. I wasn’t familiar with his earlier work, which is really rad.
Available here
Images Courtesy Erik Parker, from Erik Parker: Colorful Resistance, Skira Rizzoli, 2012
Os Gêmeos
Here is the cover of the upcoming book about the legendary Brazilian twins which will accompany their first solo exhibition at Boston’s ICA this August.
HuffPo has a nice write-up and preview of the show
You Chose Wrong
A tumblr dedicated to bad decisions in Choose Your Own Adventure-type books.
via, boingboing
WYWS Week: ESPO
We close out WYWS week with the first interview ever published in the magazine in 1997, featuring an all-time favorite, ESPO. If you haven’t bought the book yet, do it. There’s almost 500 pages of entertainment in it.
Interview by Roger Gastman
Is Elvis still alive today?
Elvis is dead. But the cool thing about Elvis is I think he is reincarnated into people. I think his soul is still around, and I think he is just taking people over, moving from host form to host form. That’s why all the Elvis appearances. He could take over your body and you could turn into Elvis. You might look the same when you look in the mirror, but when you’re walking down the street people see you as the King.
[Read more]
WYWS Week: CHRI$ NIERATKO WANTS TO KNOW IF GLENN DANZIG IS A GODDAMN SON-OF-A-BITCH!
Over the next week we’ll be pulling some interesting pages from The Worst of While You Were Sleeping in an effort to encourage you to go out and buy the book.
I’ve never been much of a fan of Glenn Danzig, the solo artist. Actually, that song “Mother” he did a few years ago was as painful to me as getting a vasectomy by a doctor with a fork and a spoon without any anesthesia. But Glenn Danzig, lead singer for Samhaim or the Misfits, that’s a whole ‘nother story. Back then, his hair was shorter, he was more pissed off and his songs made me want to fight. When I found out I was interviewing Mr. Danzig, I was hoping that I would get to interview the other guy, the ex-lead singer of the Misfits. But that’s impossible, ‘cause he’s dead. Instead, I got the “Mother” guy.
Are you originally from Lodi, NJ?
Yeah, I grew up in Lodi. I also grew up in Revere Beach in Boston and in Manhattan.
What is it they got out there?
Sluts.
[Read more]
Openings & Parties: Vice’s Dos & Don’ts
Last night at Powerhouse Arena, Vice threw a party to launch its second iteration of DOs & DON’Ts alongside hosts Cat Marnell, Fat Jew, and Jonathan Toubin. Christos Katsiaouni came through with the visual evidence.
WYWS Week: Ron Jeremy
Over the next week we’ll be pulling some interesting pages from The Worst of While You Were Sleeping in an effort to encourage you to go out and buy the book.
Interview by Roger Rock, Fat Rich, and Richard Colman
Do you think the Incredible Hulk has a bigger penis than you?
No, because usually guys that are into bodybuilding have very small penises. That’s why they go into bodybuilding in the first place.
From one stud to another, do you ever cry yourself to sleep at night?
Why? There are times that I might get sad because there is nothing romantic in my life. That’s one thing you miss being in porno. It’s hard to have a relationship because no one will take you seriously. It’s like Sam Kinison used to say, “Well honey, I really enjoyed having breakfast with you but now I gotta go fuck somebody. I’ll see you Thursday.” How many girlfriends want to hear that? It gets lonely once in a while.
[Read more]
WYWS Week: Kevin Smith Puts Slim-Fast To The Test
Over the next week we’ll be pulling some interesting pages from The Worst of While You Were Sleeping in an effort to encourage you to go out and buy the book.
This accompanied a revealing interview with the film director in WYWS #14
WYWS Week: Pat The Party Jerk
Over the next week we’ll be pulling some interesting pages from The Worst of While You Were Sleeping in an effort to encourage you to go out and buy the book.
PAT THE PARTY JERK: WALK OF SHAME
By Trevor Michaels
I heard this story the other day at a concert, and I feel that all of you would benefit from its message. And besides, it’s about a great subject: college women. In college, two things are clear. College guys are horny assholes just looking for tail, and college women are horny bitches just looking for dick. Anyway, the story goes like this.
A freshman girl sits in her room one night soon after arriving on campus. A frat guy knocks on her door. “Hey babe. Do you, uh, maybe want to go to a party with me?” At this crucial moment, the chick thinks, “I am at college. My parents aren’t here to tell me what to do. I am free and independent now. I am going to the party.”
[Read more]
WYWS Week: Americans I Most Admire
Over the next week we’ll be pulling some interesting pages from The Worst of While You Were Sleeping in an effort to encourage you to go out and buy the book.
Editor’s note:
I had been fascinated by serial killers for years, and on a trip to Milwaukee, WI, I visited the bars where Jeffrey Dahmer used to pick up his victims. One day, Ben and I were sitting around talking about awesome ways to kill people and awesome ways to dispose of their bodies, and we decided we should write about serial killers in this magazine I had somehow just started. We could write whatever we wanted; no one was going to edit us.
I can’t remember if it was supposed to be a reoccurring column or not, but it turned out to be WYWS’ longest running column, and we ended up covering more than just Americans. There are a lot of admirable European sickos out there too.
- Roger Gastman
AMERICANS I MOST ADMIRE: ED GEIN
By Ben Shupe
Picture this: La Crosse, Wisconsin, 1906. A little boy named Edward Gein was born. Heard of him? Well, let me tell you a little story about Ed.
Gein’s father was a raging alcoholic for most of the day. On the rare occasion that he was sober, he held jobs as a local tanner and a carpenter. He also found time to keep up the family farm. While Ed’s father was outside playing with the livestock, Gein’s uptight, overbearing, religious zealot of a mom was inside on her knees praying to God for the slow and painful death of her husband. She didn’t like men. Hell, she hated women just as much. Ed and his brother Henry were taught that women were nothing more than schemers and whores who would separate the family. They were also taught that premarital sex was a sin, and so was marriage. In fact, the two brothers were forbidden to marry. No problem though, Ed had masturbation! Shit, when Ed’s mom finally died in 1945—not long after his father and brother’s deaths—the coroner found cum stains all over his mothers face!
[Read more]
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Place:Central Islip, Suffolk, New York, United States
Watchers
NameCentral Islip
TypeCensus-designated place
Coordinates40.784°N 73.199°W
Located inSuffolk, New York, United States
source: Getty Thesaurus of Geographic Names
source: Family History Library Catalog
the text in this section is copied from an article in Wikipedia
Central Islip is a hamlet and census-designated place (CDP) within the town of Islip in Suffolk County, New York, United States. The population was 34,450 at the 2010 census.
Research Tips
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Australian Bureau of Statistics
Celebrating the International Year of Statistics 2013
ABS Home > Statistics > By Release Date
Statistics by Release Date
April, 1957
17/04/1957 Interim Retail Price Index, 1957 (cat no. 6401.0)
© Commonwealth of Australia 2013
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Tell me more ×
Answers OnStartups is a question and answer site for entrepreneurs looking to start or run a new business. It's 100% free, no registration required.
I recently (read: last week) started the following business:
[Business Name] is a small business IT consulting firm. We specialize in the use of Open Source Software by small businesses. Open Source Software allows a small business to lower their IT costs, while keeping their productivity the same. In fact, in some cases the change to Open Source Software can actually increase a businesses productivity.
You may be asking "What is Open Source Software though?". This is what The Open Source Initiative (OSI) has to say on the subject:
"Open source is a development method for software that harnesses the power of distributed peer review and transparency of process. The promise of open source is better quality, higher reliability, more flexibility, lower cost, and an end to predatory vendor lock-in."
~ The Open Source Initiative (OSI)
As you may have noticed, this is a local, niche business... At the moment, no one in Austin, TX (our location) knows this business exists.
How do I change that on a shoe-string budget? I already tried posting to Craigslist, but the post was buried in less than an hour, and I didn't get any calls/emails.
share|improve this question
3 Answers
up vote 5 down vote accepted
Not to be sarcastic, but what else have you tried? You really need to initiate personal contacts (and a lot of them) in order to market a business like this locally and you need to market the "crap" out of your business and your value proposition. Figure out exactly how you help your clients, what makes you the best choice, and then really push it.
Chamber of Commerce networking, Rotary, and business lead sharing networks like Le Tip and BNI are all appropriate to drum up local small business clientele.
Plus, I searched for your business with your apparent tagline "We specialize in the use of Open Source Software by small businesses" in quotes and I found absolutely nothing. Do you have a website with your mission stated clearly on the home page?
For local marketing of a B2B type business catering to general business I would favor the meatspace marketing tactics of talking to lots and lots of locals, over putting a lot of time and effort into internet marketing. Every geek thinks they're going to win business by hiding behind a screen and enhancing their SEO. This works for long tail stuff that can be sold remotely and for products, but not for personally delivered services.
To go after the small business market you probably need to market very aggressively, with standard package pricing that makes your services look like a sort of product. IE: so much per business and per employee to put the company on FOSS office tools, train the workers, integrate everything, and migrate data over from whatever the place is using now.
Just some ideas.
share|improve this answer
There is potentially a very long list which summerises as "find prospects who are local and put your pitch to them".
Working out who is going to be interested ... thats a big one and another set of questions from you, the rest assumes you know who your target market actually is ...
Some ways to go about finding local prospects:
If you are on LinkedIn their advanced search allows you to specify "within 50 miles(80 kms) of me". You can put in job titles and some other "qualifiers". This will give you a starting hit list, which you can use to start with. If you save the search LinkedIn will email you any new people within your 3 degrees of seperation. For each real candidate create a semi-personalised spam (i mean message) and either email (cheap) or mail (generally more effective). Part of the message is that you will give them a followup call next week ... you ring, you ask for the person by name, you get a chance to book a meeting. (Don't try to sell over the phone it won't work, your local, be local).
Networking
• Have a look on MeetUp.com for networking events in your city, turn up, talk to people, hand out lots of business cards. If there isn't one try starting one.
• Ask on Twitter for events and meetings in the local area, they will start to show up for you.
• Local councils often have events that you can attend, as do universities.
• There area a lot of startup events springing up everywhere, these usually cost the non-presenter but they are a good source of leads.
Tell all of your friends (but not constantly), a bit about what you do but more who you are looking for ... it might trigger something for someone.
share|improve this answer
You are a consulting firm? Why are you scared of meeting people in person? You should meet your potential customers in person. Goto their office and pitch what you have to offer.
Do some research, study case studies how other companies profited from switching to open source. Collect some data, present it in a visual way.
THIS IS A LOCAL BUSINESS, YOU SHOULD START LOCALLY! You don't even need to have a website like a user said when you get your first couple of clients. Make your website when you have something to display..
If you decide to make a website even though you have no clients, put the presentation and the data I was talking about on the website.
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Tell me more ×
Answers OnStartups is a question and answer site for entrepreneurs looking to start or run a new business. It's 100% free, no registration required.
I have developed a local upcoming events web page, that aims to deliver the events in a different way than other similar services.
The event organisers will list their events but at a cost.
My question is, what is the best strategy to follow so that it will bring revenue? I have some connections with people who work in the night so I can easily find some events to start with.
How should I do this?
• Should I charge all the customers from the beginning?
• Should I give them all a month free?
share|improve this question
6 Answers
up vote 2 down vote accepted
+25
Follow the money, the organizers and Promoters have the smallest pockets in the bunch. There are venue owners, local business, ticket services, credit card services, hotel and restraunts that have way deeper pocket.
So from your site you have information on attendance, list of venues, inbound traffic and keyword insights. Those sound like some great things to have a segmented target market group.
You can utilize localized group buying feed. Use you can partner deals with hotels and give the organizers a new way to make money. Have the organizers work for you. Set up an api for ticket seller to integrate so it is easier for the promoter and get a cut of the sales.
Congrats
share|improve this answer
I believe you should go for a premium member models, You should have a set of events( 1-2 ) posting to be free, this will help you increase your popularity.
share|improve this answer
You are going to get issues with adoption as soon as you tell the organizers they have to pay to be listed on your site. Why not make it free for anyone to post events on your site. To make money, charge organizers who are looking to have maximum exposure on your site i.e. listing stays at the top.
share|improve this answer
So I guess from your writings, that your target customer base are event managers. I also would say that it depends on the size of the event, if the managers have money or not. Therefore I would say that your primary business is here to make the "marketing" for events. So a Premium Model would not be the best model for that. Charge the managers at a hourly rate and create personal contacts with them.
Another solution would be that when you know that there will be an event in a certain area. Try to make some deals with locals and offer it as a service to event managers. So you get payed by both;)
share|improve this answer
Bill by sales volume or exposure volume.
This way:
1. Your business model is in alignment with your customers needs (More action for them = more revenue for you, a virtuous cycle)
2. Prices scale with your clients (I'm guessing you have minimal marginal cost per post, but to them the value increases with more expensive events)
If you handle the ticket sales take a percent. If you give promotion charge per action (CPC to external site? Cost per views? Targeted by audience type if you have that kind of data ala facebook ads?).
share|improve this answer
I think you have a range of potential customers, each with a range of needs.
• consumer. Person turning up to find something to do. They want a lot of choice and they want to express what they want easily. They may want to be kept up to date. They don't want to spend long and they have a range of options in your competitors.
• events organizers. These are people looking for work from your client base. You can make a margin or be their sales pipeline.
• small event. Little to no budget, they need people to know about them. There are a huge range of sites they ould use. They may need help writing engaging descriptions which if you had an ideal link you could charge for and it is included in their price. Really these guys provide you the volume the consumers are looking for.
• midsized events. They can pay something for priority of you can prove a return. They may have tickets to sell, they may have marketing materials to create, they may have staffing requirements, they may have planning needs. Presenting packages that help them get it done is a true value add if all your doing is introducing a local business to help them out.
• meetup style events. These are probably just listings and attendance items. Meetup has this covered. It there may be some value add or method of increasing your volume of offerings.
• large gigs. Have money, have a lot of options of how to spend their money, so why your site? Pretty much the other value added items listed here in "solve it for me packages"
• corporate driven events. These guys can pay, they have tickets to be sold, they have a lot of stuff to take care of in setting up the event. Your value add to them is a range of "we will take care of you" packages and you put them in contact with events organizers and planners.
• advertisers. Last but not least, people trying to get in front of any of the above subsets of people. Being able to say I'm targeting large corporates running events is worth a lot to the right advertiser
Work through the senarios and pick the useful subset that gets you volume of customers and volume of the right events, then look at how monitoring that subset first.
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Creative Commons » online course http://creativecommons.org Share, reuse, and remix — legally. Fri, 17 May 2013 00:22:14 +0000 en-US hourly 1 http://wordpress.org/?v=3.5.1 Help Build the School of Open in German http://creativecommons.org/weblog/entry/37471 http://creativecommons.org/weblog/entry/37471#comments Wed, 20 Mar 2013 18:42:35 +0000 John Weitzmann http://creativecommons.org/?p=37471 On the first weekend of March, Wikimedia Germany and CC Germany hosted a workshop around the School of Open’s official launch. Attending were professionals and enthusiasts from various fields, some lawyers but mostly teachers and education managers as well as activists of the Open Knowledge Foundation and the Internet & Society Co:llaboratory in Berlin.
School Of Open Workshop WMDE / Elly Köpf / CC BY-SA
After a quick introduction, we checked out the existing School of Open course program and all features of the P2PU user interface. The mission then was to get a first set of courses in German off the ground by either translating existing courses and/or developing new ones — and that’s what we did:
Work on three courses began, partly translating the content, partly enhancing it. One course was envisioned from scratch, aiming at giving educators an idea of how OER work, why they matter and how. Here are the courses that are in development:
• Bilder auf Wikimedia Commons hochladen – In diesem Kurs kannst du lernen, wie einfach es ist, Inhalte auf Wikimedia Commons hochzuladen und damit die große Datenbank freier Bilder weiter zu ergänzen.
English translation: Upload images to Wikimedia Commons – In this course you will learn how easy it is to upload content on Wikimedia Commons, and thus complement the large database of free images.
• Wie erstelle ich einen Kurs auf P2PU?- Du möchtest einen Kurs anlegen und mit anderen dein Wissen teilen? Hier findest du in wenigen Schritten eine Anleitung.
English translation: How to create a course on P2PU – You want to create a course and share your knowledge? Here you can find a tutorial in a few steps.
• Freie Lernmaterialien in der Schule – OER für Lehrkräfte – Mit diesem Kurs lernen Sie die Bedeutung von Open Educational Resources, kurz OER, den freien Lehr- und Lernmaterialien, kennen.
English translation: Free learning materials in schools – OER for teachers – This course will teach you the importance of open educational resources (OER) and the freedom of teaching and learning materials.
At the end of the day, a start had been made and the participants collected a lot of ideas about how to improve and develop the School of Open program. A network began to emerge of interested experts and enthusiasts, many of whom will join the School of Open discussion list (Google Group) in order to get involved.
If you would like to help us develop the courses above, or create new ones in German, please email legal@creativecommons.de or join the School of Open discussion list and introduce yourself and your interest!
For the German summary of the event, see the Wikimedia Germany blog.
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Debrief: Sprinting to Build an Open Science Course http://creativecommons.org/weblog/entry/37060 http://creativecommons.org/weblog/entry/37060#comments Wed, 27 Feb 2013 20:57:08 +0000 Billy Meinke https://creativecommons.org/?p=37060
Photo by
Billy Meinke / CC-BY
Celebrating Open Data
Open Data Day 2013 can be described as a success. Why? Because hundreds of people participated in more than 100 events distributed across six continents all over the world, celebrating open data and all that we can do with it. Here at CC, we planned and executed a community-supported event to build open learning resources around the topic of Open Science, done in a hackathon-style sprint event that gathered people with diverse backgrounds and experience levels. An undergraduate student and a post-doc researcher, both from Stanford. An instructional designer from Los Angeles and an associate professor from Auburn University, plus a handful more of very talented people. Oh, and a mother and high school-aged daughter duo that simply wanted to see what “open” is about. We all connected to help build an open course to teach others about Open Science. Here’s how we did it.
Open Content for Learning
It’s worth mentioning that the course materials that were produced during the sprint will be openly licensed CC BY and shared so that their benefit to Open Education and Open Science are not restricted by legal boundaries. The material is being curated and will undergo a review process over the next couple weeks before being ported to the School of Open, a collaborative project by Creative Commons, P2PU, and a strong volunteer community of “open” experts and organizations. Though fitting the content to P2PU’s online course platform was in the back of our minds, time and consideration were largely placed on identifying important ideas that explain what Open Access, Open Research, and Open Data mean for Open Science, and how we can engage more “young scientists” (this is an ever-broadening term) in the ways of Open.
Photo by
Billy Meinke / CC-BY
The Net Works Effect*
Adding a layer on top of open content itself, which is elastic in nature, our approach to this hackathon-style event focused on being very lean, the type of event that can be run by anyone, anywhere, and requiring very few resources. We created a Google Drive folder and a set of publicly-editable documents to collect openly-licensed resources, map out a tentative module/lesson plan, coordinate communications between participants, and generally provide a single place to collaborate on Open Science learning materials. Connecting with other event organizers at the OKFN and PLOS, mailing lists, Twitter hashtags, and other forms of communication were established so that there was a support network for those who were organizing events and those who were interested in participating in Open Data Day events on some level. David Eaves, Rufus Pollock, Ross Mounce, and many others were loud and clear on the Open Data Day mailing list, making sure news about each event was passed around.
Before the event, a registration page was created for the course sprint. We offered a handful of in-person tickets for folks to come down to our office in Mountain View, as well as a number of remote participant tickets for those who were in different geographical locations. Google Hangout “rooms” were set up on laptop computers placed in physical conference rooms at the CC HQ, allowing remote participants to work in real-time with persons on the ground. To see a more detailed description of the day’s event, see the schedule document here.
Deliverables
So what did we make? The sprinters involved in the project collected and organized resources that explain common aspects of Open Science. The main sections (access, methods, data) were helpful in searching for content, but there was a great deal of overlap between sections, which highlighted the relationhips between them. Beyond the collection of resources, sets of tasks were built that are meant to guide learners out beyond the course and into the communities of Open Science, interacting with the ideas, technical systems, and people who are opening up science. The Introduction to Open Science course on P2PU is still in a lightly-framed state, but the plan is to include the course in the launch of the School of Open during Open Education Week, March 11-15. If you’re interested in helping make this transition or to help build or review other courses that we call “open,” come introduce yourself in the School of Open Google Group. Or check out what else is happening on P2PU.
Beyond the course itself, we’re going to take a look at the sprint process we used, and work out some of the kinks. This rapid open-content creation technique is manageable, low-cost, and builds the Commons. There’s enough openly-licensed content existing on the web to produce a range of learning experiences, so now it seems that it’s a matter of developing open technology tools to the point where we can build education on the web together, easily. For more information about this and other Open Education projects being worked on by Creative Commons, see this page.
We Got Together for Open
Thanks to those who were able to participate in the Open Science course, as well as those who contributed the planning documents leading up to the event. We’ve done well.
Related Posts
PLOS Sci-Ed Blog, Guest Post: Open Data Day, Course Sprints, and Hackathons!
David Eaves’ Blog, International #OpenDataDay: Now at 90 Cities (and… the White House)
Debbie Morrison’s Blog, A Course Design ‘Sprint’: My Experience in an Education Hackathon
Also: The Flickr album from the event can be found here.
*This phrase coined by P. Kishor here, describing the interconnectedness of Open Data Day events.
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Learn how to get your Creative Commons project funded http://creativecommons.org/weblog/entry/26669 http://creativecommons.org/weblog/entry/26669#comments Tue, 01 Mar 2011 19:22:39 +0000 Jane Park http://creativecommons.org/?p=26669
by R.B. Boyer / CC BY-SA
If you are serious about a Creative Commons project idea, you may be interested in the free, online course, “Getting your CC project funded,” set to run in April. The course consists of a series of workshops and seminars that will take you through the steps from an initial idea to having a finished project proposal for submission, including assistance in identifying and finding funding bodies and collaborations relevant for your project. You provide the idea; the course provides the guidance to turn it into a proposal that can’t be refused.
The course will be run by Jonas Öberg from the Nordic CC network, a lecturer at the University of Gothenburg in Sweden with extensive grant writing and reviewing experience with the European Commission and several Nordic cultural foundations. “Getting your CC project funded” will run on the Peer 2 Peer University (P2PU) in April, and we especially invite CC Affiliates and friends to participate!
As with all P2PU courses, the course is free to take. Though only 15 active participants will be accepted into the course, the entire course, material, and other information, including the proposals which you write in the course, will be open for anyone to follow on the P2PU platform under the CC BY-SA license.
You can read more at http://p2pu.org/general/getting-your-cc-project-funded. You may start brainstorming at anytime, but official sign-up opens March 31.
If you already have experience writing and reviewing funding proposals… you may be interested in joining the team of expert external reviewers. More info on the current team is available on our wiki. If interested, please contact Jonas directly or janepark [at] creativecommons [dot] org. Though the course itself will be run in English, project proposals may be written and reviewed in English, Spanish, Swedish, Danish, Norwegian, Russian and Bulgarian. More languages may be added depending on the final team of reviewers.
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P2PU launches 3rd round of courses, with “Copyright for Educators” http://creativecommons.org/weblog/entry/23186 http://creativecommons.org/weblog/entry/23186#comments Thu, 26 Aug 2010 20:07:01 +0000 Jane Park http://creativecommons.org/?p=23186 The Peer 2 Peer University, more commonly known now as P2PU by a growing community of self-learners, educators, journalists, and web developers, launches its third round of courses today, opening sign-ups for “courses dealing in subject areas ranging from Collaborative Lesson Planning to Manifestations of Human Trafficking.”
P2PU is simultaneously launching its School of Webcraft, which is a collaboration with the Mozilla Foundation and “is a powerful new way to learn open, standards based web development in a collaborative environment. School of Webcraft courses include Beginning Python Webservices and HTML5.”
In addition, Creative Commons Counsel Lila Bailey is co-facilitating the Copyright for Educators course this round, which will focus on United States law. The course is “for educators who want to learn about copyright, open content material and licensing” and “is taught around practical case studies faced by teachers when using copyright material in their day to day teaching and educational instruction.” For more information, see the course page.
Sign-ups for all other courses are available at http://p2pu.org/course/list. The deadline to sign up is September 8, and courses will run until October 27th. All courses are free to take and openly licensed under CC BY-SA. For more information, see the full announcement, but stay tuned for more courses!
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Joi Ito to run Digital Journalism course on P2PU http://creativecommons.org/weblog/entry/22129 http://creativecommons.org/weblog/entry/22129#comments Fri, 28 May 2010 14:21:29 +0000 Jane Park http://creativecommons.org/?p=22129 Joi Ito is teaching his Digital Journalism course again at Keio University this summer, but this time with a twist. In addition to the traditional semester, where Joi will be teaching within the university, the course will also have an open and online component where anyone may apply to join via the Peer 2 Peer University (P2PU). Digital Journalism 2010 will run for seven weeks with seven physical meetings which will be webcast and allow for online participation. Additionally, asynchronous communications will continue between classes on mailing lists, the class blog, wiki, and the P2PU platform.
Digital Journalism 2010 is “an introduction to online journalism, citizen media and the use of social networks for journalism and collective action. Participants will work on self defined projects either as individuals or in groups using any combination of media types including video, photographs, illustrations and text as well as online tools such as blogs, wikis, Twitter, Facebook, Flickr and any other reasonable tool the participant or team would like to use.” In addition to learning about how the journalism landscape is rapidly changing, participants will learn to research and create news online by publishing stories of their own in teams. These stories will be presented to the class (and the world).
The course is a work in progress, so the community can contribute by suggesting readings, activities, and more. P2PU is looking for course organizers to facilitate the P2PU end of things. If interested, please contact thepeople [at] p2pu.org. To participate in the course remotely via P2PU, you can sign up to apply at www.p2pu.org/journalism. Sign-up is open now and the course will begin on Friday, 4 June.
Joi teaches Digital Journalism annually as part of the Keio Graduate School of Media Design. He has contributed pieces to the New York Times, the Asian Wall Street Journal, and Wired. He is also a prolific photographer and if you didn’t already know, the CEO of Creative Commons.
The Peer 2 Peer University is “a grassroots education project that organizes learning outside of institutional walls.” In addition to leveraging existing OER, P2PU licenses all of its own courses under CC BY-SA. For more on why P2PU chose this license, visit http://p2pu.org/license.
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WikiPremed makes money by giving away MCAT course http://creativecommons.org/weblog/entry/21440 http://creativecommons.org/weblog/entry/21440#comments Tue, 30 Mar 2010 20:44:21 +0000 Jane Park http://creativecommons.org/?p=21440 Artists have been using Creative Commons licenses in interesting ways for a while, whether it’s to encourage interesting adaptations of their work or to help boost album sales. But it’s not only the visual artists and musicians diversifying the use of CC licenses—open education initiatives like Flat World Knowledge are experimenting with innovative business models by giving away digital content while charging for services added around it. WikiPremed is another one.
WikiPremed is the result of fifteen years of hard work, founded by John Wetzel, a graduate of Stanford University who has been helping “premedical students prepare for the MCAT in small group teaching through over fifty course cycles.” The site is comprehensive in scope, basically a course “in the undergraduate level general sciences,” consisting of textbooks, flash cards, test questions, images, and more that a premed student would need to prepare for the MCAT. All materials are available for free under Creative Commons Attribution ShareAlike, which means you can translate, improve, and republish it as long as you share alike.
What’s more interesting is that the site is sustaining itself by giving away digital content for free and charging for print materials, such as its Physics flashcards and print versions of its books. There is also an ask for a one-time $25 donation that then gives students an Organic Mechanisms Pocketbook and Advanced Physiology Crosssword Puzzle Book in return as a thank you. From Glyn Moody’s short interview of John Wetzel (which got picked up by techdirt),
“Students need printed study materials, and they get sick of the computer, so I definitely think there is room for creative commons educational content supported by print publications. I think there is an ethic to not holding content hostage to purchases, but I think there are commercial advantages to the open model as well. I don’t doubt that the average customer at WikiPremed has 1000 page views before purchasing anything.
I am sure that if there were registration walls and missing chapters I would have fewer customers.
I’m not getting rich or anything, at this point, but it is working.”
If you’re interested, you can help contribute to the WikiPremed case study.
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Mozilla and CC to teach online seminar on open education http://creativecommons.org/weblog/entry/13419 http://creativecommons.org/weblog/entry/13419#comments Tue, 17 Mar 2009 20:24:08 +0000 Jane Park http://creativecommons.org/?p=13419 ccLearn is collaborating with the Peer 2 Peer University and Mozilla to teach practical open education skills to educators and anyone else who is interested. From the announcement on the course wiki:
“This six week course is targeted at educators who will gain basic skills in open licensing, open technology, and open pedagogy; work on prototypes of innovative open education projects; and get input from some of the world leading innovators along the way.
The course will kick-off with a web-seminar on Thursday 2 April 2009 and run for 6 weeks.
Weekly web seminars introduce new topics ranging from content licensing to the latest open technologies and peer assessment practices. Participants will share project ideas with a community of peers, work on individual projects, and get feedback from experienced mentors. We will also take a close look at some of the most innovative examples of open education projects, and speak to the people who designed them, including:
• The Open Source Software courses at Seneca College;
• David Wiley’s Introduction to Open Education;
• The open blog infrastructure at Mary Washington University; etc.
• The course is targeted at educators who want to help shape the open education future. Participants should have some knowledge of web technologies, or open content licensing, or open pedagogy (or all three), but don’t need to be experts.
Interested in participating? Head over to the course wiki, and submit your project idea!
Course outline: https://wiki.mozilla.org/Education/EduCourse
Sign-up page: https://wiki.mozilla.org/Education/EduCourse/SignUp
For questions about the course or the sign-up process, contact:
Philipp Schmidt
Peer 2 Peer University
philipp AT peer2peeruniversity.org”
Spaces will fill up fast, but that doesn’t prevent non-registered learners from having open and complete access to the course as it plays out. And since all Mozilla Education materials are available for reuse, redistribution, and remixing under CC BY, nothing stops users from creating a mirror wiki and developing their own projects!
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From DDO wiki
Jump to: navigation, search
Wraps of Endless Light (Level 20)
Proficiency Class Simple Weapon Proficiency
Damage and Type Handwrap
Critical threat range Handwrap
Weapon Type Handwrap / Bludgeoning weapons
Minimum Level 20
Required Trait None
Use Magical Device DC No UMD needed
Handedness Simple
Damage Mod STR
Attack Mod STR
Binding Bound to Character on AcquireBound to Character on Acquire: This item is Bound to Character on Acquire
Durability 150
Made from ClothCloth: Cloth is a very common material used in handwraps, robes, outfits. and other accessories.
Hardness 14
Base Value 00080030008,003pp
Weight 0.50 lbs
Location Mabar Endless Night Festival, Upgrade of Wraps of Endless Light (Level 16)
Enchantments
• +6 Enhancement Bonus+6 Enhancement Bonus: This weapon has been magically enhanced and gains a +6 enhancement bonus to attack and damage rolls.
• Greater Undead BaneGreater Undead Bane: A bane weapon excels at attacking one type or subtype of creature. Against Undead, this weapon's effective enhancement bonus is +4 better than it's normal enhancement bonus. It deals an extra 3d6 points of damage against the foe.
• BrillianceBrilliance: This weapon glows with the light of Irian. This weapon deals an extra 1d6 light damage on a successful hit. In addition critical hits deal an amount of extra light damage depending on the weapon's critical multiplier: x2 - 1d10. x3 - 2d10. x4 - 3d10.
• Light BringerLight Bringer: A weapon of light bringing is the bane of all undead. Vorpal effect: On an attack roll of 20 which is confirmed as a critical hit this weapon will destroy an undead outright. Undead with more than 1,000 current hitpoints will resist this vorpal effect until sufficiently wounded and will take 100 points of damage on a confirmed vorpal shrike instead of being destroyed.
• Radiant BlastRadiant Blast: This weapon stores the power of the plane of Irian deep within. Occasionally, this dynamic power comes to the surface, devastating enemies with a massive blast of radiant light.
Upgradeable?
Mabar Endless Night Festival to Wraps of Endless Light (Level 24)
Description These silken wraps blaze with an aura of light.
Tips
Note that despite the fact these are ML20, and have a purple (epic) icon-border, they do not have the hidden epic weapon tier effect granting 2[W] that most (including all post U14 lootgen versions) ML20+ wraps possess. The newer ML24 version does have it.
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Talk:Flameman
From eLinux.org
Revision as of 01:42, 28 February 2009 by Prpplague (Talk | contribs)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Jump to: navigation, search
Great looking pages! need a little more details about what you start with, i.e. more discussion of the original IPAQ and the board you started with for the Fontera
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Hamlin County, South DakotaEdit This Page
From FamilySearch Wiki
United States South Dakota Hamlin County
South Dakota
Online Records
Hamlin County, South Dakota genealogy and family history research page. Guide to genealogy, history, and courthouse sources including birth records, marriage records, death records, census records, wills, deeds and land records, Civil War records, family histories, cemeteries, churches, tax records, newspapers, and obituaries.
Hamlin County, South Dakota
Map
Location in the state of South Dakota
Location of South Dakota in the U.S.
Facts
Founded January 8, 1873
County Seat Hayti
Courthouse
Adopt-a-wiki page
This page adopted by:
SDGenWeb Project
who welcome you to contribute.
and its representative
Hamlin Co. SDGenWeb
Adopt a page today
Contents
County Courthouse
Hamlin County Courthouse
PO Box 237;
Hayti, SD 57241
Phone: 605.783.3201
Register of Deeds has birth, death, burial records from
1905, land records, marriage records from 1879;
Clerk Court has divorce and court records from 1885,
probate records from 1890, naturalization records from
1880, school census records from 1903 and school
records from 1890[1]
History
Parent County
1873--Hamlin County was created 8 January 1873 from Deuel and Hanson Counties.
County seat: Hayti [2]
Boundary Changes
Record Loss
Places/Localities
Populated Places
Neighboring Counties
Resources
Cemeteries
Census
1905, 1915, 1925 State Censuses --Unique information could include: Ethnic Background, Maiden Name, Church Affiliation & Military Service
1905 state census with images can be viewed free at FamilySearch Record Search
1915 state census with images can be viewed free at FamilySearch Record Search
1925 state census with images can be viewed free at FamilySearch Record Search
1935 state census with images can be viewed free at FamilySearch Record Search
1945 state census with images can be viewed free at FamilySearch Record Search
Church
Court
Land
Local Histories
Maps
Military
Newspapers
Probate
Taxation
Vital Records
Societies and Libraries
Family History Centers
Web Sites
• USGenWeb project. May have maps, name indexes, history or other information for this county. Select the state, then the county.
• Family History Library Catalog
References
1. The Handybook for Genealogists : United States of America, 10th ed., (Draper, UT: Everton Publishers, 2002)Hamlin County, South Dakota, p.625
2. The Handybook for Genealogists: United States of America,10th ed. (Draper, UT:Everton Publishers, 2002).
Need additional research help? Contact our research help specialists.
Need wiki, indexing, or website help? Contact our product teams.
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You're invited to explain your rating on the discussion page (you must be signed in).
• This page was last modified on 31 December 2012, at 20:45.
• This page has been accessed 1,472 times.
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