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Information for "Spectrum Digital"
Jump to: navigation, search
Basic information
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Page creatorTim Bird (Talk | contribs)
Date of page creation00:37, 13 December 2008
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Changes related to "Pennsylvania Court Records"
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Talk:United States DirectoriesEdit This Page
From FamilySearch Wiki
Contents
Why Use Directories?
• Learn the exact years your ancestor inhabited a place.
• Locate ancestor in a census that hasn’t been indexed (esp. state census).
• Estimate year of immigration.
• Learn occupation and employer as identifiers
• Find other family members.
Potential Content
• An alphabetical listing (arranged by name, address, and occupation).
• A street address listing (arranged by address, name, and occupation).
• Widows, working women, and adult children at home.
• Ward maps.
• Street locator, including cross streets.
• Street name changes.
• Removals (sometimes destinations!).
• Businesses (and index to advertisers).
• Churches, schools, funeral homes, cemeteries, post offices, courts, hospitals, benevolent associations, newspapers.
• Many early directories listed only businesspeople.
• Some directories list wife in parenthesis.
• Whether a woman is a widow (including name of husband).
• List of marriages and deaths of previous year.
• Death date.
Finding Directories
• Use the Place Search in the Family History Library Catalog. Search city and county.
• A related record which preceded city directories is minutes of town meetings including lists of inhabitants.
Finding aids
• City Directories of the United States. New Haven: Research Publications, Inc. 1971-
• City Directories of the United States, 1860-1901: Guide to the Microfilm Collection. Woodbridge, CT: Research Publications, 1983
• Spear, Dorothea N. Bibliography of American Directories Through 1860. Worcester, Mass.: American Antiquarian Society, 1961.
Websites
• UScityDirectories.com: Identifies directories by place and gives repository and call number (incl. FHL film numbers).
• DistantCousin.com/directories contains transcriptions of city directories from 18 states.
Search Steps
• Check the beginning of the directory for cutoff dates, geographical coverage, and the meaning of abbreviations.
• Check alphabetical listing or residents to find known ancestors.
• After finding a known ancestor’s address in the alphabetical listings, check the street address listing to find unknown ancestors at the same address.
Tips
• Directories list occupants (not necessarily owners).
• Major cities: Check town or county histories for outlying towns later absorbed by a city.
• Minorities were often listed separately.
• Others at your ancestor’s address may be boarders.
• Pay attention to occupations. They can give you an extra “handle” by which you can identify your ancestor in another record. If an alphabetical listing says your ancestor is “Asst. to John Doe,” see what John Doe does for a living.
• Streets were renumbered. If your ancestor’s address changes, see if his neighbors’ addresses change correspondingly.
• Second marriages: If a widow is listed at an address, then replaced by a man the next year at that address, check marriage records!
• Find ancestor in all available directories. This yields more name handles, more relatives at same address, and more occupations.
• For blank forms you can use to extract information from a directory, see www.tpl.toronto.on.ca/localhistory/directories4.html
What to Do Next
Directories serve as springboards to other records:
Church records
• To narrow down the church records to search for an ancestor, use directories to find addresses of churches near your ancestor’s residence.
• If you have a marriage certificate naming the minister who performed the marriage ceremony, find his listing in directories to learn the name of his church.
Land records
• Directory listings often mention whether the resident is an owner, renter, or boarder. If owner, see land records!
Works Referenced
Egan-Baker, Maryan. "U.S. Census & City Directories: The Dynamic Duo." Utah Genealogical Association Conference. Salt Lake City, Utah, 13 April. 2000.
Gormley, Myra Vanderpool, C.G. City Directories: Windows on the Past. <http://www.ancestry.com/columns/myra/Shaking_Family_Tree03-19-98.htm>. 19 March 1998 (Accessed 27 August 2002).
Hinckley, Kathleen W., C.G.R.S. Skillbuilding: Analyzing City Directories. <http://www.bcgcertification.org/skillbuilders/skbld965.html>. May 1996 (Accessed 27 August 2002).
Morgan, George. City Directories. <http://www.ancestry.com/columns/george/03-06098.htm?sourceid=00392187254525771865>. 6 March 1998 (Accessed 27 August 2002).
Primary Sources -- Directories. <http://www.tpl.toronto.on.ca/localhistory/directories1.html>. 27 January 2000 (Accessed 27 August 2002).
Remington, Gordon, F.U.G.A. "Needle in a Smokestack: Urban Research." Utah Genealogical Association Conference. Salt Lake City, Utah, 13 April, 2000.
• This page was last modified on 22 March 2011, at 22:28.
• This page has been accessed 188 times.
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Research
A comprehensive transcript index of the human genome generated using microarrays and computational approaches
Eric E Schadt1*, Stephen W Edwards1, Debraj GuhaThakurta1, Dan Holder2, Lisa Ying2, Vladimir Svetnik2, Amy Leonardson1, Kyle W Hart3, Archie Russell1, Guoya Li1, Guy Cavet1, John Castle1, Paul McDonagh4, Zhengyan Kan1, Ronghua Chen1, Andrew Kasarskis1, Mihai Margarint1, Ramon M Caceres1, Jason M Johnson1, Christopher D Armour1, Philip W Garrett-Engele1, Nicholas F Tsinoremas5 and Daniel D Shoemaker1*
Author Affiliations
1 Rosetta Inpharmatics LLC, 12040 115th Avenue NE, Kirkland, WA 98034, USA
2 Merck Research Laboratories, W42-213 Sumneytown Pike, POB 4, Westpoint, PA 19846, USA
3 Rally Scientific, 41 Fayette Street, Suite 1, Watertown, MA 02472, USA
4 Amgen Inc, 1201 Amgen Court W, Seattle, WA 98119, USA
5 The Scripps Research Institute, Jupiter, FL 33458, USA
For all author emails, please log on.
Genome Biology 2004, 5:R73 doi:10.1186/gb-2004-5-10-r73
The electronic version of this article is the complete one and can be found online at: http://genomebiology.com/2004/5/10/R73
Received:4 May 2004
Revisions received:7 July 2004
Accepted:16 August 2004
Published:23 September 2004
© 2004 Schadt 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
Computational and microarray-based experimental approaches were used to generate a comprehensive transcript index for the human genome. Oligonucleotide probes designed from approximately 50,000 known and predicted transcript sequences from the human genome were used to survey transcription from a diverse set of 60 tissues and cell lines using ink-jet microarrays. Further, expression activity over at least six conditions was more generally assessed using genomic tiling arrays consisting of probes tiled through a repeat-masked version of the genomic sequence making up chromosomes 20 and 22.
Results
The combination of microarray data with extensive genome annotations resulted in a set of 28,456 experimentally supported transcripts. This set of high-confidence transcripts represents the first experimentally driven annotation of the human genome. In addition, the results from genomic tiling suggest that a large amount of transcription exists outside of annotated regions of the genome and serves as an example of how this activity could be measured on a genome-wide scale.
Conclusions
These data represent one of the most comprehensive assessments of transcriptional activity in the human genome and provide an atlas of human gene expression over a unique set of gene predictions. Before the annotation of the human genome is considered complete, however, the previously unannotated transcriptional activity throughout the genome must be fully characterized.
Background
The completion of the sequencing of the human, mouse and other genomes has enabled efforts to extensively annotate these genomes using a combination of computational and experimental approaches. Generating a comprehensive list of transcripts coupled with basic information on where the different transcripts are expressed is an important first step towards annotating a genome once it has been fully sequenced. The task of identifying the transcribed regions of a sequenced genome is complicated by the fact that transcripts are composed of multiple short exons that are distributed over much larger regions of genomic DNA. This challenge is underscored by the widely divergent predictions of the number of genes in the human genome. For example, direct clustering of human expressed sequence tag (EST) sequences has predicted as many as 120,000 genes [1], whereas sampling and sequence-similarity-based methods have predicted far lower numbers, ranging from 28,000 to 35,000 genes [2-5], and a hybrid approach has suggested an intermediate number [6]. Furthermore, the availability of a completed draft sequence of the human genome has yielded neither a proven method for gene identification nor a definitive count of human genes. Two initial analyses of the human genome sequence that used strikingly different methods both suggested the human genome contains 30,000 to 40,000 genes [2,3]. However, a direct comparison of the predicted genes revealed agreement in the identification of well-characterized genes but little overlap of the novel predictions. Specifically, 84% of the RefSeq transcripts agreed with fewer than 20% of the predicted transcripts matching between the two analyses. This result suggests that, individually, these datasets are incomplete and that the human genome potentially contains substantially more unidentified genes [7].
Several recent studies have highlighted the limitations of relying solely on computational approaches to identify genes in the draft of the human genome [8-13]. Furthermore, substantial experimental data from direct assays of gene expression provide evidence for many genes that would not have been recognized in the analyses just mentioned. Saha and colleagues used a new LongSAGE technology to provide strong evidence that there are thousands of genes left to be discovered in the human genome [9]. Specifically, they sequenced over 27,000 tags from a human colorectal cell line that collapsed down to 5,641 unique groups. Interestingly, only 61% (3,419) of the tags matched known or predicted genes, whereas 10% (575) matched novel internal exons and 14% (803) appear to represent completely novel genes [9]. They extrapolate from these data to predict as many as 7,500 exons from previously unrecognized genes. A recent analysis by Camargo et al. [8] also indicates that we are far from defining a complete catalog of human genes based on the analysis of 700,000 ORESTES (Open Reading Frame ESTs) that were recently released into GenBank. Finally, Kapranov and colleagues recently constructed genome-tiling arrays for human chromosomes 21 and 22 to comprehensively query transcription activity over 11 human tissues and cell lines [10]. They detected significant, widespread expression activity over a substantial proportion of these chromosomes outside of all known and predicted gene regions.
Most current methods in widespread use for identifying novel genes in genomic sequence depend on sequence similarity to expressed sequence and protein data. For example, ab initio prediction programs operate by recognizing coding potential in stretches of genomic sequence, where the recognition capability of these programs depends on a training set of known coding regions [14]. Therefore, genes identified by ab initio prediction programs or assembled from EST data are also inaccurate or incomplete much of the time [10-12]. While ab initio prediction programs perform well at identifying known genes, predictions that do not use existing expressed sequence and protein data often miss exons, incorrectly identify exon boundaries, and fail to accurately detect the 3' and 5' untranslated regions UTRs [14]. Similarly, EST data may be biased towards the 3' or 5' UTR [13]. These deficiencies are addressed in full-length gene cloning strategies [13], but cloning is still a laborious process which could be accelerated if we were able to start from a more accurate view of a putative gene [13].
Recently, several groups have used microarrays to test computational gene predictions experimentally and to tile across genomic sequence to discover the transcribed regions in the human and other genomes [10-12,15-17]. These array-based approaches detected widespread transcriptional activity outside of the annotated gene regions in the human, Arabidopsis thaliana and Escherichia coli genomes. The recent sequencing and analysis of the mouse genome indicates extensive homology between intergenic regions of the human and mouse genomes, further highlighting the potential for other classes of transcribed regions [18]. Interestingly, recent tiling data suggests that many of these conserved intergenic regions are transcribed [15,16].
In the study reported here, we describe hybridization results generated from two large microarray-based gene-expression experiments involving predicted transcript arrays spanning the entire human genome and a comprehensive set of genomic tiling arrays for human chromosomes 20 and 22. mRNA samples collected from a diversity of conditions were amplified using a strand-specific labeling protocol that was optimized to generate full-length copies of the transcripts. Analyses of the resulting hybridization data from both sets of arrays revealed widespread transcriptional activity in both known or high-confidence predicted genes, as well as regions outside current annotations. The results from this analysis are summarized with respect to published genes on chromosomes 20 and 22 in addition to our own extensive set of genome alignments and gene predictions. Combining computational and experimental approaches has allowed us to generate a comprehensive transcript index for the human genome, which has been a valuable resource for guiding our array design and full-length cloning efforts. In addition, the expression data from the 60 conditions provides a comprehensive atlas of human gene expression over a unique set of gene predictions [19].
Results
Generating a comprehensive transcript index of the human genome
Figure 1 illustrates the process we used to generate a comprehensive transcript index (CTI) for the human genome that represents just over 28,000 known and predicted transcripts with some level of experimental validation. The first step in this process was to generate a 'primary transcript index' (PTI) by mapping a comprehensive set of computationally and experimentally derived annotations onto the genomic sequence. The computational predictions include the output of gene-finding algorithms and protein similarities, while the experimentally derived alignments are based on ESTs, serial analysis of gene expression (SAGE), and full-length cDNAs. The resulting list of transcripts in the PTI can be loosely ranked or classified into different categories, ranging from high confidence to low confidence, on the basis of the level of underlying experimental support. The advantages of a PTI are that the computations can be performed on a genome-wide scale and it incorporates the massive amounts of publicly available EST, SAGE and cDNA sequence data. However, the resulting transcript index has two significant limitations. First, the ab initio gene-finding algorithms tend to have a high false-positive rate when applied at a low-stringency setting to cast as broad a discovery net as possible. Second, gene-finding algorithms are trained on known protein-coding genes, which may limit their ability to detect truly novel classes of transcribed sequences.
Figure 1. A process to generate a comprehensive transcript index (CTI) for the human genome. The first step is the assembly of a comprehensive set of annotations to generate a predicted transcript index (PTI). Sets of microarrays capable of monitoring the transcription activity over the entire genome can then be designed on the basis of the PTI. The different microarray types that can be used in this process include predicted transcript arrays (PTA), exon junction arrays (EJA) [21] and genome tiling arrays (GTA). After hybridizing a diversity of conditions onto these arrays, the transcription data are processed to identify a comprehensive set of transcripts (the CTI) and associated probes that are capable of querying all forms of transcripts that may exist in the genome. This set of probes comprises a focused set of microarrays that can be used in more standard microarray-based experiments.
The second step towards the CTI is the use of two different types of microarrays to address these limitations (Figure 1). First, predicted transcript arrays (PTA) were used to determine experimentally which of the lower-confidence predictions in the PTI were likely to represent real transcripts. Second, genomic tiling arrays were used to survey transcriptional activity in a completely unbiased and comprehensive fashion. As shown in Figure 1, the CTI plays a central part in the subsequent design of screening arrays. These are used to monitor RNA levels for all the transcripts across a large number of diverse conditions to begin the process of assigning biological functions to novel genes based on co-regulation with known genes [20]. The CTI is also used to design exon/junction arrays that can be used to discover and monitor alternative splicing across different tissues and stages of development [21].
Generating a PTI
To generate the PTI, three distinct computational analysis steps were executed in parallel: predictions based on similarity to expressed sequences from human and mouse; predictions based on similarity to all known proteins; and ab initio gene predictions. The process resulted in mapping 91% of the well characterized genes found in the RefSeq database [22], a percentage consistent with initial genome annotation results [2,3]. The mapping results were generated by collapsing overlapping gene models and regions of similarity to define locus projections, which comprise the distinct transcribed regions making up our PTI. While the reliance on gene predictions and protein alignments biases the PTI towards protein-coding genes, the alignment of all expressed sequences should represent many of the non-coding genes reported to date. A comprehensive index of non-coding genes would require tiling arrays, as described later.
All locus projections were classified into one of eight categories on the basis of the level of underlying evidence from expressed sequence similarity, protein similarity and ab initio predictions. The categories, in decreasing order of support, are as follows: (1) known genes, taken as the set of 11,214 human genes represented in the RefSeq database when the arrays were designed; (2) ab initio gene models with expressed sequence and protein support; (3) ab initio gene models with expressed sequence support; (4) ab initio gene models with protein support; (5) alignments of expressed sequence and protein data; (6) alignments of expressed sequence data, requiring at least two overlapping expressed sequences; (7) ab initio gene models with no expressed sequence or protein support; and (8) alignments of protein data. Because of the limitations discussed in the previous section, we considered predictions with a single line of evidence (categories 6-8) as low confidence.
Table 1 provides summaries resulting from a comparison between our PTI and the published Sanger Institute data for chromosomes 20 and 22 [23,24]. Our locus projections overlap 1,177 of 1,297 (91%) Sanger genes on chromosome 20 and 854 of 936 (91%) Sanger genes on chromosome 22, and our predicted exons overlap 7,306 of 7,556 (97%) and 4,819 of 5,014 (96%) total Sanger chromosome 20 and 22 exons, respectively. This comparison highlights the fact that our annotations result in the detection of both genes and exons in genomic sequence with high sensitivity.
Table 1. Comparison of locus projections in the PTI on chromosomes 20 and 22 to Sanger-annotated genes
Predicted transcript arrays
We previously described a high-throughput, experimental procedure to validate predicted exons and assemble exons into genes by using co-regulated expression over a diversity of conditions [11]. Here we employ a similar strategy over the entire genome by hybridizing RNA from 60 diverse tissue and cell-line samples to a set of arrays designed from the PTI. For a complete list of the transcripts represented on the predicted transcript arrays and 60 tissues and cell lines hybridized to these arrays (see Additional data files 1 and 2). We designed two probes per exon, where possible, for exons containing the highest-scoring probes as described in the methods from each transcript in our PTI set (on average, a total of four probes per transcript). This was done to balance the poor specificity of ab initio gene-finding algorithms [14,25,26] against the significant microarray costs associated with large-scale gene-expression experiments. The resulting hybridization data provides experimental validation of those low-confidence predicted genes that are either unsupported or minimally supported by existing EST data, thereby providing a means of determining which transcripts are included in the CTI.
Summary of predicted transcript validation on chromosomes 20 and 22
We used an enhanced version of a previously described gene-detection algorithm to analyze the predicted transcript array dataset [11]. Basically, the hybridization data from probes each transcript from the PTI were examined to identify those transcripts with probes that appear to be more highly correlated over the 60 diverse conditions. Transcripts with probes that behaved similarly over the different conditions tested were considered to be expression-validated genes (EVGs). Unlike our original algorithm that used Pearson correlations to group similarly behaving probes, our enhanced algorithm incorporated a probe-specific model to assess the most likely set of probes making up a transcriptional unit [27] (see Materials and methods for details). We used the extensive publicly available annotations on chromosomes 20 and 22 to assess the sensitivity and specificity of our array-based detection procedure.
The sensitivity of our procedure was assessed by computing the EVG detection rate for those Sanger genes that overlap predictions (locus projections) represented in our PTI (Table 2). The average detection rate for our locus projections on chromosomes 20 and 22 is approximately 70% for those overlapping Sanger genes and just over 80% for those locus projections derived from RefSeq alignments (locus category = known) that represent Sanger genes. A true positive in this instance was defined as an expression-verified gene containing at least two probes, where at least one of the probes was contained within the exon of a Sanger or RefSeq gene.
Table 2. Summary of expression-validated genes (EVGs) from predicted transcripts over the entire human genome
This 20% false-negative rate is the result of a complex mixture of issues, including limitations in our EVG-detection algorithm, limitations in the probe design step, lack of expression in the conditions profiled, and/or alternative splicing events. While the EVG-detection algorithm provides an efficient method to assemble probes into transcript units, the detection capabilities of this model could be expected to improve as the number of samples and the number of probes targeting any given transcript increases. The use of four probes per predicted transcript was determined to be sufficient for detection of most transcripts, as supported by the overall detection rate of known genes, although in many cases the probe design step was limited by our ability to find four high-quality probes per transcript. For many transcripts, there were not four nonoverlapping probes predicted to have good hybridization characteristics for the microarray experiment carried out here. The 60 samples were chosen to represent a broad array of tissue types, as an exhaustive list of human tissues is impossible to obtain. Because no replicate tissues/cell lines were run for any of the 60 chosen samples, we relied on the replication inherent in monitoring the same transcripts over 60 different conditions. In this case, genes expressed in multiple samples provide the replication necessary to increase our confidence in the detections. However, there are clear limitations in not replicating tissues/cell lines, as genes may be expressed in only a single condition or may be switched on only under certain physiological conditions or only during a certain stages of development. In such cases, we would have reduced power to detect these genes.
Genes in the lower-confidence categories of our PTI annotations, which are not typically considered genes by Sanger, were detected at a significantly reduced rate. Interestingly, of the 337 (188 +149) higher-confidence transcripts on chromosomes 20 and 22 that did not intersect with Sanger genes, 47 (or 14%) were detected as EVGs (Table 2). These transcripts represent potential novel transcripts on these two highly characterized chromosomes.
However, before we can make claims to the discovery potential for this method over the entire genome, we need to assess the false-positive detection rates. To this end, we defined as false positives all detections made in regions with support by only a single gene model that fell outside Sanger-annotated genes on chromosomes 20 and 22. Applying this definition over all transcripts in our PTI leads to a false-positive rate of 3% (11 out of 406). Because we cannot exclude the possibility that some of the transcripts supported by a single gene model represent real genes, we consider this false-detection rate as an upper bound on the actual false-positive rate. Accepting that the Sanger annotations represent the gold standard for chromosome 22, we detected 70% of all Sanger-annotated genes, while only 4% of the chromosome 22 locus projections that did not intersect Sanger genes were detected by our procedure, highlighting the sensitivity and specificity of this approach. In addition, the enrichment for EVG detections in Sanger genes versus the non-Sanger PTI on chromosomes 20 and 22 was extremely significant with a p-value effectively equal to 0 when using the chi-square test for independence (χ2 = 3,093, with 1 degree of freedom (df)).
Summarizing EVG data over the entire genome and assessing the discovery potential. The last column of Table 2 provides the number of expression verified genes detected over the entire genome for locus projections in our PTI. This represents the most comprehensive direct experimental screening of ab initio gene predictions ever undertaken. We can use the false-positive and negative rates derived above to assess the discovery potential on that part of the genome that has not been as extensively characterized as chromosomes 20 and 22. First, we note that our detection rates over the genome were similar to that given for chromosomes 20 and 22. That is, 75% of the category 1 genes (RefSeq genes) were detected over the entire genome, compared to 80% for chromosomes 20 and 22. In total, 15,642 genes in the PTI were experimentally validated using this array-based approach. Assuming the false-positive rate of 3% defined above and a conservative false-negative rate of 30%, defined as the percentage of Sanger genes we failed to detect on chromosomes 20 and 22, these data suggest there are close to 21,675 potential coding genes represented in our PTI set. Because our PTI misses close to 10% of the Sanger genes, we corrected this number for those genes not represented in this set and provide an estimate of the total number of protein-coding genes in the human genome supported by our data to be approximately 25,000. This number is consistent with estimates given in the current release (22.34d.1) of the Ensembl database [28,29].
However, we caution that the estimate provided is based solely on the data described here, and that orthogonal sources of data [30] continue to suggest that the actual number of genes will be known only after the transcriptome has been completely characterized.
From Table 2 we note that 2,093 (1,428 + 555 + 110) of the transcripts that were detected as EVGs had only one line of evidence (EST alignment, protein alignment or ab initio prediction). These 2,093 transcripts represent a rich source of potential discoveries in our PTI. To assess the potential biological functions of this novel gene set, we annotated translations of this set by searching the domains represented in the Protein Families database (Pfam) [31]. The search results were used to assign each of the translations to Gene Ontology (GO) [32] codes as described in the methods. Figure 2 graphically depicts the breakdown of the most common GO codes for two of the three major GO categories. These data suggest there may still be a significant number of protein-coding genes with important biological functions, given that domains/motifs represented in these predicted genes are similar to those found in known genes. The 339 predictions that were validated as EVGs and that had protein domains of biological interest would be natural candidates for full-length cloning, over the 24,532 (7,170 + 16,822 + 540 from Table 2) other lower-confidence predictions in our set.
Figure 2. Gene Ontology (GO) classification of novel expression-validated genes (EVGs). EVGs not supported by the expressed sequence data (2,093) were submitted to a search against the Pfam database. Those with significant alignments (339) were assigned GO codes based on Pfam. The pie charts show the distribution of GO terms within this set of EVGs. Note that the total number of GO terms in each category is greater than the number of EVGs because of assignment of multiple GO terms to some EVGs. (a) Distribution of the different 'biological process' GO codes assigned to the EVGs with significant hits to the Pfam database: a total of 526 GO terms. (b) Distribution of the different 'molecular function' GO codes assigned to the EVGs with significant hits to the Pfam database: a total of 374 GO terms.
EVG data as an expression index
Because multiple probes in each of the approximate 50,000 predicted genes in the human genome have been monitored over 60 different tissues and cell lines, the EVG data represent a significant atlas of human gene expression that is now publicly available [19]. For each transcript, the intensity information from the corresponding probes was optimally combined as described by Johnson et al. [21] to provide a quantitative measure of the relative abundance across the panel of 60 conditions, as shown in Figure 3.
Figure 3. Utilizing PTA data as an expression index. Absolute transcript abundance over the 60 conditions described in [19] for two expression-supported transcripts. RLP09885002 represents a known gene (ATP1A1, ATPase, Na+/K+ transporting, alpha 1 polypeptide) whereas RLP10406004 was supported solely by gene model predictions before microarray validation.
Tiling arrays for chromosomes 20 and 22
To complement the use of PTI arrays, we constructed a set of genome tiling arrays comprised of 60 mer oligonucleotide probes tiled in 30 base-pair steps through both strands of human chromosomes 20 and 22. Repetitive sequences identified by RepeatMasker were ignored for probe design. These genome tiling arrays allow for an unbiased view of the transcriptional activity outside of known and predicted genes on these two chromosomes. mRNA from six (chromosome 20) or eight (chromosome 22) conditions was amplified and hybridized to the tiling arrays (see [19] and Additional data files 3 and 4). As with the PTI arrays, the amplification protocol generated strand-specific cDNA copies of the transcripts, which were full-length. Using a two-step procedure, the resulting data were analyzed to detect sequences expressed in at least one condition [33]. First, we examined probe behavior over conditions in overlapping windows of size 15,000 bp to identify windows that probably contained transcribed sequences, using a robust principal component analysis (PCA) method [33]. Second, for regions identified as likely to contain transcribed sequences, we attempted to discriminate between probes corresponding to expressed sequences (expressed 'exons') and probes corresponding to untranscribed sequences ('introns' or intergenic sequence) using a clustering procedure on variables derived from the PCA procedure [33]. All analysis results derived from this procedure were interpreted in the light of the Sanger annotations and our custom PTI set described above.
Figure 4 provides two representative examples of tiling data for two known Sanger genes, KDELR3 and EWRS1. In the first case (Figure 4a), the tiling data almost perfectly correspond to the RefSeq annotation of KDELR3, with just two potential false positives out of the 178 intron probes. The KDELR3 gene is annotated as having two alternative transcripts in the RefSeq database, given by the RefSeq accession numbers NM_006855 and NM_016657. The NCBI Acembly alternative splicing predictions further suggest the presence of additional isoforms of this gene (see Figure 4). One of the alternative forms, KDELR3.e, depicted in Figure 4a, includes a novel 5' exon. The presence of this exon is supported by the EST with GenBank accession number BM921831. The tiling data for the KDELR3 gene in two conditions clearly show expression of NM_006855 but not NM_016657, thereby reliably detecting distinct splice forms. Further, there is a significant signal 5' to exon 2 in both transcripts that seems to suggest a novel exon, as opposed to a true false positive. This putative exon exactly matches the location of the first exon given in the Acembly prediction track noted in Figure 4a (KDELR3.e).
Figure 4. Examples of tiling results for known genes. The colored bars across the bottom of the data window are color matched with the corresponding exon annotations shown in the genome viewer. (a) The KDELR3 gene shows strong agreement between the public transcript annotations and the tiling results. The top panel represents a screen shot from the UCSC genome browser [60] highlighting KDLER3. The bottom panel represents transcription activity as raw intensities (y-axis) for each probe used to tile through KDLER3 (x-axis), in one of the eight conditions monitored by the genomic tiling arrays. (b) The EWRS1 gene potentially contains a larger number of false-positive predictions, but more probably lends additional experimental support to previously predicted alternative splice forms (EWSR.b and EWSR.g), giving a more accurate representation of the putative structure of this gene. The top panel represents a screen shot from the UCSC genome browser [60] highlighting EWRS1. The bottom panel represents transcription activity as raw intensities (y-axis) for each probe used to tile through EWSR1 (x-axis), in one of the eight conditions monitored by the genomic tiling arrays. (c) Conserved regions between mouse and human upstream of the beta-actin gene. The tiling data readily detect all of the transcribed parts of the gene, but not the conserved regulatory regions. The green bars in the probe-intensity plot represent the annotated transcribed regions for the beta-actin gene, while the blue bars indicate regions that are not known to be transcribed. The lower section shows the sequence conservation between human and mouse as obtained through the program rVISTA [36,61]. Conserved coding (blue peaks) and non-coding regions (red peaks) are shown where the two genomic sequences align with 75% identity over 100-bp windows. The rows marked ELK, ETF, and SRF show binding sites for these transcription factors predicted using TRANSFAC matrix models and the MATCHTM program, which are part of the rVISTA suite. The exons for the gene are shown in blue.
Figure 4b shows the tiling data for the EWSR1 gene. In contrast to the first example, this gene has intense transcriptional activity outside of the annotated exons. Specifically, the EWSR1 gene has 43 potentially false-positive calls out of 203 intron probes. However, the EST data and alternative splicing predictions strongly suggest that these probes represent biologically relevant transcriptional activity. As with the KDELR3 gene, EWRS1 is annotated by RefSeq as having two transcripts: NM_005243 and NM_013986. The Acembly predictions identify four additional alternative splice forms; most noteworthy among these are EWSR1.b and EWSR.g, shown in Figure 4b. These predictions indicate that alternative transcripts may exist for the EWSR1 gene that essentially divide the largest transcript into two transcripts, suggesting that multiple promoter and transcription-stop signals are present in this gene. The tiling data depicted in Figure 4b shows that all exons from both RefSeq splice forms were detected. In addition, there is a region to the right of probe position 400 in Figure 4b that indicates significant transcription activity but where there are no RefSeq exons annotated. However, the green bars indicate exons that are supported by EST data as well as the EWSR.b and EWSR.g predicted alternative splice forms, providing experimental support that these predictions represent actual isoforms of this gene. In fact, these data may provide a more accurate representation of the putative structure of this gene, as they support multiple alternatively spliced transcripts in this gene, beyond what has already been annotated in the RefSeq database. In all, 5% of the probes detected as expressed in intronic sequence mapped to predicted alternative splice forms. Given the extent of alternative splicing that is yet to be characterized [21], we believe a significant proportion of the 'intron' transcriptional activity in our data may represent alternative splicing.
Summarizing the tiling results
Our genome tiling arrays consisted of 2,119,794 and 1,201,632 probes for chromosomes 20 and 22, respectively. Of these, 1,615,034 probes fell into Sanger gene regions, with 239,542 probes actually overlapping Sanger exons. Under stringent criteria 64,241 probes were detected as expressed, with 34,245 of these falling within Sanger exons, 18,551 falling within Sanger introns, and 15,835 probes falling completely outside all Sanger annotations. This widespread transcriptional activity outside annotated regions of the human genome is consistent with other reports from multiple species [10,12,15,16]. Overall, at least one exon in each of 876 Sanger genes was detected as expressed out of 1,703 total genes covered by probes (excluding annotated pseudogenes), leading to an overall gene detection rate of 52%. The bias of probes identified as exon probes that actually fall in exons is striking, given that exons comprise roughly 2% of the genomic sequence (the p-value for this enrichment using the Fisher exact test is less than 10-15). To estimate the upper bound of false-positive calls, we counted as false-positive events each probe identified as expressed by the detection process, but falling within an annotated intron of the RefSeq genes we detected as expressed. This resulted in an estimated false-positive rate of 1.3%.
As indicated in Figure 4, a percentage of these false-positive calls will be due to unannotated isoforms of genes. Others still will be due to cross-hybridization of the intron probes to genes in other parts of the genome. We consider cross-hybridization as made up of two components: specific cross-hybridization resulting from transcripts with similar, usually homologous, sequences; and nonspecific cross-hybridization resulting from the base composition of the probe sequence (J.C. and G.C., unpublished work). Of the intron probes detected as expressed, 23% had sequence similarities to known transcripts considered to render them susceptible to specific cross-hybridization, and 17% contained sequence features associated with nonspecific cross-hybridization. Accounting for probes that were positive for both specific and nonspecific cross-hybridization, we are left with 55% of the probes detected as expressed in the introns of Sanger genes that cannot easily be explained as alternative splicing or cross-hybridization. These data support recent observations that significant levels of transcription exist within the introns of known genes [15,16].
For those probes falling outside all Sanger genes, we again made use of our custom genome annotations to help interpret the extent of transcriptional activity in these regions. Table 3 summarizes the detections made for each of the categories described above. Filtering probes using the same cross-hybridization predictors described above suggests that 65% of those probes falling outside all annotations are not likely to be the result of cross-hybridization. Furthermore, for those detections that overlap low-confidence locus projections in our PTI, we used the classification procedure discussed above to assign GO codes to these transcripts. Only seven of the 297 transcribed regions detected outside of all Sanger genes via the tiling results (see Table 3) contained GO protein domains. This suggests that a large fraction of the transcriptional activity detected using tiling arrays is non-coding and of unknown biological function [15,34].
Table 3. Summary of transcription activity detected from the chromosome 20 and 22 genome tiling data
Tiling data help classify conserved sequences between species
One further advantage of the tiling data is that they can be used to discriminate between transcribed and non-transcribed sequences conserved between human and mouse, or between any other pair of species. Figure 4c highlights tiling data under one condition for the beta-actin gene, a gene that is constitutively expressed in all tissues and often serves as a positive control in mRNA and protein expression experiments. The genomic region containing the complete beta-actin mRNA and 10 kilobases (kb) of genomic sequence upstream of the transcription start, was obtained from the mouse and human genomes, aligned using the AVID program [35] and then fed into the rVista program [36]. From this, we identified the conserved regions in this gene between mouse and human, including several relevant transcription factor binding domains that are key to the transcriptional regulation of this gene [37-39]. As can be seen directly from the figure, the exons are all detected as highly expressed, but none of the conserved transcription factor regions shows activity. This combination of expressed sequence in close proximity to conserved regions that are not expressed (as determined by the tiling data), offers another powerful advantage of the tiling data in discriminating among the possible roles of conserved sequences.
Discussion
A complete understanding of the human genome will only come after all genes have been identified and the functions of those genes have been determined. There has been much recent progress in defining the human transcriptome with ab initio methods, sequencing of EST libraries, full-length gene cloning projects, and comparative analyses between fully sequenced genomes of different species. However, we are still a long way from having a comprehensive set of annotations for the human and other genomes. There is need for new high-throughput experimental approaches to accelerate the process of annotating sequenced genomes in a comprehensive and accurate fashion. Toward this goal, we have used two microarray-based experimental approaches to provide evidence of widespread transcription activity outside of any known or predicted genes in the human genome. We have also provided experimental support for many ab initio predicted genes that have no other or minimal experimental sequence support, suggesting a small but significant class of genes that have evaded all other forms of experimental detection. Similar identifications have been made recently in the first extensive comparative analysis between mouse and human genomes [18]. Despite the extent of novel discovery, our data suggest there are only 25,000-30,000 protein-coding genes in the human genome, with perhaps an equal number of non-coding transcripts that may serve important regulatory roles [34,40]. Finally, our data indicate widespread alternative splicing across known genes, providing a glimpse into the extent of transcript complexity that may exist in mammalian genomes.
We have used the expression data for the approximate 50,000 predicted transcripts hybridized to 60 diverse conditions in combination with genomic tiling data to generate a CTI containing 28,456 experimentally supported transcripts. The transcripts represented in the CTI include all computational predictions with two or more lines of evidence from our PTI (independent of microarray validation), in addition to the overlapping set of 15,642 transcripts detected as EVGs. This resulting comprehensive list of known and predicted transcripts provides the starting point for large-scale systematic studies to determine the biological function of genes in both normal and disease states. The primary goal of the CTI is to allow researchers to focus experimental efforts on a comprehensive set of genes that are likely to be real.
It is of note that between the time the predicted transcript arrays were designed and annotated using the custom genome annotations described above, and the time this work was published, more than 6,000 genes were added to the RefSeq collection. These newer RefSeq genes were represented by 5,100 locus projections in our original PTI that were not classified in the RefSeq category. Interestingly, 4,212 were detected as EVGs in the present analysis and had already been included in our CTI, a validation rate slightly greater than 82%. Only 19% of the non-RefSeq genes in our PTI had been detected as EVGs (see Table 2), yet more than 82% of the new RefSeq genes coming from this set were detected as EVGs. This result speaks to the utility of the microarray-based approach to gene validation described here (see Additional data file 5 for a complete breakdown of validation rates by category).
While using microarrays to test computational gene predictions experimentally has the advantage of being economically feasible across the whole genome, the tiling data represent a more comprehensive and unbiased view of transcription. Our data indicate widespread transcriptional activity in the introns of annotated genes and in intergenic regions, where a significant proportion of this activity can be explained by nonspecific and specific cross-hybridization. The transcriptional activity noted for our low-onfidence transcripts in the PTI indicates that 25% of the activity we observe may be coding for proteins that are at least somewhat related to existing protein data. Much of the transcription activity we have noted in the introns of genes may also represent uncharacterized alternative splicing, and potentially novel genes, in addition to specific and nonspecific cross-hybridization.
Conclusions
At present, predicted transcript arrays allow for the discovery of most protein-coding genes genome wide when many different conditions are considered. Until the discovery and characterization of these protein-coding genes is completed, this method will continue to be a cost-effective solution to drive such discovery. In contrast, genomic tiling represents a completely unbiased method for monitoring transcriptional activity in genomes, but due to cost will probably be limited to screening a smaller number of conditions. However, as novel transcription regions are identified from the tiling data, these regions can be represented on predicted transcript arrays that are hybridized over many more conditions, as described in Figure 1. As the microarray technologies have evolved, tiling the entire human genome is now possible, with such efforts presently being supported by the ENCODE (Encyclopedia of DNA Elements) project of the National Human Genome Research Institute (NHGRI) [41].
We believe the steps taken here are necessary for querying all potential transcription activity in the genome, for the purpose of identifying novel genes, more completely characterizing existing genes, and identifying a more comprehensive set of probes for these genes that can be used to monitor transcription abundances in more standard gene expression studies. Not all uses of microarrays demand an exhaustive representation of probes to all genes in the genome under study. However, experiments that seek to identify key drivers of pathways [42] or that seek to discriminate between alternative splice forms of genes within a given tissue [21] require a more comprehensive set of arrays to ensure success. These data provide an essential first step to generating a comprehensive set of arrays that are based on experimental support combined with computational annotation, instead of relying solely on the latter. These comprehensive arrays will be invaluable as we seek to better understand mechanisms of action for existing and novel drug targets and elucidate pathways underlying complex diseases. In addition, further study of the extensive noncoding RNA identified via the methods described here and elsewhere [10,12,15,16] is likely to open new fields of biology as the functional roles for these entities are determined.
Materials and methods
Data preparation
The NCBI 8/2001 assembly of the human genome was the input data for this analysis. The 4/21/1999 release of RepeatMasker [43] was used to mask for human repeats. An internal database of RNA genes and bacterial and vector sequences was aligned to the genome with BLASTN. Genomic sequences with 95% or higher identity over at least 50 bases were masked. No probes were designed from masked regions.
Gene index production
To predict genes on the basis of expressed sequence similarity, we first clustered and aligned all expressed human and mouse sequences in GenBank to create a human gene index (HGI) and a mouse gene index (MGI). Clustering and alignment were performed with the DoubleTwist Clustering and Alignment Tools (CAT) [44]. Input data included all mouse and human RefSeq mRNA sequences, and EST and mRNA sequences from GenBank release 124, masked as described above for repeats and contaminating sequences. For each species, the CAT software first clustered sequences and then defined subclusters on the basis of a multiple sequence alignment. The subclusters represent candidate alternatively spliced gene transcripts. The 644,168 human and 291,656 mouse sequences that were singleton ESTs or completely masked were excluded from the HGI and MGI.
Expressed sequence mapping
Human and mouse UniGene and RefSeq, MGI, and HGI sequences were aligned with the genome first by BLASTN 2.2.1 [45], followed by refinement of intron/exon boundaries by the sim4 algorithm (12/17/2000 release) [46]. Only the representative sequences (Hs.seq.uniq) for each UniGene cluster designated in the 3 August 2001/build 138 version of the UniGene database were used in this analysis. We only refined BLAST alignments with an E-value of less than 10-20 for human sequences and 10-8 for mouse sequences. For human UniGene and HGI, we refined only those BLAST hits where the target sequence showed greater than or equal to 92% identity to the genomic sequence over 75 bp. For human RefSeq, we refined hits with greater than or equal to 95% identity, and for MGI, RefSeq, and UniGene, we refined hits with greater than or equal to 80% identity. These thresholds were empirically determined to provide good sensitivity in aligning most sequences to the genome while limiting multiple alignments past those expected from paralogs present in the human genome. In all cases percent identity was measured over 75 bp. Individual sim4 exons of questionable confidence were then removed on the basis of percent identity and length thresholds. All sequence databases were downloaded from GenBank August, 2001.
Protein sequence mapping
The GenBank nonredundant protein database (downloaded 25 August 2001) was aligned to the genomic sequence with BLASTX 2.2.1 [45] using an E-score threshold of 10-5. Adjacent protein alignments from a single protein were grouped together as a prediction whenever the protein sequence coordinates of the alignments were consistent in direction and did not significantly overlap.
Ab initio gene prediction
GrailEXP 4.0 [47], GENSCAN 1.0 [48], FGENESH [49], and FGENESH+ [49]ab initio gene-prediction algorithms were run independently across the entire genome assembly to augment alignment-based gene identification methods. GrailEXP 4.0, GENSCAN 1.0, and FGENESH version 1.c were run with default parameters for human sequence. GrailEXP used expressed sequence evidence from RefSeq, UniGene and DoubleTwist HGI to refine gene predictions. FGENESH+ was run with protein sequences from BLASTX with E-score lower than 10-5. When multiple protein alignments overlapped, all overlapping protein sequences were clustered with BLASTClust [50] and the lowest E-score hit was used by FGENESH+.
Synthesis and analysis
Locus projections contained the union of all exons from all overlapping predictions in a contiguous region of the chromosome that were derived from sequence alignments or gene-finding algorithms. Predictions to a given strand of the genomic sequence that overlapped by even a single nucleotide were grouped into a single locus projection (antisense transcripts were not considered in defining the locus projections). The criteria for grouping predictions were intentionally kept loose, given that the intent was to include as many potential exons as possible in a given genomic region, and then use the experimental microarray-based approach to elucidate the actual gene structure. These merged overlapping predictions defined the 5' and 3' ends of the locus projections. Overlapping predicted exons were merged to form an exon prediction of maximal extent. Low-quality predicted exons from sim4 alignments that contained a high percentage of A or T were removed. We also removed sim4-predicted exons that overlapped two or more predicted exons from another sim4 alignment. Additionally, 3' sim4 and 3' or 5' FGENESH+ predicted exons that were short and/or distant from internal predicted exons were removed. Finally, locus projections that contained mRNAs from RefSeq were split at the 5' end of the RefSeq sequence.
Locus projections supported by expressed sequences alone could be portions of 3' or 5' UTRs of genes included in the other gene-prediction categories described in the text. To minimize the consequences of this potential artifact, we used a UTR filter to exclude locus projections from the expressed sequence alone category that were within 20 kb of a locus projection supported by an ab initio gene model.
All data were loaded into a relational database to count and categorize locus projections. At least one type of evidence was assigned to each predicted exon for each locus projection. Multiple types of evidence were assigned to a merged predicted exon if there was overlap between predicted exons of different types for at least 1% of the length of the merged exon prediction. One of the eight evidence categories discussed in the text was assigned to each exon on the basis of the combination of types of evidence. Locus projections inherited the highest-ranking evidence category of their constituent exons. Evidence categories were ranked in the following order: Refseq (highest); expressed sequence + protein + ab initio; expressed sequence + ab initio; protein + ab initio; expressed sequence + protein; ab initio alone; protein alone; expressed sequence alone. FGENESH+ predictions were counted as protein + ab initio. For the ab initio category, predictions from at least two of FGENESH, GENSCAN and GrailEXP were required to overlap in at least one exon to be merged.
Probe selection for the genome tiling and predicted transcript arrays
Input sequences for probe selection were masked for vector, interspersed repeats, simple repeats, poly(A) tails, Escherichia coli contamination and human non-coding RNA and mitochrondrial DNA contamination using Scylla (Paracel). For genomic tiling arrays, 60 mer probes were then selected from unmasked regions of both forward and reverse complement strands at uniform 30-base intervals. For predicted transcript arrays, up to four oligonucleotide probes were selected from the unmasked regions of each transcript using a multistep process.
The first step in the probe-selection process was the generation of a pool of candidate probes 60 nucleotides long (60 mers), where each probe was required to fall entirely within an exon from the set of exons under consideration. If there were fewer than four 60 mers then all 50 mers were considered as well. If there were fewer than four 50 mers or 60 mers then all 40 mers were considered, and so on. Stilts composed of sequence from Saccharomyces cerevisiae were added to the 3' ends of probes shorter than 60 nucleotides so that they had a total length of 60 bases when printed onto the arrays.
The second step in the probe-selection process was the classification and reduction of the probe pool on the basis of base composition and related filters. Probes were sorted into four classes on the basis of several criteria, including A, G, C and T content, GC content, the length of the longest homopolymeric run and the number of A residues at the 5' end. For example, a probe had to have GC content between 35 and 45% to be in class 1, between 15 and 55% to be in class 2, and between 10 and 60% to be in class 3. After all classifications were made, probes from lower-quality classes were discarded, keeping the number of probes per gene greater than 15. In cases where a pair of probes was overlapping by more than 50 bases, only a single probe was chosen.
The final step in the probe-selection process identified probes with minimal overlap, and predicted cross-hybridization and desirable positions in the transcript sequence. Cross-hybridization prediction was based on BLAST searching of the full collection of transcript sequences [51]. Probes with perfect matches to transcript sequences for genes other than the one undergoing design were discarded unless they were the only probes available. Otherwise the probes with the weakest predicted cross-hybridization interactions were preferred. Probes were also selected to have as little overlap as possible, and probes located in the last 500 bp of each transcript were discarded where possible to reduce the effects of impaired amplification in this region [52].
All arrays included a set of standard control probes which were used for image processing and quality control. Each array also included 30 randomly distributed copies of each of 51 negative-control probes. These probes were selected for their low intensities in previous human hybridizations. The negative controls local to each experimental probe were used for background correction. Non-control probes were added to each array such that all probes for a given input sequence were grouped together and ordered by their position on the sequence.
Preparation of labeled cDNA and array hybridization
Hybridization material was generated through a random-priming amplification procedure using primers with a random sequence at the 3' end and fixed motif at the 5' end. This amplification procedure has been fully described [52] and has been optimized to generate strand-specific cDNA copies of the mRNA transcripts that are full-length. The 60 mRNA samples from the human tissues described in Additional data files 2 and 3 were purchased from Clontech. The 60 mRNA samples hybridized to the predicted transcript set of arrays were done in duplicate with fluor reversal to systematically correct for dye bias. For tiling hybridizations, six samples were used for chromosome 20 arrays and eight samples for chromosome 22. The mRNA samples hybridized to the set of tiling arrays were not done in duplicate as the analysis carried out on these data was intensity based, and our preliminary data demonstrated reasonable results without performing the tiling experiments in fluor-reverse pairs (data not shown). Additional data files 2-4 contain the full list of samples used for each set of arrays.
Array images were processed as described [53] to obtain background noise, single channel intensity and associated measurement error estimates. Expression changes between two samples were quantified as log10 (expression ratio) where the expression ratio was taken to be the ratio between normalized, background-corrected intensity values for the two channels (red and green) for each spot on the predicted transcript arrays. An independent normalization routine was carried out on the tiling data as described [33] to correct for dye biases, given the lack of technical replicates for these data.
Analysis of predicted transcript array data
Probes from each computationally determined locus were analyzed for coordinated expression over 60 tissues by adapting an additive, probe-specific model initially developed to estimate gene expression indices [27]. The model for a single probe in a single sample pair is given by
yij = μ + φj + θi + εj,
where the yij represent the mlratio measurements for sample pair i and probe j in the current transcriptional model, μ is the grand mean term, φi is the probe-specific term for probe j in the model, θi is the sample-specific term for sample i, and εj is the probe-specific error term, which is taken to be normally distributed with mean 0 and variance . Given the above representation for an observed mlratio value, the likelihood for a single probe over N condition pairs is simply
From this, the likelihood for a given transcriptional model, where a transcriptional model in this context is defined as a set of probes that are adjacent to one another in the genomic sequence and that co-regulate over a number of conditions, is easily seen to be the product of the individual probe likelihoods defined above over the M probes comprising the current model:
The maximum likelihood estimates for the parameters of this model are obtained using standard optimization techniques.
With the likelihood model described above, probe groups making up a transcriptional model were formed by iteratively considering whether neighboring probes (within a PTI member based on genomic location) of a given probe improved the fit of the model just described. This was determined by examining the likelihood ratio statistics between the current, best transcriptional model with or without an additional probe included in the model. Thresholds for the likelihood ratio test statistic and the different model parameters were empirically determined to minimize false-positive and false-negative rates. False positives were estimated by the detection of PTI members supported by only a single ab initio prediction that fell outside annotated Sanger genes on chromosomes 20 and 22. False negatives were defined as Sanger genes on chromosome 20 and 22 that were not detected. Probe sets with a maximum likelihood statistic greater than 100 and an r2 value for fit of data to the model greater than 0.8 were considered high-confidence candidates for EVGs.
For each high-confidence EVG candidate, probes were further assessed by considering the number of conditions in which the absolute intensity of the probe was seen to be significantly above background, and the number of times the probe was seen significantly differentially expressed. Candidate EVGs with at least one probe that was: significantly above background (p-value < 0.01) in at least 10% of the samples; or significantly differentially expressed (p-value < 0.01) in at least 10% of the condition pairs, were considered validated.
Analysis of tiling array data
The analysis of the tiling data has been described in detail by Ying et al. [33]. Briefly, probes were separated into 15 kb windows along the genome with 7.5 kb overlap between the windows. For each window, a robust principal component analysis was applied to the between-sample correlation matrix for probes in the window. Windows containing transcriptional activity were characterized by comparing the distribution of the Mahalanobis distances for the probes in the window (the Mahalanobis distance for each probe was calculated from the probe location to the center of the data in the first dimensions of the principal component score (PCS)) space with the reference distribution calculated based on known intron probes. Individual probes were then classified as belonging to the transcribed unit or not on the basis of the log of the Mahalanobis distance and an approximation of the diagonal distance (slope) of the probe from the minimum first PCS and median second PCS. Using these measures for distance, the probes were clustered using standard clustering techniques as described [33].
The procedure for estimating cross-hybridization of the probes is the subject of a separate manuscript. For the analyses described in this paper, the nonspecific cross-hybridization was estimated by the presence of motifs within the probe sequence that were enriched in probes observed to have a high level of nonspecific cross-hybridization. These probes were observed to have significant intensity when hybridized to human mRNA samples despite having no EST support and falling in introns of well characterized genes on chromosomes 20 and 22. Specific cross-hybridization was estimated by the minimum predicted ΔG value for hybridization of the probe to all genes other than the intended target in the UniGene database (build 157).
Annotation of EVG and tiling data
Hidden Markov model Pfam (HMMPfam) domain predictions were run on six-frame translations of the PTIs using the HFRAME software from Paracel with an E-value cutoff of 0.01 and frameshift penalty of -12. Information on Pfam [31] domains is available [54,55]. GO terms [32] were then assigned to each locus projection using the full set of Pfam to GO mappings available from EBI FTP site [56]. The domain-to-ontology mapping is a part of the larger InterPro effort on annotating the proteome [57,58]. Multiple GO categories can be assigned to a single element of the PTI.
Additional data files
The following additional data is available with the online version of this paper and at [19]. Additional data file 1 gives a complete list of 48,614 transcripts in the PTI that were represented on the set of predicted transcript arrays. Additional data file 2 gives a complete list of 60 tissues and cell lines hybridized to the predicted transcript arrays. Additional data file 3 gives a list of six tissues and cell lines hybridized to the chromosome 20 genomic tiling arrays. Additional data file 4 lists the eight tissues and cell lines hybridized to the chromosome 22 genomic tiling arrays. Additional data file 5 contains a comparison of EVG predictions with RefSeq sequences (March 2004). Also available on our website [19] are: ratio data and body atlas data along with the EVG status, and full sequences for the locus projections in fasta format. All probe sequences and expression data are available from the GEO database [59]. The series accession numbers for the tiling and predicted transcript arrays are GSE1097 and GSE918 respectively.
Additional data file 1. A complete list of 48,614 transcripts in the PTI that were represented on the set of predicted transcript arrays
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Additional data file 2. A complete list of 60 tissues and cell lines hybridized to the predicted transcript arrays
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Additional data file 3. A list of six tissues and cell lines hybridized to the chromosome 20 genomic tiling arrays
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Additional data file 4. The eight tissues and cell lines hybridized to the chromosome 22 genomic tiling arrays
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Additional data file 5. A comparison of EVG predictions with RefSeq sequencesP
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Acknowledgements
We thank D. Kessler, M. Marton and the rest of the Rosetta Gene Expression Laboratory for sample preparation and hybridization, S. Dow for reagent and primer handling, and E. Coffey and the Array Production Team for array synthesis. We also thank M. Krolewski and S. Ezekiel for database and programming support. Finally, we would like to thank B. Bush and J. Sachs for critical evaluation of the manuscript. The authors thank J. Burchard for mapping the PTI probes to the current RefSeq sequences. Rosetta Inpharmatics LLC is a wholly owned subsidiary of Merck & Co, Inc.
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Research
Gene expression atlas of the mouse central nervous system: impact and interactions of age, energy intake and gender
Xiangru Xu1, Ming Zhan2, Wenzhen Duan1, Vinayakumar Prabhu2, Randall Brenneman1, William Wood2, Jeff Firman2, Huai Li2, Peisu Zhang1, Carol Ibe1, Alan B Zonderman2, Dan L Longo3, Suresh Poosala2, Kevin G Becker2 and Mark P Mattson1*
Author Affiliations
1 Laboratory of Neurosciences, National Institute on Aging Intramural Research Program, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA
2 Research Resources Branch, National Institute on Aging Intramural Research Program, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA
3 Laboratory of Immunology, National Institute on Aging Intramural Research Program, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA
For all author emails, please log on.
Genome Biology 2007, 8:R234 doi:10.1186/gb-2007-8-11-r234
The electronic version of this article is the complete one and can be found online at: http://genomebiology.com/2007/8/11/R234
Received:14 June 2007
Revisions received:13 July 2007
Accepted:7 November 2007
Published:7 November 2007
© 2007 Xu 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
The structural and functional complexity of the mammalian central nervous system (CNS) is organized and modified by complicated molecular signaling processes that are poorly understood.
Results
We measured transcripts of 16,896 genes in 5 CNS regions from cohorts of young, middle-aged and old male and female mice that had been maintained on either a control diet or a low energy diet known to retard aging. Each CNS region (cerebral cortex, hippocampus, striatum, cerebellum and spinal cord) possessed its own unique transcriptome fingerprint that was independent of age, gender and energy intake. Less than 10% of genes were significantly affected by age, diet or gender, with most of these changes occurring between middle and old age. The transcriptome of the spinal cord was the most responsive to age, diet and gender, while the striatal transcriptome was the least responsive. Gender and energy restriction had particularly robust influences on the hippocampal transcriptome of middle-aged mice. Prominent functional groups of age- and energy-sensitive genes were those encoding proteins involved in DNA damage responses (Werner and telomere-associated proteins), mitochondrial and proteasome functions, cell fate determination (Wnt and Notch signaling) and synaptic vesicle trafficking.
Conclusion
Mouse CNS transcriptomes responded to age, energy intake and gender in a regionally distinctive manner. The systematic transcriptome dataset also provides a window into mechanisms of age-, diet- and sex-related CNS plasticity and vulnerability.
Background
The molecular mechanisms that determine differences in structure and function among regions of the central nervous system (CNS), and their modification by internal and external environmental factors during adult life are poorly explored. CNS regions that mediate sensory and motor functions, such as the spinal cord, striatum and cerebellum, evolved before regions that mainly mediate higher cognitive functions and emotional behaviors, such as the hippocampus and cerebral cortex [1,2]. The cells of each CNS region exhibit distinct structural and neurochemical phenotypes, and electrochemical properties that presumably result from the differential expression of genes in the resident cells. Additional complexity arises from the influences of factors such as sex hormones [3], energy intake and expenditure [4], and genetic and environmental factors that affect susceptibility to aging and disease [5]. The development of technology for the simultaneous measurement of most of the mRNAs encoded by the mouse and human genomes has led to efforts to establish the transcriptomes of tissues and cells under various physiological and pathological conditions [6].
Aging in mammals is a complex and slowly progressive process that adversely affects all organ systems, resulting in morbidity and culminating in death. Because aging is the major risk factor for major CNS disorders, such as Alzheimer's and Parkinson's diseases, stroke and amyotrophic lateral sclerosis, an understanding of the molecular changes that occur during aging may reveal approaches for preventing or delaying these disorders. In this regard, it has been established that dietary energy (caloric) restriction (CR) can extend lifespan and reduce the incidence of age-related diseases [7]. CR may also protect the nervous system against age-related disease by decreasing oxidative damage to proteins, nucleic acids and lipids, and by enhancing cellular stress resistance [8]. Moreover, the CNS may control the aging process and the responses to CR that extend lifespan [9]. Effects of age and CR on gene expression in rodents have been reported for several different tissues [10-13], but were limited to experimental designs that included only two ages (young and very old), a single gender (males), no or low statistical power, and the use of arrays that included relatively small numbers of genes.
To establish the molecular basis of aging, and tissue- and gender-specific differences in cellular responses to aging, the National Institute on Aging undertook the AGEMAP (Atlas of Gene Expression in Mouse Aging Project), a comprehensive analysis of the effects of aging, CR and gender on gene expression in tissues throughout the body. Here we report the results of the CNS component of AGEMAP in which the expression levels of 16,896 genes were determined in a statistically powerful study design that included RNA samples isolated from 5 different CNS regions of male and female mice of 3 different ages (6, 16 and 24 months) that had been maintained on either normal or reduced energy diets. Five CNS regions were selected for analysis because of their well-established functions and/or their involvement in age-related diseases: cerebral cortex (sensory-motor integration and cognition; Alzheimer's disease and related dementias); hippocampus (learning and memory; Alzheimer's disease and epilepsy); striatum (control of body movements; Huntington's and Parkinson's diseases); cerebellum (coordination and balance; ataxias); spinal cord (reflexes, sensory and motor information transfer; amyotrophic lateral sclerosis).
Results
Different regions of the CNS exhibit unique transcriptomes that are independent of gender, age and energy intake
The design of our study (Table 1) provided the opportunity to establish whether each region of the CNS exhibits a unique pattern of gene expression. Gene expression profiles were generated for each of the five brain regions (5 mice × 3 ages × 2 genders × 2 diets), resulting in a total of more than five million data points (GenBank: GSE8426). A principal components analysis (PCA) of all array data revealed distinct patterns of gene expression for each CNS region (Figure 1a). These region-specific patterns were independent of age, gender and diet. We next employed PCA to evaluate the transcriptome differences between CNS regions compared to a non-CNS tissue, the lung. As expected, the CNS regional transcriptomes are more similar to each other than they are to the lung, a tissue composed of cell types distinct from neurons and glia (Figure 1b).
Table 1. Experimental design
Figure 1. CNS region-specific gene expression patterns. (a) PCA of transcriptomes of the indicated CNS regions inclusive of all ages, diets and genders. The results show that each region of CNS has its own molecular signature that is independent of age, diet and gender. (b) PCA of transcriptomes of the CNS regions and non-CNS region (lung) inclusive of all ages, diets and genders.
Age-related patterns of gene expression differ among brain regions
Previous studies of the effects of aging on gene expression in the brain typically compared only two age groups (young and old) and examined only one or two brain regions and one gender, rendering the interpretation of the results problematic in regards to whether differences between young and old animals are the result of the aging process or, instead, represent changes associated with maturation or plasticity [11-13]. To address this issue we determined the transcriptome of each CNS region for mice of three different ages (6, 16 and 24 months). For a given gene, six patterns of significant change in expression (increased or decreased) are possible during the two age intervals (6-16 months and 16-24 months): pattern 1, no change, no change; pattern 2, no change, change; pattern 3, change, change in opposite direction; pattern 4, change, reversion; pattern 5, change, continued change in same direction; pattern 6, change, no change (Figure 2a). The expression level of about 90% of the genes on the array was statistically unaffected by age. For most brain regions, more than half of the genes whose expression level changed significantly with age followed pattern 2. Overall, the least common pattern of gene expression change with age was pattern 3, indicating that it is very rare for the expression level of a gene to increase from young to middle age and then decrease from middle to old age, or vice-versa. However, in contrast to the other four regions, the striatum was notable for a relatively high percentage of genes that followed pattern 3, suggesting a dramatic switch in the regulation of these genes during the transition from middle to old age. Another outlier in the rank order of frequency of the age-related gene expression patterns was the cerebellum, whose transcriptome was quite plastic from young to middle age and stable thereafter, in contrast to the cortex, hippocampus and spinal cord for which transcriptomes changed most from middle to old age (Figure 2a).
Figure 2. CNS age-related gene expression patterns. (a) For any given gene there are six possible patterns of gene expression from young to middle-aged to old. For approximately 95% of the genes, there was no significant change (p < 0.05 and Z-ratio ≥ 1.50 or ≤ -1.50) in expression across ages (pattern 1). For most CNS regions, pattern 2 (change from middle-aged to old) was the most common. Red, upregulated; blue, downregulated. (b) Comparison of the numbers of genes that were significantly affected by age in each CNS region. The transcriptomes of the cortex, hippocampus and spinal cord were the most responsive to age, while the transcriptome of the striatum was stable over time. The spinal cord transcriptome was remarkable for the large number of genes significantly upregulated with advancing age, in contrast to other CNS regions in which most genes were downregulated with advancing age. Gene lists are in Table S2a,b in Additional data file 1.
The gene lists for patterns 2-6 include genes in a range of functional categories (Tables S2aP2-S2aP6 in Additional data file 1). Among the six patterns of gene expression, we considered only those patterns in which a significant change occurred between middle and old age as aging-associated genes (AAGs; patterns 2-5); genes that did not change between middle and old age (patterns 1 and 6) were considered unlikely to be involved in the aging process. The numbers of AAGs differed by five-fold among CNS regions (Figure 2b). Surprisingly, the spinal cord exhibited the most AAGs, with more than 600 genes affected. The cortex and hippocampus were next in line (more than 400 genes), followed by the cerebellum and striatum with less than 200 genes each. The percentage of all genes that were AAGs ranged from 0.8% in the striatum to 3.8% in the spinal cord (cortex, 2.6%; hippocampus, 2.3%; cerebellum, 1.2%). The spinal cord was remarkable for the high number of AAGs that were upregulated (more than two-thirds of total AAGs); in contrast to the other regions for which far fewer AAG were upregulated (Table S2b in Additional data file 1). More AAGs were downregulated in the cortex and hippocampus than in the other CNS regions.
To provide insight into the biological processes affected by aging, we placed AAGs into 17 functional classes (Figure 3). Among the different functional classes, genes encoding proteins involved in transcriptional regulation, protein synthesis and degradation and signal transduction were the most responsive to aging across brain regions (Table S3 in Additional data file 1). Genes involved in cell cycle regulation, and growth factor and synaptic signaling were relatively unresponsive to aging. The spinal cord was notable in that most AAGs were upregulated across functional categories, in contrast to the other CNS regions (Figure 3). For most functional categories more genes were upregulated than were downregulated during aging in the spinal cord and striatum, whereas more genes were downregulated in the cortex, hippocampus and cerebellum (Figure 3; Table S3b in Additional data file 1). A decline in the expression of energy metabolism genes (those involved in mitochondrial function and glucose metabolism) is a shared feature of aging in all CNS regions examined (Figure S1 in Additional data file 1). This is consistent with other data suggesting that downregulation of mitochondrial gene expression may be central to the process of aging in the CNS [14].
Figure 3. Functional categorization of CNS age-related genes. Numbers above bars are the actual numbers of genes affected by aging in that gene category/CNS region. Functional categories (FC): FC1, DNA damage and repair; FC2, transcription regulators; FC3, RNA editing/processing; FC4, protein synthesis/degradation; FC5, signal transduction; FC6, growth factors and signaling; FC7, channels and transporters; FC8, cytoskeleton; FC9, trafficking; FC10, other synaptic function related ; FC11, stress response; FC12, immune responsive; FC13, mitochondrial function; FC14, cell cycle; FC15, glucose metabolism; FC16, lipid metabolism; FC17, amino acid metabolism. Gene lists are in Table S3 in Additional data file 1.
Transcriptome responses to caloric restriction are CNS region- and age-specific
Fewer than 0.5% of the genes in any of the CNS regions examined were significantly affected by CR, and the effects of CR on these genes did not exhibit a progressive change from young to old animals (Table 2; Table S1 in Additional data file 1). However, several interesting findings were evident in the analysis of age- and region-specific transcriptome responses to CR. Relatively few genes in any brain region were responsive to CR in six-month-old mice (Figure 4a; Table S4a-6M in Additional data file 1). Across ages, the transcriptomes of cells in the striatum and cerebellum were insensitive to CR, while cells in the other three brain regions were significantly more responsive to CR. The transcriptome of the hippocampus exhibited a dramatic increase in sensitivity to CR in middle-aged mice compared to young or old mice, with most of the affected genes exhibiting upregulation (Figure 4a; Table S4a-16M in Additional data file 1). The transcriptome of the spinal cord exhibited a progressive increase in sensitivity to CR with increasing age, with most genes being downregulated in middle and old age (Tables S4a-16M and S4a-24M in Additional data file 1). Genes in multiple functional categories were affected by CR in mice of each age, with those involved in amino acid and lipid metabolism, and signal transduction being notable for their responsiveness to age in all five CNS regions (Figure 4b). Interestingly, across CNS regions most CR-responsive genes are downregulated in young mice and upregulated in middle-aged mice; the CNS transcriptomes of old mice exhibited more variability among regions, suggesting impaired control of CNS region-specific processes. We next identified age-sensitive genes in each CNS region for which the effect of aging was negated by CR. In general, CR prevented age-dependent changes in the expression of only a small percentage of genes in each CNS region (Figure 4c; Table S4c in Additional data file 1). An exception was the spinal cord, where nearly 50% of the genes upregulated during aging were reverted in mice maintained on CR. Interestingly, however, fewer than 5% of the genes downregulated in the spinal cord during aging were reverted by CR.
Table 2. Genes consistently responsive to CR across advancingage in mouse CNS
Figure 4. CNS transcriptomes respond to caloric restriction in a region- and age-dependent manner. (a) CNS gene expression responses to CR. (b) Metabolic and signaling pathways in which genes were significantly affected by CR in all five CNS regions. Red, upregulated; blue, downregulated. (c) CNS AAGs reverted by CR. The percentage of AAGs reverted by CR is shown at the top of each bar. Note that the spinal cord exhibits a much larger percentage of upregulated AAGs that are reverted by CR compared to the other CNS regions. Gene lists are in Table S4a,c in Additional data file 1.
Analysis of CR-responsive genes for each age group revealed striking age-dependent differences within and between CNS regions (Figure 4c; Table S4c in Additional data file 1). In the case of the spinal cord, more than 90% of the CR-responsive genes were downregulated in 6- and 24-month-old mice, whereas in 16-month-old mice less than 40% of the CR-responsive genes were downregulated. In the cerebral cortex, most CR-responsive genes were downregulated in 6-month-old mice, whereas in 24-month-old mice most CR-responsive genes were upregulated. Similarly, most CR-sensitive genes in the hippocampus were downregulated in young mice, whereas in 16- and 24-month-old mice most CR-sensitive genes were upregulated. Very few genes were responsive to CR in striatum and cerebellum, regardless of age.
CNS transcriptomes of males and females are differentially affected by age and diet
The influence of gender on CNS transcriptomes is largely unknown, though relevant studies have been conducted [15-17]. The lists of genes that were differentially expressed in males and females regardless of age and diet (Table S5a in Additional data file 1) showed that the transcriptomes of the cerebral cortex, hippocampus and spinal cord were the most sensitive to gender (Figure 5a). As was the case with responses to age and diet, very few genes in the striatum were affected by gender. The hippocampus, which exhibited more genes affected by CR than any other CNS region (Figure 4a), was also notable for a very high number of genes that were differentially affected by CR in males and females (Figure 5b; Table S5b in Additional data file 1). In the hippocampus of young mice, relatively few genes were responsive to CR in either gender, with approximately twice as many genes responding in females compared to males (Figure S2 in Additional data file 1). There was a dramatic increase in the number of CR-responsive genes in the hippocampus of 16-month-old mice compared to 6-month-old mice; this increase occurred in both males and females, but was of much greater magnitude in males. There was also a marked increase in the percentage of the total number of CR-responsive genes that were upregulated in 16-month-old mice compared to 6-month-old mice. The number of CR-responsive hippocampal genes remained elevated in 24-month-old mice, but at this age females exhibited more than twice as many CR-responsive genes as males. In young mice the majority of CR-responsive genes were downregulated, whereas in 16- and 24-month-old mice most CR-responsive genes were upregulated (Figure S2 in Additional data file 1).
Figure 5. Gender-specific modulation of CNS transcriptomes by age and diet. (a) Numbers of genes that are differentially expressed in males and females in an age-dependent manner. (b) Genes differentially affected by CR in males and females. (c) Gene cluster analysis comparison of the effects of CR on hippocampal transcriptomes of male and female mice of different ages. Note the similarities in responses of young and middle-aged male and female mice, and the striking differential response of males and females to CR in old mice. (d) Age-responsive genes that were reverted by CR in males compared to females. Gene lists are in Tables S5a-d in Additional data file 1.
Gene cluster analysis was employed to further elucidate the interactions of age, diet and gender on CNS transcriptomes (Figure 5c; Table S5c in Additional data file 1). CR exerted generally similar effects on patterns of CNS gene expression in young and middle-aged male and female mice. In contrast, patterns of CR-sensitive gene expression in males and females were very different in old mice. We next calculated the percentage of age-responsive genes for which the age effect was abolished by CR, comparing males with females. This revealed that many more age-responsive genes were reverted by CR in males compared to females (Figure 5d; Table S5d in Additional data file 1), indicating that although many genes were responsive to aging and CR in both males and females, CR affected primarily genes that were age-insensitive in females. Thus, CR effects are not restricted to AAGs.
Chromosome mapping of age-, diet- and gender-responsive genes
Different genes that encode proteins that function in the same or similar biochemical processes can be located in physical proximity to each other in the genome, which may facilitate transcriptional co-regulation in the context of evolutionary selection [18,19]. We therefore generated and analyzed maps of the chromosome locations of genes that were significantly affected by age, diet and gender. Age-responsive genes and CR-responsive genes were scattered among chromosomes, with chromosomes 9 and 19 exhibiting the highest densities of age- and CR-responsive genes (Figure S3a,b in Additional data file 1). There were hot spots of age-responsive upregulated genes on chromosomes 4, 5, 6, 11 and 15, and of downregulated genes on chromosomes 5, 7 and 11. Clusters of CR-responsive upregulated genes were present on chromosomes 5, 6, 8 and 9, and of downregulated genes on chromosomes 4, 5, 6 and 11. Previous quantitative trait loci mapping of human populations identified the D4S1564 region of Homo sapiens chromosome 4 as a possible locus of genes that confer exceptional longevity [20], with a microsomal transport carrier protein as the possible locus [21]. The syntenic region in the mouse genome is located on chromosome 3 (Figure S3c in Additional data file 1); however, the mouse ortholog of the human microsomal transport carrier gene was not included in our array. Studies of cancer, aging and cellular senescence have identified a region of human chromosome 9 that includes genes coding the linked genes p16INK4a/ARF, which regulate the retinoblastoma (Rb) and p53 pathways [22]. Studies of the syntenic locus on chromosome 4 in mice (Figure S3d in Additional data file 1) have suggested roles for this region of the genome in mammalian aging. Two genes, coiled-coil domain containing 2 (ccdc2) and a functionally undefined gene RIKEN cDNA 6230416J20 (6230416J20Rik), located within p16INK4a/ARF locus were upregulated by CR in the 24 M cortex region. Age- and CR-responsive genes that were differentially expressed in males and females were scattered throughout the genome (Figure S3e,f in Additional data file 1).
Pathways involved in CNS aging and adaptive plasticity
A goal of transcriptome analysis is to identify individual genes and functional groups of genes that interact with each other to regulate physiological or pathological responses of cells, tissues and organisms. Many genes critical for tissue-specific or development-specific regulation are transcription factors and genes located on the cell signaling pathway hubs that play key roles in determining cell phenotypes. However, their changes are often subtle and hidden, and may be missed when routine array analysis statistics are employed. In the present study we identified three pathways of interest in CNS aging and neurodegenerative disorders (Figure 6a) that exhibited relatively high levels of age responsiveness compared to other pathways by hypergeometric function analysis; these pathways were analyzed using PathwayPro, our newly developed systems biology approach that is based on the Markov chain model for simulating dynamical changes of a pathway in response to intrinsic processes or external simulation [23].
Figure 6. Pathway transcriptomes responsive to age, diet and gender. (a) Left: the ubiquitin-proteasome protein degradation pathway. E1, E2 and E3 are ubiquitin activating, conjugating and ligating enzymes, respectively. Ub, ubiquitin; 19S and 20S are proteasome subunits. Middle: the Wnt β-catenin signaling pathways. Fzd, frizzled; LRP, lipoprotein-related protein; DKK1, dickkopf 1; Frat, frequently rearranged in advanced T-cell lymphomas-1; GSK3β, glycogen synthase kinase-3β; Nlk, nemo-like kinase; NF-AT, nuclear factor of activated T cells; TCF, T cell-specific transcription factor; Tab1, TGF-beta activated kinase-1 binding protein-1; Tak1, TGF-beta-activated kinase 1. Right: Werner (WRN) interacting proteins involved in DNA repair and telomere homeostasis. PARP-1, poly (ADP-ribose) polymerase 1; FEN-1, flap endonuclease 1; DNAPKcs, DNA-dependent protein kinase catalytic subunit; Ku, Ku p70/p80 antigen; MRN, MRE11-RAD50-NBS1 protein complex; BLM, Bloom syndrome; TRF1 and TRF2, telomere repeat-binding factors 1 and 2. (b) Frequency of UPS pathway gene sensitivity to age, gender and diet in the striatum (genes identified based on a threshold of perturbation probability ≥ 0.10). (c) The perturbation probability of Uchl1 gene and/or its combinations in different CNS regions during aging and in response to CR.
Impaired function of the ubiquitin-proteasome system (UPS) has been implicated in normal aging and the pathogenesis of neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis (ALS), in which abnormal proteins accumulate within neurons [24]. Numerous UPS genes were responsive to aging and CR, including those encoding ubiquitin E1, E2 and E3 ligases, and proteasome subunits (Table S6a in Additional data file 1). Particularly striking was the disproportionate number of UPS genes affected during the transition from middle to old age in the striatum compared to the other CNS regions, in contrast to the overall low responsiveness of the striatal transcriptome to aging and CR (Figure 6b). Most of the striatal age-related changes were reverted by CR, an anti-aging intervention shown to protect neurons in a mouse model of Huntington's disease [25] and a monkey model of Parkinson's disease [26], suggesting that they may be fundamental to the aging process. The PathwayPro analysis identified ubiquitin C-terminal hydrolase 1 (Uchl-1) as the most highly age- and CR-responsive UPS gene. Although the latter gene did not show significance by differential expression analysis, the network analysis indicated that it does contribute to the state change between ages/diets; therefore, PathwayPro analysis added a dimension of systems biology to this array-based study. These findings suggest that age-related changes in the UPS in the striatum and its input neurons in the substantia nigra may play a role in the vulnerability of these brain regions to Huntington's disease and Parkinson's disease, a possibility consistent with evidence from studies of animal models of these diseases [27]. UCHL-1, a susceptibility gene for Parkinson's disease [28], exhibited CNS region-specific age- and CR-related changes in expression (Figure 6c; Table S6c in Additional data file 1). Expression of UCHL-1 in the striatum was particularly sensitive, decreasing from middle to old age and increasing in response to CR.
The Wnt signaling pathway plays major roles in embryogenesis, including development of the CNS [29], but its involvement in CNS aging is unknown. Genes that encode proteins involved at multiple levels of the Wnt signaling pathway were affected by aging and CR, including the Wnt receptor frizzled, α-catenin, nemo-like kinase and calcium/calmodulin-dependent kinase II (Figure 6a; Table S6 in Additional data file 1). In addition, there were significant gender differences in the expression of Wnt signaling genes in the cerebral cortex and hippocampus.
Several different proteins involved in DNA repair and telomere function have been associated with the aging process, and with age-related cancers [30,31]. Among these, Werner was remarkable for its highly significant downregulation with aging in multiple CNS regions, and by the ability of CR to prevent the effect of aging on Werner expression (Tables S6 in Additional data file 1). Because loss-of-function mutations in Werner cause a premature aging syndrome [31], our findings suggest a role for Werner in normal aging of the CNS. Whereas approximately 3% of genes in the CNS transcriptome were significantly responsive to age, approximately 30% of the telomere-associated genes in the array were affected. These included those encoding telomeric repeat binding factor 1 (TRF1), telomerase binding protein p23, tankyrase 1, tankyrase-binding protein 1 and Werner. Werner, tankyrase 1 and several other telomere-associated proteins (Figure 6a) are known to play important roles in DNA damage response and repair processes.
Discussion
Our PCA established that each region of the mouse CNS possesses its own unique transcriptome signature that distinguishes it from other regions regardless of the age, gender or diet of the animal. Although previous studies have shown that different CNS regions have unique patterns of gene expression [32-34], our study demonstrates that the region-specific patterns of gene expression are maintained regardless of the age of the animal, its dietary energy intake and its gender. These signature transcriptomes presumably represent the molecular basis of the phenotypic differences in the cells that comprise the different CNS regions and, by extension, regional functionality. Changes in many different common and cell type-specific genes in the CNS are known to occur in response to environmental factors, including age, diet, exercise, activity in neuronal circuits, and injury or disease [11,35-40]. However, our findings suggest that such epigenetic responses to the environment do not alter the fundamental transcriptome 'fingerprints' that distinguish different regions of the CNS.
The false discovery rate (FDR), an approach widely applied to microarray data analysis, allows the researcher to balance the size of the candidate gene list against its quality in order to enhance confidence in the validity of the data, and is particularly well-suited to large datasets such as ours. However, we found that the variability in gene expression was considerably larger in samples from old compared to young animals (Figure S4 in Additional data file 1), a result consistent with a recent study [40]. The increased variation with advancing age results in higher p values, and q-values as well since the q-value is computed from the p value. Inasmuch as aging is considered a stochastic process, it should be expected that the effect of natural aging on the gene expression is more variable when compared with gene expression effects of more well-defined and dramatic experimental manipulations or disease states. Our PCA analysis (Figure 1) also provided evidence that the factor of age has only minor effects on the CNS regional transcriptomes. FDR is typically applied to large (robust) effects of factors on gene expression [41]. Because many of the genes that were significantly affected by age in our study exhibited relatively small changes, the FDR was not, therefore, applied to this dataset. However, it should be noted that the increased variance in p values in the old cohort may confound findings concerning the numbers of genes identified as changed in the old group compared to the younger group.
A change in the expression of a gene during aging might contribute to a decline in function and degeneration of neural cells or, instead, might be an adaptive response to aging. The greater number of genes upregulated by aging in the spinal cord may represent a superior ability of cells in the spinal cord to adapt to aging, perhaps because it is the most primitive part of the CNS and the most essential for survival. On the other hand the relatively greater proportion of white matter (olidodendrocytes) in the spinal cord may also contribute to the greater effect of aging on the spinal cord transcriptome compared to the four brain regions examined. Interestingly, many genes downregulated during aging in brain regions were upregulated in the spinal cord, including genes of the UPS, for example (Table S3 in Additional data file 1). In contrast to the spinal cord, very few genes in the striatum were affected by aging and CR and most of those that did respond, including those in the UPS, were downregulated with age and upregulated by CR. These findings suggest the striatum may be prone to age-related diseases, such as Huntington's and Parkinson's, because its transcriptome does not respond adaptively during aging.
Prior gene expression studies of brain aging included only young and old animals of one gender, typically used microarrays with relatively few genes and often analyzed pooled RNA samples resulting in negligible statistical power [11-13]. We therefore analyzed RNA isolated from five different CNS regions from male and female mice of three different ages and two different diets (three to five mice analyzed for each age, gender and diet) using a large mouse gene array. Inclusion of the middle-aged group revealed a caveat with previous studies of 'aging' in which comparisons are made between young and old individuals only. We found that many genes that would have been considered sensitive to aging in a young versus old comparison are, in fact, changed only between young and middle ages with no further change between middle and old age. Indeed, in the cerebellum, 64% of the age-responsive genes followed the latter pattern (Figure 2a). In the hippocampus, 27% of the genes that were significantly affected by age changed between young and middle age, and then returned to the young level in old age. On the other hand, we found that it was extremely rare for the expression level of a gene to change in one direction between young and middle age, and in the opposite direction between middle and old age.
A comparison of our data with those of previous gene array analyses performed on RNA samples from the cerebral cortex of mice [11] and humans [36] revealed only five genes that were significantly affected by age in all three studies (Table S7a in Additional data file 1). Two of the genes (vimentin and GFAP) encode astrocyte cytoskeletal proteins previously shown to be upregulated in aging and neurodegenerative disorders [42]. The other three genes encode a cell adhesion molecule (ICAM2), a protein that interacts with SIRT1 and p53 in cellular stress response signaling (NDRG1) [43] and a putative energy and nutrient sensor (FRAP1) [44]. An additional nine genes were common to the cerebral cortex datasets of the two mouse studies and included those encoding proteins involved in cell senescence, mitochondrial translation initiation and protein phosphorylation (Table S7b in Additional data file 1). A comparison of our mouse cerebellum dataset with that of Lee et al. [11] identified eight genes significantly affected by aging (Table S7c in Additional data file 1), including those encoding proteins involved in proteolysis (ubiquitin-specific peptidase 46 and cathepsin Z), transforming growth factor-β signaling (TGFβ receptor 3) and nitric oxide signaling (endothelial nitric oxide synthase). A comparison of our mouse cortical dataset with the human frontal cortex data [36] identified nine genes significantly affected by aging (Table S7d in Additional data file 1), including those involved in calcium signaling (voltage-dependent calcium channel beta-2 subunit and calcium/calmodulin-dependent kinase 3), neurotransmitter signaling (gamma-aminobutyric acid (GABA-A) receptor subunit beta 3) and oxidative stress responses (thioredoxin interacting protein). A comparison of hippocampal gene expression profiles in young (2 month old) and middle-age (15 month old) C57BL/6 mice identified 35 genes as being significantly upregulated; they included genes related to synaptic plasticity, inflammation, oxidative stress and protein processing [45]. Blalock et al. [37] performed microarray analyses of approximately 2,000 genes in hippocampi from young, middle-aged and old rats, and correlated changes in gene expression with performance of the rats on learning and memory tasks. Many of the AAGs in the latter two studies were in the same functional categories as AAGs in our study, including calcium signaling, oxidative stress and proteolysis.
In addition to functional classes of genes documented in previous studies of brain aging and neurodegenerative disorders, our findings identified Werner/telomere-interacting proteins and the Wnt signaling pathway as being highly responsive to both aging and dietary energy restriction throughout the CNS. Werner is a DNA helicase that plays a pivotal role in DNA repair and telomere function [31]. Loss-of-function mutations in Werner cause a premature aging syndrome that includes neurological abnormalities. A brain imaging study of two siblings with Werner's syndrome provided evidence for reduced cerebral energy metabolism compared to age-matched control subjects [46]. The latter findings, evidence for reduced energy metabolism in the brain during normal aging [47], and our finding of significantly reduced expression of Werner in the brain during normal aging suggest a possible role for Werner in age-related compromise of brain function. Werner interacts with several telomere-associated proteins also implicated in cellular senescence (Figure 6a). Increasing evidence suggests that telomerase and other telomere-associated proteins play roles in neuronal plasticity and survival [48,49]. The responsiveness of several telomere-associated proteins to aging and CR suggests roles for telomere modifications and DNA damage and repair processes in CNS aging. In addition to playing major roles in CNS development [29], the Wnt signaling pathway has been implicated in adult neural plasticity and the pathogenesis of neurodegenerative disorders [50]. Several genes in the Wnt signaling pathway were prominently affected by aging and CR in several CNS regions in our study, including those encoding nemo-like kinase, α-catenin and calcium/calmodulin-dependent kinase 2. Each of the latter proteins is involved in mechanisms of signaling associated with the pathogenesis of neurodegenerative disorders [51-53], suggesting a role for age-related perturbation in Wnt signaling in the disease processes.
Chromosome mapping of genes that were differentially expressed in mice of different ages and/or in response to CR revealed a wide distribution of genes with some physical clustering of responsive genes within the genome. The latter findings are consistent with the concept that aging is a complex process and that evolutionary adaptations to aging, if they exist, may or may not involve geographic clustering of functionally related genes.
Despite the existence of many phenotypic differences between males and females, the vast majority of gene expression analyses have been performed on males, and direct comparisons of CNS transcriptome responses of males and females to aging and environmental factors are lacking. However, analysis of the expression of 4,000 genes in trained and untrained muscles of young and old men and women revealed that more genes were affected by gender than by age or training [54]. In our study, numerous genes, spanning functional categories, were differentially expressed in the CNS of males and females (Tables S5a-d in Additional data file 1). In general, genes involved in protein degradation, oxidative stress resistance and cell survival were expressed at higher levels in females compared to males, suggesting a superior ability of brain cells in females to resist age-related oxidative and metabolic stress. Interestingly, there was considerable variability in the numbers of genes affected by gender among CNS regions, with the hippocampal transcriptome being the most sensitive to gender and the striatum and cerebellum the least sensitive. The proteins encoded by genes differentially expressed in the CNS of males and females may determine gender-specific differences in behaviors, responses to dietary energy intake and susceptibility to age-related dysfunction and disease.
Conclusion
The factors of age, gender and energy intake significantly affected less than 10% of examined genes but did not considerably influence the unique transcriptome of any CNS region. The transcriptomes of each CNS region showed distinctive responses to the age, diet and gender. This systematic transcriptome dataset provides a window into mechanisms of age-, diet- and sex-related CNS plasticity and vulnerability.
Materials and methods
Mice and dietary manipulations
All mice (male and female C57BL/6 mice) were obtained from the same breeding colony [55], and were maintained in the same facility on either ad libitum (AL) or CR diets. CR was initiated at 14 weeks of age at 10% restriction, and then changed to 25% at 15 weeks and 40% restriction at 16 weeks onward. Mice were housed individually in standard cages with free access to water, and were maintained on a 12 hour light/12 hour dark cycle. Cohorts of male and female mice were euthanized at 6, 16 and 24 months of age (Table 1). Mice were sacrificed at the same time of day to control for diurnal variation.
Tissue dissection, RNA extraction and cDNA-array analysis
Mice were euthanized by cervical dislocation and five CNS tissues: cerebral cortex, cerebellum, hippocampus, striatum, and spinal cord were removed and flash frozen. All samples were processed by the same investigator (XX) in balanced batches. The tissue was processed using a Bead Beater (Bio-Spec, Bartlesville, OK, USA) followed by RNA purification using RNEasy Mini Kits (Qiagen, Valencia, CA, USA). The RNA was evaluated for quantity and quality using a Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Five micrograms of each RNA sample were used in a PCR reaction with 32P-dCTP (Valeant, Costa Mesa, CA, USA). Radiolabeled cDNA was allowed to hybridize overnight at 43°C to the mouse NIA 17K cDNA filters [56]. Details on these methods are available elsewhere [57]. The hybridized filters were washed and placed under imaging screens for three days to allow for sufficient exposure. The images were developed and scanned, and the data were extracted using ArrayPro Software (Media Cybernetics, San Diego, CA, USA).
CNS transcriptome data normalization and differentially expressed gene selection
All data were first processed by Z score transformation [58] and then genes that were differentially expressed were identified by a multi-step process in which: only genes for which the average intensity between the two conditions was greater than zero - this eliminates spurious selection of genes of low expression level compared to the background; and the remaining genes were then tested to identify those for which z-ratios were greater than 1.5 or less than -1.5 and a p value < 0.05. The z-ratio is a measure of fold change between comparisons, and the p values test for reproducibility of a gene's intensity among biological replicate arrays. Z-ratio (between condition A and B) = z(A) - z(B)/SD deviation). Remaining genes were analyzed by two-way ANOVA to establish the statistical significance of differential levels of expression between ages, genders and diets (p < 0.05). This analysis was performed on DIANE 1.0, a new microarray analysis tool developed by VV Prabhu (see SDIANE in Additional data file 1). All gene lists were annotated with the recently updated RefSeq (RefSeq-release23) and UniGene database9 (May 2007) [59]
Gene expression pattern recognition
PCA was performed using Partek software (Partek Inc., St. Louis, MO, USA) to establish CNS region-specific transcriptome patterns that were independent of age, diet and gender. Hierarchical clustering analysis and heatmap were conducted using Cluster and TreeView [60].
PathwayPro analysis of pathways
With a Java-based interactive computational tool, PathwayPro, we analyzed the ubiquitin-proteasome system, Wnt signaling pathways and Werner-related DNA repair/telomere systems. PathwayPro (PP in Additional data file 1) is a novel computational algorithm we developed for systematic characterization of network dynamic behavior by modeling the correspondence between network activity for specific interventions, or perturbation applied to a network [23]. Intervention was simulated mathematically by altering the expression of each gene or gene combination, while perturbations were external variables such as age, diet and gender. See PP in Additional data file 1 for a description of the PathwayPro analysis methods.
Real time RT-PCR validation of microarray results
The same RNA samples used for microarray analysis were used to validate the microarray results for selected genes using real-time RT-PCR methods. Standard protocols were used for the generation of cDNA from RNA following elimination of genomic DNA contamination using DNA-free (Ambion, Austin TX, USA). Oligonucleotide primers (Promega, Madison WI, USA) and PowerScript reverse transcriptase (Clontech, Mountain View CA, USA) were added to the RNA samples to prepare the first-strand cDNAs. Samples from the latter mixture were diluted with distilled water and subjected to real-time RT-PCR analysis. The real-time PCR reaction (20 μl) consisted of template, gene specific primers, and SYBR Green PCR master mix. The mixtures were added to wells of a 96-well plate and analyzed in an OPTICON real-time PCR analyzer (MJ Research, Waltham MA, USA). The sequences of the primers used and the results of the validation of microarray data by real-time RT-PCR are listed in SPrimers in Additional data file 1. The results for each gene analyzed (those encoding HSPc-α, HIF1-α, Werner, Jagged-1, ELOVL family member 6 and Nemo-like kinase) are shown in SRTPCR in Additional data file 1; these show that more than 85% of gene expression alterations in all given conditions are consistent between RT-PCR and array data.
Abbreviations
AAG, aging-associated gene; AL, ad libitum; CNS, central nervous system; CR, caloric restriction; FDR, false discovery rate; PCA, principal components analysis; UPS, ubiquitin-proteasome system.
Authors' contributions
MMP, LDL, and BKG designed research; XX, DW, BR, WW, ZP, and IC performed research; ZM, PV, FJ, PS, and ZAB contributed new reagents or analytical tools; XX, ZM, and LH analyzed data; XX, and MMP wrote the paper.
Additional data files
The following additional data are available with the online version of this paper. Additional data file 1 contains supplemental figures, tables and files. Figure S1 shows one feature of aging shared across CNS regions, namely, the decline of energy metabolism. Figure S2 shows age-related changes in responsiveness of the hippocampal transcriptome to CR in males and females. Figure S3a-3f shows chromosome mapping of CNS age- and diet-responsive genes and loci. Figure S4 shows age-dependent gene expression variation analysis. Table S6 lists the perturbation probabilities of UPS, Wnt, and WRN pathways in different sexes of CNS regions during aging and in response to CR. Overview Of DIANE 1.0 provides an outline of this program. PathwayPro describes the PathwayPro computational tool Gene-specific primers used for Real-Time PCR analyses and microarray quality validation lists the primers for genes validated by quantitative RT-PCR Table S2aP2-6 lists genes of mouse CNS age-related gene expression patterns 2-6. Table S2b lists mouse CNS AAGs. Table S3 lists functional categories of known mouse CNS AAGs. Stable 1 shows genes consistently responsive to CR across advancing age in mouse CNS. Table S4a-6M-16M-24M lists CR responsive genes of 6M-16M-24M mouse CNS. Table S4c lists the impact of CR on the reversion of AAG expression. Table S5a lists age-dependent differentially expressed genes in males and females. Table S5b,c lists genes differentially affected by CR in males and females. Table S5d lists age-responsive genes that were reverted by CR in males compared to females. Table S7a-d lists results of cross comparison between this study and previous published data.
Additional File 1. Figure S1 shows one feature of aging that was shared among CNS regions, namely, the decline of energy metabolism. Figure S2 shows age-related changes in responsiveness of the hippocampal transcriptome to CR in males and females. Figure S3a-3f shows chromosome mapping of CNS age- and diet-responsive genes and loci. Figure S4 shows age-dependent gene expression variation analysis. Table S6 lists the perturbation probabilities of UPS, Wnt, and WRN pathways in different sexes of CNS regions during aging and in response to CR. Overview Of DIANE 1.0 provides an outline of this program. PathwayPro describes the PathwayPro computational tool Gene-specific primers used for Real-Time PCR analyses and microarray quality validation lists the primers for genes validated by quantitative RT-PCR. Table S2aP2-6 lists genes of mouse CNS age-related gene expression patterns 2-6. Table S2b lists mouse CNS AAGs. Table S3 lists functional categories of known mouse CNS AAGs. Stable 1 shows genes consistently responsive to CR across advancing age in mouse CNS. Table S4a-6M-16M-24M lists CR responsive genes of 6M-16M-24M mouse CNS. Table S4c lists the impact of CR on the reversion of AAG expression. Table S5a lists age-dependent differentially expressed genes in males and females. Table S5b,c lists genes differentially affected by CR in males and females. Table S5d lists age-responsive genes that were reverted by CR in males compared to females. Table S7a-d lists results of cross comparison between this study and previous published data.
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Acknowledgements
We thank Dr Zhu Chen for his critical comments on the manuscript, and members of the NIA Laboratory of Neurosciences for their support of this project. This research was supported by the National Institute on Aging Intramural Research Program.
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Neuromolecular Med 2003, 3:65-94. PubMed Abstract | Publisher Full Text
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37. Blalock EM, Geddes JW, Chen KC, Porter NM, Markesbery WR, Landfield PW: Incipient Alzheimer's disease: microarray correlation analyses reveal major transcriptional and tumor suppressor responses.
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38. Cavallaro S, D'Agata V, Manickam P, Dufour F, Alkon DL: Memory-specific temporal profiles of gene expression in the hippocampus.
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51. Phiel CJ, Wilson CA, Lee VM, Klein PS: GSK-3alpha regulates production of Alzheimer's disease amyloid-beta peptides.
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54. Roth SM, Ferrell RE, Peters DG, Metter EJ, Hurley BF, Rogers MA: Influence of age, sex, and strength training on human muscle gene expression determined by microarray.
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56. Nadon NL, Mohr D, Becker KG: National Institute on Aging microarray facility--resources for gerontology research.
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57. Tanaka TS, Jaradat SA, Lim MK, Kargul GJ, Wang X, Grahovac MJ, Pantano S, Sano Y, Piao Y, Nagaraja R, et al.: Genome-wide expression profiling of mid-gestation placenta and embryo using a 15,000 mouse developmental cDNA microarray.
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Seth says he wants to banish anonymous communications.
Seth Godin
Virus writers are always anonymous.
Vicious political lies (with faked photoshop photos of political leaders, or false innuendo about personal lives) are always anonymous as well.
Spam is anonymous.
eBay fraudsters are anonymous too.
It seems as though virtually all of the problems of the Net stem from this one flaw, and its one I’ve riffed on before. If we can eliminate anonymity online, we create a far more civil place.
I disagree. Although most vicious attacks I have received have been anonymous, I still believe there is a role for anonymity and that the value outweighs the cost.
The American Association for the Advancement of Science has a project on anonymous communication on the Internet. They list a few of cases where we might need anonymous communication on the Internet.
AAAS
Case 1 - The Crimesolvers Website
Case 2 - Chatting Online About Addiction
Case 3 - The Case(s) of the Hot News Tips
Case 4 - An Anonymous Computer Hotline: Is it Worth the Costs?
Case 5 - Terror in Elb!
Case 6 - Good Communication Gone Bad
Case 7 - His Word Against Whose?
Remember that the Internet is one of the few tools for a variety of people who are at risk including whistle-blowers and human rights workers. It is very difficult or impossible to "fix" the Internet without breaking it for others.
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About this Archive
This page is an archive of recent entries in the Business and the Economy category.
Books is the previous category.
Computer and Network Risks is the next category.
Find recent content on the main index.
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[Date Prev][Date Next][Thread Prev][Thread Next][Date Index][Thread Index]
Re: [tdf-discuss] Copyright Assignments & the Document Foundation
On Thu, Oct 28, 2010 at 9:18 AM, Charles-H. Schulz <
charles.schulz@documentfoundation.org> wrote:
> Hello all, (apologies for this quite long email)
>
> I would like to discuss a bit the position of the Document Foundation
> with respect to copyright assignments. I understand there have been
> questions here and there about this topic, and it's perhaps necessary
> to explain our position.
>
> We initially agreed not to request the assignment of copyright for code
> contributions, and we can only witness that it's been so far the right
> decision: Many developers have joined us and contribute to the
> LibreOffice codebase or extend it by localizing it and testing
> LibreOffice.
>
> We knew ever since the beginning that imposing a copyright assignment
> would be a big minus for developers. For one thing, it represents
> complexity for developers, and on the other hand, the experience we had
> with the copyright assignment under the stewardship of Oracle speaks
> for itself. It is also worth noting that in practical terms, the bulk
> of the LibreOffice codebase, that is, everything except our new
> patches, our new code, the localizations, the hacks, etc. is still
> under copyright from Oracle. Also, as a warning of sorts, keep in mind
> that copyright assignments are not the same thing as software licenses.
>
> I am going to write below some of the reasons why I also think that not
> having a copyright assignment is either a good idea or does not really
> matter at all.
>
> 1) no one has yet been able to clearly articulate what advantage we
> would gain by having one for TDF. For instance, it's not at all clear,
> and is in fact quite likely than any major software vendor would be
> shunned away from our project if we had a copyright assignment: it
> would basically mean that we would own their "intellectual property",
> and I'm not so sure it flies well with corporate lawyers in charge of
> protecting it.
>
> 2) the state of the art in terms of such assignments is changing
> rapidly. We stand at a corner of FOSS history, where the realization
> that projects led by one vendor only tend to fail, unless the vendor
> itself puts others in charge of the projects and gives free reins to
> its community. Look at what's happening with Fedora with respect to
> its ditching of copyright assignments. Experiences in other projects
> show that the "protection" that such assignments provide is at best
> minimal, and most of the times quickly abused, most of the time by its
> steward.
>
> 3) copyright assignments are not blocking the reuse of code or
> anything similar; there are several reasons for this, but one which is
> practical: a few years ago, you had a central branch with a tool like
> CVS. In the CVS (and even SVN) there was a real hierarchy. There was my
> branch and you were contributing to it. Now, many projects use similar
> tools, except that they are in fact quite different: they are
> distributed: there are as many different copies as there are
> developers; and the choice is social (people agree on what's best or
> respect the guy who has the biggest beard or something like this). So
> people create a big heap of code, and if they want to create their own
> stuff in their own corner, they do it; they don't deal with
> hierarchies, and paperwork. If they're not happy, they leave. That's
> how it works today. BTW; LibreOffice uses Git, which is a distributed
> SCM.
>
> 4) the notion that we cannot change license because we don't have
> copyright assignment needs to be put to rest once and for all today.
> There is a very simple explanation with respect to this issue; ask any
> lawyer and he/she will confirm this: Sun/Oracle has licensed the OOo
> code under LGPL v3. They could have put "LGPL v3 or later" or "LGPL v3
> or +". But they didn't. And that's what makes impossible to turn OOo
> into a different license unless the sole copyright owner agrees to
> change it, which is unlikely with Oracle.
>
> 5) based on my 4) point, you can object that without a copyright
> assignment, we would be stuck with the same license for ever, since we
> would not be able to decide to change the license of our lines of
> code. In fact, the problem lies in the heap of code we would have to
> change in order to be able to turn the whole code into something
> else... but here's what the developers of LibreOffice did: they simply
> didn't change the license, they started to license their own changes
> under the same license (LGPL V3)... and added : "or +" after it. So the
> license will change or at least be modified that way. But what if we
> want to change the whole thing? well, we'll contact all the authors who
> got their code into LibreOffice. We have their emails, etc. And if some
> of them don't agree with us, then perhaps we'll have to redevelop their
> own code.
>
> 6) there is also a confusion between copyright assignment and copyright
> protection. True, when you assign code to the FSF, you do expect to be
> legally protected against unpleasant surprises. But developers can also
> decide they don't like the FSF so you will have lost your
> effective control over what you develop. One might object, then, that
> if someone sues you, you would be better off with an entity
> protecting you. Usually, the patent trolls and the suers on code of
> this world don't attack individuals. They attack entities with money.
> On to the money question...
>
> 7) what if we had money to protect our code? Well, we may still not want
> to lose developers for that. But we could do something else: acting as
> the defenders of all the copyright owners. And then, it does not
> require a copyright assignment, it only requires, if a problem arises,
> that enough contributors or all the contributors signs a small paper
> saying "The Document Foundation is representing us legally in xyz case".
> That's all.
>
> Your questions are welcome, but I hope it helped clarified this
> question.
>
I understand not requiring any form of copyright assignment, but what about
voluntary copyright assignment?
--
Unsubscribe instructions: Email to discuss+help@documentfoundation.org
Posting guidelines: http://netmeister.org/news/learn2quote.html
Archive: http://www.documentfoundation.org/lists/discuss/
*** All posts to this list are publicly archived ***
References:
[tdf-discuss] Copyright Assignments & the Document Foundation"Charles-H. Schulz" <charles.schulz@documentfoundation.org>
Privacy Policy | Impressum (Legal Info) | Copyright information: Unless otherwise specified, all text and images on this website are licensed under the Creative Commons Attribution-Share Alike 3.0 License. This does not include the source code of LibreOffice, which is licensed under the GNU Lesser General Public License (LGPLv3). "LibreOffice" and "The Document Foundation" are registered trademarks of their corresponding registered owners or are in actual use as trademarks in one or more countries. Their respective logos and icons are also subject to international copyright laws. Use thereof is explained in our trademark policy.
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Biomod/2012/TeamSendai/Idea
From OpenWetWare
< Biomod | 2012
Revision as of 10:46, 26 October 2012 by Naito Ushio (Talk | contribs)
Jump to: navigation, search
Team Sendai Top
Project
motivation
We want to make an artificial channel.
If we make an artificial channel, it is desirable that the channel can carry selectively
and actively only what we want to carry.
Also, it is desirable that the channel can change its function and shape artificially.
Think of these things, we think it is best to make this structure with DNA-origami.
If we use DNA, the shape of the channel is changeable as we like.
Also, the carry structure that through the channel is possible by using DNA’s complementarity,
so we decided to use DNA to make the structure.
Experiment goal
When we test the effect of the Cell-gate, we use liposome as a model of a cell membrane.
We have to make liposome and confirm the effect of the Cell-gate.
So finally, we will make molecular robot which is Cell-gate on liposome and transport the object inside or outside liposome.
In short, our goal in this experiment is following. (D－Heart画像)
Three experiment parts
But on experiment, it is not smart that proceeing our project in order.
Luckily, large number of people in our team(and most of us are fresh!).
So we decided to separate our project into several part and do experiment parallelly.
Our experiment separates three parts; Gate part, Porter part, and Membrane part.
(３つのグループに分かれてる画像端っこにシミュレーション班もいるよ的な感じで)
Gate part is the group making the Cell-gate itself.
Porter part is the group making the function to transport the target in the channel.
Membrane part is the group making liposome by using lipid.
To separate our project and finally mix, we aim to gain our achievement.
And we also establish simmulation group that verifies each structure theoretically.
Gate
We will make the gate made of DNA origami! DNA origami is the way how to fold DNA and make structure investigated by Paul Rothemund.
So we decided to make the tube structure as the Gate using DNA origami. Consideration for the form of the Gate is written on Design page "Gate".(ここにデザインページGATEへのリンクを!..)
In the Gate, "Porter" which transport the target is planted. So Gate can connect the inside and outside.
We can use the Gate as an injector or extractor.
The simulation that the targets actually enter in the Gate is here.(simlationへのリンクを！..)
We considered about annealing situation of the Gate and did electrophoresis and AFM for observing the Gate.
Consideration for annealing situation and experiment results is here(ここにexperimentのリンクを！..)
Porter
We thought to make DNA Porter which is function to transport the target in channel. One of the characteristic of DNA is to bind another DNA comprementary sequence to it. If we design that the DNA binds the target more stable than former one, and if next DNA binds more stable than it…, the target moves to most complementary sequence DNA. We thought this characteristic of DNA can be utilized the power of channel. This channel can make us transport the object selectively and actively independent of concentration gradient. We deceided to make the Porter made of DNA. DNA sequence of Porter is here.（Designページporterへのリンクを!...） And simulation of suitable Porter length.（simulationへの以下略） We did electrophoresis to confirm working of DNA. Experiment method and result is here.(experiment he no rinku wo!)
Membrane
reference from "the CELL" (画像の下に置きたいセリフ)
In this project, We make the model of cell membrane and aim that the channel penetrate it.
Because using cell membrane immediately is hard.
We make liposome by using lipid.
Liposome is the membrane made of lipid and utilize liposome as model of cell-membrane.
Consideration making situation of liposome is here.(リンクおね)
And experiment method and result is here.(ここからリンクよろ)
Application in future
Finally, this project aims to attach to real cell and transport a substance to cell and from cell. Of cause, this channel can be applied to medical use. Also, it can be used for bring some substance which it is difficult to bring back now from cell. In this experiment, we used liposome as a model of a cell membrane, but if we consider the channel attached liposome as one robot, the robot can use to cleaner robot or medical sprinkling robot.
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IGEM:Peking/2007/Count:Gel-Extraction
From OpenWetWare
Jump to: navigation, search
quick gel purifcation
according to Transgen kit protocol
1. Agarose gel electrophoresis,cut the DNA band as thin as possible.pute into a clear EP tube.weighing,if the weight is 100mg,the volume can be considered as 100uL,add solution GSB,put into 55C water bath until complently merge,it takes about 5 minutes
2. cool the tube with water to adjust the tempertature of the solution to room temperature
3. Add the solution into the collection tube, standing for 1 minute,13000rpm for 1 min, discard the flow-through
4. Add 650 uL WB,13000rpm for 1 min,discard the flow-through
5. 13000rpm for 2 min,discard the flow-through
6. Put the collection tube into a clean EP tube, Add 50 uL 60C ddH2O in the center of the collection tube,standing for 1 min
7. 13000rpm for 1 min.
8. store the DNA solution at -20C
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laurabar24's bookmarks
"Progress is impossible without change, and those who cannot change their minds cannot change anything."
Shaw, George Bernard on change
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My book as a pdf
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Welcome parties explain difference between Boston & Miami
John - Red's Army June 7, 2012 Celtics News 6 Comments
CSNNE does a wonderful job getting us ready for tonight’s game with a nice, four-minute video contrasting the introductions of each newly constructed Celtics and Heat team. It’s pretty much all you need to know about each team, and why one has found success while the other is… well… struggling with the whole thing right now.
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Posts: 57 | Thanked: 13 times | Joined on Jun 2012
#1
Hi All
I dont suppose Nokia will ever release them, but I am asking if anyone maybe from Noka, Jolla or a dev has the tools that would enable us to extract and repackaged the firmware for the N9 / N950 please make them availbale to us. This would enable us to be able to modify the firmware and allow modders and devs to make our amazing phones even better as we will proably never recieve any more firmware updates from Nokia. So anyone that may have access to the tools please could you make them available to us.
Regards
Adam
Moderator | Posts: 5,845 | Thanked: 5,278 times | Joined on Nov 2011
#2
I don't mean to interrupt your party but are you going to be the one who will mod the firmware files?
If the devs here knew it was possible wouldnt they have already done it?
Lastly, i would refer you to rainisto's post which was on a thread titled "unpack firmware": http://talk.maemo.org/showpost.php?p...61&postcount=4 Read thro' the whole thread and you will get all your answers...
Isn't it easier to search, read and discover instead of starting a new thread which is not helpful to you as you will not be modding the fw?
Edit: Firmware updates don't need you to unpack the fw so a CSSU-type implementation is already in discussion if you searched it out...
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Should Southland really be there? Alot of the stores have shut down and I don't know if people are going to be going there anymore.
Based on the traffic jam to get off the Freeway during the recent Christmas shopping season, I'd have to say there is still plenty of life in that antique mall. -- Colin 03:25, 2 January 2007 (EST)
Being a crappy mall, there is still a lot of shopping that goes on there. I try to avoid it but everytime I go there on the weekend it is still fairly busy (busier than Newpark Mall). GammaRei 13:37, 8 March 2007 (EST)
As of 2012, the mall is definitely active, with a full complement of anchor stores, smaller stores, and food, including an Elephant Bar. It can stay here.Mercurywoodrose 14:17, 9 June 2012 (EDT)
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• Close to a third (32%) of people participating in the labour force were mature age (those aged 45-64 years), up from 24% in 1983.
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• The Education industry employed the highest proportion of mature age workers, with 47% employed in this industry aged 45-64 years. Other industries with higher proportions of mature age workers include Agriculture, forestry and fishing (44%) and Health and community services (42%).
• The unemployment rate for 45-64 year olds was 3.5% representing 114,000 people in this age group who were looking and available to work, compared with an overall unemployment rate of 5.4%.
• Among unemployed 45-64 year olds, the proportion who were long-term unemployed (i.e. had been unemployed for 52 weeks or more), was nearly twice as high (40%) as the proportion for all unemployed people (23%).
For further details, refer to the article 'Mature age workers' included in
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Emin Valley steppe
Ecoregions:
Emin Valley steppe
Satellite view of the Emin Valley, on the border of Kazakhstan and China Photograph by USGS
This article has been reviewed by the following Topic Editor: Mark McGinley
This grassland and steppe-dominated ecoregion on the border between northwest China and Kazakhstan includes cold temperate mountains and a system of shallow saline lakes that provide breeding habitat for many waterfowl including two globally threatened bird species, Dalmation pelicans (Pelecanus crispus) and relict gulls (Larus relictus). The main threat to this ecoregion is mineral and oil extraction; Kazakhstan is well known for its mineral deposits.
Location and General Description
The Emin Valley is located along the China-Kazakhstan border, separated by dry mountains from the Junggar Basin to the east. Landscapes of this ecoregion are predominantly montane grassland with shrub-steppe below 2,000 meters (m) and meadow steppe and alpine meadow above. Grasses include the Central Asian steppe species, Festuca ovata, Stipa capillata, and S. glareosa.
Kazakhstan has a continental climate with seasonal temperature extremes. Winters are long and cold with snow cover lasting from 50 to 150 days depending on the region while summers are short hot and dry with an annual precipitation varying from less than 100 millimeters (mm) to 400 mm. Average temperature ranges in the north are from -18?C (degrees Celcius) in January to 19?C in July, while in the south it ranges from -3?C in January to 30?C.
The western end of the Emin Valley ecoregion is perhaps the most biologically distinctive part. It includes two large and shallow saline lakes, Alakol and Sasakol. These lakes and their surrounding wetlands comprise 6,000 square kilometers (km2) in the extreme southeast of Kazakstan, south of the Tarbagatai Mountain Range. Lake Alakol has several small, low islands that provide important breeding grounds for wetland birds and are protected as part of the Alakol State Sanctuary (Kazakhstan). The lakes are surrounded by steppe vegetation and separated from one another by a large expanse of Phragmites spp. reedbeds.
Biodiversity Features
This ecoregion provides habitat for several mammal species of conservation significance, although it is not certain that they are currently present within the ecoregion. These include ibex (Capra ibex) and Saiga antelope (Saiga tartarica), and possibly their predators, snow leopards (Uncia uncia), and wolves (Canis lupus).
Both Alakol and Sasakol lakes have historically supported breeding populations of the globally threatened relict gull (Larus relictus) and globally threatened Dalmatian pelican (Pelecanus crispus). Surveys in 1998 located a large number of pelicans, but failed to record any relict gulls. The little bustard (Otis tetrax) breeds in the Tarbagatai Mountains on the northern side of Emin Valley.
Tacheng Nature Reserve (15 km2) supports riparian communities that contain wild almond (Prunus amygdalus) and its habitat.
Current Status
Concern has been raised about the status of relict gull breeding populations on Lake Alakol. None were observed during a visit in 1998, although team members suggest that they may have visited the site too late in the season and they recommend a return visit. In the late 1940s, Russia used parts of Kazakstan as sandpits for nuclear weapons, and it is unknown how much damage to the surrounding environment this has caused.
Types and Severity of Threats
The main threat to this ecoregion is mineral extraction. Oil, coal, iron ore, manganese, chromite, lead, zinc, copper, titanium, bauxite, gold, silver, phosphates, sulfur, iron, and steel, are mined from this area and the consequent contamination and destruction of habitat is of serious concern.
Justification of Ecoregion Delineation
This ecoregion consists of broad plains and montane slopes of Emin Valley along the China-Russia border. Boundaries in China are adapted from CVMCC vegetation map classes of grassland and shrub-grassland. This area corresponds closely to the Mackinnon et al. biogeographic classification of Emin Valley in the Turanian Steppe vegetation province. The adjoining area in Central Asia desert is comprised of steppes sagebrush-grass and semishrub semideserts of the North Tian Shan region as depicted in Pereladova’s map of Central Asian ecosystems.
Further Reading
• Chinese Vegetation Map Compilation Committee. 1979. Vegetation map of China. Map (1:10,000,000). Science Press, Beijing, China. ISBN: 7030089561
• Mackinnon, J., M. Sha, C. Cheung, G. Carey, Z. Xiang, and D. Melville. 1996. A biodiversity review of China. World Wide Fund for Nature, Hong Kong.
• MacKinnon, J., and K. Phillipps. 2000. A Field Guide to the Birds of China. Oxford University Press, New York. ISBN: 0198549407
• Pereladova, O., V. Krever, and M. Williams. 1997. Biodiversity Conservation in Central Asia. Moscow.
Disclaimer: This article is taken wholly from, or contains information that was originally published by, the World Wildlife Fund. Topic editors and authors for the Encyclopedia of Earth may have edited its content or added new information. The use of information from the World Wildlife Fund should not be construed as support for or endorsement by that organization for any new information added by EoE personnel, or for any editing of the original content.
Citation
World Wildlife Fund (Lead Author);Mark McGinley (Topic Editor) "Emin Valley steppe". 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 March 20, 2007; Last revised Date August 9, 2012; Retrieved May 18, 2013 <http://www.eoearth.org/articles/view/152403/China/?topic=49597>
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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User Profile
Name: Osamu Saito
Member Since: May 11th, 2007
Member Name: Osamu.saito
Biography:
Osamu Saito currently works for the Research Institute for Sustainability Science, Osaka University as assistant professor. His main research interests include: Environmental systems analysis and management in relation to forest ecosystem and biomass resources in Japan and Asia; Studies and environmental risk assessment and management of natural resources; Environmental policy and decision making; Mitigation and regulation of greenhouse gas emission; Environmental education in higher education; Human capacity building and empowerment; Development of sustainability assessment methodology and indicators.
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Title Role Type Website Date
Pathways to sustainable industrial societies Author Article Encyclopedia of Earth 2007-05-14 13:52:11
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"warc_filename": "<urn:uuid:26589aeb-6ed5-4cba-b8dc-26f2c772b2d0>",
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Georgia Native RacesEdit This Page
From FamilySearch Wiki
The term "Native Races" is a broad term used to mean any or all races living in a locality prior to some defined date in history. In this Wiki, the term "American Indians" is used to describe those who were residing in this locality prior to European contact. It is also used to describe their descendants, especially those recognized as such by the various levels of government in the United States. It is used as the preferred term in this Wiki and is based upon accepted usage by the Library of Congress.
For a more detailed description of the natives of this locality, see Indians of Georgia.
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• This page was last modified on 6 February 2010, at 23:04.
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Changes related to "Indonesian Maps"
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Lassen County, CaliforniaEdit This Page
From FamilySearch Wiki
Revision as of 07:01, 7 January 2011 by 127.0.0.1 (Talk)
United States > California > Lassen County
Contents
County Courthouse
Quick Facts
Wikipedia has more about this subject: Lassen County, California
Parent Counties
1 April 1864: Lassen County was created from Plumas and Shasta Counties.
County seat: Susanville [1]
Boundary Changes
Record Loss
Places/Localities
Populated Places
Neighboring Counties
Resources
Archives and Libraries
Cemeteries
Church History and Records
LDS Ward and Branch Records
• Susanville
• Westwood
Court Records
History
Land
Maps
Military History and Records
Newspapers
Probate Records
Taxation
Vital Records
Societies, Libraries and Museums
Websites
References
1. The Handybook for Genealogists: United States of America,10th ed. (Draper, UT:Everton Publishers, 2002).
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About this Journal Submit a Manuscript Table of Contents
International Journal of Differential Equations
Volume 2012 (2012), Article ID 173634, 18 pages
doi:10.1155/2012/173634
Research Article
Direct Method for Resolution of Optimal Control Problem with Free Initial Condition
1Department of Mathematics, Faculty of Sciences, University Mouloud Mammeri of Tizi-Ouzou, Tizi-Ouzou, Algeria
2Laboratoire de Conception et Conduite de Systèmes de Production (L2CSP), UMMTO Tizi-Ouzou, Algeria
Received 22 September 2011; Accepted 3 November 2011
Academic Editor: Sabri Arik
Copyright © 2012 Louadj Kahina and Aidene Mohamed. 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.
How to Cite this Article
Louadj Kahina and Aidene Mohamed, “Direct Method for Resolution of Optimal Control Problem with Free Initial Condition,” International Journal of Differential Equations, vol. 2012, Article ID 173634, 18 pages, 2012. doi:10.1155/2012/173634
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Bibliography: Galactic Business
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Title: Galactic Business
Author: Eliot Fintushel
Year: 1998
Type: SHORTFICTION
Storylen: novelette
ISFDB Record Number: 83305
User Rating: This title has fewer than 5 votes. VOTE
Current Tags: None Add Tags
Publications:
Copyright (c) 1995-2011 Al von Ruff.
ISFDB Engine - Version 4.00 (04/24/06)
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Red Shell
From the Super Mario Wiki
Jump to: navigation, search
Red Shell
Description
• First Appearance
Super Mario Bros. (1985)
• Latest Appearance
New Super Mario Bros. U (2012)
“Throw a red shell, and it will zero in on the enemy!”
Penguin, Super Mario Galaxy
Red Shells are red Koopa Troopa shells that first appeared in Super Mario Bros. Although not their first specific use, they were worn by Koopa Troopas. Unlike the green-shelled Koopa Troopas, they did not fall off cliffs and simply turned around when reaching a ledge. They are the second most common shell, after Green Shells.
Contents
[edit] History
[edit] Super Mario series
[edit] Super Mario Bros.
The Red Shell's first appearance was in Super Mario Bros. They are found being worn by Koopa Troopas. Red-shelled Koopa Troopas appeared later than green-shelled Koopa Troopas. Despite their different color, they act in the same manner as Green Shells. Once tossed, it will continue until jumped on or dropped down a cliff. This may prove as a valuable projectile, defeating most enemies, but may damage the player when it is touched while moving.
[edit] Super Mario Bros.: The Lost Levels
Super Mario Bros.: The Lost Levels returned Red Shells along with Green Shells. Little or nothing was changed about the behavior of Red Shells in this game from Super Mario Bros.
[edit] Super Mario Bros. 2
In Super Mario Bros. 2 (which calls them "Turtle Shells"), they are the only type of shells and are found in grass rather than on Koopa Troopas. The player can throw it at enemies just like the vegetables in the game. It will continue sliding until it hits a wall, then it will disintegrate rather than bounce back. It is possible, with some difficulty, to actually ride the shells by getting on top of it.
[edit] Super Mario Bros. 3
Red Shells returned in Super Mario Bros. 3. They acted in the exact same manner as in Super Mario Bros., except they could now be carried and thrown unlike Super Mario Bros., where they can only be kicked.
[edit] Super Mario World
During the events of Super Mario World, Yoshis can spit out fire while in possession of the Red Shell. Any type of Yoshi will spit out fire if they eat a red shell. Also, Red Shells can only be obtained by stomping a Koopa Troopa out of its shell, making it become a Beach Koopa. If a Beach Koopa returns to its shell, it once again gains possession of the red shell.
[edit] New Super Mario Bros.
Red Shells appear once again in New Super Mario Bros. Again, they are only obtained by defeating Red Koopa Troopas who hold the shell. No change has occurred between New Super Mario Bros. and in Super Mario Bros. regarding functions.
[edit] Super Mario Galaxy
Red Shells reappear in Super Mario Galaxy as an offensive weapon. Like the Green Shells in the game, Mario can only hold the Red Shell, not ride it. If Mario throws the Red Shells near an enemy, it will home in that enemy until it hits something. Underwater, it allows Mario to swim faster and with greater ease. It can also be thrown at underwater creatures and home in on them as well. The other variety of shell that appear in Super Mario Galaxy are Gold Shells, which act like Red Shells when thrown.
[edit] New Super Mario Bros. Wii
Red Shells reappear in New Super Mario Bros. Wii as a projectile. Little or no change has occurred between the game and its predecessor regarding functions.
[edit] New Super Mario Bros. 2
Red Shells appear again in New Super Mario Bros. 2, once again as a throwable weapon. Nothing major has changed, though if the player runs through a Gold Ring and there is a Red Koopa Troopa in the area, they will turn Golden, as does their shell. Once thrown, the shell produces a trail of coins behind it.
[edit] New Super Mario Bros. U
Red Shells appear again in New Super Mario Bros. U, where they act in the same way as in the prequels, in the game are found giant Red Shells from Reds Gargantua Koopa Troopas these Red Shells have the ability to destroy Giant Brick Blocks and can also destroy normal Brick Blocks without bouncing on them.
[edit] Mario Kart series
In the Mario Kart series, Red Shells have the ability to home in on the opponent whose rank is one higher than the user's. Red shells can be picked up in sets of one or three. When the triple variant is used, three Red Shells rotate around the driver's kart upon usage, (all games except Mario Kart: Double Dash!!) protecting him or her from Bananas and other shells. If the driver collides with another driver using Triple Red Shells, he or she will be hit by one, and if the other driver also has another set of Triple Green Shells or Triple Red Shells, both drivers lose their shells. Triple Red Shells are not used in Mario Kart DS Wi-Fi mode due to the fact that items cannot be equipped in that mode.
In Mario Kart: Super Circuit, single Red Shells can be laid behind the player and act as a land mine, chasing after the first kart that passes by, even the user.
In Mario Kart: Double Dash!!, the Triple Red Shells are a special item exclusive to Koopa Troopa and Paratroopa (and Petey Piranha and King Boo, due to their ability to have any special item). Also, in Double Dash!!, the three shells are carried in their hands rather than having them circling around the kart. When the kart is hit by an item, the three shells are reduced to one.
In Mario Kart 7, red shells are able to trail in the sky, unlike older Mario Kart games, where Red Shells easily fall down when thrown in the air, similar to what happens when a Green Shell is thrown in the air.
[edit] Yoshi series
[edit] Super Mario World 2: Yoshi's Island / Yoshi's Island: Super Mario Advance 3
Unlike the main stream Mario games, Koopa Troopas appear in rarer cases in Super Mario World 2: Yoshi's Island. A Red Koopa Shell could be used by spitting it out, but it cannot be used for making eggs.
[edit] Yoshi's Island DS
Just like its preceding game, Koopa Troopas wearing the red shell are a rare find in Yoshi's Island DS. They are used in the exact same manner as in Super Mario World 2: Yoshi's Island.
[edit] Super Smash Bros. series
Red Shells also appeared in the Super Smash Bros. series. They were in both Super Smash Bros. and Super Smash Bros. Melee. Unlike the Green Shell, which went in a straight path until it fell off, the Red Shell will stay on one platform, and bounce back and forth hitting anyone who hits it (including the user). They disappear over time, however. Notably, the Red Shell is absent from Super Smash Bros. Brawl.
[edit] Paper Mario series
[edit] Paper Mario: Sticker Star
Red Shells apear as the shiny variant of Green Shells. Also, every Paratroopa in the game has a Red Shell on its back
[edit] Mario and Luigi series
[edit] Mario & Luigi: Partners in Time
In Mario & Luigi: Partners in Time, the Red Shell is a Bros. Item that is used as a weapon against enemies. When the item is chosen, one of the brothers will kick the Shell towards the foe. It will then bounce off the enemy, cause damage, and come to the other player. After each kick, the shell starts moving faster along the ground. Unlike with the Green Shell, which is only capable of dealing 16 hits, the brothers can then repeat this indefinitely until the player misses a kick. Also unlike the Green Shell, the Red Shell will move on and target another enemy if the chosen enemy is defeated. When the adult and toddler Mario brothers fight together, the babies can sit over the shell and spin upon hitting the enemy, causing extra damage to the foe.
[edit] Mario Strikers series
Red Shell icon in Super Mario Strikers.
In the Mario Strikers series, the item works very similarly to the Green Shells, which are also found in the games, however, they differ slightly in appearance and effect. They are red, and instead of moving in one direction, they home in on the opposing players. Every time this item is collected, normally is obtained in a group of three. This allows it to home in on three opposing players, however, one will not get hit. These can be used when trying to perform a Super Strike, a Mega Strike, or a Skillshot. There is also a very small chance that the shells might miss the opposing team members, and start bouncing around the stage like a Green Shell. The giant variety of this shell also appeared in the games. It can be useful for clearing room for a Super Strike or Mega Strike.
[edit] Mario Sports Mix
Red Shells also appear in Mario Sports Mix. When used normally, they'll chase after an opponent, and temporarily knock out anyone in their path. When combined with a ball/puck, the object will do a curved path at high speeds. The curve can occasionally throw off opposing players.
[edit] Trophy Information
Name Image Game Description
Red Shell Super Mario Bros.
10/85
Throw a Red Shell, and it will home in on random characters and send them flying straight up. Red Shells moving around the surface are dangerous to all players, but they may also present attack chances for players with good timing and strategy. Red Shells won't leave the platform they're on once they start moving.
[edit] Gallery
[edit] Names in Other Languages
Language Name Meaning
Japanese あかこうら
Aka koura
Red Shell
Spanish Caparazón rojo Red Shell
French Carapace rouge Red Shell
Dutch Rood Schild Red Shell
German Rote Panzer Red Shell
Italian Guscio rosso "Guscio" (Shell), "Rosso" (Red)
Portuguese Casco Vermelho Red Shell
Russian Красный панцирь Red Shell
Chinese 红龟壳
Hónɡ Guīké
Red Shell
Personal tools
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Nano Express
Investigations of the pore formation in the lead selenide films using glacial acetic acid- and nitric acid-based electrolyte
Sergey P Zimin1*, Egor S Gorlachev1,2, Viktor V Naumov2 and Fedor O Skok1
Author Affiliations
1 Microelectronics Department, Yaroslavl State University, Yaroslavl, 150000, Russia
2 Yaroslavl Branch of the Institute of Physics and Technology of Russian Academy of Sciences, Yaroslavl, 150007, Russia
For all author emails, please log on.
Nanoscale Research Letters 2012, 7:338 doi:10.1186/1556-276X-7-338
Published: 22 June 2012
Abstract
We report a novel synthesis of porous PbSe layers on Si substrates by anodic electrochemical treatment of PbSe/CaF2/Si(111) epitaxial structures in an electrolyte solution based on glacial acetic acid and nitric acid. Electron microscopy, X-ray diffractometry, and local chemical microanalysis investigation results for the porous layers are presented. Average size of the synthesized mesopores with approximately 1010 cm−2 surface density was determined to be 22 nm. The observed phenomenon of the active selenium redeposition on the mesopore walls during anodic treatment is discussed.
Keywords:
Porous semiconductors; Lead selenide; Anodic electrochemical treatment; Electrolyte; Mesopores; 81.05.Rm; 71.20.Nr; 81.65.Cf.
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Contributions
No Contributions Found
About Me
Managing "Plan-B for OpenOffice".org, video based technical support.
Honorary organizer of the Boston Java Meetup.
My current employer has just acquired Oloh.net - 10/2010
Homepage
http://plan-b-for-openoffice.org/index
Follow @
Location
Boston, MA, USA
Ohloh Activity
Joined Ohloh 20 Apr 2009
38 Ohloh website edit(s)
1 post(s)
1 project review(s)
Kudos
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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Contributions
OrientDb c client 91 commits Oct 2010 to Present
Object Query 124 commits Jul 2012 to Present
C++ Testing Framework 72 commits Feb 2009 to Present
OrientDB 1 commits Dec 2012 to Present
windows-alt-tab 20 commits Dec 2011 to Present
Roma Meta Framework 784 commits Jan 2009 to Present
Roma Framework 262 commits Jan 2009 to Jan 2012
ICT Romulus 45 commits May 2009 to Apr 2010
Cornelius Project Management 49 commits May 2009 to Jan 2010
About Me
Ohloh Activity
Joined Ohloh 12 Jan 2009
47 Ohloh website edit(s)
No forum posts.
No project reviewed.
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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Low Activity
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Settings : Managers
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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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Activity Not Available
Project Summary : Factoids
Analyzed about 1 year ago based on code collected about 1 year ago.
Very large, active development team
Over the past twelve months, 30 developers contributed new code to GNOME-DB. This is one of the largest open-source teams in the world, and is in the top 2% of all project teams on Ohloh.
For this measurement, Ohloh considers only recent changes to the code. Over the entire history of the project, 437 developers have contributed.
Mature, well-established codebase
The first lines of source code were added to GNOME-DB in 2000. Projects with recent activity, and a code base more than five years old are likely solving vital problems and delivering consistent value, and may be organized to reward sustained effort by an engaged team of contributors.
Such a lengthy source control history in conjunction with recent activity may indicate that this code base and community are important enough to attract long-term commitment, and may also indicate a mature and relatively bug-free code base.
Note: The source code for GNOME-DB might actually be older than the source control history can reveal. Many new projects begin by incorporating a large amount of source code from existing, older projects. You might be able to tell whether this is the case by looking for a rapid rise in the amount of code early in the project's history.
Increasing Y-O-Y development activity
Over the last twelve months, GNOME-DB has seen a substantial increase in activity. This may be a sign that interest in this project is rising, and that the open source community has embraced this project.
Ohloh makes this determination by comparing the total number of commits made by all developers during the most recent twelve months with the same figure for the prior twelve months. The number of developers and total lines of code are not considered.
See all possible factoids
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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SBB10AssayTeam1-Notes
From OpenWetWare
Jump to: navigation, search
Contents
General
Potential Timeline
Day 1: make master mix for 40 constructs, 2 arabinose, 2 non-arabinose for each construct. and transform by heat shock, grow overnight.
• JH - when are you putting the arabinose? Picking step, or transformation? What if the part is toxic (you can't tell why transformation failed?)
• JK - the protocol we found said to combine the arabinose with the LB media after we pick the colonies.
• JK - we need to make enough master mix to have controls as well
• AL- check inventory for toxicity for the master mix for each batch.
Day 2: pick colony and grow to saturation with LB media.
Day 3: make arabinose and non-arabinose samples out of saturated bacteria mixture, apply antibiotics (K and C), grow.
Day 4: run TECAN with cell aliquot.
Day 2-5: repeat Day 1-4 for the rest of the constructs.
Contact
• Jenna's e-mail: jenna.kelleher@gmail.com
• Maz's email: fuzzybulldozer@gmail.com
• Jim's email: jimhsu@berkeley.edu, jimhsu77479@gmail.com
• Apple's email: xiaoyan207@gmail.com, xliu@berkeley.edu. phone # is 415 627 7226.
PowerPoint
http://docs.google.com/present/edit?id=0AYl_c3UjUjSqZGZ3Z2o5eHpfMjdmandiZzNjZg&hl=en
Parts list
Media:140L_parts1_032910.xls
Vector
Toxicity
Toxicity Protocol
Controls: DH10B (blank), and pBca????-Bca1144 (plasmid effects.)
Parts: 2 arabinose, 2 non-arabinose
• Take 2 tubes of 280ul of cell and add 60ul KCM and 100ul water to each.
• Add 20ul of the KCM/water/cell solution into each construct.
• Transform (heat shock, etc.)
• Grow plates overnight.
• Pick 6 colonies from each sample.
• Grow to saturation in 96 well blocks with 400 uL LB media in each well. (16 parts total per well)
• Then from each of the 6 unique liquid cultures, make 3 arabinose samples and 3 non-arabinose samples
• Add 50 uL of LB media or LB Media+100ug/mL arabinose See note below per well in 384 well plate.
You should make cocktails of LB with whatever antibiotics or arabinose you need. Kanamycin and Chloramphenicol are stored at 25mg/mL. Ampicillin is stored at 100mg/mL. Arabinose is stored at 100mg/mL. At these concentrations, they are 1000x. You want to start with the same batch of LB for all samples in your plate. Calculate what total volume of each mixture of additives you'll need, and make up the mixtures in Eppendorf plates, mix well, then transfer the aliquots to the 384-well plates.
So assuming everything is at 1000X concentrations, we need to dilute everytion to 1x in a 50ul LB media. This means that everything needs to be 1000x more dilute than it presently is. In order words we need just need to add a concentration that is 1000 times less than 50ul which is: 0.05ul of each in the 50 ul lb or 0.4ul of each in the 400ul lb
Lastly, for quantities of everything, we need (1/1000*400ul*19) or 7.6 ul of arabanose. We need (400ul*19) or 7.6ml (7600ul) of LB. All of our parts are CA (JH - really?! makes our life easier.) so we need (1/1000*400ul*19) or 7.6 ul of both Ampicillin and Chloramphenicol.
So in short:
Chloramphenicol 6.8ul
Ampicillin 7.2ul
arabanose 7.6ul
LB 7.6ml
Plasmids DH10B (no antibiotics), and pBca????-Bca1144.
• We made 2 tubes of LB. One 850ul tube with 0.85ul of arabinose and one with no added arabanose. We did not need to add antibiotics as it was premixed.
• We added 50ul of LB (no arabinose) to # wells
• We added 50ul of LB with arabinose to # different wells
• In each of the wells, we added 1ul of the appropriate vector
• We put our sample in the TECAN for 36 time points, 10 minutes between each.
Reagent inventory
Toxicity
http://spreadsheets.google.com/ccc?key=0AsHu1Wyunvu3dHhoSGxrVVFCc0hJM2xZNmNEUFUtUVE&hl=en
Notes:
• JH - not strictly necessary but we might confirm that both tag and untagged variants that are otherwise identical are needed.
• JH - Only 8 units are specified for transformation. Is this enough, because Template:SBB-Protocols_Micro1 states to use 70uL per ligation. If we do the math, (200+50+30)/70 = 4, or 3 to allow for inexact volumes (alternatively, you could bump up the amount used by a bit, or bump down the amount to put in ligation to 65 uL, etc). If you read carefully, you will see 2YT (100) is needed for EACH ligation/transformation. Putting it as part of MM will not work (well).
Insertformulahere
Transformation
Competent cells are stored as 280uL aliquots in the -80 freezer as a communal stock.
Volumes below have been corrected.
1. Thaw 1 tubes of 10 uL aliquot of cells on ice
2. Add 2.5 uL of water to each tube
3. Add 1.5 uL of KCM salts to each tube
4. Add 0.5 ul of the constructs to 10 uL of the cell cocktail. Pipette up and down gently to mix
5. Let sit on ice for 10 min
6. Heat shock for 2 min at 42
7. Put back on ice for 1 min
8. For ampicillin selection, you can plate immediately, otherwise:
9. Add 100uL of 2YT, let shake in the 37 degree incubator for 40 min
10. Plate on selective antibiotics, let incubate overnight
TECAN Safire II operation protocol (96-well)
A TECAN Safire II machine at Architecture et Fonction des Macromolécules Biologiques
Running a TECAN analysis:
The procedure below only works for black 96-well flat bottom plates.
1. Turn TECAN power on (wait for light to stop flashing)
2. Open XFluor4 Safire II XLS spreadsheet
3. Load plate:
1. Have 96-well plate with media+cells
2. Goto XFluorSafireII menu > movements > out
3. Load plate
4. Goto XFluorSafireII menu > movements > in
4. Load the program to run:
1. Select Multi Labeling Kinetic
2. Load multi labeling kinetic parameter > "\iGEM 2007\My Documents\Weston\gabe's experimental folder\kinteticmodified_no gfp reading.mps"
3. Click "Run"
5. Wait for data collection. Operation can only be cancelled when machine is performing measurements.
Questions
• What are the parts to analyze? Basically everything on the parts list. Expect to use 3-4 plates
• What plasmid are we using? Antibiotic selection markers? A R6K - Kanamycin - Chloramphenicol plasmid with various selectable antibiotic markers (see "vectors" section).
• Number of replicates to do? 2 is sufficient.
Powerpoint comments by JCA
• If we have R6K plasmid, we need DH10B with pir+. ...right? AFAIK MC1061 pir+ is the default pir strain
• All samples can be done at once - high throughput ftw.
Expression
• Plate washer: improves data quality and reproducibility
• rbs-prepro - periplasmic - deoxycholic acid
• rbs - cytoplasmic - "Bugbuster"
Questions
• Theoretical:
1. Tags to use? (i.e. Myc, 6His, etc). Everything. The ones I know of: Myc, His, HA, FLAG
2. Antibodies on plate? We will likely be doing a sandwich ELISA, so yes.
3. How to make this quantitative? How much protein DO you have? Most likely reporting OD should be sufficient. Ask JCA for more details.
• Experimental:
1. Available primary antibodies? Concentrations? JCA - we may have 2nd antibody covalently attached to HRP
2. Is the first antibody bound to the plate already? Do as protocol indicates. The first blocking step is the crucial one.
3. Experimentally "sound" concentration BSA / time to block?
4. Detection - ABTS: how much, how long?
Powerpoint comments - JCA
• Don't need 2 batches.
• All samples can be done at once - high throughput ftw.
• Use PBS (+ Tween) instead of CB1 or stupid proprietary buffers. This will depend on the kit though.
• We will talk about using positive controls ... they probably have single-tag purified proteins, and protein fusions can create "dual tags", though not identical to our dual tag configuration.
General protocol
Sample Prep
• Combine cell aliquots, KCM, and water
• Transform (heat shock, etc.)
• Grow plates overnight
• Pick colonies from each sample
• Lyse cells: Add correct reagent to blocks, shake at 37C for 1 hour
Reagent inventory
ELISA
http://spreadsheets.google.com/ccc?key=0AvbXytCFRyFCdFdnbW15cENsQll0ZUJpYXFhbWRrX3c&hl=en
Notes:
• JH - Please put explanations of color coding on Sheet 1 somewhere (comments, or here)
ELISA
JH - I think I finally got all the controls figured out for ELISA after a bit of pondering. Check the powerpoint.
Possible protocol (ABTS concentration): http://www.southernbiotech.com/techbul/5300-05.pdf
Another protocol (with some notes on concentration of target protein): http://www.abcam.com/index.html?pageconfig=resource&rid=11388
1. JH - And ... how do we know the concentration of protein if we haven't done the ELISA? Unless I'm reading this wrong, it's a Catch-22.
1. JCA - CB1 is 99% likely to just be PBS. Use PBS
2. Adding some Tween (detergent) to PBS may improve results.
• Add appropriate antibody to each dilution, to a concentration of .1-10 ug/mL (maybe one dilution of CB1 per type of tag?)
• Pipette solution into the plates, between 50 and 300 uL per well
• Incubate 3-24 hrs at room T, protect from light, minimize evaporation by covering the top of each well
• After incubation, dump coating solution out of wells (the antibodies are now coating the bottom of each well)
• Wash 2-4 times with ELISA wash buffer
• Add 300-400 uL block buffer 1 to "minimize nonspecific adsorbtion and protect the antibodies from harsh external conditions, while blocking uncoated regions of the well"
1. JCA - just use BSA or milk.
• Incubate as before (3-24 hr, room T, no light etc)
• Aspirate block buffer
• Add cell lysate into wells on plate
• Incubate
• Wash off unbound lysate and rinse with PBS
• Block with BSA
1. JCA - this 2nd block is not critical, but can be helpful. Mix the block in dilute lysate.
• Wash with PBS
• Add anti (something) antibodies
• Incubate
• Wash with PBS
• Add 2nd antibody conjugated with streptavidin/HRP, incubate
• Wash with PBS
• Add detection solution, incubate for a specific time
• Add stopping solution
• Read absorbance with ELISA plate reader
Personal tools
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3. But Aristarchus of Samos, a mathematician of great powers, has left a different explanation in his teaching on this subject, as I shall now set forth. It is no secret that the moon has no light of her own, but is, as it were, a mirror, receiving brightness from the influence of the sun. Of all the seven stars, the moon traverses the shortest orbit, and her course is nearest to the earth. Hence in every month, on the day before she gets past the sun, she is under his disc and rays, and is consequently hidden and invisible. When she is thus in conjunction with the sun, she is called the new moon. On the next day, reckoned as her second, she gets past the sun and shows the thin edge of her sphere. Three days away from the sun, she waxes and grows brighter. Removing further every day till she reaches the seventh, when her distance from the sun at his setting is about one half the extent of the firmament, one half of her is luminous: that is, the half which faces toward the sun is lighted up by him.
This work is licensed under a Creative Commons Attribution-ShareAlike 3.0 United States License.
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Sitecore Selected to Power Digital and Mobile Customer Engagement Strategies for Procter & Gamble
Printer-friendly versionPDF version
Sitecore’s Customer Engagement Platform will be used to deliver a personalized customer experience to 4.6 billion P&G consumers
San Francisco February 28, 2013 – Sitecore, a leading web content management and customer experience management software company, today announced that its Customer Engagement Platform (CEP) has been chosen by Procter & Gamble, one of the strongest portfolios of trusted, quality, leadership brands, including Pampers®, Tide®, Oral-B®, Duracell®, Olay®, Head & Shoulders®, Wella® and Gillette®, to help deliver relevant, personalized and meaningful customer interactions.
Sitecore’s Customer Engagement Platform adds customer insights to a proven web content management platform. This powerful combination of data and content delivers a complete view of a customer or prospect, enabling marketers to sense and adapt real-time to a consumer’s behavior. The outcome is a personalized experience most relevant to their audiences throughout the engagement.
“Today’s consumers demand personalized communication in every aspect of their lives,” said Darren Guarnaccia, senior vice president product marketing, Sitecore. “Accordingly, organizations are rethinking the ways that they acquire, grow and retain customers through increased focus on delivering an experience that is highly relevant and predictive of customer and prospect’s needs. Sitecore’s platform makes every interaction more personalized, more relevant, more engaging and more compelling. That’s what delivers measurable results – and what drives conversions and sales.”
Sitecore has received industry accolades from leading media and industry analyst organizations, including Leader in 2012 Gartner *Magic Quadrant for Web Content Management, Visionary in 2012 Gartner **Magic Quadrant for CRM Multichannel Campaign Management, Stratecast Frost & Sullivan 2012 Best Practices Award for Technology Innovation in Customer Engagement Platforms, Software Magazine Software 500 ranking of the world’s largest software and service providers, and finalist in the 2013 SIIA CODiE Awards for Best Content Management Platform.
Additional Resources:
About Sitecore
Sitecore is a global software company that creates products to deliver the most relevant experience and content to customers at any moment of interaction and via any communications channel – the web, email, mobile, social and offline. Our customer experience management platform combines proven web content management with customer intelligence to create a single view of a customer that drives meaningful interactions, increases conversions and builds lifetime customers. Global brands, including American Express, Carnival Cruise Lines, easyJet, Heineken, LEGO, Microsoft, and Nestle rely on Sitecore to get and keep loyal customers who engage more and drive revenue growth.
Gartner, Inc., *Magic Quadrant for Web Content Management, Mick MacComascaigh, et al, September 6, 2012; **Magic Quadrant for CRM Multichannel Campaign Management, Adam Sarner, May 22, 2012.
Gartner does not endorse any vendor, product or service depicted in its research publications, and does not advise technology users to select only those vendors with the highest ratings. Gartner research publications consist of the opinions of Gartner's research organization and should not be construed as statements of fact. Gartner disclaims all warranties, expressed or implied, with respect to this research, including any warranties of merchantability or fitness for a particular purpose
News Source : Sitecore Selected to Power Digital and Mobile Customer Engagement Strategies for Procter & Gamble
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The Ex-Blocker
For the stalky OCD girl or the weak dude who just needs to stop obsessing.
The Ex-Blocker is a Firefox and Chrome plug-in that not only removes your ability to look at your exe’s facebook profile and twitter account, but removes his or her name from the internet entirely.
Not a bad idea…and since all you’re doing is putting in a name, you can go crazy. George W. Bush? gone. Heidi Montag? evaporated. Justin Bieber? buh-bye. Miley Cyrus? Well, we’re keeping that one.
(Big ups Alix)
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Place:Braddock, Allegheny, Pennsylvania, United States
Watchers
NameBraddock
TypeBorough
Coordinates40.404°N 79.869°W
Located inAllegheny, Pennsylvania, 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
Braddock is a borough located in the eastern suburbs of Pittsburgh in Allegheny County, Pennsylvania, 10 miles (16 km) upstream from the mouth of the Monongahela River. The population was 2,159 at the 2010 census. The borough is represented by the Pennsylvania State Senate's 45th district, the Pennsylvania House of Representative's 34th district, and Pennsylvania's 14th congressional district in the United States House of Representatives.
Research Tips
This page uses content from the English Wikipedia. The original content was at Braddock, Pennsylvania. 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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Australian Bureau of Statistics
Celebrating the International Year of Statistics 2013
ABS Home > Statistics > By Release Date
1306.5 - Western Australia at a Glance, 2003
Previous ISSUE Released at 11:30 AM (CANBERRA TIME) 04/04/2003
Page tools: Print Page Print All RSS Search this Product
• About this Release
ABOUT THIS RELEASE
Previously: Western Australia in Brief (ISSN: 0727-2022)
Contains a wide range of statistics on Western Australia, including physical data, population, vital statistics, employment and wages, price indexes, mineral production, manufacturing, building, foreign trade and tourism.
There was no 1994 issue.
© 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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215 reputation
22
bio website abusinessmentor.com
location Alabama
age 61
visits member for 3 years, 3 months
seen Apr 15 '10 at 5:52
stats profile views 50
I completed my BA, and most of my first Masters Degree on discharge from the Army in 1981. (Public Administration) I finally obtained my second as an MBA in Management in 1995. I've been involved in building or managing business operations all my life. I managed my first kitchen at the age of 15. I write business plans. I train sales people. I provide remote training/management by webinar and VoIP conferencing. I have a number of areas I consider myself expert in, including Event Marketing and Promotions, Telemarketing Operations, and Start-up operations.
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Thursday, September 27, 2012
86 Million Americans Use a Smartphone to Shop
comScore released the results from the latest market study of U.S. mobile smartphone user shopping behavior, which found that 4 in every 5 people -- 85.9 million in total -- accessed retailer related content on their device in July, 2012.
Amazon Sites led as the top retailer with an audience of 49.6 million visitors, while multi-channel retailers including Apple (17.7 million visitors), Wal-Mart (16.3 million visitors), Target (10 million visitors) and Best Buy (7.2 million visitors) also attracted significant mobile audiences.
"With nearly 86 million Americans now shopping on their smartphones, this pronounced shift in consumer behavior is simply too large for retailers to ignore, with the future of their business depending on how well they adapt to the new environment," said Mark Donovan, comScore SVP of mobile research.
comScore says that adapting to today's online retail environment requires optimizing the experience across multiple platforms -- for both mobile websites and apps. Moreover, retailers that understand how people are engaging in mobile shopping behaviors are designing better user experiences.
As an example, Ticketmaster attracted 5.7 million visitors as summer concerts and performances saw fans turn to their smartphones for tickets and information, while seasonal home improvements fueled visitation to the Home Depot (4.4 million visitors) and competitor Lowes (3.2 million visitors).
As mobile becomes an increasingly important channel for retailers to reach current and potential customers, other companies are recognizing the opportunity smartphones present throughout the shopping process.
Shopkick, a shopping rewards app that provides points for consumers who visit retail partners’ physical stores, saw its mobile audience reach more than 4 million visitors in July -- demonstrating one way consumers are turning to their mobile devices as part of their in-store shopping experience.
Among both iPhone and Android users, Amazon ranked as the top retailer attaining a reach of 43 percent among iPhone users and 55 percent among Android users, with visitation to the Amazon Appstore largely accounting for the higher reach among Android users.
Analysis of smartphone shoppers versus those visiting on desktop or laptop computers revealed new insights into audience demographics. Across both smartphones and desktop computers, males and females represented nearly equal proportions of retail category visitors.
However, females accounted for a higher share of time spent on retail destinations at 53.4 percent of minutes on desktop computers and an even greater share of retail minutes on smartphones at 56.1 percent.
Smartphone shoppers were also more likely to be younger than their desktop counterparts -- with 70.7 percent of smartphone retail visitors under the age of 45 compared to 61.1 percent of desktop users. Engagement among these audiences showed even greater disparity with visitors under the age of 45 accounting for nearly 3 in every 4 minutes spent on retail content via smartphones -- compared to 61.6 percent of retail minutes on desktop computers.
Smartphone retail audiences were more likely to reside in higher income households compared to desktop computer users, likely as a result of smartphone ownership skewing towards higher income segments compared to an average consumer.
Among smartphone audiences accessing retail destinations, nearly 1 in every 3 had a household income of $100,000 or greater, with this income segment driving a comparable 31.2 percent of minutes spent on retail sites and apps.
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Investigating The Psychological Effects Of Sustainable Buildings On Human Life
Hanie Okhovat, Aryan Amirkhani, Mohammad Reza Pourjafar
Abstract
As the environmental impact of buildings becomes more apparent, a growing field called sustainable design is leading the way to reduce that impact at the source. Sustainable design is the practice of creating healthier and more resource efficient models of construction, renovation, operation, maintenance, and demolition. Nowadays architects are seeking to construct more sustainable buildings whereas there is still little research on social response and human behavioral interactions in sustainable buildings. This paper addresses the psychological benefits that affect the occupant's life in green buildings. The social, psychological and applied behavior of occupants in these structures are also investigated.
The results show that behavioural aspects of sustainability and how people interact with these structures can be important in sustainable design. Therefore these issues are needed to be addressed in facility management of sustainable buildings.
Full Text: PDF
This work is licensed under a Creative Commons Attribution 3.0 License.
Journal of Sustainable Development ISSN 1913-9063 (Print) ISSN 1913-9071 (Online)
Copyright © Canadian Center of Science and Education
To make sure that you can receive messages from us, please add the 'ccsenet.org' domain to your e-mail 'safe list'. If you do not receive e-mail in your 'inbox', check your 'bulk mail' or 'junk mail' folders.
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ELC 2012 Presentations
From eLinux.org
Revision as of 12:07, 23 February 2012 by Wmat (Talk | contribs)
Jump to: navigation, search
Presenters, Demo-ers, Participants: Thanks very much for your participation in Linux Foundation's Embedded Linux Conference 2012.
This page is for collecting the presentations that were made at the conference. During and after the conference we will collect materials from the presenters and place them here. Please watch this page if you are interested in a particular presentation - and if it doesn't show up, please send me and email and we'll try to track it down.
Contents
Videos
Videos for ELC2012 from the Linux Foundation can be found at ELC2012 Videos. Free Electrons also took video recordings of the talks, and plans to make them available shortly.
Instructions
Presenters: Please post your technical conference presentations on this page. (See Instructions below the tables)
Table of Presentations
NOTE: If you add a wikilink to your presentation and attempt to upload it via the link, it may fail. If it does, use the Special:Upload page to upload your file.
Keynotes
Keynotes
Presenter(s) Session Description Presentation
Jon Corbett, Editor at LWN.net The Kernel Report The Kernel Report ELC2012
Mike Anderson, CTO at The PTR Group The Internet of Things The Internet of Things
Presenters
Presentations
Presenter(s) Session Description Presentation
Day 1, 9:30am
Loïc Pallardy Saving the Power Consumption of the Unused Memory PDF
Bernhard Rosenkränzer, Linaro What Android and Embedded Linux Can Learn From Each Other PDF
Ricardo Salveti de Araujo, Linaro Ubuntu on ARM: Improvements and Optimizations Done By Linaro PDF
Day 1, 11:30am
Zach Pfeffer, Linaro Binary Blobs Attack PDF
Hisao Munakata, Renesas Electronics Close Encounters of the Upstream Resource PDF
Daniel Hursh, IBM Open Source Automated Test Framework PDF
Day 1, 2:00pm
Saul Wold, Intel The Yocto Project Overview and Update PDF
Embedded Linux Pitfalls Sean Hudson, Mentor Graphics, Inc. PDF
Vincent Guittot, Linaro Comparing Power Saving Techniques For Multicore ARM Platforms PDF
Day 1, 3:00pm
Tim Bird, Sony Network Entertainment Status of Embedded Linux PDF
Bruce Ashfield, Wind River A View From the Trenches: Embedded Functionality and How It Impacts Multi-Arch Kernel Maintenance PDF
R Durgadoss, Intel PeakCurrent Management in x86-Based Smartphones PDF
Day 1, 4:15pm
Matt Porter, Texas Instruments Passing Time With SPI Framebuffer Driver PDF
Wookey, Linaro Multiarch and Why You Should Care: Running, Installing and Crossbuilding With Multiple Architectures PDF SOURCE
Amit Daniel Kachhap, Linaro/Samsung A New Simplified Thermal Framework For ARM Platforms PDF
Day 1, 5:15pm
Tsugikazu Shibata, NEC On The Road: To Provide the Long-Term Stable Linux For The Industry PDF
Thomas P. Abraham, Samsung Electronics Experiences With Device Tree Support Development For ARM-Based SOC's PDF
Paul E. McKenney, IBM Making RCU Safe For Battery-Powered Devices PDF
Day 2, 10:30am
Thomas Petazzoni, Free Electrons Buildroot: A Nice, Simple, and Efficient Embedded Linux Build System PDF
Steven Rostedt, Red Hat Automated Testing with ktest.pl (Embedded Edition) PDF
David VomLehn, Cisco Intricacies of a MIPS Stack Backtrace Implementation PDF
Day 2, 11:30am
Edward Hervey, Collabora GStreamer 1.0: No Longer Compromise Flexibility For Performance PDF
Steven Rostedt, Red Hat Automated Testing with ktest.pl (Embedded Edition) (Cont.) PDF
Tim Bird, Sony Network Entertainment Embedded-Appropriate Crash Handling in Linux PDF
Day 2, 2:00pm
Arnd Bergmann, Linaro ARM Subarchitecture Status PDF
Mark Gisi, Wind River Systems The Power of SPDX - Sharing Critical Licensing Information Within a Linux Device Supply Chain PDF
Yoshitake Kobayashi, Toshiba Ineffective and Effective Ways To Find Out Latency Bottlenecks With Ftrace PDF
Day 2, 3:00pm
Ohad Ben-Cohen, Wizery / Texas Instruments Using virtio to Talk With Remote Processors PDF
Elizabeth Flanagan, Intel Embedded License Compliance Patterns and Antipatterns PDF
David Anders, Texas Instruments Board Bringup: LCD and Display Interfaces PDF
Day 2, 4:15pm
Rob Clark, Texas Instruments DMA Buffer Sharing: An Introduction PDF
Ken Tough, Intrinsyc Linux on eMMC: Optimizing For Performance PDF
Paul Larson, Linaro LAVA Project Update PDF
Day 2, 5:15pm
Jeff Osier-Mixon, Intel Yocto Project Community (BoFs) No Slides
Frank Rowand, Sony Network Entertainment Real Time (BoFs) PDF
Mike Turquette, Texas Instruments Common Clock Framework (BoFs) PDF
Day 3, 9:00am
Hunyue Yau, HY Research LLC Userland Tools and Techniques For Linux Board Bring-Up and Systems Integration PDF
Matt Weber, Rockwell Collins Inc. Optimizing the Embedded Platform Using OpenCV PDF
Greg Ungerer, McAfee M68K: Life in the Old Architecture PDF
Day 3, 10:00am
Gary Bisson, Adeneo Embedded Useful USB Gadgets on Linux PDF
Jason Kridner, Texas Instruments GUIs: Coming To Uncommon Goods Near You PDF
Mike Anderson, The PTR Group Adapting Your Network Code For IPv6 Support PDF
Day 3,11:30pm
Koen Kooi, The Angstrom Distribution Producing the Beaglebone and Supporting It PDF
Danny Bennett, basysKom GmbH HTML5 in a Plasma-Active World PDF
Marcin Mielczarczyk, Tieto Getting the First Open Source GSM Stack in Linux PDF
Day 3, 2:00pm
Pierre Tardy, Intel PyTimechart Practical PDF
Linus Walleij, ST-Ericsson Pin Control Subsystem Overview PDF
Khem Raj, OpenEmbedded Project OpenEmbedded - A Layered Approach PDF
Day 3, 3:00pm
Lucas De Marchi, ProFUSION Embedded Systems Managing Kernel Modules With kmod PDF
Jean Pihet, NewOldBits A New Model for the System and Devices Latency PDF
Pintu Kumar, Samsung Controlling Linux Memory Fragmentation and Higher Order Allocation Failure: Analysis, Observations and Results PDF
Workshops
Workshops
Presenter(s) Session Description Presentation
Karim J. Yaghmour Embedded Android Workshop PDF
Instructions for Presenters
Please create a link in the table for your presentation, copying the style of other links. (You may need to create an account in order to edit the wiki or upload files.)
When you have created the link, click on it to upload the file containing your slides.
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Harrison County, OhioEdit This Page
From FamilySearch Wiki
Guide to Harrison County Ohio genealogy. Birth records, marriage records, death records, census records, family history, and military records.
Ohio
Online Records
Harrison County, Ohio
Map
Location of Ohio in the U.S.
Courthouse
Address Harrison County Courthouse
100 West Market Street
Cadiz, Ohio 43907
Harrison County Website
United States Ohio Harrison County
Contents
Historical Facts
Beginning dates for major county records
Birth
Marriage
Death
Tax
Land
Probate
1867
1813
1867
1812
1813
*For earlier dates, try... Church | Obituaries | Cemeteries
Boundary Changes
See an interactive map of Harrison County boundary changes.
Record Loss
Resources
Bible Records
Biography
Business Records and Commerce
Cemeteries
Cemetery records often reveal birth, marriage, death, relationship, military, and religious information.
Online Grave Transcripts Published Grave Transcripts County Cemetery Directories
Findagrave.com
Findagrave.com
Interment.net
WorldCat
Names in Stone
USGenWeb
Billion Graves
Billion Graves
Epodunk
Ohio Gravestones
Names in Stone
See Ohio Cemeteries for more information.
Census
Church Records
Church records and the information they provide vary significantly depending on the denomination and the record keeper. They may contain information about members of the congregation, such as age, date of baptism, christening, or birth; marriage information and maiden names; and death date. For general information about Ohio denominations, view the Ohio Church Records wiki page.
Finding Church Records at Other Repositories
Additional church records can sometimes be found using search phrases such as Harrison County, Ohio Church Records in online catalogs like:
Court Records
Emigration and Immigration
Ethnic, Political, or Religious Groups
Gazetteers
Genealogy
History
Local histories are available for Harrison County, Ohio. County histories may include biographies, church, school and government history, and military information. For more information about local histories see the wiki page section Ohio Local Histories.
Land and Property
Land and property records can place an ancestor in a particular location, provide economic information, and reveal family relationships. Land records include: deeds, abstracts and indexes, mortgages, leases, grants and land patents.
See Ohio Land and Property for additional information about early Ohio land grants. After land was transferred to private ownership, subsequent transactions were usually recorded at the county courthouse and where records are currently housed.
Maps
Military
• Civil War
Civil War service men from Morgan County served in various regiments. Men often joined a company (within a regiment) that originated in their county. Listed below are companies that were specifically formed in Morgan County.
- 98th Regiment, Ohio Infantry, Companies C and F
Naturalization and Citizenship
Newspapers
Harrison County, Ohio newspapers may contain genealogical value including obituaries, births, marriages, deaths, anniversaries, family gatherings, family travel, achievements, business notices, engagement information, and probate court proceedings.
To access newspapers, contact public libraries, Ohio Genealogical Society chapters, college or university libraries, the Library of Congress, Google News, or the Ohio Historical Society. The Ohio Genealogical Society Obituary Database is another source of newspaper information.
For more Ohio newspaper information see the Newspaper Guides on the wiki page Ohio Newspapers.
Obituaries
Periodicals
Probate
Probate records created after 1852 are held by the Harrison County, Ohio Probate Court. From 1797 or the creation of the county, probate records were held by the Court of Common Pleas. Most counties transferred all records to the Probate Court, but in some circumstances, Court of Common Pleas records should be searched for records prior to 1852. Most records are housed at the Harrison County, Ohio Courthouse. Some records are on microfilm at the Ohio Genealogical Society and the Family History Library. For more complete information about the location of county probate records see:
See the wiki page Ohio Probate Records for information about how to use probate records.
Content: Probate Records may give the decedent's date of death, names of his or her spouse, children, parents, siblings, in-laws, neighbors, associates, relatives, and their place of residence.
Record types: Wills, estates, guardianships, naturalizations, marriage, adoption, and birth and death records (1867-1908 only).
Public Records
Repositories
Courthouse
Harrison County Courthouse
100 W Market Street
Cadiz, OH 43907-1132
Phone: 740.942.4623
Clerk Court has divorce and court records from 1813;
Probate Judge has birth records to 1917, marriage and
probate records; County Health Office has birth records
from 1917, death and burial records; County Recorder
has land records[1]
Family History Centers
Libraries
Museums
Societies
Taxation
• 1800–1850 Ohio Tax Records, 1800-1850 at FamilySearch Historical Records – free. Name index of tax records as recorded with the County Auditor of each county. Entries are recorded in voucher books, one person per page. The majority of the tax records in this collection are for the years 1816 through 1838. Currently (September 2012) this collection is 17% complete. Additional records will be added as they are completed.
Finding Tax Records at Other Repositories
Additional tax records can sometimes be found using search phrases such as Harrison County, Ohio Tax Records in online catalogs like:
Vital Records
Vital records consist of birth, death, marriage and divorce records. Although Ohio enacted a statute in 1856 -1857 requiring registration of births, deaths and marriages, many did not comply. A second law was written in 1867 but, again, was not always followed. By 1908, the law was more clearly defined and kept. Any existing birth and death records from 1867 through December 19, 1908 are located at the Harrison County, Ohio Probate Court. The Ohio Department of Health has birth records filed after December 20, 1908 and death records filed after January 1, 1954 while the Ohio Historical Society houses death records from December 20, 1908 through December 31, 1953.
Original marriage records are held at the office of the Harrison County, Ohio Probate Court with divorce records located with the Harrison County, Ohio Clerk of Courts.
Birth
Marriage
Death
Ohio Deaths, 1908-1953 Free name indexes and images at FamilySearch. Records include such information as birth date of deceased, city, county, and state of death, name of spouse if married, names of parents, maiden name of mother, name of informant, if deceased was single, married, windowed or divorced, occupation of deceased.
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
Places
Populated Places
References
1. 1.0 1.1 Handybook for Genealogists: United States of America, 10th ed. (Draper, Utah: Everton Pub., 2002), [FHL book 973 D27e 2002].
2. Wikipedia contributors, "Harrison County, Ohio" in Wikipedia: the Free Encyclopedia at http://en.wikipedia.org/wiki/Harrison_County,_Ohio (accessed 10 May 2012).
3. Carol Willsey Bell, Ohio Wills and Estates to 1850: An Index (Columbus, Ohio: the author, 1981). FamilySearch Books Online - Free online copy.
Need additional research help? Contact our research help specialists.
Need wiki, indexing, or website help? Contact our product teams.
Did you find this article helpful?
You're invited to explain your rating on the discussion page (you must be signed in).
• This page was last modified on 17 May 2013, at 01:47.
• This page has been accessed 2,042 times.
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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.
Item1370: Implementation of feature proposal DelegateMoreProcessingToSearchAlgorithm
Priority: CurrentState: AppliesTo: Component: WaitingFor:
Enhancement Closed Engine
see DelegateMoreProcessingToSearchAlgorithm
-- SvenDowideit - 26 Mar 2009
the changes for foswiki 1.1 are done, new ones will be a new task.
-- SvenDowideit - 14 May 2010
Topic revision: r14 - 04 Oct 2010, 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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Research
Genetic diversity in India and the inference of Eurasian population expansion
Jinchuan Xing, W Scott Watkins, Ya Hu, Chad D Huff, Aniko Sabo, Donna M Muzny, Michael J Bamshad, Richard A Gibbs, Lynn B Jorde* and Fuli Yu*
Genome Biology 2010, 11:R113 doi:10.1186/gb-2010-11-11-r113
No comments have yet been made on this article.
Post a comment
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Comment
The Columnist Manifesto
Gregory A Petsko
Author Affiliations
Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454-9110, USA
Genome Biology 2011, 12:136 doi:10.1186/gb-2011-12-12-136
Published: 28 December 2011
First paragraph (this article has no abstract)
A specter is haunting Europe - the specter of the euro. As I write this, the likelihood that European monetary unity will break apart like a dropped porcelain doll looms larger than at any time in the past 2 years. The resulting financial upheaval would certainly send both that continent, and much of the rest of the world, into a deeper recession, if not into an actual economic depression. Even if the single currency manages to survive, the austerity measures that are being implemented - foolishly, in my opinion - throughout most of the EU member nations (and that even Britain appears to be hell-bent on following, at least in part) almost certainly guarantee that the worst recession in half a century will continue for several more years.
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[maemo-users] [maemo-users] Random reboots
From: Eero Tamminen eero.tamminen at nokia.com
Date: Tue Feb 13 11:02:49 EET 2007
Hi,
ext James Sparenberg wrote:
> On Monday 12 February 2007 09:51:17 Andrew Flegg wrote:
>> On 2/12/07, Sergey Udaltsov <sergey.udaltsov at gmail.com> wrote:
>>>>> /usr/sbin/dsp_dld -p --disable-restart -c /lib/dsp/dsp_dld_avs.conf :
>>>>> 1 *
>> [snip]
>>
>>> It loolks like a bit odd solution - reboot the device if one command
>>> restarts too many times. It is linux after all;)
>> It makes sense from a consumer point of view: if something's
>> repeatedly dying in user space which is critical to the end-user
>> experience, reboot.
>
> Not really as this could cause critical loss of data, or other corruption.
> Besides this is Linux a reboot is not the answer, a reboot resets the kernel
> not just user space. Since this is a user space flaw, it should be addressed
> with user space 'repairs' IMHO.
dsp_dld restarts fail because there's a (DSP related) issue on
the kernel side. It shouldn't (couldn't?) cause data loss, but
in that case reboot is the only way to recover one of the main uses
(sounds/music/videos) for the device...
>> Personally, I'd prefer a confirmation first "A critical process
>> [dsp_dld] has failed. It is recommended you restart your Internet
>> Tablet. <Restart> <Continue>", and we've seen from the reboot loops
>> with broken RSS handling that it needs some sanity to prevent infinite
>> reboots and returns.
>
> This, I agree, would at least would give the user the chance to save data from
> open processes and then restart at a convenient moment in time. Also it
> would assist the developers in that if everyone and his sister is seeing the
> same cause of this message the noise pointing out the culprit would focus
> their attention on the right cause rather than causing the frustration of not
> knowing where to start.
There are many services in the device which death would prevent user
from doing about anything with the device UI:
- X server
- Window manager and Desktop (to switch to apps)
- sapwood server (needed by sapwood theme engine)
- D-BUS
etc.
The lifeguard service doesn't know which of these services are crucial
for the user (or the 3rd party applications user uses). Developers can
disable the automatic reboots with flasher, but this enables also R&D
mode which has some other effects.
>>> Anyway, I am still puzzled - why would idle and charging n800 make
>>> dsp_dld restart many times...
>> It's a known bug:
>>
>> https://maemo.org/bugzilla/show_bug.cgi?id=976
>>
>> Or, rather, a series of bugs - apparently. I suggest you vote on it so
>> Nokia can get an update out quickly.
- Eero
More information about the maemo-users mailing list
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User:Bridget Wall
From OpenWetWare
Jump to: navigation, search
I am a new member of OpenWetWare!
Contents
Contact Info
Bridget Wall (an artistic interpretation)
I work in the Niles Lab.
Education
• Expected graduation in 2013 with a PhD in Bioengineering
• Bachelor of Arts in Biology; Illinois Wesleyan University
Research interests
1. Interest 1
2. Interest 2
3. Interest 3
Publications
1. Goldbeter A and Koshland DE Jr. . pmid:6947258. PubMed HubMed [Paper1]
2. JACOB F and MONOD J. . pmid:13718526. PubMed HubMed [Paper2]
leave a comment about a paper here
3. Mark Ptashne. A genetic switch. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 2004. isbn:0879697164. [Book1]
All Medline abstracts: PubMed HubMed
Useful links
Personal tools
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helgigud's bookmarks
"Work banishes those three great evils: boredom, vice and poverty."
Voltaire on work
8 fans of this quote
"A competent and self-confident person is incapable of jealousy in anything. Jealousy is invariably a symptom of neurotic insecurity."
Long, Lazarus on self-confidence
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But wait... my book has more: prev 1, 2, 3, 4, 5, 6, 7, 8, 9 next
Helgi I Gudmundsson's quote collection
I'm male and made my book on 1st October 2009.
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Scarface: The World Is Yours/Nacho's Tanker
From StrategyWiki, the video game walkthrough and strategy guide wiki
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Reputation +268,050
Exotics N/A
Turf [%] N/A
Balls +3,610
Drugs [g] N/A
Total Cash N/A
The aim here is to keep the support chopper intact from the attack boats. You may not manage to keep it alive, but try regardless, as the longer it stays up the less chance they'll shoot you. The helicopter will make a few laps around the tanker; take out everybody you can see to prevent yourself from taking damage. After two runs it'll land on the bow.
You've reached a check point. There's a bomb and you've got a minute to defuse it. Take out the guy that is just in front of you, go down the right hand stairs, and shoot the guy going up the other side. Now shoot the guy coming along the side. Defuse the bomb now. After that, rush up to the bow of the ship and use the mounted gun to take out the two boats once they appear in range.
You now have three minutes to defuse the bomb in the cargo hold; things start to get difficult now. Make your way down to the cargo hold using the stairs, but don't hang around as you'll be shot at. Once you're down in the cargo hold, use your blind rage and duck in and out of the crates, there are a few health kits laying around so find them if you're running low. Once you've cleared the floor defuse the bomb.
You have three minutes to defuse the last bomb and it's right back at the stern. Head on up and don't worry about the boats that have appeared. Rush up, killing those that get in your way, and defuse the last bomb without worrying about wiping out all the gang members out, as they spawn infinitely. After you've defused the bomb, the captain will appear, so go up and take him out. Rage is good to use around this time, as you'll be surrounded. There are quite a few health kits lying around though.
You'll end up back in a dock in Miami. Once you reach the top of the ramp, you'll hit a checkpoint. Head towards the foreman and fill your rage meter. Once full, use it and you'll clear the dock in no time flat.
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File Type Information
Supports embedding license URL: Yes
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Supports embedding more permissions: No
Metadata Location: WCOP, WOAF tags
Metadata Format: ID3
This is a developer-oriented document. Read our non-technical account with explanatory comics first.
This document describes how to apply our non-web content guidelines to MP3s.
MP3 embedded metadata is placed in ID3 frames. Two of these will be used for embedding Creative Commons license claim metadata:
WCOP (Copyright/Legal information) should include the license URL. For example, http://creativecommons.org/licenses/by/2.5/.
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TCOP (Copyright Text) should include a human-readable license and license claim information. The contents of this frame are displayed by many programs in a "file info" window. Example value: 1995 Example Band. Licensed to the public under http://creativecommons.org/licenses/by/2.0/ verify at http://example.com/cclicenses.html
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Help Wikitravel grow by contributing to an article! Learn how.
Difference between revisions of "Port Huron"
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(Get out)
(Sleep)
Line 26: Line 26:
==Sleep==
==Sleep==
* <sleep name="Holiday Inn Express" address="1720 Hancock St
* <sleep name="Holiday Inn Express" address="1720 Hancock St
Port Huron, MI 48060" phone="1-810-987-5999" url="http://www.hiexpress.com/h/d/ex/1/en/hotel/PHRMI/welcome?start=1"Chain hotel located at the end of Interstate 94, and near the Blue Water Bridge></sleep>
+
Port Huron, MI 48060" phone="+1 810 987-5999" url="http://www.hiexpress.com/h/d/ex/1/en/hotel/PHRMI/welcome?start=1"Chain hotel located at the end of Interstate 94, and near the Blue Water Bridge></sleep>
==Get out==
==Get out==
Revision as of 02:10, 29 August 2009
Port Huron [1] is a city in Southeast Michigan, the second-busiest border crossing (with Sarnia, Ontario) in the state.
Contents
Get in
From the south take I94 east
From the west take I69 east
From Ontario take highway 402 west crossing the St. Clair River into the United States via the Blue Water Bridge
Get around
Blue Water Area Transit (810) 987-7373 BWAT
See
The annual Port Huron to Mackinac sailboat race one of the longest fresh-water races in the world with over two hundred boats entering the race each year.
Do
Buy
Eat
Drink
Sleep
• Holiday Inn Express, 1720 Hancock St Port Huron, MI 48060, +1 810 987-5999.
Get out
Sarnia is across the Blue Water Bridge in Ontario, Canada.
Routes through Port Huron
Flint W E END
DetroitRoseville W E → becomes Ontario 402 → Sarnia
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 Release Date
5368.0 - International Trade in Goods and Services, Australia, May 2004
Previous ISSUE Released at 11:30 AM (CANBERRA TIME) 29/06/2004
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ABOUT THIS RELEASE
Absorbs: 5301.0 , 5320.0 and 5422.0.
Provides estimates for 15 months of the major aggregates for, and the balance on, international trade in goods and services (balance of payments basis) in both seasonally adjusted and trend estimates terms. Longer term graphs are provided, in seasonally adjusted and trend estimates terms, for the goods and services aggregates, and the balance on goods and services. In addition it provides more detailed commodity statistics for both goods and services in original terms, with goods provided on balance of payments and international trade basis, together with year-to-date information. More detailed services statistics, in seasonally adjusted and trend terms on a monthly basis as well as original terms on a quarterly basis are also provided. Merchandise imports and exports are provided at one and two digit SITC level with selected commodities at three digit level. Merchandise trade data are provided by country and by state. Data on exchange rates and analytical comments are included.
See also 5439.0 and 5372.0.55.001
© 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
7506.0 - Agricultural Industries, Financial Statistics, Australia, Preliminary, 1996-97
Previous ISSUE Released at 11:30 AM (CANBERRA TIME) 03/02/1998
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MEDIA RELEASE
February 3, 1998
Embargoed 11:30am (AEST)
9/98
Farm finances hold steady
Preliminary farm finance estimates for the financial year 1996-97, released by the Australian Bureau of Statistics today, showed little change in farm finances at the national level compared to the previous year. This follows a substantial improvement in 1995-96 after the easing of the drought in the eastern States.
However, performance at the State level was mixed, with Queensland showing strong growth while Western Australia and South Australia showed significant declines.
Turnover in 1996-97 was estimated at $26.1 billion, a 2.3 per cent decrease on 1995-96 turnover. Turnover in the beef cattle industry fell 17.9 per cent to $2.5 billion, while in the sheep-beef cattle industry turnover fell 16.4 per cent to $1.2 billion.
Turnover from sales of livestock decreased from $6.3 billion in 1995-96 to $5.5 billion in 1996-97, reflecting lower beef prices. Sales from livestock products increased 5.4 per cent to $5.2 billion in 1996-97. Turnover from the sales of crops was $13.0 billion.
Farm business cash operating surplus in 1996-97 was $5.6 billion, a decrease of 13.6 per cent on the 1995-96 level of $6.4 billion. The average cash operating surplus per farm business in 1996-97 was $52,400, 11.6 per cent lower than in 1995-96.
Gross indebtedness of the farming industry was $19.1 billion at the end of June 1997, compared to a debt of $19.6 billion at the end of June 1996. The average debt per farm business at June 1997 was $180,100. Farm businesses paid $1.7 billion in interest during 1996-97, a marginal increase of 4.3 per cent on the previous year.
Details are available in Agricultural Industries, Financial Statistics, Australia, Preliminary 1996-97 (cat. no. 7506.0) available from ABS bookshops.
© Commonwealth of Australia 2013
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Australian Bureau of Statistics
Celebrating the International Year of Statistics 2013
ABS Home > Statistics > By Catalogue Number
5487.0 - Information Paper: International Merchandise Trade Statistics, Australia: Data Confidentiality, 1999
Latest ISSUE Released at 11:30 AM (CANBERRA TIME) 20/12/1999
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• About this Release
ABOUT THIS RELEASE
International trade data may be suppressed to prevent the identification of the activities of exporters and importers. This paper provides details concerning: why data may not be disclosed; the ways in which data may be suppressed; how individuals or businesses can register objections to the disclosure of data they consider confidential; how the ABS will handle these objections and the effect of confidentiality on output.
© Commonwealth of Australia 2013
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Trying to find free opportunities to put your business’ name out there? Well, here’s a great one that you may not have known about: The online Yellow Pages.
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<D <M <Y
Y> M> D>
: Rachel and I went to the Newport Pier thinking that it was the place where they filmed the boardwalk scenes for Arrested Development. That's basically where those scenes were set, but they were actually shot at a more photogenic pier, many miles northwest in Marina del Rey. But we ate a frozen banana anyway. (As things you buy on a pier go, frozen banana is pretty healthy.)
I originally tried to write this entry in the style of an Arrested Development episode, but though it was formally correct there were no jokes in it. This is how you repay me for how I repay you?!?!
[Main]
Unless otherwise noted, all content licensed by Leonard Richardson
under a Creative Commons License.
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Research
Gastrointestinal lymphomas in a North American population: clinicopathologic features from one major Central-Midwestern United States tertiary care medical center
Joshua Warrick1, Jingqin Luo2, Diane Robirds1, Julie Branson1, John L Frater1, Friederike Kreisel1, Anjum Hassan1 and TuDung T Nguyen1*
Author Affiliations
1 Department of Pathology & Immunology, Washington University Medical School, 660 S. Euclid Ave, Campus Box 8118, St. Louis, MO 63110, USA
2 Department of Biostatistics, Washington University Medical School, St. Louis, MO, USA
For all author emails, please log on.
Diagnostic Pathology 2012, 7:76 doi:10.1186/1746-1596-7-76
The electronic version of this article is the complete one and can be found online at: http://www.diagnosticpathology.org/content/7/1/76
Received:2 March 2012
Accepted:22 May 2012
Published:28 June 2012
© 2012 Warrick 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
Gastrointestinal (GI) lymphomas are very common types of extranodal lymphomas, and we hypothesize there are regional differences in subtype, distribution in the GI tract, and epidemiological features among the different populations.
Methods
We retrospectively evaluated the clinical, molecular and histologic features of North American primary and secondary GI lymphomas diagnosed from 2000–2009 seen at our institution. We utilized immunohistochemistry and fluorescence in situ hybridization to further evaluate a subset of the gastric lymphomas.
Results
Extranodal marginal zone lymphomas of mucosal associated lymphoid tissue (MALTs) and diffuse large B cell lymphomas (DLBCLs) were the most common subtypes of GI lymphomas. Select gastric DLBCLs (N = 6) and MALTs (N = 13) were further examined for API2-MALT1 and IGH translocations, and P16 and P53 protein expression. Gastric MALTs showed frequent API2-MALT1 (38%) but not IGH translocations (0%), and the DLBCLs showed neither translocation. Expression of P16 and P53 proteins and the proliferative index were compared between high grade gastric lymphomas (gastric DLBCLs) and low grade gastric lymphomas (gastric MALTs). P53 overexpression (P = 0.008) and a high proliferation index [Ki-67] (P = 0.00042) were significantly associated with gastric DLBCL, but no statistically significant difference was observed in P16 expression (p = 0.108) between gastric DLBCL and gastric MALT.
Conclusion
Our study revealed that GI lymphomas from a Central-Midwestern North American population showed differences and similarities to non-North American cohorts. In addition, API2-MALT1, P16 and P53 abnormalities occurred frequently in gastric lymphomas from this North American population.
Virtual slides
The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1415505838687793 webcite
Keywords:
Gastrointestinal lymphoma; Secondary versus primary; Molecular features; Locations
Introduction
Gastrointestinal (GI) lymphomas are a relatively common type of extranodal lymphoma, accounting for up to 30-50% of extranodal lymphomas in some series [1,2]. However, the majority of population based studies have been performed on Asian or European cohorts [2-9] with only one recent study from a Canadian-North American cohort [10]. Though studies from the United States (US) have been performed, most are several decades old, and use nomenclature that predates the current World Health Organization (WHO) classification for lymphomas [11,12]. It is established that the prevalence of many gastrointestinal malignancies differs between continents. For example, the incidence of gastric adenocarcinoma is known to be substantially higher in Japan than in the US [13]. Therefore, it stands to reason that the distribution of different lymphoma subtypes in the GI tract may differ between North America and other continents.
Specific genetic and epigenetic factors play a role in the pathogenesis of many lymphomas, including translocations (i.e. API2-MALT1CCND1-IGH) [14], defects in tumor suppressor genes (i.e. RB1TP53p15/CDKN2B) [15-19], and promoter hypermethylation of tumor suppressor genes (i.e. p16/CDKN2A) [20-22]. Promoter hypermethylation of p16/CDKN2A occurs quite frequently in gastric lymphoid follicles with Helicobacter pylori infection, gastric marginal zone lymphomas of mucosal associated lymphoid tissues (MALTs), and gastric diffuse large B cell lymphomas (DLBCLs), accounting for 10%, 41.7% and 72.7% of these cases, respectively in a Korean study [22]. Some genetic perturbations have been shown to be characteristic of specific lymphomas of the GI tract. For example, the t(11;18)(q12;q21); API2-MALT1 is relatively common in gastric MALTs, while it is less common in pulmonary MALTs, and is virtually never found in MALTs from other locations [14]. Epidemiologic factors, such as Helicobacter pylori infection in gastric MALTs [14], are also known to be associated with some lymphoma subtypes.
Systemic lymphomas may secondarily involve the GI tract. However, the subtypes of lymphomas that affect the GI tract primarily versus secondarily have not been comprehensively detailed.
In the current study, we evaluated primary and secondary GI lymphomas diagnosed at Washington University Medical Center over the 10 year period from 2000–2009. We determined the frequency of involvement of different anatomic locations in the GI tract by different lymphoma subtypes. Gastric DLBCLs and MALTs were further studied to determine P16 and P53 expression, and the presence of IGH and API2-MALT1 rearrangements, to establish frequencies of these alterations in cases from a Central North American population. Our studies highlight there are distinct clinicopathologic features of primary and secondary GI lymphomas from a Central-Midwestern North American cohort.
Material and methods
Patient selection
All aspects of this study were approved by an internal ethics committee at Washington University.
We retrospectively examined cases of GI lymphomas diagnosed at Washington University from January 2000 to December 2009. A total of 242 cases were included in this study, including 216 primary and 26 secondary GI lymphomas, among which 64 were initially diagnosed in our institution and 178 were referred from outside institutions. Primary GI lymphoma was defined as cases presenting with GI symptoms or predominant tumor(s) location in the GI tract [23]. Secondary GI lymphomas were diagnosed when patients had a prior diagnosis of lymphoma that later recurred in the GI tract (esophagus, stomach, small intestine, large intestine). Lymphoma cases from patients in the post-transplant setting were excluded. All patients were classified according to the World Health Organization (WHO) 2008 criteria [24]. Staging was performed according to the Ann Arbor classification modified by Musshoff for the digestive tract [25,26]. Clinical history, including Helicobacter pylori (HP) infection (by positive histology or serologic testing), imaging studies, and patients’ chart review were performed to categorize these cases as primary or secondary GI lymphomas. Sites of involvement were recorded. Among the 64 primary GI lymphomas initially diagnosed at our institution, there were 30 DLBCLs, 19 MALTs, 4 follicular lymphomas (FLs), 3 anaplastic large cell lymphomas (ALCLs), 2 mantle cell lymphomas (MCLs), 4 Burkitt lymphomas (BLs), and 2 other non-ALCL T cell lymphomas. 12 gastric DLBCLs and 16 gastric MALTs were initially diagnosed at our institution, and sufficient tissue was available on 6 gastric DLBCLs and 13 gastric MALTs to perform additional tests (immunohistochemistry for P16, P53, and Ki-67 and fluorescence in sit hybridization studies [FISH]). 1 duodenal MALT was also evaluated by immunohistochemistry for P16, P53, and Ki-67 and FISH. 3 benign chronic gastritis biopsies with lymphoid hyperplasia were also evaluated by immunohistochemistry for P16 and P53.
Immunohistochemistry
All tissues were obtained by endoscopic biopsy or surgery, fixed in 10% neutral buffered formalin, and then sectioned at 4 μm to evaluate by immunohistochemistry. The following antibodies were analyzed: P16 (Clone INK4a, MTM laboratories, prediluted), P53 (Bp-53-11, Ventana, Prediluted), and Ki-67 (Clone 30–9, Ventana, prediluted). All immunohistochemical stains were blinded and independently reviewed by two pathologists (J.W. and T.N.) and discrepancies were settled over a multiheaded scope.
P16 and P53 immunostaining was scored as follows: 0, no to ≤ 5% of lesional cells positive; 1, >5% to < 20% of lesional cells positive; 2, ≥ 20% of lesional cells positive, regardless of staining intensity. Staining was performed using the BenchMark XT (Ventana, Tusco, AZ), and antibody detection with UltraView Universal 3,3’-diaminobenzidene detection system (Ventana). Ki-67 was scored by the presence of positive nuclear staining in the lesional cells.
Interphase fluorescence in situ hybridization analysis
The presence of immunoglobulin heavy chain (IGH) and API2-MALT1(Vysis LSI BIRC3/MALT1 dual color dual fusion) translocations were examined by a bicolor FISH detection system, using a dual color break apart probe and dual color fusion probe respectively (Abbott Molecular, Downer’s Grove, IL, USA), according to manufacturer’s recommendations and laboratory protocol previously described [27]. Nuclei were then counter-stained with DAPI (0.5 l/ml) and the sections were examined using an Olympus BX51 or BX61 fluorescent microscope with appropriate filters (Olympus, Melville, NY, USA). At least one hundred non-overlapping nuclei were examined for each probe. The IGH break apart rearrangement probe was scored positive if a split signal pattern with a split of red and green signals were seen in at least 10% of the cells analyzed. The API2-MALT1 probe was read as positive if at least 5% cells with 2 fusion signals (yellow), 1 red signal and 1 green signal were seen.
Statistical analysis
Fisher’s exact test was conducted to test association between P16 and P53 expression with lymphoma grade, while Ki-67 proliferation index between patients of low and high grade lymphoma was compared using Wilcoxon rank sum test. Low grade lymphomas were defined as gastric MALTs for our statistical analysis. High grade lymphomas were cases that met criteria for gastric DLBCL. Gastric MALTs with admixed large B cells were classified as low grade lymphomas in our statistical analysis. Statistical significance was deemed at the 5% level. All analyses were conducted in R2.13.1.
Results
Subtype and site of involvement for the primary GI lymphomas
The primary GI lymphomas in our series are depicted by anatomic site and lymphoma subtype in Additional file 1 Table S1. Intestinal lymphomas (n = 116) were more common than gastric lymphomas (n = 97), with the large intestine (excluding the rectum) being the most common site of involvement in the intestine. In analyzing both the in house primary GI lymphomas and primary GI lymphomas diagnosed initially at an outside institution which were referred to our medical center for treatment, gastric DLBCLs and gastric MALTs accounted for the greatest numbers of lymphomas. Although DLBCL was the most common lymphoma type in the intestine, accounting for 33% of cases, follicular lymphoma [FL] (21%) and mantle cell lymphoma [MCL] (22%) also comprised a considerable fraction of cases in this site. Burkitt lymphoma (9%) appeared to be primarily an intestinal lymphoma among our cases. MALTs were less common (10% of intestinal cases), and the majority of cases occurred in the duodenum. Similar to the intestinal cases, DLBCL was the most common lymphoma type in the stomach, accounting for 48% of cases. In contrast to the intestine, MALT was the second most common type (44%) in the stomach. Gastric FL (1%), T-cell non-Hodgkin lymphoma (2%), and MCL (2%) were distinctly uncommon. Excluding MCL, which tends to involve many sites of the intestinal tract, we identified three cases of primary GI lymphomas involving multiple, non-contiguous sites. Two were DLBCLs involving the stomach and cecum, and the other was an anaplastic large cell lymphoma (ALCL) involving the duodenum and large intestine.
Additional file 1. Table S1. Subtype and site of involvement of gastrointestinal lymphomas seen at a North American Medical center from 2000-2009.
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Patient demographics
The median age and sex distribution of the primary GI lymphoma cases are listed in Additional file 2 Table S2. A male predominance was seen in intestinal MALT (5:1 male:female), intestinal DLBCL (1.7:1 male:female), and intestinal Burkitt lymphoma (1:0 male:female). The remaining lymphoma subtypes showed either no considerable sex predominance or were present in insufficient numbers to make a determination. Gastric and intestinal DLBCLs, MALTs, and FLs tended to affect patients in the sixth and seventh decade, while intestinal Burkitt lymphomas tended to affect a younger cohort, with a median age of 41 years. Review of the clinical charts revealed Helicobacter pylori infection occurred in approximately 24% of gastric MALTs and 9% of gastric DLBCLs by positive serology or histology. Stage of diagnosis was examined in all gastric MALTs and DLBCLs that had adequate radiographic data available. Gastric DLBCLs presented more frequently at a higher stage (i.e. stage III or IV) compared to the gastric MALTs (72% vs. 3%). The majority of gastric MALTs were stage IE or IIE.
Additional file 2. Table S2. Demographics of primary gastrointestinal lymphomas seen at a North American medical center from 2000-2009.
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Immunophenotype, expression of P16 and P53, and proliferation index in gastric lymphomas
Six gastric DLBCLs had sufficient tissue for evaluation with immunohistochemistry, and the results are summarized in Additional file 3 Table S3. All 6 cases were negative for CD10 and CD5. All gastric DLBCLs showed a high proliferative index, with a Ki-67 index ≥80%. In contrast, the gastric MALTs had lower Ki-67 rates (≤20%) in the majority of cases. Rare (2/13) cases of gastric MALT showed a higher proliferative index of ~50%, which were those with increased admixed larger cells (see Additional file 3 Table S3, MALT cases noted by **).
Additional file 3. Table S3. Immmunophenotype, P16 and P53 expression, proliferative index, clinical and molecular features in gastric lymphomas.
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Expression of P16 and P53 in 3 benign chronic gastritis biopsies was evaluated to establish a baseline. P16 expression was low or absent in the chronic gastritis cases, while P53 was not overexpressed in any of these benign cases. All gastric DLBCLs showed P53 expression, and frequent overexpression (score 2 in 83%) was observed in the gastric DLBCLs. 3 gastric DLBCLs (50%) also showed P16 expression. Among the 13 gastric MALTs evaluated, only two (15%), which also had increased large cells and a higher Ki-67 index, showed overexpression of P53 (defined as score 2 or ≥ 20% tumor cells positive), while the majority (85%) lacked P53 over-expression. P16 expression was seen in only 2 (15%) of gastric MALTs. We compared the P16 and P53 expression and proliferative index between high grade gastric lymphomas (gastric DLBCLs) and low grade gastric lymphoma (gastric MALTs). A statistically significant association between P53 overexpression and gastric DLBCL was noted (p = 0.008). Ki-67 level was also associated with gastric DLBCL using a Wilcoxon rank sum test (p = 0.00042). However, no statistically significant difference was observed in P16 expression between gastric DLBCL and gastric MALT (p = 0.108). Figure 1 demonstrates the histology, Ki-67 expression, and P53 expression in a gastric MALT and a gastric DLBCL showing the most common expression profile for each type.
Figure 1. A,C, and E depict a low grade gastric marginal zone lymphoma of mucosal associated lymphoid tissue (MALT). B, D, and F depict a gastric diffuse large B cell lymphoma (DLBCL). (A) MALT in a gastric biopsy (H&E, 400X). (B) DLBCL in a gastric biopsy (H&E, 600X). (C) Ki-67 shows a low proliferative index in a low grade MALT (Ki-67 immunohistochemistry, 200X). (D) Ki-67 shows a high proliferative index, 100%, in this DLBCL. The negatively stained cells are the glandular epithelial cells. (Ki-67 immunohistochemistry, 400X). (E) P53 protein is absent in the tumor cells of a MALT, while weak nuclear P53 staining is seen in the adjacent benign glandular epithelium (P53 immunohistochemistry, 1000X). (F) P53 protein overexpression is noted in the tumor cells of this DBLCL, while the cells negative for nuclear P53 immunostaining are benign glandular epithelial cells. (P53 immunohistochemistry, 1000X).
FISH evaluation for API2-MALT1 and IGH translocations in gastric lymphomas
5 of 13 (38%) cases of gastric MALTs were positive for the API2-MALT1 translocation, while all 13 cases were negative for the IGH translocation. A single duodenal MALT was positive for the IGH translocation, but negative for the API2-MALT1. All 6 gastric DLBCLs were negative for IGH and API2-MALT1 translocations.
Subtype and site of involvement of secondary GI lymphomas
Secondary involvement of the GI tract by lymphoma was considerably less common than primary GI lymphoma (26 secondary vs. 216 primary cases). DLBCL was the most common lymphoma type to secondarily involve the GI tract, accounting for the majority of intestinal (57%), gastric (80%), and esophageal (100%) cases. Gastric involvement was more common than intestinal or esophageal involvement. A handful of cases of FL, ALCL, Burkitt lymphoma, small lymphocytic lymphoma, and T-cell non-Hodgkin lymphoma also showed secondary involvement of the GI tract, while no secondary cases of MALT or B or T lymphoblastic lymphoma were identified in our series. The results are summarized in Additional file 1 Table S1.
Discussion
We undertook this 10 year retrospective study to evaluate the clinicopathologic and molecular features of GI lymphomas at a major US tertiary care medical center to determine the features of GI lymphomas in North America in comparison to other non-North American lymphoma cohorts. No previous extensive US study describing the primary and secondary GI lymphomas presenting in a Northern American population has been published that utilizes the current World Health Organization lymphoma classification. We show that there are distinct differences and similarities in our cases compared to other population based studies. Although many types of GI lymphomas were diagnosed, most of the cases presenting to our facility were referral cases initially diagnosed as lymphoma at an outside facility which were later referred to our facility for additional treatment. Thus, many of these patients had been treated with many different therapeutic regimens, so treatment regimens varied greatly among our cases and were too heterogenous to correlate treatments with clinical outcome in our analysis.
Several series from Europe, Canada-North America, and Asia have reported on primary GI lymphomas by lymphoma type and anatomic site [2-5,7,9,28-30]. Several trends emerge when reviewing the data collectively. First, primary gastric lymphomas tended to be more common than primary intestinal lymphomas in the European and Asian studies. Intestinal cases accounted for only 14%, 19%, 21%, 10%, 30%, 34%, 50% and 43% of cases in the Greek, Japanese, German, Austrian, Serbian, Turkish, Canadian-North American and Danish studies, respectively [2-5,7,9,28-30]. Gastric lymphomas accounted for >50% of total cases in most of these series. Our study stands in contrast to these, in that intestinal cases out-number gastric cases, accounting for 54% of total primary GI lymphomas. This difference noted in our study could be due to lower prevalence rates of HP infection in the US resulting in fewer gastric MALTs diagnoses, earlier treatment for HP infection resulting in fewer cases progressing to gastric MALTs, or that US patients with intestinal involvement have more severe symptoms such as intestinal obstruction and/or easier health care access resulting in more frequent visits to our tertiary care surgery facilities than patients with only solitary gastric involvement. The second trend to emerge is that high grade gastric lymphomas (i.e. gastric DLBCLs) tend to occur in approximately equal proportion to low grade gastric lymphomas (i.e. gastric MALTs). In this regard, our data are consistent with the European and Asian studies. Third, MALTs are much more common in the stomach than other sites, while DLBCLs tend to affect all segments of the GI tract. Our series also shows this trend to hold true.
Our study is also consistent with a recent study of primary GI FL from France by Damaj et al., which showed that primary FL of the GI tract tends to be a disease of the small intestine [31]. Similar findings were reported in a recent large US study which revealed small intestinal FLs made up 63% of cases while gastric FLs accounted for only 3% of primary gastrointestinal FLs [32]. Similarly, 14 of the 25 (56%) of primary FLs in our series were found in the small intestine. No other low grade B lymphoma type in our series showed such a strong propensity for small intestinal involvement. In contrast to the study by Damaj et al., which showed a female predominance, the small intestinal FLs in our series were equally distributed among men and women.
In examining the stage of presentation in our gastric MALTs and gastric DBLCLs, we also noted that gastric DLBCLs tended to have higher stage disease at presentation, while gastric MALTs often showed lower stage disease at presentation. Previous studies have shown that stage of lymphoma is an important prognostic predictor of clinical outcome, and that the lower grade gastric MALTs tend to have low stage while higher grade DLBCLs tend to have higher stage at presentation [3,4,28,33]. Thus, our study reveals that this association also occurs in the lymphomas from a Central North American population.
Environmental factors, especially Helicobacter pylori (HP) infection, have been noted to play a prominent role in the development of gastric lymphomas [34]. In 2 Italian studies, HP has been reported to occur in 88% of low grade gastric MALTs identified by histologic examination, while it occurred at lower frequency (52-63%) in high grade gastric lymphomas [28,35]. Interestingly, both Italian studies revealed a statistically significant association between HP infection and low grade gastric lymphoma (p < 0.0001) [28,35]. Similarly, a serologic study performed at a New York center showed HP seroprevalence up to 67% in gastric MALTs, and increased seropositive rates were associated with increased age and country of birth outside the US or Canada (p = 0.0001) [34]. Interestingly, HP infection in our study was seen in only 24% of the gastric MALTs and 9% of the gastric DLBCLs by positive serology or histology, and we also noted higher frequency of HP infection in the low grade gastric MALTs compared to high grade gastric DLBCLs. A 2008 Canadian-North American study also showed HP infection in 20% of gastrointestinal non-Hodgkin lymphomas, of which 44% were in MALTs and 13% were in DLBCLs [10]. The less frequent HP infection rates in our cases could relate to our patient population having a higher percentage of patients born in the US or Canada, differences in the ethnicities of the Central-Midwestern compared to the New York population, or lower prevalence of HP infection in the Midwest. Alternatively, the absence of serologic testing in all our cases, which may be more sensitive than histology in detecting HP infections, could also contribute to a lower HP prevalence rate in our gastric MALT patients.
Because of the high prevalence of genetic defects in p16/CDNK2A in non-Hodgkin B cell lymphomas, we also evaluated P16 expression in our gastric MALTs and DLBCLs. Previous studies have shown that P16 is underexpressed more frequently in gastric MALT than in other lymphoma types [20,21]. This has been shown to primarily relate to p16 promoter hypermethylation in non-transformed MALTs [20,36,37]. As such, p16 hypermethylation, and the resulting loss of P16 protein, is a high-frequency event in gastric MALTs occurring in up to 75% to 79% of cases in some studies [20-22,38,39]. One Korean study by Min et al. also showed increased frequency of p16 hypermethylation in gastric DLBCLs (N = 11, 72.7%) compared to gastric MALTs (N = 24, 41.7%) and proposed that malignant transformation of gastric MALTs was attributed in part to p16 hypermethylation [22]. Our results confirm the prior findings that p16 is frequently inactivated in gastric MALTs, since P16 expression was absent in 11 of 13 (85%) gastric MALTs, and expression was scored as 0 (i.e. less 5% of tumor cells positive) in these cases. In addition, P16 was expressed in only 3 of the 6 (50%) gastric DLBCLs. Most p16 hypermethylation has been previously seen in HP dependent cases. This fact may explain the weak P16 expression seen in one HP + gastritis case we tested, but does not provide a mechanism for P16 loss in the our HP- gastritis cases and HP- gastric MALTs. Although the overall numbers of cases analyzed for P16 expression is low, our study showed no statistically significant difference was observed in P16 expression between gastric DLBCL and gastric MALT (p = 0.1078). Our result differs from the Korean study by Min et al. that showed hypermethylation of p16 (which can consequently downregulate P16 expression) to be more frequently associated with gastric DLBCLs compared to low grade gastric MALTs [22]. Since the number cases in our study is only half as many as studied by Min et al., other studies would be needed to determine if this association between p16 hypermethylation (P16 expression loss) in high grade gastric DLBCLs holds true in other populations.
P53 over-expression has been shown to correlate with mutations in TP53 [40]. This may relate to decreased rate of degradation of the mutant protein, leading to elevated cytoplasmic accumulation. P53 immunohistochemistry may be useful in evaluating primary GI lymphomas, because P53 accumulation has been shown to be more common in high grade lymphomas than low grade lymphomas [41]. Moller et al. showed that P53 protein overexpression, defined as ≥ 20% of tumor cells positive for P53, was 80-90% sensitive and 100% specific in predicting TP53 mutations in DLBCLs [42]. Moller et al. also reported that p53 overexpression was an independent predictor of poor outcome in both T and B non-Hodgkin lymphomas (n = 199), and that P53 predicted poorer overall survival in indolent and aggressive non-Hodgkin lymphomas in addition to being associated with treatment failure and relapse-free survival. Using the same scoring criteria, high grade gastric DLBCLs were more significantly associated with P53 overexpression than low grade gastric MALTs in our series (p = 0.008). Thus our data corroborates the prior studies which have suggested mutations in TP53 may relate to progression from low grade to high grade lymphoma, as well as increased risk of relapse [19,42].
Increased Ki-67 index has also been shown to correlate with increased grade of lymphoma [41]. Our results were consistent with these previous observations, since all of our tested gastric DLBCLs had high Ki-67 index ≥80%, while the majority (85%) of the gastric MALTs had Ki-67 ≤20%. Statistical analysis on our lymphomas also showed higher Ki-67 levels were significantly associated with high grade lymphoma, specifically gastric DLBCL (p = 0.00042). Of note, the two MALTs with the highest Ki-67 index (50%) both showed strong P53 expression and an increased admixture of large B cells. This suggests they are not typical low grade MALTs, and likely are intermediate or transitional cases between a low grade and a high grade gastric B cell lymphoma. Alternatively, since these two gastric MALTs were diagnosed from small endoscopic biopsies, undersampling of a DLBCL on a small biopsy could also explain the P53 overexpression in these two outlier cases. Identification of a high proliferative index (Ki-67 >80%) on small gastric biopsies is also a helpful feature in diagnosing DLBCL, since all low grade gastric MALTs lacking evidence of large cell transformation (or admixed large cells) tend to show lower Ki-67 levels (≤20%). Assessment of the proliferative index may be a useful feature to study in small gastric biopsies with significant crushed features where morphologic evaluation is limited, and optimal grading and subtyping of the B cell lymphomas can be challenging due to the suboptimal quality of the biopsy.
The API2-MALT1 translocation has been shown to be relatively frequent in gastric MALTs, and has been shown to predict both lack of resolution with H. pylori eradication [43] and resistance to further genetic damage [44,45]. Perhaps unsurprisingly, this translocation has been reported to be a rare event in gastric DLBCLs [20,45-47], indicating a gastric MALT with this translocation rarely progress to gastric DLBCL. Our series confirm these previous findings, as none of our gastric DLBCLs showed the API2-MALT1 translocation, and none of the gastric MALTs with this translocation had concomitant HP infection. In addition, this translocation was quite frequent (38%) in the gastric MALTs in our series, similar to the 17%-40% reported in other studies [17,18,30,48,49].
Although IGH translocations such as t(1;14)(p22;q32);BCL10-IGH are reported in 3% of gastric MALTs and 5-10% of intestinal MALTs [17,18], we did not detect frequent IGH translocations in this series, as our tested gastric MALTs were all negative. Only one duodenal MALT tested showed the IGH translocation. Unfortunately, due to the absence of karyotype analysis, we could not further determine the partner gene for this case. Nevertheless, our data corroborates the prior data that an IGH translocation (such as the IGH-MALT1IGH-BCL10IGH-FOXP1), which is not the same as a clonal IGH gene rearrangement detected by polymerase chain reaction analysis, is an infrequent genetic alteration in the pathogenesis of gastric MALTs.
In summary, our study highlights the differences in distribution and subtype of GI lymphomas at a large North American medical center. Secondary GI lymphomas were less frequent than primary GI lymphomas, and there were clear differences in frequency and subtype between primary and secondary cases. We confirm that certain genetic alterations occur frequently in GI lymphomas, such as loss of P16 protein expression and API2-MALT1 translocations in gastric MALTs, and P53 overexpression in gastric DLBCLs. However, we also found differences in the subtype, molecular, and clinical features in our Central-Midwestern North American patient population compared to other non-North American series.
Abbreviations
GI: Gastrointestinal; MALT: Extranodal marginal zone lymphoma of mucosal associated lymphoid tissue; DLBCL: Diffuse large B cell lymphoma; IGH: Immunoglobulin heavy chain gene; HP: Helicobacter pylori; FL: Follicular lymphoma; ALCL: Anaplastic large cell lymphoma; MCL: Mantle cell lymphoma; BL: Burkitt lymphoma; FISH: Fluorescence in situ hybridization; US: United States.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
JW helped design the study, performed the epidemiological and immunohistochemical data analysis, and drafted the manuscript. JL performed statistical data analysis. DR and JB performed the cytogenetic data analysis. JLF helped design the project, contributed cases to the study, and helped write the manuscript. FK and AH contributed cases to the study, and helped write the manuscript. TN designed the study, coordinated the research efforts, carried out the histologic, immunohistochemical, and epidemiologic data review, and drafted the manuscript. All authors read and approved the final manuscript.
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24. Swerdlow SHCE, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW: WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Lyon: IARC; 2008.
25. Rohatiner A, d'Amore F, Coiffier B, Crowther D, Gospodarowicz M, Isaacson P, Lister TA, Norton A, Salem P, Shipp M, et al.: Report on a workshop convened to discuss the pathological and staging classifications of gastrointestinal tract lymphoma.
Ann Oncol 1994, 5:397-400. PubMed Abstract | Publisher Full Text
26. Ruskone-Fourmestraux A, Delmer A, Hennequin C: Gastro-intestinal lymphomas.
Gastroenterol Clin Biol 2006, 30(2):2S81-2S90. PubMed Abstract | Publisher Full Text
27. Fallon KB, Palmer CA, Roth KA, Nabors LB, Wang W, Carpenter M, Banerjee R, Forsyth P, Rich K, Perry A: Prognostic value of 1p, 19q, 9p, 10q, and EGFR-FISH analyses in recurrent oligodendrogliomas.
J Neuropathol Exp Neurol 2004, 63:314-322. PubMed Abstract | Publisher Full Text
28. Andriani A, Zullo A, Di Raimondo F, Patti C, Tedeschi L, Recine U, Caruso L, Bonanno G, Chiarenza A, Lizzani G, Miedico A, Romanelli A, Costa A, Linea C, Marrone C, Mirto S, Mistretta A, Montalbano L, Restivo G, Vinci M, Bibas M, Hassan C, Stella F, Cottone M, Morini S: Clinical and endoscopic presentation of primary gastric lymphoma: a multicentre study.
Aliment Pharmacol Ther 2006, 23:721-726. PubMed Abstract | Publisher Full Text
29. Erkurt MA, Aydogdu I, Kuku I, Kaya E, Basaran Y: Clinicopathologic characteristics and therapeutic outcomes of primary gastrointestinal non-Hodgkin's lymphomas: 10 years of experience from a single center in eastern Anatolia.
Med Princ Pract 2009, 18:399-406. PubMed Abstract | Publisher Full Text
30. Remstein ED, Dogan A, Einerson RR, Paternoster SF, Fink SR, Law M, Dewald GW, Kurtin PJ: The incidence and anatomic site specificity of chromosomal translocations in primary extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) in North America.
Am J Surg Pathol 2006, 30:1546-1553. PubMed Abstract | Publisher Full Text
31. Damaj G, Verkarre V, Delmer A, Solal-Celigny P, Yakoub-Agha I, Cellier C, Maurschhauser F, Bouabdallah R, Leblond V, Lefrere F, Bouscary D, Audouin J, Coiffier B, Varet B, Molina T, Brousse N, Hermine O: Primary follicular lymphoma of the gastrointestinal tract: a study of 25 cases and a literature review.
Ann Oncol 2003, 14:623-629. PubMed Abstract | Publisher Full Text
32. Misdraji J, Harris NL, Hasserjian RP, Lauwers GY, Ferry JA: Primary follicular lymphoma of the gastrointestinal tract.
Am J Surg Pathol 2011, 35:1255-1263. PubMed Abstract | Publisher Full Text
33. Zullo A, Hassan C, Cristofari F, Perri F, Morini S: Gastric low-grade mucosal-associated lymphoid tissue-lymphoma: Helicobacter pylori and beyond.
World J Gastrointest Oncol 2010, 2:181-186. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
34. Cuttner J, Werther JL, McGlynn P, Chen A, Bodian C, Ogata S, Strauchen J, Troy K, Itzkowitz S: Seroprevalence of Helicobacter pylori infection in patients with lymphoma.
Leuk Lymphoma 2001, 40:591-597. PubMed Abstract | Publisher Full Text
35. Zullo A, Hassan C, Andriani A, Cristofari F, Cardinale V, Spinelli GP, Tomao S, Morini S: Primary low-grade and high-grade gastric MALT-lymphoma presentation.
J Clin Gastroenterol 2010, 44:340-344. PubMed Abstract | Publisher Full Text
36. Kondo T, Oka T, Sato H, Shinnou Y, Washio K, Takano M, Morito T, Takata K, Ohara N, Ouchida M, Shimizu K, Yoshino T: Accumulation of aberrant CpG hypermethylation by Helicobacter pylori infection promotes development and progression of gastric MALT lymphoma.
Int J Oncol 2009, 35:547-557. PubMed Abstract | Publisher Full Text
37. Neumeister P, Hoefler G, Beham-Schmid C, Schmidt H, Apfelbeck U, Schaider H, Linkesch W, Sill H: Deletion analysis of the p16 tumor suppressor gene in gastrointestinal mucosa-associated lymphoid tissue lymphomas.
Gastroenterology 1997, 112:1871-1875. PubMed Abstract | Publisher Full Text
38. Huang Q, Ai L, Zhang ZY, Fan CY, Weiss LM: Promoter hypermethylation and protein expression of the p16 gene: analysis of 43 cases of B-cell primary gastric lymphomas from China.
Mod Pathol 2004, 17:416-422. PubMed Abstract | Publisher Full Text
39. Yang Y, Wei ZJ, Zhai Y: The significance of p16 protein and Ki-67 antigen expression in gastric mucosa-associated lymphoid tissue lymphoma.
Zhonghua Nei Ke Za Zhi 2008, 47:743-745. PubMed Abstract
40. Hall PA, Lane DP: p53 in tumour pathology: can we trust immunohistochemistry?-- Revisited!
J Pathol 1994, 172:1-4. PubMed Abstract | Publisher Full Text
41. Li HL, Sun BZ, Ma FC: Expression of COX-2, iNOS, p53 and Ki-67 in gastric mucosa- associated lymphoid tissue lymphoma.
World J Gastroenterol 2004, 10:1862-1866. PubMed Abstract | Publisher Full Text
42. Moller MB, Gerdes AM, Skjodt K, Mortensen LS, Pedersen NT: Disrupted p53 function as predictor of treatment failure and poor prognosis in B- and T-cell non-Hodgkin's lymphoma.
Clin Cancer Res 1999, 5:1085-1091. PubMed Abstract | Publisher Full Text
43. Liu H, Ruskon-Fourmestraux A, Lavergne-Slove A, Ye H, Molina T, Bouhnik Y, Hamoudi RA, Diss TC, Dogan A, Megraud F, Rambaud JC, Du MQ, Isaacson PG: Resistance of t(11;18) positive gastric mucosa-associated lymphoid tissue lymphoma to Helicobacter pylori eradication therapy.
Lancet 2001, 357:39-40. PubMed Abstract | Publisher Full Text
44. Starostik P, Patzner J, Greiner A, Schwarz S, Kalla J, Ott G, Muller-Hermelink HK: Gastric marginal zone B-cell lymphomas of MALT type develop along 2 distinct pathogenetic pathways.
Blood 2002, 99:3-9. PubMed Abstract | Publisher Full Text
45. Schreuder MI, Hoeve MA, Hebeda KM, Verdijk MA, Ligtenberg MJ, Bot FJ, Chott A, van Krieken JH: Mutual exclusion of t(11;18)(q21;q21) and numerical chromosomal aberrations in the development of different types of primary gastric lymphomas.
Br J Haematol 2003, 123:590-599. PubMed Abstract | Publisher Full Text
46. Nakamura S, Ye H, Bacon CM, Goatly A, Liu H, Kerr L, Banham AH, Streubel B, Yao T, Tsuneyoshi M, Savio A, Takeshita M, Dartigues P, Ruskone-Fourmestraux A, Matsumoto T, Iida M, Du MQ: Translocations involving the immunoglobulin heavy chain gene locus predict better survival in gastric diffuse large B-cell lymphoma.
Clin Cancer Res 2008, 14:3002-3010. PubMed Abstract | Publisher Full Text
47. Huang X, Zhang Z, Liu H, Ye H, Chuang SS, Wang J, Lin S, Gao Z, Du MQ: t(11;18)(q21;q21) in gastric MALT lymphoma and diffuse large B-cell lymphoma of Chinese patients.
Hematol J 2003, 4:342-345. PubMed Abstract | Publisher Full Text
48. Barth TF, Bentz M, Leithauser F, Stilgenbauer S, Siebert R, Schlotter M, Schlenk RF, Dohner H, Moller P: Molecular-cytogenetic comparison of mucosa-associated marginal zone B-cell lymphoma and large B-cell lymphoma arising in the gastro-intestinal tract.
Genes Chromosomes Cancer 2001, 31:316-325. PubMed Abstract | Publisher Full Text
49. Wang G, Auerbach A, Wei M, Dow N, Barry TS, Hodge L, Schaffer D, Sobin LH, Aguilera NS: t(11;18)(q21;q21) in extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue in stomach: a study of 48 cases.
Mod Pathol 2009, 22:79-86. PubMed Abstract | Publisher Full Text
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Template:Move
From eLinux.org
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Move proposal : It has been suggested that this page be moved to a new name.
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United States Bible RecordsEdit This Page
From FamilySearch Wiki
United States Bible Records
See also United States, How to Find Genealogy Records
Many families have traditionally recorded births, marriages, and deaths in a family Bible, family record book, or book of remembrance. Family Bibles that are no longer in the possession of the family may be at a historical or genealogical society. They are sometimes transcribed and published in genealogical periodicals.
The Daughters of the American Revolution have collected and transcribed many Bible records. These transcripts are at the DAR Library in Washington, DC, or at local DAR chapters. Most are on microfilm at the Family History Library. Partial indexes to these records are:
• Genealogical Records Committee Index a free online index in the DAR Genealogical Research System (GRS Search).
• Kirkham, E. Kay. An Index to Some of the Bibles and Family Records of the United States: 45,500 References as Taken from the Microfilm at the Genealogical Society of Utah. Logan, Utah: Everton Publishers, 1984. (Family History Library book FHL 973 D22kk; fiche FHL Collection 6089184.) WorldCat entry.
• National Genealogical Society (Arlington, Virginia). Bible Records, ca. 1747-1982. Salt Lake City, Utah: Genealogical Society of Utah, 1995. (On six Family History Library films FHL Collectionstarting with 1987737].) No circulation to Family History Centers. Includes index. Bible records collected from members of the society.
The DAR and other Bible records at the Family History Library are generally listed in the Locality Search of the Family History Library Catalog under one of the following:
[STATE] - BIBLE RECORDS
[STATE], [COUNTY] - BIBLE RECORDS
[STATE] - VITAL RECORDS
[STATE], [COUNTY] - VITAL RECORDS
Copies, or abstracts of old family Bibles that are no longer known to exist, may survive in Revolutionary War Pension application files at NARA, Washington, D.C., which are available online at three commercial websites: Ancestry, Footnote, and Heritage Quest Online.
You can find further information about Bible records in Wiki pages for each state under the topic of "Bible Records.".
• From Stalkin' Kin In Old West Texas, Vol XVI, No. 2.(San Angelo Genealogical and Historical Society, Inc. Aug 1988):
"BIBLE RECORDS: The National Archives and the Library of Congress in Washington, D.C. have collections of Bible Records sent as evidence in various claims against the U.S. Government. These are arranged in alphabetical order. Lists of these Bible records are available upon request."
Web Sites
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• This page was last modified on 11 December 2012, at 23:16.
• This page has been accessed 3,550 times.
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Help:Create an internal linkEdit This Page
From FamilySearch Wiki
Revision as of 05:26, 4 March 2010 by Tomhuber (Talk | contribs)
How to ...
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Template:TOC
Types of links
A link is a hyperlink or method for navigating to another page in the wiki or on the internet. There are two kinds of links that you can use in the FamilySearch wiki:
1. An internal link provides a link to other pages in the FamilySearch wiki.
2. An external link provides a link to pages that are not part of the FamilySearch wiki. To create external links, including a list of templates to use for linking to pages on specific internet sites, see Create an external link.
Does the text on the page have to match linked page's title?
It is not required, but the linking text should cleary convey the content of the linked page to the researcher and may 1) expand on the title of the linked page or 2) be more concise.
Example 1:
Page Title: Mennonties
Linking Text: Researching Mennonite Records
Example 2:
Page Title: Create an external link
Linking Text: External links
How to create an internal link
The wiki editor provides a set of tools for creating both internal and external links.
1. Log in to the FamilySearch Research Wiki.
2. Navigate to the page where you want to add the internal link. Click the Edit link in the navigation bar page options. This will open the editing window.
3. In the editing window, type the text you want to create the link with. Remember, this does not need to be the same as the title of the article to which you are linking.
4. Using your mouse, position your cursor in front of the text you just typed. While holding down the left mouse button, drag the cursor across the text until it is highlighted and then release the mouse button (the text should still be highlighted as shown in the image below).
5. On the editing toolbar, click the hyperlink icon. It looks like a tiny globe/earth with two chain links in front of it.
6. After clicking the hyperlink icon, the "Link" window will open.
7. At the top of the "Link" window, locate the "Link" box. Here you will type the name of the article you want to link to. Notice that after typing the first three letters of the article title, possible matches will appear in the "Automatic Search Results" box.
8. If the title of the article you want to link to appears in the "Automatic Search Results" box, left-click on the title of the article and the name of the article will appear in the "Link" box above. Once the correct article title appears in the "Link" box, click OK to save the link in the editing window.
9. The text that you highlighted in step 4 will now appear blue in the editing window.
10. Save your changes by scrolling down using the outside scroll-bar until you locate the Save page button located below the editing window.
11. After clicking Save page, you will be able to click on the newly created link between two Wiki articles. NOTE: If the link appears red on the page, the title of the linked article was typed in incorrectly. Repeat the steps above, paying close attention to steps 6-7.
Other Helpful Hints:
• To link to an article in this wiki, the link text must exactly match the article title (including spelling and punctuation). If there are any differences, the link will create a new blank article with the title you entered.
• If the page exists, the link will be blue.
• If the page has not yet been created, the link will be red.
• Click on the red link to create the page and begin adding text.
To create an internal or external link using wikitext see Advanced Linking.
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IrelandEdit This Page
From FamilySearch Wiki
Revision as of 16:04, 23 January 2013 by Bromaelor (Talk | contribs)
The Research Forums have been closed. For a limited time the Ireland Research forum will be available in read-only mode.
Topics
Genealogy courses: Learn how to research from an expert in Ireland courses.
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Contents
Introduction
Prior to 1922 the island of Ireland was one country, comprised of thirty-two counties within the four provinces of Connaught, Leinster, Munster and Ulster.
Today the island is divided into two countries:
• the Republic of Ireland, also known as Eire, made up of twenty-six counties
• Northern Ireland, a constituent part of the United Kingdom, made up of six of the counties of the Province of Ulster.
For further historic information, see Counties of Ireland.
Counties
The map below shows the whole of Ireland. The six counties of Northern Ireland are divided from the rest by a heavy black line.
Click on a county to go to that county's page:
Or click on a county name below:
Antrim § Dublin Limerick Roscommon
Armagh § Fermanagh § Londonderry (Derry) § Sligo
Carlow Galway Longford Tipperary
Cavan Kerry Louth Tyrone §
Clare Kildare Mayo Waterford
Cork Kilkenny Meath Westmeath
Donegal Laois (Queens) Monaghan Wexford
Down § Leitrim Offaly (Kings) Wicklow
§ These counties make up Northern Ireland
Beginners Corner
Research Guidance
If you are just beginning to research your Irish ancestors, here are some helps to get you started. Choose an event to learn about in the life of your Irish ancestors:
BYU's Ireland Research Guide
You may also wish to review the Ireland Research Guide. This 538-page resource is from the BYU Family History Library at Brigham Young University in Provo, Utah, and is a collection of many reference sources and articles (far more than just the Ireland Research Outline that appears first). The information is out of date and does not include online sources, but it is still valuable.
Records Available on FamilySearch
Wiki articles describing online collections are found at:
News & events
Did You Know?
• Between 1831 and 1841 in Ireland, 34,090 recruits joined the Army.
• The Princess Grace Irish Library provides online biographical & bibliographical information on 4,500 Irish writers on its EIRData website. EIRData, which stands for Electronic Irish Records Dataset, was compiled by Dr. Bruce Stewart of the University of Ulster. The site also contains primary and secondary bibliographies, commentaries, quotations and notes.
• The term 'Census Strays' refers to people who are born in one place whose name appears in a census in another place. A page on the North of Ireland Family History Society website contains details of over 15,000 records of persons living in households with one or more people of Irish origin. These "strays" compiled by the Society were sent in from the UK and the rest of the world. office
• Records of Officers from first Irish Police force on internet. Record of more than 80,000 officers from the 1st Irish Police Force are being released on line. It will contain personal details of every man that enlisted in the Irish Constabulary between 1816-1921 including their name, year and place of birth, age of enlistment and marital status. It is on the Ancestry Subscription site.
• The National University of Ireland in Galway, in conjunction with the Moore Institute for Research in the Humanities and Social Sciences, has created an online index to the landed estates of Connaught Province covering c.1700-1914. Though the project was not primarily intended as a genealogical tool, it is that, and they are to be commended for creating this great resource. The index can be searched alphabetically by the names of estates, families, and houses, and the website includes maps and images of the great houses. The index is found at the Landed Estates website.
Useful websites
More Ireland Websites ....
Need additional research help? Contact our research help specialists.
Need wiki, indexing, or website help? Contact our product teams.
Did you find this article helpful?
You're invited to explain your rating on the discussion page (you must be signed in).
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About this Journal Submit a Manuscript Table of Contents
Hepatitis Research and Treatment
Volume 2013 (2013), Article ID 125398, 6 pages
http://dx.doi.org/10.1155/2013/125398
Research Article
HD-03/ES: A Herbal Medicine Inhibits Hepatitis B Surface Antigen Secretion in Transfected Human Hepatocarcinoma PLC/PRF/5 Cells
Research and Development, The Himalaya Drug Company, Bangalore 562 123, India
Received 31 January 2013; Revised 5 March 2013; Accepted 19 March 2013
Academic Editor: Yoichi Hiasa
Copyright © 2013 Sandeep R. Varma 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
HD-03/ES is a herbal formulation used for the treatment of hepatitis B. However, the molecular mechanism involved in the antihepatitis B (HBV) activity of this drug has not been studied using in vitro models. The effect of HD-03/ES on hepatitis B surface antigen (HBsAg) secretion and its gene expression was studied in transfected human hepatocarcinoma PLC/PRF/5 cells. The anti-HBV activity was tested based on the inhibition of HBsAg secretion into the culture media, as detected by HBsAg-specific antibody-mediated enzyme assay (ELISA) at concentrations ranging from 125 to 1000 μg/mL. The effect of HD-03/ES on HBsAg gene expression was analyzed using semiquantitative multiplex RT-PCR by employing specific primers. The results showed that HD-03/ES suppressed HBsAg production with an IC50 of 380 μg/mL in PLC/PRF/5 cells for a period of 24 h. HD-03/ES downregulated HBsAg gene expression in PLC/PRF/5 cells. In conclusion, HD-03/ES exhibits strong anti-HBV properties by inhibiting the secretion of hepatitis B surface antigen in PLC/PRF/5 cells, and this action is targeted at the transcription level. Thus, HD-03/ES could be beneficial in the treatment of acute and chronic hepatitis B infections.
1. Introduction
Hepatitis B virus (HBV) infection is a major health problem throughout the world, affecting more than 350 million people who are carriers of this virus that can cause chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma [1]. A variety of serological markers appear following the infection with HBV, and first among these is HBsAg (hepatitis B surface antigen), which is observed two to three weeks before the clinical and biological symptoms appear. Prevalence of HBsAg in India varies from 1 to 13 percent with an average of 4.7 percent [24]. The molecular diagnosis which detects the HBsAg in the serum samples plays a significant role in the early diagnosis during hepatitis B (HB) infection.
PLC/PRF/5 is a continuous human hepatocarcinoma cell line whose genome contains integrated HBV DNA and secretes two of the hepatitis B virus envelope proteins [5]. The cells could secrete HBsAg continuously into the culture medium [6, 7]. These cells are suitable to study the effects of drugs on HBsAg expression and secretion [6]. Since the cells do not produce infectious virion particles, it is safe to handle the cell line with biosafety level 2 containment [8].
Several antivirals are currently available for the treatment of HBV, which include IFN-α, lamivudine, entecavir, telbivudine, and tenofovir. However, interferon therapy has limited efficacy, is slow-acting, and frequently causes adverse effects [9]. Interferon therapy is effective only for about 30 to 40 percent of the patients with chronic HBV infection. Undesirable side effects of interferon treatment are found such as fever, malaise, fatigue, depression, hair loss, neutropenia, and thrombocytopenia [10]. Lamivudine also produces response in a modest proportion of patients and causes a few side effects [11]. Moreover, antiviral drugs and interferon are expensive.
Herbal compounds from plant origin are leading for new drug discovery for infectious and noninfectious diseases. Several hundred plant species have been reported to possess antiviral properties and some have been utilized to treat patients [12]. HD-03/ES is a novel herbal formulation used for the treatment of HBV infections and is marketed as Liv.52 HB. HD-03/ES is a capsule formulation consisting of 125 mg each of hydroalcoholic extracts of the roots of herbs, Cyperus rotundus and Cyperus scariosus. The anti-HBV activity of HD-03/ES has been reported by several workers [1315]. However, the molecular mechanism behind the anti-HBV activity of HD-03/ES has not been studied well in vitro. The present study investigated the effect of HD-03/ES on the inhibition of HBsAg secretion and its gene expression in PLC/PRF/5 cells.
2. Materials and Methods
2.1. Materials
PLC/PRF/5 cells were obtained from National Center for Cell Science (NCCS), Pune. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), TRI reagent, and custom-prepared oligonucleotides, were obtained from Sigma Chemical Co. (St Louis, MO, USA). HBsAg Ultra ELISA kit was purchased from Bio-Rad, France. Penicillin and streptomycin were from Hi-media, Mumbai, India. Moloney murine leukemia virus (MMLV) reverse transcriptase, dNTP, and Taq DNA polymerase were from MBI Fermentas (Glen Burnie, MD, USA).
2.2. Extraction of HD-03/ES
HD-03/ES granules were obtained from the Formulation and Development Department, The Himalaya Drug Company, India. About 100 g of HD-03/ES granules was packed in a Soxhlet extraction apparatus. The material was extracted using methanol for 8 hours at 80°C. The extract was concentrated using a rotary evaporator. The dry residue was subjected to in vitro studies.
2.3. Cell Culture and Cytotoxicity Assay
PLC/PRF/5 cells were cultured in DMEM high glucose medium supplemented with 10% FBS, 100 IU penicillin, and 100 μg streptomycin per mL at 37°C and 5% CO2. PLC/PRF/5 cells at density of 2 × 105 mL were seeded in 96-well plates and incubated overnight at 37°C and 5% CO2. HD-03/ES was dissolved in 0.5% DMSO in DMEM high glucose medium and used for the experiments. The cells were treated with different concentrations of HD-03/ES in culture media and incubated for 24 h at 37°C and 5% CO2 to determine cytotoxicity of the extract. Cell control and vehicle control were also maintained. Cell viability was tested by MTT assay after exposing the cells to 1 mg/mL MTT for 3 h at 37°C. The blue formazan product was solubilised in DMSO and optical density measured at 540 nm [16]. Nontoxic concentrations of HD-03/ES were used for further experiments.
2.4. HBsAg Detection
PLC/PRF/5 cells at the concentration of 5 × 104 mL were seeded in a 24-well plate and incubated overnight at 37°C. The cells were treated with four nontoxic concentrations of HD-03/ES and incubated for 24 h at 37°C and 5% CO2. At the end of the incubation period, the supernatant was collected by centrifugation at 1000 rpm for 10 min at 4°C. The supernatant was collected in a fresh 1.5 mL microfuge tube and stored at −20°C for ELISA. The cell pellet was stored at −80°C for RNA isolation.
The diagnostic kit for HBsAg (ELISA) from BioRad, France, was used for the detection of HBsAg in the culture medium. The assay was carried out according to the manufacturer’s protocol. The absorbance was measured at 450 nm for determining the HBsAg present in the samples. The percentage inhibition of HBV by HD-03/ES was calculated over the cell control.
2.5. RNA Isolation and RT-PCR
Total RNA was isolated from control cells and HD-03/ES-treated cells using TRI reagent and the RNA was stored at −80°C. RNA was subjected to DNase I treatment (10 μg DNase I for 5 min at 65°C and cooled in ice for 1 min). RNA was quantified using spectrophotometer and the quantity of RNA was determined. One microgram of RNA was reverse-transcribed using Oligo-dT Primer at 42°C as described by us earlier [17]. The cDNA was stored at −20°C for further PCR reactions. A semiquantitative multiplex PCR was designed to compare the RT-PCR products of S gene and pre-S gene with GAPDH gene products to determine the relative levels of expression of HBsAg. PCR was carried out to amplify the HBsAg (S and pre-S genes) using specific primers in the second-strand synthesis. The GAPDH primers were also added to the same tube for each PCR reaction. Since the annealing temperatures of HBsAg gene and GAPDH were similar (60°C), the annealing temperature of the reaction was fixed at 60°C. The primer sequence for S gene was 5′-CCCAATACCACATCATCC-3′ (sense) and 5′-GGATTGGGGACCCTGCGC-3′ (antisense). The primer sequences used for pre-S were 5′-GGGTCACCATATTCTTGG-3′ (sense) and 5′-GTCCTAGGAATCCTGATG-3′ (antisense). For GAPDH gene, the primer sequence used was 5′-ACCACAGTCCATGCCATCAC-3′ (sense) and 5′-TCCACCACCCTGTTGCTGTA-3′ (antisense). The PCR reaction was subjected to 36 seconds of denaturation at 95°C, followed by 25 cycles of denaturation at 95°C for 36 sec, annealing at 60°C for 30 sec and extension at 72°C for 90 sec. A final extension at 72°C for 10 min completed the PCR programme. The PCR products were analyzed on 2% agarose gel stained with ethidium bromide and photographed under exposure to UV light. A standard molecular weight marker was resolved along with the samples to differentiate the cDNA amplicons in the agarose gel. Densitometric analysis (Image J software, Rasband, USA) was carried out to find the differences in the expression of the selected genes.
3. Statistical Analysis
Data were analyzed to determine mean ± SD. Statistical analysis of the data was done by Student’s unpaired t-test using GraphPad Prism software, (San Diego, USA). value of less than 0.05 was considered significant.
4. Results
Prior to the investigation of the anti-HBV effects, any putative cytotoxic effects of the HD-03/ES extract on PLC/PRF/5 cells were studied by MTT assay. The result of the cytotoxicity measurement of HD-03/ES extract on PLC/PRF/5 cells is shown in Figure 1. The percentage toxicity of HD-03/ES at 2000, 1000, 500, 250, and 125 μg/mL was found to be 8.75, 1.25, 0.75, 0.25, and 0.00, respectively, in PLC/PRF/5 cells. The amount of HBsAg secreted into the cell culture medium was determined by ELISA. Four nontoxic concentrations (1000, 500, 250, and 125 μg/mL) were used for HBsAg detection in PLC/PRF/5 cells. The optical density (OD) was read at 450 nm using an ELISA plate reader and the percentage of HBsAg secretion by the drug-treated cells was calculated over cell control. Each experiment was repeated three times and the results showed that HD-03/ES at concentrations of 1000, 500, and 250 μg/mL inhibited HBsAg secretion by 86.38, 71.17, and 17.1%, respectively, as compared to the control (Figure 2). At 125 μg/mL, HD-03/ES did not inhibit HBsAg in PLC/PRF/5 cells. However, at higher concentrations, HD-03/ES suppressed HBsAg production in PLC/PRF/5 cells with an IC50 of 380 μg/mL.
Figure 1: Cytotoxicity of HD-03/ES on PLC/PRF/5 cells. PLC/PRF/5 cells were incubated for 24 hr with different concentrations of HD-03/ES and the cell viability was then determined using an MTT assay. Data are expressed as percentage of control ().
Figure 2: Effect of HD-03/ES on HBsAg secretion in PLC/PRF/5 cells by ELISA. PLC/PRF/5 cells were treated with four nontoxic concentrations of HD-03/ES for 24 hrs and the supernatant was assayed for ELISA using HBsAg Ultra ELISA kit. The percentage inhibition of HBsAg was calculated over control. Data is representative of three experiments. *.
In order to check whether the inhibitory effect of HBsAg by HD-03/ES is targeted at the transcription level, semiquantitative multiplex RT-PCR was carried out using the RNA isolated from HD-03/ES-treated/untreated cells. Both S-gene- and pre-S-gene-specific primers were employed to amplify the gene encoding HBsAg in PLC/PRF/5 cells. The amplification yielded specific cDNAs corresponding to S gene (625 bp) and pre-S gene (553 bp) (Figures 3 and 4). Densitometric analysis compared the gene expression levels of the amplicons in comparison with GAPDH, the internal control. The results showed that HD-03/ES at 1000 and 500 μg/mL suppressed the expression levels of HBsAg as compared to the cell control. The HBsAg expression levels in the cells treated with HD-03/ES extract were less than the control (Figures 3 and 4). The internal control, GAPDH, was uniformly amplified in all the samples.
Figure 3: The effect of HD-03/ES on HBsAg gene expression. PLC/PRF/5 cells were treated with or without HD-03/ES at three nontoxic concentrations. RNA was isolated from drug-treated and -untreated cells and multiplex RT-PCR was performed using specific primers as described in the text. (a) RT-PCR product of S-gene and GAPDH resolved in 2% agarose gel. (b) Densitometric analysis of the gene transcripts and the values depict arbitrary units. Data is representative of two experiments.
Figure 4: The effect of HD-03/ES on HBsAg gene expression. PLC/PRF/5 cells were treated with or without HD-03/ES at three nontoxic concentrations. RNA was isolated from drug-treated and -untreated cells and RT-PCR was performed using specific primers as described in the text ((a) RT-PCR product of pre-S gene and GAPDH resolved in 2% agarose gel; (b) densitometric analysis of the gene transcripts and the values depict arbitrary units). Data is representative of two experiments.
5. Discussion
Chronic HBV infection remains a major public health problem worldwide as well as a therapeutic challenge. Various treatments for chronic HBV infections have had only limited success [18]. The long-term effects of the recent advanced techniques employed to eliminate the virus, including therapy with nucleoside analogs and other virus-replication inhibitors [19], are yet to be determined. Since HBV reverse transcriptase lacks proof-reading function, the virus shows rapid mutagenesis thus creating a large number of variants, some of which show resistance to antiviral drugs. This phenomenon is responsible for the low efficacy of the current drugs and the high rates of drug resistance [20, 21]. Therefore, there is an urgent need to develop new anti-HBV drugs.
HD-03/ES is a herbal medicine used for curing hepatitis B and contains the extracts of Cyperus rotundus and Cyperus scariosus roots. Several clinical trials and in vitro studies have been carried out which confirmed the anti-HBV activity of HD-03/ES [1315, 22]. A previous study reported that HD-03/ES inhibited alanine aminotransferase and HBV DNA [14]. A recent study on HD-03/ES showed that the drug at 5 and 2.5 mg/mL inhibited 1.5 pg/mL of the HBV virus, and the drug prevents HBV infection by possibly interfering with the viral entry [13]. However, the molecular mechanism behind the anti-HBV activity of HD-03/ES has not been studied well. Though previous studies have shown that HD-03/ES suppresses HBsAg, [13, 14] the effect of HD-03/ES on the HBsAg gene expression has not been studied in vitro. The present study investigated the cellular and molecular effects of HD-03/ES on the HBsAg using PLC/PRF/5 cells.
Stable cell lines with integrated HBV genomes, namely, PLC/PRF/5 cells, are commonly used for assessing the action of drugs on HBsAg secretion [23, 24]. The property of PLC/PRF/5 cells to secrete HBsAg in the supernatant was used in the present study to evaluate the anti-HBV properties of HD-03/ES. HD-03/ES extract did not produce cytotoxic effect on PLC/PRF/5 cells within a reasonable dose range. It was seen that HD-03/ES at 1000 and 500 μg/mL concentrations inhibited the secretion of HBsAg by 86.38 and 71.17%, respectively, for a period of 24 hours. However the lower concentrations were not successful in inhibiting the HBsAg in PLC/PRF/5 cells. The lack of cytotoxicity on PLC/PRF/5 cells, at the concentrations tested, indicates that the decrease in HBsAg is not due to an adverse effect of the drug on cell viability. In order to further confirm that the inhibition is HBsAg specific, the secretion of albumin in cell culture supernatants was checked (data not shown). The results showed that the albumin content in the cell supernatants of drug-treated/untreated cells was similar (data not shown). This study showed that the secretion of other cellular proteins like albumin was not altered by HD-03/ES at the doses tested. This result suggested that the antiviral effect of HD-03/ES might be more specific to the HBV.
PLC/PRF/5 cells contain six hepatitis B viral genomes integrated into the high molecular weight host DNA. The cells secrete only sAg and do not produce hepatitis B core antigen or free viral particles [25]. Both pre-S1 and pre-S2 proteins are expressed on the surface of HBsAg particles and are the essential components of complete virions and HBsAg filaments [26]. The expression of these envelope proteins originates from the HBV DNA coding for the respective genes, which is integrated into the cellular genome [23]. The pre-S2 mRNA encodes albumin receptors, which bind to pHSA and mediate viral attachment to the hepatocytes [27]. Pre-S1 and pre-S2 proteins are detectable in the serum of patients with acute and chronic hepatitis B virus infection when there are high levels of viral replication, and the clearance of these antigens from serum usually correlates with the prognosis of hepatitis B virus infection.
In order to assess whether the antiviral effect is due to the suppression of HBsAg gene, semiquantitative multiplex RT-PCR was carried out to amplify the regions coding the HBsAg gene in PLC/PRF/5 cells. The results showed that in drug-treated/control cells, HD-03/ES dose dependently suppressed the HBsAg gene expression. Densitometric analysis of the transcripts of S gene and Pre-S gene showed that HD-03/ES at higher concentrations, namely, 1000 and 500 μg/mL, has downregulated the HBsAg gene expression in PLC/PRF/5 cells. Thus, it could be concluded that HD-03/ES inhibits HBV by inhibiting HBsAg at the transcription level.
This study suggests that in liver cells which have integrated HBV DNA, both S and pre-S antigen secretion could be inhibited by HD-03/ES. Since truncated pre-S antigen has been shown to be a transactivation protein possibly involved in the oncogenic process, the effect of HD-03/ES on pre-S gene is of therapeutic relevance. The impact of HD-03/ES on HBV inhibition by proteins, such as core and polymerase associated with full replication, deserves further study.
6. Conclusion
In conclusion, we have studied the in vitro anti-HBV effect of HD-03/ES in transfected human hepatocarcinoma cells. HD-03/ES suppressed HBsAg production with an IC50 of 380 μg/mL in PLC/PRF/5 cells for a period of 24 h. HD-03/ES also downregulated HBsAg gene expression in PLC/PRF/5 cells. Previous reports have clearly indicated the anti-HBV activity of HD-03/ES. The main thrust of the present study is that, besides other methods of interference, HD-03/ES is capable of suppressing HBsAg, and the action is targeted at the transcription level.
Disclosure
The authors alone are responsible for the content and writing of the paper.
Conflict of Interests
The authors report no conflict of interests.
Acknowledgment
The authors are thankful to Dr. Shyam Ramakrishnan, Chief Scientific Officer, R&D, The Himalaya Drug Company, Bangalore, India, for his constant support and encouragement during this study.
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The free office suite
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Paper: Armenian MFA building to be sold to Kempinski hotel group
PanARMENIAN.Net - According to Zhoghovurd daily, Yerevan’s chief architect confirmed the rumors on the Armenian Foreign Ministry building to be transformed into a hotel.
“The ministry will have a new building where the Justice Ministry premises used to be,” Narek Sargsyan said.
On September 23, 2011, the paper divulged the government’s plans to sell the Foreign Ministry building to Kempinski Hotels S.A for USD 500 mln, with RA authorities denying the information then.
After Azerbaijani assassin Ramil Safarov’s extradition, it would be cleverer to close the ministry altogether, the daily says.
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Earlier, ArmRosgasprom CJSC addressed Armenia’s Public Services Regulatory Commission with an offer to reconsider natural gas price.
Armenian defense ministry’s spokesman described the maneuvers as ordinary exercises conducted several times a year.
Participants will learn basic skills in protecting IT systems and data as well as how to investigate computer-facilitated crimes.
“I wish to further promote the beauty of Armenian art and its principles of tolerance and respect to diversity,” Mnatsakanyan said.
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Infusionsoft Acquires Social Media Marketing Company GroSocial
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Acquisition Brings Easy-to-Use Social Media Lead Generation Tools to Small Businesses and Further Expands Infusionsoft Sales and Marketing Software
(Chandler, Ariz. - Jan. 22, 2013) - Infusionsoft today announces it has acquired social media marketing software company GroSocial. GroSocial's web-based software allows small businesses to easily build and track social media marketing campaigns that generate leads across popular social networks, including Facebook and Twitter. Earlier this month, Infusionsoft announced $54 million in growth capital financing led by Goldman Sachs. Infusionsoft acquired GroSocial to meet the growing customer and market demand for an effective, easy-to-use social media marketing software tool that helps small businesses generate qualified leads. See a demo of GroSocial here.
As the leading all-in-one sales and marketing software, Infusionsoft is riveted on helping small businesses manage the entire sales and marketing lifecycle, from attracting and capturing leads to converting new sales and generating referrals from existing customers. With the addition of GroSocial, Infusionsoft will provide small businesses the ability to easily convert the leads they generate through social platforms into customers. GroSocial will be integrated with Infusionsoft's CRM, marketing automation and e-commerce tools so that small businesses can automatically move prospects through the entire customer lifecycle. Today, there is an Infusionsoft widget in the GroSocial app that allows users to capture leads and trigger automatic follow-up. Small businesses can also use GroSocial to create a professional social media presence, schedule and post Tweets and updates, and to engage followers and fans through contests and promotions.
Small businesses face a myriad of choices when it comes to where to invest time, money and effort in their sales and marketing strategy and tactics. They are pragmatic and want social media tools that will generate leads and sales, in addition to improving customer engagement and brand value.
How GroSocial Helps Small Businesses With Social Media Lead Generation
• Create a Professional Presence
Publish professional Facebook tabs, timeline covers and Twitter backgrounds using stylish design themes and a friendly drag and drop editor.
• Capture Leads through Opt-In Forms
Turn Facebook fans and followers into leads with simple, attractive opt-in forms.
• Attract Followers and Engage Fans
Increase followers and social engagement with contests and promotions.
• Connect with Customers on Facebook, Twitter, YouTube, Pinterest, Google+
Expand your social footprint by connecting with customers on multiple social networks.
• Schedule and Post Tweets and Updates
Save time by scheduling social updates directly from GroSocial. Monitor conversations with custom saved searches and RSS feeds.
• Measure the Results of Your Social Media Efforts
Track the effectiveness of your social media marketing with intuitive reports.
Comments on the News
"GroSocial is helping small businesses get real results from their social media marketing efforts. The company has proven its commitment to customer success with an amazing product that drives social media leads for tens of thousands of growing small businesses. Their team is talented and passionate, and the company is a clear winner and the ideal choice for us in the social media marketing space," says Infusionsoft co-founder and CEO Clate Mask. "An Infusionsoft and GroSocial combination will lead to innovation and powerful results for small businesses."
Zach Mangum, co-founder of GroSocial goes on to say, "Social media marketing isn't just for big brands. We see a huge opportunity to change the way small businesses get results from social media platforms. Our tools make it easy for small businesses to have a big-business presence on social media and to actually generate leads and convert sales from their social media marketing efforts. We're excited to catapult our innovation for small businesses with Infusionsoft even further."
"As we considered an acquisition in the social media marketing space we were looking for three key factors," says Hal Halladay, Infusionsoft SVP of corporate development. "We wanted an affordable but powerful product that made social media lead generation simple for true small businesses and that could easily integrate with our software. We wanted a team that could bring to Infusionsoft deep knowledge and expertise in social media marketing. Finally, we wanted a company that shared our passion to help small businesses succeed. GroSocial was the undisputed leader in all three areas. I don't know if we could have found a better strategic and cultural fit than the GroSocial team."
Founded in 2010 by Zach Mangum, Kevin Kirkland and Chris Wright, GroSocial currently has more than 30,000 users. The 19-person GroSocial team will join Infusionsoft and push the company's total employee count to 370, but will continue to operate as a dedicated product team in Utah. Mangum will continue to lead the Utah operation and will oversee, with Kirkland, the social product strategy for GroSocial.
This is Infusionsoft's second acquisition. In Nov. 2011, Infusionsoft announced the acquisition of CustomerHub, an online membership site and customer portal tool.
Today, there are more than 12,000 small businesses that use Infusionsoft to attract and capture leads, nurture and convert prospects automatically, grow sales and referrals, and save time.
For more information about the GroSocial acquisition, check out the Infusionsoft blog or this free upcoming webinar.
About Infusionsoft
Infusionsoft provides an all-in-one sales and marketing software for small businesses. Its web-based system helps small businesses automatically market to get more customers, grow sales and save time. The privately held, six-time Inc. 500/5000 company is based in Chandler, Ariz. and is funded by Goldman Sachs, Mohr Davidow Ventures and Signal Peak Ventures. For more information, visit www.infusionsoft.com.
About GroSocial
GroSocial is the tool of choice for thousands of small businesses to generate leads through social media. Over 30,000 users worldwide turn to GroSocial's social media marketing software to look professional and easily create social media marketing campaigns and contests on Facebook and Twitter. Founded in 2010, GroSocial is based in Orem, Utah. GroSocial can be found at www.grosocial.com or on Facebook.
News Source : Infusionsoft Acquires Social Media Marketing Company GroSocial
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Google+ "Share" Link Added To Google Search Results
Jul 19, 2012 • 8:50 am | (10) by | Filed Under Google Search Engine
Now when you search in Google and you are logged in, you should see a "share" link when you mouse over Google search results. Here is a picture:
This "Share" link only shows up when you hover your mouse over the search result.
This news was first covered by Search Engine Land via a hat tip from @berianreed.
Google's Sean Liu then officially announced it on Google+ saying:
Starting today, we’re rolling out a new experiment to show a Share link in Google search in place of the +1 button, making it easier for you to share a great website with your friends. Now when you click the Share link next to a result, you can add a comment about why you found it useful. You can then choose if you want to share it publicly or just to your Circles and it will post to your Google+ stream, making it easier for you to share directly from the search results page. This will appear for those searching in English.
Prior, Google had +1 buttons in the search results and then after that, they tried thank them buttons in the search results.
I wonder how long this one will last?
Forum discussion at Google+.
Previous story: Google 404s The Old Google Analytics
blog comments powered by Disqus
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Place:Conselheiro Lafaiete, Minas Gerais, Brazil
Watchers
NameConselheiro Lafaiete
Alt namesCampo Alegre dos Carijóssource: Family History Library Catalog
Lueluzsource: Encyclopædia Britannica (1988) III, 553
Queluzsource: Rand McNally Atlas (1994) I-141
Real Vila de Queluzsource: Family History Library Catalog
TypeCity
Coordinates20.667°S 43.8°W
Located inMinas Gerais, Brazil (1700 - )
source: Getty Thesaurus of Geographic Names
source: Family History Library Catalog
the text in this section is copied from an article in Wikipedia
Conselheiro Lafaiete is a city of the state of Minas Gerais, Brazil. It was known as Queluz until 1934, when it was renamed by decree, as a tribute to Counselor Lafayette Rodrigues Pereira
It is situated 96 km south from Belo Horizonte, capital of Minas Gerais, in one of the most important economic areas of Brazil. The population was 113.019 inhabitants, in 2006. It's one of the most populated cities of MG.
Research Tips
This page uses content from the English Wikipedia. The original content was at Conselheiro Lafaiete. 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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Australian Bureau of Statistics
Celebrating the International Year of Statistics 2013
ABS Home > Statistics > By Release Date
8635.5.55.001 - Tourist Accommodation, Small Area Data, Western Australia, Sep 2012
Previous ISSUE Released at 11:30 AM (CANBERRA TIME) 11/01/2013
Page tools: Print Page Print All RSS Search this Product
TOURIST ACCOMMODATION, SMALL AREA DATA
This product presents results from the September quarter 2012 Survey of Tourist Accommodation (STA) for the following categories of establishments:
• hotels and resorts with 15 or more rooms
• motels, private hotels and guest houses with 15 or more rooms
• serviced apartments with 15 or more units
Data provide information on the supply of, and demand for, tourist accommodation facilities. Data include number of establishments, capacity and employment for the quarter and occupancy and takings from accommodation for each month; by type of establishment and by star grading.
Geography changes
The September quarter 2012 release of STA data is the third to incorporate the Australian Statistical Geography Standard (ASGS) as the geographical framework for the collection. Detailed information on the transition of the STA to the ASGS and how the data has been impacted by this change is contained in Information Paper: Future Changes to Tourist Accommodation, Australia (cat. no. 8635.0.55.003).
A summary of the main changes resulting from the move to ASGS are:
• Small area data is now produced at Statistical Area Level 2 (SA2), replacing Statistical Local Areas (SLA).
• Tourism Regions (TRs) are constructed from allocations of SA2s and as a result have changed. For some TRs, these changes are minimal.
• Data for Local Government Areas (LGAs) and Special area - Brisbane City Core are no longer produced.
To assist users transition to the new geography, this product contains data on both the ASGS and the ASGC geographic frameworks.
Further information
For further information on Tourism Accommodation statistics, including implementation of the ASGS in the Tourist Accommodation collection or changes to outputs, please contact the National Information and Referral Service on 1300 135 070.
© 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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This article is part of the supplement: Basic science, rationale, background and future of denosumab: a RANK ligand inhibitor
Review
Aseptic loosening of total joint replacements: mechanisms underlying osteolysis and potential therapies
Yousef Abu-Amer1*, Isra Darwech2 and John C Clohisy2
Author Affiliations
1 Department of Orthopaedic Surgery and Department of Cell Biology & Physiology, Washington University School of Medicine, Barnes Hospital Plaza, Saint Louis, Missouri 63110, USA
2 Department of Orthopaedic Surgery, Washington University School of Medicine, Barnes Hospital Plaza, Saint Louis, Missouri 63110, USA
For all author emails, please log on.
Arthritis Research & Therapy 2007, 9(Suppl 1):S6 doi:10.1186/ar2170
The electronic version of this article is the complete one and can be found online at: http://arthritis-research.com/content/9/S1/S6
Published:29 June 2007
© 2007 BioMed Central Ltd
Abstract
Total joint replacement, although considered an excellent surgical procedure, can be complicated by osteolysis induced by implant particles and subsequent aseptic loosening of the implant. The pathogenesis of implant-associated osteolysis includes inflammatory and osteolytic processes. The sustained chronic inflammatory response initiated by particulate debris at the implant-bone interface is manifested by recruitment of a wide array of cell types. These cells include macrophages, fibroblasts, giant cells, neutrophils, lymphocytes, and – most importantly – osteoclasts, which are the principal bone resorbing cells. The 'cellular response' entails secretion of osteoclastogenic and inflammatory cytokines that favor exacerbated osteoclast activity and enhanced osteolysis. An appreciation of the complex network that leads to these cellular and inflammatory responses will form a foundation on which to develop therapeutic interventions to combat inflammatory periprosthetic bone loss.
Introduction
Aseptic loosening of joint implants is a disabling condition that can affect patients 10 to 20 years after joint replacement surgery (Figure 1). Total joint replacement for end-stage joint diseases such as osteoarthritis and inflammatory rheumatoid arthritis is an effective surgical intervention [1]. Unfortunately, wear debris, primarily generated from the prosthetic joint articular surface, remains the major factor limiting the survival of joint implants. Subtle progression of tissue destruction around the implant imposes a major challenge, because signs and symptoms may not be clinically apparent until late stages of failure.
Figure 1. Radiographic appearance of osteolysis. Radiograph of the right hip of a 68-year-old woman 10 years after undergoing cementless primary total hip replacement shows inflammatory osteolysis; the periarticular bone loss is indicated by arrows.
The basis of periprosthetic tissue destruction, the so-called 'biologic response' to implant debris, is complex. However, this response is the outcome of multiple factors, including physical and biologic components [2,3]. At the core of the biologic response that leads to osteolysis is activation of the receptor activator of nuclear factor-κB (RANK)/RANK ligand (RANKL) axis, which is indicated by expression of RANK, RANKL, and osteoprotegerin (OPG) in periprosthetic membranes [4,5]. This activation culminates in enhanced osteoclast recruitment and activity adjacent to bone-implant interfaces, leading to osteolysis [6,7].
Aseptic loosening and osteolysis: etiology, pathogenesis, and cellular responses
Wear debris is formed at prosthetic joint articulations, modular interfaces, and nonarticulating interfaces [1,8]. Although a wide range of particles has been found, the majority of particles formed are less than 5 μm in diameter and are randomly shaped. Studies have suggested that the cellular response to particles may vary with size, shape, composition, charge, and number of particles [9,10]. Furthermore, it was proposed that particle phagocytosis represents an important component of the cellular response to implants; hence, the size of these particles is significant. In this regard, several reports have estimated that particles ranging from 0.2 to 10 μm in diameter undergo phagocytosis by macrophages [11]. In vitro studies of macrophage cultures clearly indicated that smaller polymethylmethacrylate (PMMA) and polyethylene particles (< 20 μm) elicited a significantly greater inflammatory cytokine response, as indicated by increased release of tumor necrosis factor (TNF), IL-1, IL-6, prostaglandin (PG)E2, matrix metalloproteinases, and other factors [9,12-14]. Although particle phagocytosis has been identified as a critical component of this biologic response, recent studies in human macrophages indicate that direct interactions between particle and cell surface are sufficient to activate osteoclastogenic signaling pathways [9,15].
Aseptic loosening can be the result of inadequate initial fixation, mechanical loss of fixation over time, or biologic loss of fixation caused by particle-induced osteolysis around the implant. The causes of particle accumulation vary from implant interface wear, micromotion occurring in response to corrosion, oxidative reactions, and minor pathogen contaminations [2,3]. In general, the initial response is a localized anti-inflammatory response that is characterized by formation of fibrous tissue that encapsulates the implant. Often, synovial fluid and synovial lining membranes are also formed, and granulomatous tissue is established. Immunohistochemical studies of these tissues have revealed an abundance of macrophages, fibroblasts, giant cells, neutrophils, and lymphocytes [16-18]. However, aseptic loosening is characterized by poorly vascularized connective tissue dominated by fibroblasts and macrophages [15]. Subsequently, secretion of proinflammatory factors, gelatinases, and proteases contributes to periprosthetic osteolysis and to failure of the joint implant [19]. Perhaps the most important aspect of the response to particles is its sustained duration, caused by its resistance to enzymatic degradation and digestion [20,21]. This characteristic fuels and continuously builds up the pathologic response, leading to aggressive osteolysis.
The rate at which particles accumulate is also considered an important factor in the occurrence of osteolysis. Various studies found osteolysis to be associated with increased wear rates [3,12,20,22]. Specifically, areas of increased lysis were found to contain significantly greater concentrations of particles (of similar size and shape) as compared with nonosteolytic regions of the loosened implant at revision [10,20]. These observations were further supported in vitro studies in which induction of transcriptional activity and cytokine release were found to be particle dose dependent. An effect of particle concentration on secretions by various types of cells has also been reported [12,21-24]. In primary human monocytes, release of cytokines, PGE2, and hexosaminidase was found to depend on size, concentration, and surface area of particles [12,21-24]. Other studies using macrophage cell lines found dose-dependent apoptosis of the cells when they were treated with ceramic and polyethylene particles [21]. In studies of other cells such as synovial fibroblasts and chondrocytes, treatment with increasing doses of cobalt and vanadium was found to have toxic effects [25].
Clearly, particulate debris load and particle composition are important factors in the osteolytic process. Therefore, ongoing investigations into and evaluations of alternative weight-bearing surfaces are critical if we are to identify materials that are optimal in terms of minimizing particle generation over time [15,26]. Ceramics, and use of highly cross-linked polyethylene and of metal-on-metal articulations show promise as approaches that may markedly reduce the production of wear particles.
Biologic and mechanical factors have been incriminated in the early and late stages of the development of osteolysis following joint replacement. Inflammatory reactions develop at early stages as part of the resolution process. These reactions primarily include increased circulation and elevated fluid levels in the vicinity of the affected tissue [26,27]. In addition, apart from massive recruitment of macrophages to the site of injury, some studies identified recruitment of lymphocytes, indicating that the immune system may be involved in this inflammatory response [15,26,28-32]. Perhaps the most complex aspect of this reaction is secretion of various cytokines and factors by these lymphocytes and other cells, leading over time to adverse effects [7,33,34]. The cellular and associated inflammatory response is not restricted to the initial healing process, but rather it appears to take center stage at progression and in the late stages of periprosthetic osteolysis.
Several co-factors have been described as facilitating propagation of inflammatory and eventual osteolytic events. One major factor relates to poor implant fixation and subsequent motion. In this regard, ample evidence indicates that loosening of implants is associated with excessive motion and physical stress, which result in accelerated release of particulate debris [24]. This is apparent from the progressive nature of bone resorption around loose screws associated with wear debris. In recent years compelling evidence has been reported indicating that the release of such debris leads to inflammatory responses that ultimately result in bone loss. Evidence implicating particulate debris as a major component in osteolysis is derived from studies conducted in animal models exposed to particulate debris, studies with macrophages [35,36] and osteoclasts conducted in vitro and in vivo, and evaluations of osteolysis and co-factors in clinically successful and failed total joint prostheses [21,37-41]. The amounts of particulate debris around implants in most cases exhibit a fair correlation with the degree of aseptic loosening, although certain patients have an exaggerated biologic response to particulate debris. However, particles have been documented in remote tissues as well. Factors that contribute to particle distribution include (but are not limited to) particle number and size, fluid flow, implant design, and joint space [10,26].
Biology of the osteolytic response
The cellular response is dominated by phagocytes and macrophages [26,35,42,43]. However, the osteolytic response includes various cell types, such as osteoclasts, fibroblasts, and osteoblasts/stromal cells. This is supported by the wide range of factors that are secreted by various cells, including cytokines, growth factors, metalloproteinases, prostanoids and lysosomal enzymes, among others [44,45]. The mechanisms that underlie induction by particles of cellular responses and osteolysis are largely unclear and remain subject to vigorous investigation. It is believed that recognition of particles relies on phagocytosis of small-sized particles by macrophages and unidentified cell surface interactions. The latter interactions may include nonspecific physical induction of transmembrane proteins or recognition of cell surface molecules by particles or proteins/factors that are adherent to the surface. However, the precise nature of stimulation of cells by particles remains unknown. Furthermore, it is well accepted that host defense cells, which comprise the core of the inflammatory reaction, recognize particles and release large quantities of proinflammatory cytokines and factors [15,26,28-30,32], including TNF, IL-1α and IL-1β, IL-6, RANKL and PGE2, among others [34,37,38,42,46-48]. These factors stimulate bone loss by targeting osteoclasts and their monocytic precursors. In this regard, RANK/RANKL is the principal axis that regulates the osteoclastogenic event.
The RANK/RANKL pathway was discovered during the late 1990s and resolved the mystery regarding osteoclast differentiation from hematopoetic monocyte/macrophage precursors [7,38]. Osteoclast differentiation is primarily controlled by the stromal/osteoblast-derived proteins RANKL (also known as OPG ligand and osteoclast differentiation factor) and macrophage colony-stimulating factor [49]. A major breakthrough in our understanding of the regulation of osteoclastogenesis was achieved with the identification of OPG, a soluble protein of the TNF receptor family [50] that binds RANKL and acts as a decoy molecule (for review [6,7,51]). Several studies have demonstrated that OPG is a potent inhibitor of bone loss, thus regulating bone density and mass in mouse and humans. This secreted cytokine was also shown to be effective in blocking metabolic, pathologic (including periprosthetic osteolysis), and metastatic bone loss. A similar approach using soluble extracellular RANK protein, which is similar to OPG, neutralizes RANKL by sequestering it in an inactive complex [52]. This approach was further proven to be effective in vitro and in vivo by studies in which Fc-RANK fusion protein was found to block bone loss pathologies. Also important to regulation of osteoclastogenesis and bone loss is that many proinflammatory and anti-inflammatory cytokines act directly to enhance or inhibit the RANK/RANKL axis. For example, the effect of TNF as a stimulator and activator of osteoclastogenesis appears to be entirely dependent on an active RANK/RANKL pathway [53]. In this regard, signaling by TNF appears to be superimposed over pathways initiated by RANKL in osteoclast precursors. A similar synergistic pattern has been observed for other pathologic insults such as implant particles and pathogens. These observations are in discord with studies indicating that TNF, in the absence of RANK, is sufficient to induce osteoclastogenesis [54].
Studies conducted in animal models and in vitro cell cultures have shown that TNF plays a crucial role as a mediator of particle-induced osteoclastogenic and osteolytic events [15,42,47]. In this regard, PMMA particles fail to elicit aggressive osteolysis in TNF receptor null animals or in the presence of TNF neutralizing agents, for instance various soluble TNF binding proteins. Likewise, animals that lack RANK or RANKL resist induction by particles of osteolysis, which is similar to their protection from arthritic bone erosion [34,40,47]. This is not unexpected, considering that TNF family members, especially RANKL, are prerequisites for osteoclast formation.
Further downstream signaling by wear particles, unsurprisingly, overlaps with that of TNF and RANKL. In this regard, activation of kinases and recruitment of molecules that are essential for osteoclast differentiation and activation have been documented [13,15,42,47,55]. Notably, particle-induced pathways lead to activation of kinases and transcription factors that are essential for osteoclastogenesis. Among these are activation of the tyrosine kinase c-src, mitogen-activated protein kinases, and the nuclear factor-κB (NF-κB) cascade [7,13,34]. Although activation of these pathways might be secondary to other events, selective blockade of these downstream pathways reduces particle transmitted effects.
Potential therapeutic intervention in inflammatory osteolysis: molecular approaches to arrest osteoclast activity
The biologic mechanisms of debris-mediated osteolysis are complex and involve various cell types and inflammatory cytokines of the periprosthetic membrane. Particles inhibit bone formation by targeting osteoblasts and activating macrophages and osteoclasts, which leads to significant periprosthetic bone loss [15,56]. Therefore, in combination with improvements in implant integration, strategies to target the cellular components (osteoblasts and osteoclasts) that contribute to implant failure represent potentially effective therapeutic interventions. In this regard, it should be noted that differentiation of bone marrow macrophages (osteoclast precursors) into mature osteoclasts requires recognition and binding of the osteoblast, fibroblast, and T cell secreted factor RANKL by its cognate receptor, RANK, which is expressed on the surface of osteoclast precursors [6,38,57]. This process is regulated by another factor secreted by osteoblasts, namely OPG, which acts as a decoy receptor by binding to RANKL and reducing its bioavailability. On the other hand, binding of RANKL to RANK prompts induction of several intracellular pathways by this receptor, leading to activation of key transcription factors, most notably NF-κB.
It has been reported that the NF-κB family of transcription factors is central to pathologic responses and is essential for osteoclast differentiation (for review [7,51]). It consists of several family members that primarily form heterodimers. These dimers are found bound to an inhibitory protein (inhibitor of NF-κB [IκB]) that, in the absence of stimulation, retains the complex in the cytoplasm. Stimulation by RANKL or other specific stimuli leads to activation of upstream IκB kinases (IKKs), which in turn phosphorylate IκB, leading to its dissociation from the NF-κB complex and eventually to its degradation by the proteosome system. IκB-liberated NF-κB then translocates to the nucleus, binds to specific DNA sites, and induces basal transcription (for review [7,58]).
Knowledge has accumulated of the NF-κB activation mechanisms that eventually lead to osteoclast formation and, when induced by factors such as TNF and PMMA particles, exacerbate osteoclastogenesis and inflammatory responses. Several approaches that exploit this knowledge have been designed to perturb this pathway and hence alleviate the deleterious effects of inflammatory osteolysis. A number of approaches to osteoclast-based therapy may be considered. The first involves targeting osteoclast precursor cells, which are recruited to inflammatory sites by circulating cytokines. The second entails targeting precursors that are stimulated by the particle-mediated cellular response to differentiate and form bone resorbing osteoclasts. The third approach involves targeting activation mechanisms of mature osteoclasts.
There are various approaches to inhibiting differentiation of precursor cells to osteoclasts. First and foremost, direct inhibition of osteoclast differentiation may be achieved by application of RANKL decoy molecules such as OPG and the soluble fusion protein RANK-Fc [52,59]. Indeed, studies in animal models and in vitro osteoclast cultures have identified significant inhibition of osteoclastogenesis and reduced hallmarks of osteolysis [16,52]. Other approaches target key intracellular signal transduction pathways that are essential for osteoclast differentiation and inflammatory exacerbation of this process. The transcription factor NF-κB is at the center of this process, and is essential for both inflammatory and osteolytic responses; inhibition of NF-κB hinges on disrupting its translocation to the nuclei, as noted above. Transduction of a dominant-negative form of the NF-κB inhibitory protein, IκB, which retains NF-κB in the cytoplasm, was indeed sufficient to block osteoclast formation and activity [39,55,60]. Another viable approach is to block activation of the upstream IKK complex, which is responsible for phosphorylation of IκB and subsequent activation of NF-κB. This was possible by introducing a small peptide that perturbs assembly of the IKK complex and attenuates activation of NF-κB [61]. More importantly, administration of the dominant-negative IκB protein or the IKK inhibitory small peptide to arthritic mice significantly was found to block bone erosion associated with inflammatory arthritis and particle-induced osteolysis of calvaria in mice (for review [7]). Because NF-κB is central to most host immune and inflammatory responses and induces expression of a large number of genes encoding proteins that are associated with bone pathology, efforts have been directed toward identifying specific NF-κB-mediated genes and their products. Steps in this direction should lead to the design of a future generation of selective inhibitors that might be effective in alleviating inflammatory bone loss.
Several genes have been shown to be critical for osteoclast differentiation. Among these are c-fms, c-fos, RANKL, NF-κB, c-src, and the proton ATPase [51]. Recent studies have revealed that proinflammatory cytokines such as TNF act directly on some of these genes and their products, in particular c-src and NF-κB, to accelerate osteoclast formation and cause a potent osteoclastic response (for review [7,62]). Selective inhibitors of the c-src tyrosine kinase have shown great promise in halting osteoclast activity, and future studies should be directed toward testing the effect of such inhibitors on inflammatory osteolysis [63,64]. Another promising approach involves the use of bisphosphonates [65]. These compounds inhibit osteoclast function and induce their apoptosis. Animal models have been used to study the efficacy of an oral bisphosphonate in inhibiting bone resorption mediated by wear debris [26,66,67]. The findings indicate reduced radiographic periprosthetic radiolucency, although the levels of PGE2 and IL-1 remained elevated in tissue cultures from these implants. These studies have served as the basis for clinical trials using alendronate in patients with radiographically evident osteolytic lesions. In other studies, bone loss around implant caused by intra-articular injection of particles was prevented and treated by alendronate [68-71]. Thus, bisphosphonates may be useful in preventing particle-induced osteolysis around total joint implants.
Inhibiting recruitment of osteoclast precursor and other cells to the inflammatory site entails use of anti-inflammatory approaches to neutralize mediators such as TNF, IL-1, IL-6, and others. It has been proposed that pharmacologic intervention targeted at the macrophage may provide the means to slow the response to wear debris. Indeed, local cytokine inhibition may reduce inflammation in the periprosthetic tissue, and several biologic mediators have been identified as useful for clinical application. In particular, the IL-1 receptor antagonist protein has been reported to reduce inflammation, and the anti-inflammatory cytokine IL-10 appears able to reduce cell mediated reactions in inflammation [28,72]. Using particle-stimulated murine air pouch and calvarial models to evaluate the potential of gene therapy to treat inflammation induced by orthopedic wear debris, recent studies showed that retroviral vectors encoding human IL-1 receptor antagonist, human TNF receptor, and viral IL-10 led to marked decreases in inflammation, decreased pouch fluid accumulation, and reduced macrophage influx [40,48]. In the murine air pouch study, histologic assessments revealed that pouches transduced with viral IL-10 or IL-1 receptor antagonist exhibited a 40% reduction in inflammatory cell infiltration when compared with nonviral controls or LacZ transduced membranes. The calvaria study corroborated these findings by independently demonstrating that gene delivery of viral IL-10 inhibits three processes that are critically involved in periprosthetic osteolysis: proinflammatory cytokine production induced by wear debris, osteoclastogenesis, and osteolysis.
Anti-inflammatory cytokines secreted by T lymphocytes such as IFN-γ, IL-4, and IL-10 have also been shown to be effective in antagonizing the actions of proinflammatory cytokines (for review [7,40]). These cytokines are being now exploited in erosive inflammatory responses because they arrest osteoclast formation. The finding that activated T-helper-1 cells (which secrete RANKL and proinflammatory cytokines) and T-helper-2 cells (which secrete IL-4) are present in the synovium strongly implicates the immune system as a key regulator of inflammatory bone disease. Of significance is a recent finding that IL-4 mRNA was more frequently identified in nonerosive than in erosive disease (38% versus 15%). These findings provide indirect evidence that IL-4 has bone sparing effects in vivo. Additionally, IL-4 adenoviral gene therapy has been shown to be effective in reducing inflammation, inhibiting proinflammatory cytokine secretion, and sparing bone destruction in a model of adjuvant-induced arthritis [73].
Clarifying the molecular mechanisms that underlie the anti-osteoclastogenic actions of these anti-inflammatory cytokines should unveil useful molecular targets. In this regard, recent progress toward elucidating the anti-osteoclastogenic action of IL-4 and IFN-γ was reported. Specifically, IL-4 binds to membrane receptors expressed on the surface of osteoclast precursors, and activates key transcription factors, most notably the signal transducer and activator of transcription (STAT)6. The mechanism responsible entails activation by IL-4 of Janus kinases, which phosphorylate STAT6, prompting its dimerization and translocation to the nucleus, where it binds to DNA and regulates target genes [74]. In osteoclasts and their precursors, STAT6 was found to be crucial for IL-4 inhibition of osteoclastogenesis and inhibits inflammatory bone erosion. Although the precise mechanism is unknown, STAT6 is presumed to inhibit NF-κB and mitogen-activated protein kinase activation and thus inhibits transcription of genes, including those encoding inflammatory cytokines such as TNF, IL-6, NF-κB family members, and more [75]. These observations indicate that STAT6 is a potentially useful target for anti-erosive drug design.
IFN-γ is another major product of immune cells that potently inhibits bone resorption (for review [7,58]). Recent reports illustrated that IFN-γ interferes with the RANK\RANKL signal transduction in osteoclasts and their precursors. It induces rapid degradation of TNF receptor associated factor 6, a RANK adaptor protein. This action results in arrest of RANK downstream signals, such as NF-κB and c-Jun\Janus kinase pathways. In another study [76] it was reported that RANKL-induced secretion of IFN-γ by osteoclast precursors counterbalances bone resorption by blocking osteoclastogenesis in an autoregulatory manner. Although a direct role for IFNs in particle-induced inflammatory osteolysis has not yet been established, the possibility that these cytokines will block the bone loss associated with this disease is worthy of further investigation.
Conclusion
Aseptic loosening secondary to wear debris mediated inflammatory osteolysis is a serious health problem and will have an impact on increasing numbers of patients over the coming decades. The initial response to particulate debris includes a subtle inflammatory response, which becomes more pronounced as osteolysis progresses. The inflammatory environment provokes a cellular response characterized by elevated levels of secreted factors such as TNF, RANKL, IL-6, IL-1, and IL-11. Most of these cytokines affect osteoclast differentiation and activity directly, and result in enhanced osteolysis. Clarifying the molecular details of these signaling cascades and cellular responses represents, and will continue to represent, an enormous therapeutic challenge.
Abbreviations
IFN = interferon; IκB = inhibitor of NF-κB; IKK = IκB kinase; IL = interleukin; NF-κB = nuclear factor-κB; OPG = osteoprotegerin; PG = prostaglandin; PMMA = polymethylmethacrylate; RANK = receptor activator of nuclear factor-κB; RANKL = receptor activator of nuclear factor-κB ligand; STAT = signal transducer and activator of transcription; TNF = tumor necrosis factor.
Competing interests
The authors declare that they have no competing interests.
Acknowledgements
The authors acknowledge the generous support by the National Institute of Health (NIH; grants # DE13754, AR47443, AR49192, and AR47096) and by the Shriners Hospital for Children. These funds were instrumental for completing studies by the authors cited in this review.
This article is published as part of Arthritis Research & Therapy Volume 9 Supplement 1, 2007: Basic science, rationale, background and future of denosumab: a RANK ligand inhibitor. The full contents of the supplement are available online at http://arthritis-research.com/supplements/9/S1.
Publication of the supplement has been supported by an unrestricted grant from Amgen Inc.
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Ask Your Question
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Easy way to make a chart full sheet size
asked 2012-06-29 23:13:58 +0200
mdwy62
1 1 1 2
updated 2013-01-28 09:13:52 +0200
qubit
5693 3 48 41
The only way I can make a full page chart - which I use constantly - is to continuously fiddle with print ranges, Print Preview, and dragging and repositioning chart and margins to fit the whole page. Excel does this in one click with the option to save a chart to a new sheet. Is this option patented? Is there some other reason why it is missing from LO?
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answered 2012-06-30 01:46:32 +0200
mariosv
4599 20 45
Making the chart big enough and create a page style with the Sheet - Scale - Scaling Mode to "Fit print range(s) on number of pages" to 1. Always adjust the chart to a page.
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Kurobox Media Ripper
From NAS-Central Buffalo - The Linkstation Wiki
Revision as of 19:56, 12 July 2007 by Ramuk (Talk | contribs)
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Contents
Kurobox Media Ripper
Submitted for June/July Contest. Contest Closed
Introduction
The KuroRipper is an addition to the Kurobox NAS system. It allows automatic copying of images,video and audio files from USB media such as USB keys, Compact FLASH, and SD cards. Also, it will rip entire audio CDs to mp3 format and, if avaialable will add the correct tags from the CDDB database to your Kurobox shares. Target directories, along with supported media types is all configurable in usr/local/bin/ripper.conf or from the webmin interface.
The Disk Full Led on your Kurobox will flash when ripping is in operation. It is safe to remove any USB adapters only when the Disk Full LED is not blinking.
Distribution Installation
The media ripper is based off of the Debian install from June 2006. To install this release, use the instructions at Debian_sylver and the file http://www.kurobox.com/sylver/debian/debian-sarge-2.6.17.1-mh1_kuro-box-20060623.tgz] or use the Sylver Auto Installer at <ADD LINK>
Install the Media Ripper
Install support packages
apt-get install samba hdparm udev usbmount cdparanoia mpg123 cddb libcddb2 toolame id3v2
Download the Mediaripper at MediaRipper Uncompress the program at /
cd /
tar zxvf mediaripper.tgz
chkconfig add mediaripper
Configure the rest of the system
ln -s /usr/bin/toolame /usr/bin/lame
mkdir -p /mnt/share/Images
mkdir -p /mnt/share/Music
mkdir -p /mnt/share/Videos
Configuring Media Ripper
Via Webmin
Log in to the webmin interface, choose the hardware tab. Select the Hardware Tab and Select the "Rip USB media to your shares" icon
Via Command Line
With your favorite editor, edit /usr/local/bin/ripper.conf
Additional tips
Install Wizd
Wizd is a program to share your media on your Kurobox with your Buffalo Linktheater.
Share the drives via Samba
Of course all good NASes should be sharing their data. Why not use webmin!
To Do
• Make hdparm spin the drive down
• Allow the ripper to rip from Data CDs
• Allow the ripper To rip DVD isos
Personal tools
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.NET Debugging Demos - Information and setup instructions (IIS)
0
kicks
.NET Debugging Demos - Information and setup instructions (IIS) (Unpublished)
This is a series of debugging demos aimed to help you get some hands on experience in debugging the most common types of Hang/performance, memory and crash scenarios in .net applications. The demos are written in ASP.NET which means that you do need IIS and .net framework 2.0 installed on the development machine where you install the demos.
Kicked By:
Drop Kicked By:
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Success!
MIX 2011: Back to Square One
0
kicks
MIX 2011: Back to Square One (Unpublished)
MIX 2011: Back to Square One This session was delivered by Nishant Kothary. In this session Nishant tries to solve the $64000 question: Why do we suck at predictably building great software?
Kicked By:
Drop Kicked By:
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Information for "Category:Historical Development"
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Difference between revisions of "RPi Programming"
From eLinux.org
Jump to: navigation, search
(Uncategorised)
(Add Scratch reference)
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* Python <ref>http://ntoll.org/article/baking-with-raspberrypipy</ref>
* Python <ref>http://ntoll.org/article/baking-with-raspberrypipy</ref>
+
* [[Scratch_on_RPi|Scratch]]
* Java
* Java
* PHP
* PHP
Revision as of 21:35, 4 January 2013
Contents
Back to the Hub.
Software & Distributions:
Software - an overview.
Distributions - operating systems and development environments for the Pi.
Kernel Compilation - advice on compiling a kernel.
Performance - measures of the Pi's performance.
Programming - programming languages that might be used on the Pi.
Programming languages, IDEs, etc
Tested on Alpha board
Tested on RPi
Expected to work
See also: RaspberryPiBoard/EducationalLinks for education-friendly languages.
Graphical Programming
• Gambas - possibly a good choice; easy like old visual basic
• Scratch
• Alice
• Android App Inventor
• Kodu
• Star Logo
• PrimerLabs CodeHero
• Lazarus I was working on LaZorOS4Pi a while back to provide a on-device IDE but I'm stuck with only a VGA monitor + chroot. Will pick it back up asap for it is a great combination. (Used: Fedora, Razor-QT desktop and Lazarus-QT+FreePascal. 2nd attempt => Funtoo)
• YAD 'Yet Another Dialog' for fast GUI scripting in BASH.
• EasyBashGui Want to keep it fast and simple than this is your tool. Goes even faster with the code snippets from Komodo Edit + BASH Menu plugin.
• BlueGriffon is a very slick WYSIWYG editor for HTML5.
• fpGUI Toolkit has been tested and fully working on the RPi. fpGUI is a custom drawn, full featured, cross-platform GUI toolkit that has been in development since 2006. fpGUI can be use for Desktop or Embedded environments, and is suitable for commercial and open source projects. Implemented 100% in Object Pascal, and producing small executables and very low library dependency (just the standard X11 - no Qt or GTK etc required). For a very quick setup of the Free Pascal Compiler & fpGUI Toolkit, download the starter archive (3.5MB download) from http://www.turbocontrol.com/easyfpgui.htm. Unzip, and you are ready to go! fpGUI includes lots of ready made widgets, a documentation viewer (docview), a Visual GUI Forms Designer (uidesigner), and an IDE (maximus). The IDE is still in the very early stages of development, but any programmer editor could be used with fpGUI too.
• Pi3D hardware accelerated 3D (and 2D) for python - still being improved but works now
• Adafruit Learning System Raspberry Pi WebIDE - allows programs to be written, compiled, and executed on the RPi via a web interface.
Robotics
Would Be Great If These Worked
• wvdial -- Dialer for Land-line, GSM, UMTS modems and other serial devices.
• VHDL -- VHDL (VHSIC hardware description language) is a hardware description language used in electronic design automation to describe digital and mixed-signal systems such as field-programmable gate arrays and integrated circuits.
• Verilog -- Verilog, standardized as IEEE 1364, is a hardware description language (HDL) used to model electronic systems. It is most commonly used in the design and verification of digital circuits at the register-transfer level of abstraction.
Uncategorised
See also Category:Education
References
1. http://www.vanhaarlem.eu/assembler
2. http://ntoll.org/article/baking-with-raspberrypipy
3. http://ntoll.org/article/baking-with-raspberrypipy
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For the half-year to 30 June 2013, the IPKat's regular team is supplemented by contributions from guest bloggers Stefano Barazza, Matthias Lamping and Jeff John Roberts.
Two of our regular Kats are currently on blogging sabbaticals. They are Birgit Clark and Catherine Lee.
Sunday, 16 September 2007
Doctor Danny and the Roaring Dragon; Spicy IP
The IPKat is delighted to learn that his learned friend Danny Friedmann has gained his doctorate. Under the watchful eye of thesis advisors Professor P. Bernt Hugenholtz and Professor Peter K. Yu, Danny has created the intriguingly-titled "Paper Tiger or Roaring Dragon: China's TRIPs Implementations and Enforcement".
The thesis asks whether China’s IPR enforcement laws are TRIPs-compliant. According to its author:
"At least on paper most of China’s IPR procedural laws are compliant with specific TRIPs provisions. China’s IPR laws are certainly non-compliant to the more general provisions of TRIPs, due to incompatible extra-judicial factors. Nevertheless, no unequivocal preference for a WTO case against China can be given. Another option, although more complicated, to tackle China’s IPR enforcement challenges is to be preferred: to address China’s transparency, market access, uniform application of law, integrity and impartiality of the courts and expertise in and respect for IPR".
You can read the thesis here in pdf format. Danny welcomes your comments if you email them to him here.
CORRECTION! Danny has since told the IPKat that his thesis is "only" a Masters dissertation, not a doctorate. The Kat has told him to hurry up with his doctorate!
The exciting and well-informed Spicy IP blog is one of the best things to have come out of India in a long time. It has a strong team of writers and a wealth of lively and controversial subject-matter to discuss. Notwithstanding this, the blog is looking for further co-bloggers. According to Spicy IP activist Shamnad Basheer,
"We are particularly interested in folks that can blog on non patent related IP matters (copyright, trade marks, traditional knowledge, GIs) or even "innovation" more generally or any other IP/innovation theme that has some relevance for India. The understanding between us now is that each blogger post at least once a week.
We intend to make this blog a one stop shop for leading IP news/cases/comments pertaining to India. The scope of the blog has expanded in the recent past to include a regular column on recent news (SpicyIP tidbits), comments (SpicyIP comments). It will soon feature updates on IP events (SpicyIP events), IP Scholarship (SpicyIP Scholar), IP Jobs (SpicyIP Jobs) and an online journal for IP articles (SpicyIP journal)
The blog is read by a fairly wide audience comprising practitioners, academics, students, NGO's, government folks etc".
So if you wish to be part of his exciting endeavour and make your voice heard, please let Shamnad know by emailing him here.
Subscribe to the IPKat's posts by email here
Just pop your email address into the box and click 'Subscribe':
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User:Pranav Rathi
From OpenWetWare
Revision as of 12:48, 1 March 2013 by Pranav Rathi (Talk | contribs)
Jump to: navigation, search
Contents
About Me
Education
• Planning on getting PhD, Optical Science&Engineering, University Of New Mexico, Albuquerque-NM working at Koch Lab at UNM.
• 2010, MS, Optical Science&Engineering, University Of New Mexico, Albuquerque-NM
• 2005, MS, Applied-Physics, Northern Arizona University, Flagstaff-AZ
• 2003, MA, Mathematical Sciences, MJP-Rohalkhand University, Uttar Pradesh-UP, India
Experience
Skills & Expertise
Research interests
Optical Trap
1. Optics: nano, nonlinear, photonics, Bio-optics and optoelectronics.
2. Design and construction of optical, opto-mechanical, opto-electrical and other devices.
3. Material research through AFM, confocal, near and farfield microscopy and FTIR-spectroscopy.
4. Design and construction of devices for bio-engineering research.
Research & Work
• [Optical Tweezers[1]]
• [Design & Construction of devices for Optical Tweezers[2]]
• [Other research projects [3]]
• [Project page [4]]
Reccomendations
Contact Info
This user is a DNA unzipper
Personal tools
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User talk:Jason R. Kelly
From OpenWetWare
(Redirected from User talk:Jasonk)
Jump to: navigation, search
Hello test
Contents
first
second
3rd
oww sc time
Jennyn 13:42, 12 June 2006 (EDT): Hey Jason, for the conference calling, do they need to call in before 12 as opposed to 4? I was thinking this was the text from previous meetings that have been held at 4.
inviting new users to OWW
Hoatlinm 16:24, 20 June 2006 (EDT)Hi Jason. My lab supplies antibodies and other reagents to lots of labs working on Fanconi anemia. I was going to let the other labs know later this week about our updated information on our wiki and encourage them to join up. I think it is a fantastic tool for collaborations. How many labs can you take on OWW? If 20 Fanconi labs joined (I don't know if they would, but one can always hope) would that clog things up?
Software download section
Hi Jason, thanks for your comments. I like the idea of just putting up a page in OWW and linking it to another place so I'll probably do that for now. If I can help with the development of a software section, please let me know. Thanks. --Najaf 12:31, 23 June 2006 (EDT)
Recent changes
Hey Jason, thanks for the Smolke-specific changes section. One thing, though - Christina edited Smolke:Lab Members this morning, and it didn't show up on the Smolke:Recent_changes page. Any idea why? -Jkm
Repressilator
Federico_C 22:01, 17 January 2008 (EDT) Thanks a lot! I've barely started to learn how to design devises and I often make mistakes. I appreciate a lot the advice and I'll use weaker LVA tag. Keep in touch
Whoisonline extension
Vincent 13:28, 11 February 2008 (CST): Hey, the 'whoisonline' extension is cool. Is there any plan to bring it to the OWW front page ? sidebar ?
Thanks you for the light... --John Cumbers 08:54, 19 March 2008 (CDT)
Personal tools
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Quotation added by staff
Why not add this quote to your bookmarks?
The hardest part has been maintaining a small head -- remaining down to earth. So many people try to make you more than you are. This business has changed a lot of good people and a lot of good families, and I don't want that to happen to me. Brandy
This quote is about acting and actors · Search on Google Books to find all references and sources for this quotation.
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Quotation added by staff
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Great intellects are skeptical. Nietzsche, Friedrich
This quote is about skepticism · Search on Google Books to find all references and sources for this quotation.
A bit about Nietzsche, Friedrich ...
Friedrich Wilhelm Nietzsche (October 15, 1844 August 25, 1900) was a German philosopher, whose critiques of contemporary culture, religion, and philosophy centered around a basic question regarding the foundation of values and morality. Beyond the unique themes dealt with in his works, Nietzsche's powerful style and subtle approach are distinguishing features of his writings. Although largely overlooked during his short working life, which ended with a mental collapse at the age of 44, and frequently misunderstood and misrepresented thereafter, Nietzsche received recognition during the second half of the 20th century as a highly significant figure in modern philosophy. His influence was particularly noted by many existentialist and postmodern philosophers.
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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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The influence of individual character extends from generation to generation. Macleod, Iain
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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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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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The greatest ability in business is to get along with others and influence their actions. A chip on the shoulder is too heavy a piece of baggage to carry through life. Hannah, John A.
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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°
Click here to buy this »
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Quotes by Moodie, Susanna
We don't have a biography. Please consult wikipedia.
"Ah, Hope! what would life be, stripped of thy encouraging smiles, that teach us to look behind the dark clouds of to-day, for the golden beams that are to gild the morrow."
Moodie, Susanna on hope
"What a wonderful faculty is memory! -- the most mysterious and inexplicable in the great riddle of life; that plastic tablet on which the Almighty registers with unerring fidelity the records of being, making it the depository of all our words, thoughts and deeds -- this faithful witness against us for good or evil."
Moodie, Susanna on memory
"When things come to the worse, they generally mend."
Moodie, Susanna on change
"The want of education and moral training is the only real barrier that exists between the different classes of men. Nature, reason, and Christianity recognize no other. Pride may say Nay; but Pride was always a liar, and a great hater of the truth."
Moodie, Susanna on class
Take a look at recent activity on QB!
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European Union
From RationalWiki
Jump to: navigation, search
How the EU grew, shrank a bit and grew over 50 years.
The European Union (EU) is a grouping of 27 European-ish countries. It is primarily an economic union aiming to harmonise financial and business activity between its member countries according to reasonable folk. The EU is often popularly ridiculed in the United Kingdom for its silly rules about square strawberries, straight bananas and designating the carrot as a fruit, although most of these "rules" do not exist, or were exaggerated or misinterpreted by Eurosceptics.[1]
There is some debate about the what direction the EU should take in the future, with some looking for a "wider" association and some for a "deeper" one. Those who wish a wider union are typically in favour of European expansion without any increased integration of the countries involved. Those who wish a deeper union are more interested in increased integration of the existing countries. These differing objectives can cause some conflict, as the wider the union becomes the more difficult it is to achieve integration; and the deeper the union becomes, the more difficult it is for new member countries to join.
Not to be confused with the Council of Europe.
Critics[Who?] claim the security and peace enjoyed by western European countries following the Second World War (The first time in history that Europe wasn't trying to kill itself) was almost entirely down to the threat of annihilation offered by the Soviet Union, and the military alliance with NATO.
Contents
[edit] Member countries
In case you aren't confused enough already.
[edit] Potential future members
[edit] Official candidates
The following countries are recognised as candidate countries. [2]
• Turkey has been trying to get in for twenty years, but has been refused on every application. It seems that it may have some way to go before it meets all EU standards, particularly as it only barely meets the "being in Europe" one.[3]
• Croatia
• The former Yugoslav Republic of Macedonia, which Greece plans to reject if they get close to membership because the northern part of their country is also called "Macedonia." Rappers have had more substantive feuds.
• Iceland is having its application to join hastened after its banking system collapsed in 2008.
[edit] Potential candidates
The following countries are recognised as pre-candidate countries.[4]
[edit] Other countries of note
Norway and Switzerland have both been invited but turned the offer down, meanwhile Morocco attempted to join the EU, but were also upset to be informed they weren't part of Europe.
Despite its non-membership in the EU, Norway is displayed as part of the map of Europe on some modern Euro coins, serving the vital purpose of making the rest of Scandinavia look less like a giant dong.
[edit] The Antichrist
The peaceful European Union will later become the dreadful Beast of the Apocalypse, that of the number 666! This is a common, though disputed, interpretation of Revelation.[5][6][7][8] They claim the EU is the successor to the Roman Empire and the Fifth Empire prophesied by Daniel. There were, obviously, other empires between the Babylonians and Romans, so there's no reason to assume that the Romans were the Fourth. But alas, enjoy the fundie shoehorning.
[edit] See also
[edit] Footnotes
1. BBC News: Guide to the best euromyths
2. EU website on expansion
3. Turkey status EU
4. EU Pre-candidate countries
5. Rapture Ready, European Union In Prophecy - This one says very near the bottom of a very long web page that we can't be sure the EU is the empire of the Antichrist but gives the impression beforehand that we are certain: "The great question of our time, prophetically speaking, might be: Is the European Union the prophesied governmental body that will eventuate in becoming the ten kingdoms that will provide Antichrist with his beastly power? No one can say for certain..."
6. Rapture Ready, Will the Antichrist come out of the European Union? - This one contradicts the one above though they are from the same fucking website. It claims "the European Union is clearly the fulfillment of this prophetic event." Do they hope their readers are too silly or too lazy to read through everything and find the contradiction? Apparently so.
7. The Antichrist, European Union, and Rome - This site recognizes that the EU is often seen as Antichrist but interprets the Bible differently.
8. Islam, Antichrist, and the European Union - This one expects Europe to become Islamic and then become the Antichrist.
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10 Oct 2005 marcin » (Journeyer)
Okay, so I was a bit premature in giving myself kudos for getting a nice subsumption/arbitration thing going there. I've been refactoring the code a little while writing the rotating ranger code (ie servo+SRF as a single entity), and as it turns out, one of the conditional compiles wasn't #defined previously. Now that I have enabled it ... well subsumption has a little way to go.
That being said, though, I have to say that the architecture works as designed, it is just that my behaviours are too simple - they should be state machines, not simple conditional outputs. It does raise the question though, once the behaviours are FSMs, what will happen once a higher-priority tasks interrupts a lower priority one, since not all behaviours are mutually exclusive.
Cheers, Marcin.
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Wikitravel:Using discussion pages
From Wikitravel
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Contents
Every article on Wikitravel has an associated discussion page for discussing that article. Discussion pages are not chat boards or comment areas; they're for coordinating editorial decisions, suggesting new material that should be considered, and generally collaborating on making a great article. For questions, comments, or personal stories about a destination or topic, check out Wikitravel Extra.
If you have new information for an article, by all means plunge forward and edit that page. But if there are multiple people working on an article, it can help to use that article's discussion page to divide up tasks and hammer out differences.
[edit] Discussion page formatting
You can get to the discussion page for an article by clicking on the discussion tab when reading an article.
You edit discussion pages just like editing any other page in Wikitravel; see how to edit a page for instructions. When using discussion pages, add new paragraphs at the bottom of the page.
If there are already conversations going on on that page, it can help to add a new heading. You can either click on the [ + ] button next to the "edit" tab if it's there, or create the heading manually like this:
==Names in Chinese==
...so that there's a visual distinction between topics of discussion.
You should sign your posts on discussion pages, so people know who they're talking to. Just enter four tildes at the end of your comments ("~~~~"), and it will turn into a signature with your user name, time and date.
Responses to other people's postings should be indented. You can use a colon (":") at the beginning of a paragraph to indent that paragraph. Responses to responses should be indented twice (two colons); responses to responses to responses get indented three times. Hopefully we don't get that much farther in a discussion, but if so, well, continue in that pattern.
[edit] User talk pages
The discussion page for a user page is special; it's called a user talk page. You can use user talk pages to leave someone in particular a personal message. Don't forget that they're publicly accessible, and anyone can read them. Try to keep discussions about a particular article on that article's talk page, too.
[edit] Travellers' pub
If you have something to discuss about Wikitravel as a whole, the Wikitravel:Travellers' pub is a good place to put your comments. You can also add a talk-page style note to the Logbook by clicking on "Today's Log" in the left-hand nav bar.
[edit] Requests for comment
If you think an issue needs more attention, you can add it to the requests for comment page to get more attention on it.
[edit] Etiquette
There are some points of etiquette in using discussion pages that have built up over the years. Here are a few:
• Unlike everything else in Wikitravel, it's considered bad form to change someone else's posts on a discussion page — even to correct spelling or grammar.
• It's usually perfectly OK, though, to change something you wrote on a discussion page, for any reason. If you made spelling or grammatical errors, feel free to change them.
• If, in the heat of the moment, you said something you regret, go ahead and change that, too.
• And, ForgiveAndForget when someone changes a nasty comment to something more civil and productive.
• As an exception, it's impolite to remove a comment if someone's responded to it. It makes them look ridiculous.
• In general, conversations aren't deleted from discussion pages but are instead archived when they are old or no longer relevant. To archive discussions simply create a new page such as User talk:Mypage/Archive and copy the old discussions to it.
• It's best to wait until the page has grown quite long before archiving, and such archives should always be clearly linked from the principal discussion page, so that everything is easy to find. Avoid archiving discussions on destination and policy article talk pages. Archives should not be edited.
[edit] Rants
If you must rant, there are endless venues to do so in the internet. While everyone is entitled to a good one now and then, off topic rants on discussion pages will usually be reverted.
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Hatay
From Wikitravel
Europe : Turkey : Mediterranean Turkey : Hatay
Revision as of 11:20, 17 January 2009 by KEREMCANN (Talk | contribs)
Jump to: navigation, search
Hatay Province is in southeastern part of Mediterranean Turkey, bordering Syria.
Regions
Cities
• Antakya, also known as Antioch – the provincial capital
• Iskenderun, also known as Alexandretta – the biggest city of the province, situated on the shore of Mediterranean Sea
Other destinations
Understand
The province, which was then known as Sandjak (county) of Alexandretta, was under French control as a part of the French Mandate of Syria between 1920 and 1938. In 1938, it appeared on the maps as the independent State of Hatay. Following a plebiscite a year later, it joined Turkey as the Hatay Province in 1939.
Talk
Almost all people in the province can speak Turkish, although with a heavy accent in most cases. Also Syrian dialect of Arabic is the native tongue for many people in many parts of the province.
Get in
by air
airport for both international and domestic flights is located in HATAY. 25 km to City Center.
by train
The only significant settlement in the province with a railline is Iskenderun, which has several daily train connections with Adana and Mersin.
by car
The main highway connecting the province to the rest of Turkey to the north is the motorway O-53 (toll-road). There are also secondary highways as well, some of which eventually reaches one of many border posts located on the Turkish-Syrian border.
Get around
See
Itineraries
Do
Eat
Drink
Sleep
Stay safe
Contact
The telephone code of the province is 326, which should be prefixed with 0 when calling from elsewhere in Turkey, or with +90 when calling from out of Turkey.
Get out
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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Romantic Road
From Wikitravel
Europe : Central Europe : Germany : Romantic Road
Revision as of 21:39, 25 January 2006 by Brendio (Talk | contribs)
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The Romantic Road ("Romantischestrasse" in German) is a popular tourist route through historical towns in southern Germany, from Würzburg to Füssen.
Cities
Other destinations
Understand
The term "Romantic Road" was first used in around the 1950s in the American-occupied section of a Germany. The name was initially known predominantly among American soldiers, who took their families on vacation there. The route is now popular with all nationalities. Signs can even be seen in Japanese.
More info can be obtained from the Touristik Arbeitsgemeinschaft Romantische Strasse, Waaggässlein 1, D-91550 Dinkelsbühl, phone +49 9851 551387, (info@romantischestrasse.de). [1]
Talk
• German is spoken along the Romantic road, though it may be strong on the Schwabish dialect in the region around Donauwörth.
Get in
• Depending on which direction you travel, Frankfurt and Munich make good entry and exit points to the Romantic Road.
Get around
• The Europabus[2] operates along the Romantischestrasse between April and October. €58.50 is the adult fare one way from Würzburg to Füssen.
• Those with more time on their hands can travel at a more leisurely pace by bicycle along the 420-km stretch of road in about ten days. Guided tours are also available to take some of the stress out of the journey—they will take care of your luggage and accommodation, and your can travel in their bus when it rains.
• Most of the destinations along the Romantic Road can also be reached by train,[3] but what would be the fun in that?
See
The best-known places along the route are Rothenburg ob der Tauber, Baroque city of [[Würzburg] (a major wine region), Füssen (at the foot of the Alps), Augsburg, and the small but delightful town of Dinkelsbühl.
Do
• Walk around the old towm walls, visit the medieval tourture museum, and buy Christmas decorations in Rothenburg ob der Tauber
Eat
• German food
Drink
• German beer. Try the Radler (beer mixed with lemonade/soda) if you are cycling.
• Sample wine from the distinctive Bocksbeutel in Würzburg.
Stay safe
• Observe the road rules
Get out
This is a usable article. It has information for getting in as well as some complete entries for restaurants and hotels. An adventurous person could use this article, but 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 Release Date
8635.4.40.001 - Tourist Accommodation, Small Area Data, South Australia, Dec 2000
Previous ISSUE Released at 11:30 AM (CANBERRA TIME) 23/03/2001
Page tools: Print Page Print All RSS Search this Product
• About this Release
ABOUT THIS RELEASE
Contains the results from the on-going quarterly Survey of Tourist Accommodation. Data provide information on the supply of, and demand for, tourist accommodation facilities. Data include number of establishments, capacity and employment for the quarter and occupancy and takings from accommodation for each month; by type of establishment and by star grading. This is a useful reference for policy makers and industry monitors and advisers. Details are provided for local areas and tourism regions in the State.
Since March quarter 1998 all hotels, motels and guest houses and serviced apartments with 15 or more rooms/units have been included in the survey. Every third year, beginning in 2000, the survey also includes caravan parks with 40 or more powered sites or cabins, visitor hostels with 25 or more bed spaces and holiday flats and units operated by owners, managers or real estate agents with sole letting rights to 15 or more units.
The State and Territory Tourist Accommodation publications, from 8635.1.40.001 to 8635.7.40.001, have been discontinued.
These have been continued by Data Cubes, in Excel format, from 8635.1.55.001 to 8635.8.55.001
© 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
1301.0 - Year Book Australia, 2006
Previous ISSUE Released at 11:30 AM (CANBERRA TIME) 20/01/2006
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Contents >> Chapter 3 - International relations >> Australia's credentials and place in the international system
AUSTRALIA'S CREDENTIALS AND PLACE IN THE INTERNATIONAL SYSTEM
Australia’s international interests are shaped in part by its geography and history, which have underpinned its active engagement in Asia and the Pacific and its close ties with North America and Europe.
Australia has a liberal, competitive economy marked in recent times by consistent growth, low inflation and low unemployment. Between 1998 and 2004, Australia’s gross domestic product (GDP) grew by an average of 3.6% each year - well above the global annual average of 2.6%. Australia is the 13th largest economy in the world. A modern and open economy, strong skills base and modern physical infrastructure support Australia's efforts to advance its economic interests overseas.
Australia's cultural diversity, record of constructive international engagement, strong political institutions and liberal democratic values - including commitment to the rule of law, freedom of the press and accountability of government - inform its involvement in world affairs.
The Australian Government published a second foreign and trade policy White Paper - Advancing the National Interest - in 2003. It is a comprehensive assessment of Australia’s place in the world and outlines how Australia can best use its political, strategic and economic assets to advance its national interests on the international stage.
Previous PageNext Page
© 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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Meet the Mansumer
Posted by NetworkSolutions under Marketing
From http://www.networksolutions.com 140 days ago
Made Hot by: BIZantium on January 3, 2013 12:38 am
Do you want your retail business to enjoy continued sales success long after the holiday shopping season ends? Then you need to know about a new kind of shopper that’s on the rise and will be increasingly important in 2013 and beyond: the “mansumer.”
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