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Thread Tools Search this Thread
Posts: 223 | Thanked: 38 times | Joined on Jul 2007 @ home
#1
There have been many complaints as of late of Nokia's decision to move the 810 to MiniSD (exclusively). However, I've been doing some reading, and that may not be so after all.
I was watching a video (http://www.nseries.com/index.html?l=...ucts,n810,demo) and at the end of the video some text popped up saying that the N810 will support up to 8GB cards. I thought, that's strange, miniSD goes up to 2 GBs, and MiniSDHC cards up to 4 GBs. 8GBs? huh?
Digging a little more I found this: http://europe.nokia.com/A4568593
Storage
* Up to 2GB internal memory
*Support for compatible miniSD and microSD memory cards (with extender). Supports cards up to 8GB. (SD cards over 2GB must be SDHC compatible.)
According to those specifications, miniSD and microSD are supported with adapters, with regular SD support up to 8GB, SDHC that is.
So the N810 is not ditching the full SD memory card slot, just a bit of poor wording has been floating around?
Comments?
--Jon
Posts: 751 | Thanked: 521 times | Joined on Mar 2007 @ East Gowanus
#2
No. Users who have final devices have opened up the flap and it is indeed Mini-SD, there is no full size SD support sorry
Posts: 3,096 | Thanked: 1,492 times | Joined on Jan 2006 @ Michigan, USA
#3
That was how I read that also, extender would imply full sd card, if they made my 8GB card worthless, after I managed to keep them from destroying it, that upgrade is looking less and less desirable
Posts: 223 | Thanked: 38 times | Joined on Jul 2007 @ home
#4
Well that sucks.
I was hoping Reggie would confirm my hypotheses... I guess I'll just have to find a way to cram a fullsize SD card if I ever get an 810 (likelihood just dropped 34.7%).
Of course, one has to remember that the devices *may* (though probably are ) not be final yet. One month is plenty of time.... oh who am I kidding? I dunno. Nokia, surprise me.
Thanks for the info mobileD
Posts: 228 | Thanked: 20 times | Joined on Oct 2007
#5
Definitely miniSD. Look at the engadget picture here: http://www.engadget.com/gallery/noki...nds-on/443962/
Posts: 1,674 | Thanked: 171 times | Joined on Mar 2007 @ Anderson, IN
#6
just because there is a mini sd in the pic doesnt mean that there isnt a full sd , plenty of room on the sides... lest we forget that the 800 came with a mini sd???
Posts: 2,669 | Thanked: 2,535 times | Joined on Apr 2007 @ Halifax, Nova Scotia, Canada
#7
Originally Posted by penguinbait
That was how I read that also, extender would imply full sd card, if they made my 8GB card worthless, after I managed to keep them from destroying it, that upgrade is looking less and less desirable
The N810 isn't meant to be an upgrade, but basically a more consumer friendly N800.
Posts: 223 | Thanked: 38 times | Joined on Jul 2007 @ home
#8
That's what I'm afraid Zero
They're (Nokia) trying to wean us (the hackers) off the tablet for the eventual mainstream, step-5, end product.
When the final product will be release, what will it be like? Will it be just another ugly iPhone gadgety thing? Will us hackers move on to the next hacker-centric mobile device? I hope that never happens
Posts: 3,096 | Thanked: 1,492 times | Joined on Jan 2006 @ Michigan, USA
#9
Well I just sold a car and earmarked 500$ for an upgrade (sneaking past the warden, uh I mean my beautiful wife ), but I am going to take a serious look at the market before considering another tablet. I would rather spend 1000$ and get a real processor.
All I have to do is keep the 1000$ device 2 years and it would be equal to the 500$ a year I spend on tablet and accessories each year. And a UMPC would probably be supported for 3 years.
I already have a n800, although KDE on a n810 with a keyboard would be so awesome
I guess I keep the n800 and look at UMPC's the SD card is very disappointing. They may be shooting themselves in the foot with this one.
Last edited by penguinbait; 10-20-2007 at 12:28 AM.
Posts: 223 | Thanked: 38 times | Joined on Jul 2007 @ home
#10
By KDE you're talking about KDE 4.0, right?
*nudge* *nudge*, *wink* *wink*
A UMPC sounds tasty, but I have yet to find one that really excites me. When A UMPC sporting a Core Duo and some nvidia G80 chip comes out.... for a reasonable price...
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Australian Bureau of Statistics
Celebrating the International Year of Statistics 2013
ABS Home > Statistics > By Catalogue Number
1216.0 - Australian Standard Geographical Classification (ASGC), July 2011
Latest ISSUE Released at 11:30 AM (CANBERRA TIME) 05/10/2011 Final
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PURPOSE OF THE ASGC
The main purpose of the ASGC is for collecting and disseminating geographically classified statistics. These are statistics with a ‘where’ dimension.
The ASGC provides a common framework of statistical geography which enables the production of statistics that are comparable and can be spatially integrated.
In practice, statistical units such as households and businesses are first assigned to a geographical area in one of the ASGC structures. Data collected from these statistical units are then compiled into ASGC defined geographic aggregations which, subject to confidentiality restrictions, are then available for publication.
The purposes of this publication are to outline the ASGC structures, describe the codes and names of geographical areas used and depict the statistical relationship between different types of geography used in the classification.
ENQUIRIES
For information regarding the ASGC and the ASGS, please refer to the ABS Geography web portal at abs.gov.au/geography or, contact ABS Geography by email geography@abs.gov.au or by writing to Locked Bag 10, Belconnen, ACT 2616.
For information about related statistics, 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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Australian Bureau of Statistics
Celebrating the International Year of Statistics 2013
ABS Home > Statistics > By Release Date
1318.3 - Qld Stats, Dec 2009
Previous ISSUE Released at 11:30 AM (CANBERRA TIME) 21/12/2009
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HOUSING FINANCE
HOUSING FINANCE COMMITMENTS
In Queensland, the number of owner occupied housing commitments in trend estimate terms decreased to 13,391 (down 0.4%) from September 2009 to October 2009, the fourth monthly decrease after ten consecutive monthly increases.
Housing Finance Commitments, (Owner Occupation)(a), Queensland: Trend
The trend estimate of the total value of housing finance commitments for owner occupation in Queensland decreased by 0.5% to $3,544 million, from September 2009 to October 2009. Over the year to October 2009, in trend estimate terms, the value of housing finance commitments showed an increase of 30%.
Since October 1999, the average home loan commitment for owner occupied dwellings in Queensland has more than doubled from $122,500 to $269,100.
In October 2009, the average loan size for first home buyers ($273,500) increased 3.4% from the previous month. The October 2009 average loan size for non-first home buyers ($267,700) increased 0.6% from the previous month.
Average Loan Size by Type of Buyer (Owner Occupation)(a), Queensland: Original
Further information on this topic is available in Housing Finance, Australia (cat. no. 5609.0) or by contacting the National Information and Referral Service on 1300 135 070. This publication is released monthly.
© 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
8752.1 - Building Activity, New South Wales, Dec 1994
Previous ISSUE Released at 11:30 AM (CANBERRA TIME) 13/04/1995
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• About this Release
Number of dwelling units and value of residential buildings (houses and other residential), value of alterations and additions to residential buildings and value of non-residential building by class of building (eg. hotels, offices, etc.); private sector/public sector ownership; stage of construction (commenced, under construction, completed); value of work done during period, value of work yet to be done; number of new houses by class of builder. Includes a summary of building activity (value of work: commenced, under construction, completed, done during period and yet to be done) classified by class of building (excluding houses) and stage of construction for Sydney Statistical Division. Also includes seasonally adjusted and constant price data.
This publication has been converted from older electronic formats and does not necessarily have the same appearance and functionality as later releases.
© 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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Software
TreeDyn: towards dynamic graphics and annotations for analyses of trees
François Chevenet1*, Christine Brun2, Anne-Laure Bañuls1, Bernard Jacq2 and Richard Christen3
Author Affiliations
1 Laboratoire de Génétique et Evolution des Maladies Infectieuses, UMR CNRS/IRD 2724, IRD, 911 avenue Agropolis, BP 64501, 34394 Montpellier Cedex 5, France
2 Institut de Biologie du Développement de Marseille-Luminy, CNRS UMR 6216, Parc Scientifique et Technologique de Luminy, Case 907, 13288 Marseille Cedex 9, France
3 Laboratoire de Biologie Virtuelle, CNRS UMR 6543, Université de Nice Sophia Antipolis, Centre de Biochimie, Campus Valrose, 06108 Nice, France
For all author emails, please log on.
BMC Bioinformatics 2006, 7:439 doi:10.1186/1471-2105-7-439
Published: 10 October 2006
Abstract
Background
Analyses of biomolecules for biodiversity, phylogeny or structure/function studies often use graphical tree representations. Many powerful tree editors are now available, but existing tree visualization tools make little use of meta-information related to the entities under study such as taxonomic descriptions or gene functions that can hardly be encoded within the tree itself (if using popular tree formats). Consequently, a tedious manual analysis and post-processing of the tree graphics are required if one needs to use external information for displaying or investigating trees.
Results
We have developed TreeDyn, a tool using annotations and dynamic graphical methods for editing and analyzing multiple trees. The main features of TreeDyn are 1) the management of multiple windows and multiple trees per window, 2) the export of graphics to several standard file formats with or without HTML encapsulation and a new format called TGF, which enables saving and restoring graphical analysis, 3) the projection of texts or symbols facing leaf labels or linked to nodes, through manual pasting or by using annotation files, 4) the highlight of graphical elements after querying leaf labels (or annotations) or by selection of graphical elements and information extraction, 5) the highlight of targeted trees according to a source tree browsed by the user, 6) powerful scripts for automating repetitive graphical tasks, 7) a command line interpreter enabling the use of TreeDyn through CGI scripts for online building of trees, 8) the inclusion of a library of packages dedicated to specific research fields involving trees.
Conclusion
TreeDyn is a tree visualization and annotation tool which includes tools for tree manipulation and annotation and uses meta-information through dynamic graphical operators or scripting to help analyses and annotations of single trees or tree collections.
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Research article
The endocrine stress response is linked to one specific locus on chromosome 3 in a mouse model based on extremes in trait anxiety
Mariya Gonik1,2, Elisabeth Frank1, Melanie S Keßler1, Darina Czamara1, Mirjam Bunck1, Yi-Chun Yen1, Benno Pütz1, Florian Holsboer1, Thomas Bettecken1, Rainer Landgraf1, Bertram Müller-Myhsok1, Chadi Touma1 and Ludwig Czibere1*
Author affiliations
1 Max Planck Institute of Psychiatry, Munich, Germany
2 Current address: Institute for Stroke and Dementia Research, Ludwig Maximilian University, Munich, Germany
For all author emails, please log on.
Citation and License
BMC Genomics 2012, 13:579 doi:10.1186/1471-2164-13-579
The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1471-2164/13/579
Received:19 August 2011
Accepted:29 October 2012
Published:31 October 2012
© 2012 Gonik 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 hypothalamic-pituitary-adrenal (HPA) axis is essential to control physiological stress responses in mammals. Its dysfunction is related to several mental disorders, including anxiety and depression. The aim of this study was to identify genetic loci underlying the endocrine regulation of the HPA axis.
Method
High (HAB) and low (LAB) anxiety-related behaviour mice were established by selective inbreeding of outbred CD-1 mice to model extremes in trait anxiety. Additionally, HAB vs. LAB mice exhibit comorbid characteristics including a differential corticosterone response upon stress exposure. We crossbred HAB and LAB lines to create F1 and F2 offspring. To identify the contribution of the endocrine phenotypes to the total phenotypic variance, we examined multiple behavioural paradigms together with corticosterone secretion-based phenotypes in F2 mice by principal component analysis. Further, to pinpoint the genomic loci of the quantitative trait of the HPA axis stress response, we conducted genome-wide multipoint oligogenic linkage analyses based on Bayesian Markov chain Monte Carlo approach as well as parametric linkage in three-generation pedigrees, followed by a two-dimensional scan for epistasis and association analysis in freely segregating F2 mice using 267 single-nucleotide polymorphisms (SNPs), which were identified to consistently differ between HAB and LAB mice as genetic markers.
Results
HPA axis reactivity measurements and behavioural phenotypes were represented by independent principal components and demonstrated no correlation. Based on this finding, we identified one single quantitative trait locus (QTL) on chromosome 3 showing a very strong evidence for linkage (2ln (L-score) > 10, LOD > 23) and significant association (lowest Bonferroni adjusted p < 10-28) to the neuroendocrine stress response. The location of the linkage peak was estimated at 42.3 cM (95% confidence interval: 41.3 - 43.3 cM) and was shown to be in epistasis (p-adjusted < 0.004) with the locus at 35.3 cM on the same chromosome. The QTL harbours genes involved in steroid synthesis and cardiovascular effects.
Conclusion
The very prominent effect on stress-induced corticosterone secretion of the genomic locus on chromosome 3 and its involvement in epistasis highlights the critical role of this specific locus in the regulation of the HPA axis.
Keywords:
F2; Corticosterone; Stress response; HPA axis; QTL
Background
To warrant for adequate stress reactions, the hypothalamic-pituitary-adrenal (HPA) axis is activated upon exposure to stressors. As a consequence of such a reaction, corticosterone (CORT) and cortisol, respectively, are secreted in mammals. This is the first phase of the physiological stress response, which already initiates the depletion of further stress hormone secretion via negative feedback mechanisms [1-3]. As demonstrated by various preclinical and clinical studies, exaggerated responses of the HPA axis can lead to continuously elevated stress, considered as one underlying cause in the aetiology of anxiety and depression disorders in humans [3,4]. Anxiety is also reflected at a cognitive level by the individuals' behaviour and strictly connected to physiological responses of central nervous, neuroendocrine and cardiovascular systems [5].
To shed light on the genetic underpinnings of the polygenic, multifactorial trait of anxiety, including its neuroendocrine correlates, multiple approaches have been applied up to date. Genetic engineering has helped to create knock-out and knock-in mice. These models have contributed a considerable amount of knowledge to describe the effects of neurotransmitter and neuromodulator systems, e.g., the corticotropin releasing hormone (CRH), vasopressin (AVP), tachykinin-class neuropeptides or serotonin (5-HT) [6-9]. However, the high number of regulatory mechanisms that are required to maintain the stress response and its return to homeostasis suggests that each molecular player contributes slightly to each phenotype, making it difficult to highlight all the mechanisms involved [10,11].
Compared to genetic engineering, selective breeding strategies have the advantage of keeping the integrity of the genome and simulating the interplay between multiple systems, thus being closer to the clinical situation. In our laboratory, mice of the outbred CD-1 strain were selectively bred for > 20 generations, based on their anxiety-related behaviour on the elevated plus-maze (EPM), resulting in high (HAB) vs. low (LAB) anxiety-related behaviour lines. In addition to differences in anxiety-related behaviour, these mice also exhibit comorbid phenotypic differences in other behavioural tests, such as the dark/light box, open field (OF), tail-suspension (TST) or forced swim tests (FST) [12], the latter two being indicative of depression-like behaviour. Hence, using the HAB/LAB mouse model furthered the identification of candidate genes of trait anxiety, including Avp, Enoph1, Glo1 and Tmem132d[13-16].
We here generated a HAB x LAB F2 intercross and focussed on endocrine system responses as a trait of interest. To reveal the link of genomic loci with the modulation of CORT responses upon stress exposure, we performed linkage analysis followed by an association study to specify the localisation of the QTL. Implementation of multipoint oligogenic segregation and linkage analysis by use of Bayesian Markov chain Monte Carlo (MCMC) method in the presence of missing values increases the power of the study by including the information from a total three-generation pedigree dataset and by sophisticated IBD sharing probability estimations [17]. In the present study, the focus is on the genetic analysis of endocrine phenotypes, while other findings of the linkage of anxiety-related and depression-like behaviour phenotypes (Czibere, unpublished) are largely neglected at this stage. The findings of this study have translational potential for human studies dealing with the dysregulation of the HPA axis, which is regarded as one of the key phenotypes in major depression [4].
Methods
Animals
All experimental procedures were conducted in accordance with the European Community Council Directive (10/24/1986; 86/609/EEC) and approved by local authorities. All animals tested were bred in the animal facilities of the Max Planck Institute of Psychiatry in Munich as described previously [12-16]. Briefly, mice derived from an outbred mouse strain (CD-1) were bidirectionally inbred, based on their anxiety-related behaviour measured in the EPM test for each generation at an age of seven weeks. After 20 generations of selective breeding, 16 HAB and 16 LAB mice (F0 generation: 8 males and females from each line) were reciprocally cross-mated in order to generate genetically heterozygous animals (F1 generation). F1 animals (N = 58 females and N = 45 males) were further bred with littermates to obtain the F2 generation (N = 534, all males, thereof 273 with a HAB F0 mother and 261 with a LAB F0 mother; Figure 1). In this generation, all differing alleles from the heterozygous F1 parents would segregate freely, thus providing the basis for linkage analysis of the known loci with the behavioural traits. Only male F2 mice were subjected to further analysis to avoid any effects of the oestrous cycle on behavioural and neuroendocrine indices.
Figure 1. Breeding scheme of the F2 generation from 16 HAB and 16 LAB F0 mice.
Animals were kept under standard housing conditions in groups of two to four per cage (clear plastic, Makrolon type II, 23 × 16 × 14 cm) with a 12-h light/dark cycle (lights on at 07:00 am). Breeding pairs and newborn litters were housed in Makrolon type III (38 × 22 × 15 cm) cages until weaning of the pups at four weeks of age.
Phenotyping
All behavioural tests were carried out between 08:00 am and 01:00 pm to minimise circadian variations of HPA axis activity. To investigate different traits including anxiety-related behaviour, exploratory drive, locomotor activity, CORT secretion profile and depression-like behaviour, various test paradigms were used [18]. Starting at the age of seven weeks, all animals (F0, F1 and F2) underwent a series of tests including EPM (5 min, 300 Lux on the open arms), TST (6 min), OF (5 min, 60 Lux in the central compartment), elevated platform test (EPF; 5 min), HPA axis reactivity test (HPA-RT; also called stress reactivity test), and FST (6 min, water at 23°C), as already described previously [12,19-21]. Animals were tested once in each paradigm in the order described above with an intertest interval of 48 h. Throughout the test battery, we tried to avoid exposure of animals to two similar tests on two consecutive days. Therefore, as a test for depression-like behaviour, TST was performed 48 h after the first anxiety-related behaviour test (EPM) on day one. To examine long-lasting effects of the relatively stressful TST on the OF test, we exposed 9 CD-1 mice to TST stress two days prior to OF and compared them to 10 unexposed controls. An independent sample of 12 HAB and 11 LAB mice from generation 18 was used to reassure the phenotypic divergence of HAB vs. LAB mice in the HPA-RT.
Described just briefly, the procedure for the HPA-RT was as follows. Mice were taken out of their home cages and a first blood sample was taken from their tail veins using glass capillaries. The procedure was limited to 2 min to allow acquisition of basal (i.e., unstressed) values for CORT. The animals were then immobilised for 15 min in 50 ml plastic tubes (with holes at the front and end), and the tubes were covered by a lid to ensure darkness for the restraint stress period. After this 15-min stressor, a second blood sample was taken from a fresh cut less caudally than the first one. Blood samples were processed to obtain at least 10 μl of blood plasma, which was then quantified for CORT using a radioactive immunosorbent assay (DRG Diagnostics, Marburg, Germany) according to the manufacturer’s protocol. All of the samples were assayed in the same batch at the same time. According to the manufacturer’s recommendation, intra-assay variability was reduced to the least possible extent by measuring duplicates. Single measurement values were considered valid, if they were within the range of the standards supplied by the manufacturer.
All behavioural analyses were performed by an experimenter blind to the background of the animals and videotaped for evaluation. Body weight was measured directly after EPM testing. At the age of 14 weeks, animals were single-housed for four days, and their 24-h water consumption was measured as described previously [16]. Then, the animals were decapitated under isoflurane anaesthesia and tail tips were collected for further analysis. Videotapes were scored by an observer blind to the background of the animals using “plus-maze” software for the EPM (E. Fricke, Munich, Germany), Anymaze (Stoelting Co, Wood Dale, IL) for the OF, and Eventlog 1.0 (EMCO software, Reykjavik, Iceland) for TST, EPF and FST, respectively. In the EPM test, an open arm entry is an entry with the forepaws only, corresponding to an entry of > 30% of the full body size, whereas a full open arm entry is an entry with the fore- and hind paws, thus corresponding to > 95% of the full body size.
The results of this study will focus on the phenotypic analyses and on the genetic linkage of CORT-related measurements from the HPA-RT, comprising the CORT concentrations before (basal CORT) and after (stress CORT) the 15-min restraint and the increase in CORT levels (stress CORT minus basal CORT) due to stressor exposure. As the increase in CORT is calculated as mentioned above, it is likely to be correlated, though not identical, with stress CORT, i.e., the two values will not be truly independent.
Selection of suitable single-nucleotide polymorphisms (SNPs) for HAB vs. LAB mice
In an unbiased whole-genome assay, SNPs that consistently differ between HAB and LAB animals (i.e., each line carries the opposite alleles homozygously) were identified, thus allowing for genotyping of F2 mice. To assess a high number of SNPs, the Mouse Medium Density Linkage Panel (Illumina, San Diego, CA) was chosen to determine 1,449 genotypes simultaneously, giving an overview of SNPs available as specific markers for HAB vs. LAB animals. Providing the basis for subsequent genotype-phenotype associations in F2 mice, all male and female F0 (N = 16 each) and some F1 mice (N = 18) were genotyped, with the latter serving as controls. For SNPs, where HAB and LAB mice would show the opposite genotype homozygously, F1 mice should exclusively show heterozygous genotypes.
DNA samples were isolated from tail tips using the NucleoSpin Tissue kit (Macherey-Nagel, Düren, Germany). Samples were processed according to the manufacturer’s instructions (Illumina Golden Gate Assay for Sentrix Array Matrix workflow sheets). Fluorescence signals of hybridised samples were captured in the Illumina BeadStation, and genotypes were called from fluorescence intensity clusters using the Illumina BeadStudio software Ver. 3.1.0.0 with the genotyping module Ver. 3.1.12. All intensity clusters were inspected individually and adjusted manually, if necessary. Analysis of genotypes focussed on the detection of valid genotypic SNP markers to distinguish HAB from LAB mice.
Genotyping of F2 mice
Based on the previously described experiment using the Medium Density Linkage Panel, a custom designed oligo pool (384 SNPs, Golden Gate Assay, Illumina) was set up for genotyping of all 521 F2 mice and, in addition, the 16 HAB and 16 LAB F0 mice, respectively. Therefore, 267 SNPs were chosen from the HAB vs. LAB mouse SNP identification experiment and another 116 SNPs were selected from the MGI database to fill up unmapped gaps and to screen selected genes from previously published or unpublished own studies (Additional file 1: Table S1). One SNP (rs3659551) was genotyped twice serving as internal control. Evaluation was performed as described for the Medium Density Linkage Panel. All genomic locations mentioned refer to genome assembly NCBI37/Mm9.
Additional file 1. Table S1. Single-nucleotide polymorphisms (SNP) tested using the custom designed oligo pool (Illumina). Custom designed SNP pool for Illumina Golden Gate Assays to genotype the F2 mice for the current study. Source (1) refers to SNPs, chosen from the Mouse Medium Density Linkage Panel, (2) additional SNPs selected from the MGI database based on genes known from previously published or unpublished studies. Gene association is assumed, if a SNP is located 10 kbp around a gene locus.
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Statistical analyses
Two groups comparison (Mann-Whitney-U test) was conducted for the parental F0 HAB and LAB mice to examine differences in the endocrine stress measures.
A principal component (PC) analysis was applied to examine the phenotypic structure of the F2 mice. PC analysis was used to reveal the phenotypes giving the highest loadings to the total phenotypic variance and to check for correlations between the HPA axis reactivity and the behavioural phenotypes. PCs selected by the stricter broken-stick criterion [22] were kept for analysis. PC analysis was followed by an orthogonal varimax rotation, which resulted in each retained factor bearing a small number of large loadings and a large number of zero (or small) loadings, i.e., the variance of the loadings was maximised. Loadings with absolute values > 0.7 were treated as major loadings. PC analysis and varimax rotation were performed using the open-source statistical software package R ( http://www.r-project.org/ webcite). PC analysis was carried out using the normalised phenotypic data of male mice only.
The Loki package [23,24] for multipoint IBD estimations and oligogenic model for segregation and linkage analysis were used to benefit from the entire three-generation pedigree and to implement reversible jump MCMC techniques for likelihood estimations and Bayesian approaches. A quantitative trait was modelled as being genetically controlled by diallelic QTL and affected by the sex covariate. The description by Cox et al. served as the basis for conversion of physical to genomic distances on the autosomal chromosomes [25].
The Oligogenic model suggests the presence of multiple putative QTLs. The advantage of the Bayesian strategy, in contrast to other linkage methods, is that the number of putative QTLs is not fixed a priori. In contrast, this number was treated as an unknown model parameter. The posterior distribution for the number of underlying QTLs was calculated by using an iterative MCMC sampling technique [26].
The Bayes factor [27], or L-score, was computed to determine linkage as follows. For each sampling iteration (100,000 iterations were used for each analysis), the prior probability of finding a QTL linked to a 1 cM bin was 1 / t, where t was the total map length of the genome (approximately 1400 cM) [28]. If, for a particular iteration, there were n QTLs in the model, the prior probability, P, of at least one QTL located in the bin was 1 − (1 – 1 / t) n. The posterior probability, Q, is 1 or 0 depending on whether at least one QTL is located in the 1 cM bin. The score for each bin was estimated by averaging Q / P over an interval of iterations, which is supposed to perform a relatively stable outcome (75,000 – 100,000 iteration interval was used). Genomic regions that have a high probability of containing a QTL were expected to have considerably elevated L-scores compared to surrounding regions. The Bayes factors (L-scores) cannot be quantified in terms of a LOD score, but guidelines for estimating the importance of the Bayes factor are given by Kass and Raftery [27]. Twice the natural logarithm of the Bayes factor is on the same scale, as are the familiar device and likelihood ratio test statistics. 2 log (L-score) scores of a range of 2 to 6 were considered positive signals, 6 to 10 were considered strong, and over 10 very strong [27].
To quantify the linkage signal in terms of more common LOD scores, multipoint parametric linkage analysis of quantitative traits was performed by MQScore_SNP software package [29]. A priori, a diallelic autosomal QTL is supposed to be placed near each marker. Parameters of the model are estimated during linkage analysis by maximisation of the LOD score. This score compares the null hypothesis that SNP and QTL segregate independently with the alternative hypothesis, i.e., close linkage between QTL and each marker.
To estimate the location of the identified QTL in terms of common confidence interval and to test for epistatic interactions, QTL mapping in the F2 intercross was performed using the open-source statistical software package R/qtl [30]. Since there is no standard procedure available to determine confidence intervals in multipoint linkage analysis, the single-QTL scan was performed by standard interval mapping (maximum likelihood algorithm). The confidence interval of QTL location was estimated by 95% Bayes credible intervals, which are supposed to depend less on marker density [31]. The QTL-by-QTL epistatic interactions were characterized by the p-values for the LOD scores of corresponding epistatic models. The interactions were accepted to be significant if they did not exceed the adjusted threshold of 0.01. To assess the adjusted p-values, a traditional permutation test (N = 1000) was performed for the genomic region of interest [31].
To localise the most prominent markers more exactly, we additionally implemented the WG-Permer software ( http://www.wg-permer.org webcite) for rapid association analysis on the autosome chromosomes and R ( http://www.r-project.org webcite) for association analysis on X chromosome in male F2 mice. The Bonferroni correction for multiple testing was applied. The level of significance for all association tests was set to 10-4. An allelic model was used to make the results comparable with those of the linkage analysis mentioned above [23].
The gene functional classification tool available at the “Database for Annotation, Visualization and Integrated Discovery” (DAVID; http://david.abcc.ncifcrf.gov/ webcite) [32,33] was used to retrieve the functional gene categories overrepresented among the genes situated in the region of the identified QTL. Analysis of the enriched functional-related gene groups was carried out with the DAVID-based Fisher's exact test, and results were Bonferroni corrected.
Results
Phenotypic assessment of the parental F0 HAB and LAB animals (group comparisons) confirmed the previously described data on comorbid anxiety-related and depression-like behaviours [12-15]. In addition, a nominally significant difference was found for the neuroendocrine parameters stress CORT and increase in CORT as measured in the HPA-RT in males (p < 0.05), but not females. As correction for multiple testing abolished this statistic effect (Table 1), the group comparison was replicated in an independent batch of HAB and LAB mice. The significant difference between the two lines could be confirmed (p < 0.001; Figure 2), with LAB mice clearly displaying elevated CORT levels after restraint stress compared to their HAB counterparts. Due to the fact that HABs avoid entering open arms of the EPM and LABs usually spend so much time on the open arm that they rarely enter closed arms, both closed and total arm entries are supposed to be rather unreliable indices of locomotor activity. Accordingly, locomotion is test-dependent (Table 2). Therefore, we rather focus on the total distance travelled in the OF to assess locomotor activity with LAB travelling significantly more than HAB mice. Independent of locomotor activity, the EPF shows nominally significant differences in male mice for explorative behaviour (frequency of head dips; Table 1).
Table 1. Characteristic phenotypes of HAB vs. LAB mice (F0 mice, N = 8 per gender and line); values are represented as mean ± standard error of means (SEM); corrected p-values refer to Bonferroni correction and are sorted by p-values of comparison in male mice
Figure 2. Corticosterone levels in the blood plasma of male HAB vs. LAB mice (A) under basal conditions, (B) after a 15-min restraint stressor. Data are shown as mean ± standard error of the mean (SEM) with N (HAB) = 12 and N (LAB) = 11; *** p < 0.001.
Table 2. Principal component (PC) analysis of behavioural phenotypes in HAB x LAB derived F2 mice sorted by behavioural test paradigms
Importantly, even if the preceding test is stressful, it is unlikely to have any impact on behaviour two days later. Indeed, CD-1 mice tested in the TST and CD-1 controls without prior testing showed similar behavioural indices in the OF (Additional file 2: Figure S1).
Additional file 2. Figure S1. Effect of prior tail-suspension test exposure on the behaviour in the open field test. Key parameters assessed in the open field test showed no significant difference in CD-1 mice between control (N = 10, only exposed to the open field test) and tst (N = 9, exposed to the tail-suspension test 48 h before the open field test) mice. Data are shown as means +SEM.
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To structure the phenotypic data obtained from all 44 test parameters in the male F2 mice, PC analysis was performed. Seven PCs were qualified as important and independent according to the selection criterion (Table 2). These 7 PCs accounted for 62% of the total phenotypic variance and included 24 phenotypes providing the highest (i.e., major) loadings of absolute values > 0.7 (Table 2). Basic data distribution is exemplarily shown for the F2 phenotypes HPA-RT (increase in CORT, Figure 3A), EPM (percent time spent on the open arm, Figure 3B) and OF (total time spent in inner zone, Figure 3C).
Figure 3. Histograms of the main phenotypes considered for the F2 mice and the PC analysis. These included (A) the corticosterone (CORT) increase after a 15-min restraint stressor, (B) the percent time spent on the open arm of the elevated plus-maze and (C) the time spent in the inner zone of the open field.
The comorbidity of anxiety-related and depression-like phenotypes observed in HAB vs. LAB mouse comparisons was not reflected by the PC analysis in our F2 animals, leading to high loadings of anxiety-related behaviour as measured in the EPM in PC2, in the OF in PC3 and of depression-like behaviour in PC4 (Table 2). In a similar manner, phenotypes reflecting locomotor activity or exploratory drive in the EPM and OF showed their highest loadings on two separate anxiety-independent PCs (PC5 and PC1, Table 2).
The PC analysis clearly indicated that two of three endocrine parameters, which both reflect the CORT response upon restraint stress (stress CORT and increase in CORT), showed a high correlation (adjusted R2 = 0.99). These two endocrine parameters demonstrated the highest loadings (−0.97 and −0.96, respectively) on PC6. Hardly any other PC bore comparably high loadings except for the inner/outer zone time spent in the OF (PC3) and entries to the middle open arm in the EPM (PC1). Thus, PC6, accounting for 4.6% of the total phenotypic variance, is representative of CORT responses to a stressor as measured in the HPA-RT with basal CORT measurements, largely not affecting the overall outcome in F2 mice, leading to similar stress CORT and increase in CORT values. Based on these findings, any of these two endocrine measurements (stress CORT or increase in CORT) might be used as a compound score for the CORT response.
Genotyping of the F2 animals at 384 SNPs (average call rate 93%) resulted in 27 unidentifiable loci, 89 showed no informative value concerning the F2 panel, and one SNP was genotyped twice (showing identical results), leaving 267 SNPs for further testing with informative value for genotype-phenotype associations. From these markers, 233 SNPs were taken from the HAB vs. LAB mouse SNP identification experiment and 35 SNPs from those that were additionally screened on the 384 Sentrix Array Matrix (more than 25% of the newly added SNPs were informative for our mouse lines). Eleven markers were only specific for one LAB-subline, thereof two (rs29341895, rs29354500) from the list of SNPs added after the initial screening experiment. These 11 SNPs were not included in the phenotype-genotype linkage due to a shift of allele frequencies, leaving a total of 256 SNPs. This resulted in an average SNP marker density of 1 SNP per 11 Mbp. There were one genomic region of 53 Mbp on chromosome 3 and further five regions between 40 and 45 Mbp in size on chromosomes 2, 4, 10 and 6 for which we did not have any SNP markers. According to their genotypes, all F0 animals could clearly be clustered to their respective HAB or LAB origin.
The stress CORT phenotype was used as a basis for genetic linkage analyses to find the chromosomal regions, which segregate with the trait. First, the MCMC technique was applied genome-wide to take advantage of Bayesian approaches, including a correction for sex in the model. The Bayes factor values (L-scores) were obtained with a step of 1 cM, permitting a better resolution of the QTL for stress CORT. This highlighted a unique region from 40.5 cM (93.1 Mbp) to 45.2 cM (102.4 Mbp) on chromosome 3, showing a very strong positive evidence for linkage (the highest 2ln (L-score) value of > 10 between positions rs13477268 and rs4138887, Figure 4A). Next, a parametric linkage analysis was carried out to estimate the corresponding LOD values on chromosome 3. Stress CORT showed a strong positive linkage signal across chromosome 3 with the highest LOD value of > 23 (Figure 4B).
Figure 4. Linkage of stress corticosterone (CORT) in HAB x LAB-derived three-generation mouse pedigree on chromosome 3. (A) L-score (Bayes factor) multipoint oligogenic linkage analysis of stress CORT using Bayesian Markov chain Monte Carlo approach. (B) LOD score two-point parametric linkage analysis of stress CORT.
The maximum likelihood estimate of the QTL location was found at 42.3 cM with the 95% confidence interval indicating the ends of the QTL at 41.3 cM (closest marker: rs13477268 at 40.5 cM) and 43.3 cM (closest marker: rs4138887 at 45.3 cM). The genomic position of the linkage peak (42.3 cM / 97.5 Mbp) is covered by the protein-coding gene Pde4dip. The two-QTL scans on chromosome 3 provided evidence for the epistasis (chromosome-wide adjusted p < 0.004), between the identified locus at 42.3 cM and the locus at 35.3 cM (Figure 5).
Figure 5. Two-QTL epistasis on chromosome 3. (A) The upper left triangle contains the LOD scores for the epistasis, e.g., log10 likelihood ratio comparing the full model (two QTLs plus interaction) to the additive model (two QTLs); the lower right triangle contains the LOD scores for the full model, e.g., log10 likelihood ratio comparing the full model to the null model. (B) The phenotype means for each group defined by the genotypes at two epistatic loci.
Finally, to check the co-occurrence of traits and markers for CORT-release upon restraint stress, the association analysis of autosomes and the X chromosome was performed for all CORT-related phenotypes: stress CORT, increase in CORT and basal CORT. The significant associations were found for stress CORT and increase in CORT, but not basal CORT. The highest association peaks were placed on chromosome 3 for rs13477268 and rs4138887, followed by rs6376008 with p < 10-28 (Figure 6). These 3 SNPs were shown to be highly correlated with R2 > 0.8. The pronounced local association minima for stress CORT and increase in CORT (Figure 6) resulted from the shifted allele frequency of the homozygotic genotypes (for rs6376008, rs6211610 and rs13477268). Additionally, besides the strong association signal on chromosome 3, the only other association signals reaching nominal statistical significance (10-4 < p < 0.05) were located on the X chromosome, with two pronounced peak regions, the first around 92 Mbp and a second smaller peak around 136 Mbp (Table 3).
Figure 6. Association of three phenotypes (stress CORT, increase in CORT, basal CORT) in HAB x LAB-derived F2 mice on chromosome 3. The correlations R2 between the top rs13477268 and surrounding SNPs are coded by a colour bar.
Table 3. Association analysis of markers on the X chromosome for hypothalamic pituitary adrenal axis activity-related phenotypes (p-values for increase in CORT, stress CORT and basal CORT)
Importantly, to exclude parent-of-origin effects, calculations made separately for all F2 mice with a HAB F0 mother (N = 273) and with a LAB F0- mother (N = 261) did not show any differences in the effects shown on chromosome 3 (nominal p-value for offspring of HAB F0 mother rs13477268: p < 10-28; for offspring of LAB F0 mother: p < 10-25).
Combination of linkage and association analyses offered the possibility to approximate the genomic location for the HPA axis response upon restraint stress. Indeed, the linkage study could highlight a single genomic locus, a 5-cM region between markers rs13477268 (40.5 cM / 93.1 Mbp) and rs4138887 (45.2 cM / 102.4 Mbp) on chromosome 3. In addition, the association study revealed the presence of another highly correlated SNP: rs6376008 (37.9 cM / 86.5 Mbp). Therefore, we hypothesised that a set of genes located within that QTL region (37.9 cM - 45.2 cM) might show a well-defined enrichment in specific functional annotation categories. Within the 7-cM QTL, we could identify 391 known mouse genes and conducted a functional classification applying the DAVID analysis tool. Finally, among these 391 genes the analysis revealed a single significantly enriched (Bonferroni corrected p < 0.01) cluster of 5 genes (Table 4). It represents a specific functional class for androgen, oestrogen and progesterone biosynthesis (for linkage to CORT see KEGG enzyme EC 5.3.3.1). Another cluster, including 3 genes and related to cholesterol biosynthesis, was identified (Table 4), but showed only nominal significance (uncorrected p < 0.05).
Table 4. Panther functional enrichment analysis of the genomic region strongly linked to the CORT response phenotype
Discussion
Our results highlight a specific genomic region on chromosome 3 as a major contributor to the variation of CORT response derived from the HAB/LAB mouse model. The strong contribution of genomic factors of this region seems to be essential for regulating stress-related reactivity of the HPA axis.
Here, we described first of all a difference in CORT secretion in HAB vs. LAB mice upon a 15-min restraint stressor, with LAB mice secreting more CORT compared to HAB mice. Thus, HAB mice appear to have a rather blunted stress response. Although, in general, high levels of CORT response are described to be associated with high anxiety states [34,35], the opposite is true for HAB vs. LAB mice. This finding was replicated in CD-1 mice bred for differences in CORT responses to a stressor, with lower HPA axis reactivity mice showing more passive exploratory behaviour [20]. In addition, a stronger increase in CORT has been demonstrated after social defeat in LAB than in HAB rats, probably due to the more active coping style of the former in response to any kind of stressor [36].
We could not detect any major difference in locomotion of HAB vs. LAB mice in the EPM, indicating that the animals’ anxiety-related phenotype is mainly independent of effects based on differing locomotion. Though, we cannot completely reject a confounding influence of locomotion on behaviour, especially as HAB vs. LAB male mice exhibited nominally significant differences in locomotion in the OF. On the other hand, we could also detect nominally significant differences in exploratory behaviour on the EPF for HAB vs. LAB males, a test much less affected by differences in locomotion.
Linkage studies were applied in rats and mice to a wide variety of complex phenotypes, including alcohol preference, anxiety-related, depression-like and exploratory behaviours or HPA axis function [37-40]. Based on the study by Williams et al., where a comparable breeding strategy led to the identification of about 300 genotypic markers useful for complex trait analysis [41], our mouse population seems to fulfil the requirements for linkage analysis. In addition, loading of the phenotypes of different behavioural tests in F2 on independent PCs underlines the good segregation of characteristic traits from the original HAB and LAB mouse lines.
Anxiety phenotypes of EPM and OF tests mainly loaded on two separate PCs showing limited comparability and predictability of measurements from different anxiety tests in freely segregating F2 animals. This is consistent with findings in other studies [42,43] supporting the idea that different paradigms reflect – at least partially – different facets of trait anxiety. Similarly, in the F2 generation, phenotypes representing anxiety, depression and endocrine stress response loaded on separate PCs, reflecting strongly reduced comorbidity of these traits compared to F0 HAB and LAB mice. This resembles findings of another F2 study, where a connection between depression-like behaviour and stress reactivity was rejected [39]. However, as solely based on low loadings of other behavioural phenotypes in the stress-related phenotypic cluster, a connection between these phenotypes cannot be completely denied, being in line with the idea of anxiety and stress reactivity representing multifactorial and polygenic traits with minor contributions of many genes. It is, therefore, likely that associated phenotypes and analogous comorbidities in the clinical situation such as those between anxiety, depression-like behaviours and HPA axis/stress reactivity share contributing genetic factors that are hardly detectable. This is also underlined by a recent study, proposing a network approach to analyse overlapping symptoms in the concept of comorbidity [44].
As stress CORT and increase in CORT are highly correlated (due to the low levels of basal CORT, resulting in almost near-to-identical values for the two parameters) and placed on the independent PC6 representing the only prominent loadings, both might be considered equally representative of the neuroendocrine system response. Stress CORT is exemplarily shown (Figure 4) for genetic analysis. Identified by sequential linkage analysis of trait cosegregation in a full three-generation pedigree and F2 intercross mapping, a CORT-related QTL was detected on mouse chromosome 3 between 41.3 cM (closest marker: rs13477268 at 40.5 cM) and 43.3 cM (closest marker: rs4138887 at 45.3 cM) with a linkage peak at 42.3 cM. Two-QTL scans showed an evidence for epistasis between the region of the peak location at 42.3 cM and the locus at 35.3 cM (closest marker: rs6376008 at 37.9 cM). The association analysis of freely segregating F2 mice derived from a HAB x LAB cross identified a prominent trait co-occurrence with rs13477268 and two highly correlated nearby markers, rs4138887 and rs6376008. The high correlation of nearby SNPs (rs13477268, rs4138887 and rs6376008) points to the presence of a broad linkage disequilibrium (LD) block, indicating that the identified group of significant markers represents the effect of the true locus underlying the neuroendocrine stress response.
Taken together, our findings highlight the region from 37.9 cM to 45.2 cM of mouse chromosome 3 as an endocrine stress response-specific locus. The additionally identified regions on the X chromosome, only showing nominally significant association with the traits stress CORT and increase in CORT, provide an impetus for the involvement of sex-specific mechanisms in influencing the respective trait, which is supported by other studies [45].
Comparative genome analyses of mouse chromosome 3 showed rat and human homologues on chromosomes 2 (rat) and 1 and 4 (human), respectively [46]. The markers that were linked to stress CORT and increase in CORT are not located near to any neurotransmitter receptor gene or other candidate genes for HPA axis regulation mapped to date in mice (according to NCBI, Ensembl and UCSC databases).
A particularly interesting candidate gene for the trait of neuroendocrine stress response is Pde4dip, a locus characterised by the maximum likelihood estimate of QTL location and involved in epistasis. The encoded protein, myomegalin, is responsible for cardiac contractility during adrenergic stimulation [47]. The rat homologues of our identified QTL (Figure 4A) are highly overlapping with previously identified QTLs for cardiovascular disease-related phenotypes. These overlaps include rat QTLs for blood pressure and heart rate [48] and bradycardia [49]. As these rat QTLs also include the genomic locus of Pde5a, which is capable of modulating acute and chronic cardiac stress responses [50,51], represented by rs13477379 in our study, it makes this gene an interesting additional candidate, although not included in the region of strongest linkage. Particularly as acute CORT responses show a pronounced effect on the cardiovascular system [52], the previously described cardiovascular QTLs in this genomic region are in line with our findings.
The identified QTL is located in a region that also overlaps with a QTL for ethanol-induced CORT responses in mice [53] and is homologous to a QTL for fasting cortisol levels in humans [54]. Both ethanol administration and fasting have been shown to provoke an increase in CORT secretion and glucocorticoid production, similar to acute stress responses [55,56]. Finally, the 7-cM region, identified to be linked to stress CORT in the present study, is completely homologous with one of the QTLs (Srcrt-1) Solberg et al. described to influence stress CORT in their rat model [40], with the difference that the linkage in our study can be limited to a single, smaller region, which is strongly linked to the stress CORT phenotype. In case of Solberg et al., Srcrt-1 shows the second strongest effect on stress CORT, behind a marker on rat chromosome 6 (homologous to mouse chromosome 12) of five linked genomic loci altogether [40].
The strongly linked 7-cM genomic region on chromosome 3 harbours at least five genes involved in steroid, most prominently glucocorticoid, synthesis (Table 4) [57], emphasising a strong effect on the stress CORT phenotype. Although these genes are primarily focussed on androgen, oestrogen and progesterone synthesis (Table 4), their enzyme products are known to play a vital role in the synthesis of all glucocorticoids, among them CORT (KEGG pathway mmu00140; http://www.genome.jp/kegg/ webcite). This strongly supports a conserved genomic and, thereby, heritable effect on stress reactivity. Interestingly, a recent study indicates that corticosteroids can be synthesised endogenously in the brain [58]. Therefore, the genomic region identified in our study might also affect corticosteroids directly in the brain. The second functionally enriched group (Table 4), albeit showing only nominal significance, is related to cholesterol biosynthesis, which is the first metabolite required for glucocorticoid synthesis [59]. Another genomic site containing genes coding for glutamate receptors and other transmembrane proteins (NCBI) is related to the locus at 35.3 cM, which is in epistatic interaction with the identified locus at 42.3 cM.
While many mechanisms of genomic and non-genomic regulation remain largely unclear in terms of activation vs. inhibition of the HPA axis [45], our results strongly point to a genetic influence of the 7-cM region. The additionally identified regions on the X chromosome, only showing nominally significant linkage to the stress CORT trait, provide an impetus for the involvement of sex-specific mechanisms in influencing the respective trait, which is supported by other studies [60].
Conclusion
Taken together, based on a HAB x LAB mouse intercross panel, we have identified one major QTL on chromosome 3 linked to both stress-induced CORT level and the level of increase in CORT, providing a strong basis for the heritability of neuroendocrine stress reactivity and emphasising a critical role of the genes relevant for steroid synthesis. In addition to the neuroendocrine effect, Pde4dip covering the position of the linkage peak and Pde5a placed in a nearby region overlapping with known cardiovascular QTLs, suggest a comorbid cardiovascular effect driven by the identified locus. Further supported by epistatic effects, our findings highlight a novel aspect towards the understanding of the well-orchestrated mechanisms of stress response regulation. They will contribute to further facilitate the search for candidate genes that are important for the regulation of neuroendocrine systems under stress conditions and, simultaneously, provide deeper insights into their phenotypic outcome, behaviour.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MG planned and carried out the statistical analyses, EF, MSK and RL conceptualised and carried out the F2 breeding, behavioural assessment and analysis of behaviour, DC participated in the statistical analyses, MB and YCY carried out the behavioural assessment and analysis, BP participated in conceptualising the statistical analysis, FH contributed to shaping the project, TB contributed to planning and carrying out the genotyping, RL conceptualised and supervised the project, BM conceptualised and supervised the statistical assessment, CT participated in planning and carrying out the phenotyping and provided additional neuroendocrine data, LC carried out the behavioural assessment, conceptualised and carried out the genotyping and wrote the manuscript together with MG and RL. All authors have read and approved the final manuscript.
Acknowledgements
The authors would like to thank Markus Nußbaumer, Marina Zimbelmann, Sabine Damast, and Susann Sauer for perfect technical assistance and Ramona Zeh, Hendrik Stein, Julia Baier, Jan-Michael Heinzmann and Kathrin Halfter for their help with phenotyping. Last but not least, the authors would like to thank the anonymous reviewers for their suggestions to improve this manuscript.
All authors and the study were funded by the Max Planck Society. The funder had neither a role in study design, collection, analysis and interpretation of data, in writing the manuscript, nor in the decision to submit the manuscript for publication.
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A computational tool for the design of live attenuated virus vaccine based on microRNA-mediated gene silencing
Duangdao Wichadakul*, Wuttichai Mhuantong, Anan Jongkaewwattana and Supawadee Ingsriswang
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Research article
A large sustained endemic outbreak of multiresistant Pseudomonas aeruginosa: a new epidemiological scenario for nosocomial acquisition
Cristina Suarez1, Carmen Peña1,6*, Olga Arch1, M Angeles Dominguez2, Fe Tubau2, Carlos Juan3, Laura Gavaldá4, Mercedes Sora5, Antonio Oliver3, Miquel Pujol1 and Javier Ariza1
Author Affiliations
1 IDIBELL, Infectious Diseases Service, Hospital Universitari de Bellvitge, University of Barcelona, Barcelona, Spain
2 IDIBELL, Microbiology Service, Hospital Universitari de Bellvitge, University of Barcelona, Barcelona, Spain
3 Microbiology Service, Hospital Son Espases, Palma de Mallorca, Spain
4 IDIBELL, Preventive Medicine Service, Hospital Universitari de Bellvitge, University of Barcelona, Barcelona, Spain
5 IDIBELL, Pharmacy Service, Hospital Universitari de Bellvitge, University of Barcelona, Barcelona, Spain
6 Infectious Diseases Service, Hospital Universitari de Bellvitge, L'Hospitalet de Llobregat, Barcelona, Spain
For all author emails, please log on.
BMC Infectious Diseases 2011, 11:272 doi:10.1186/1471-2334-11-272
The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1471-2334/11/272
Received:7 December 2010
Accepted:13 October 2011
Published:13 October 2011
© 2011 Suarez 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
Studies of recent hospital outbreaks caused by multiresistant P.aeruginosa (MRPA) have often failed to identify a specific environmental reservoir. We describe an outbreak due to a single clone of multiresistant (MR) Pseudomonas aeruginosa (PA) and evaluate the effectiveness of the surveillance procedures and control measures applied.
Methods
Patients with MRPA isolates were prospectively identified (January 2006-May 2008). A combined surveillance procedure (environmental survey, and active surveillance program in intensive care units [ICUs]) and an infection control strategy (closure of ICU and urology wards for decontamination, strict compliance with cross-transmission prevention protocols, and a program restricting the use of carbapenems in the ICUs) was designed and implemented.
Results
Three hundred and ninety patients were identified. ICU patients were the most numerous group (22%) followed by urology patients (18%). Environmental surveillance found that 3/19 (16%) non-ICU environmental samples and 4/63 (6%) ICU samples were positive for the MRPA clonal strain. In addition, active surveillance found that 19% of patients were fecal carriers of MRPA. Significant changes in the trends of incidence rates were noted after intervention 1 (reinforcement of cleaning procedures): -1.16 cases/1,000 patient-days (95%CI -1.86 to -0.46; p = 0.003) and intervention 2 (extensive decontamination): -1.36 cases/1,000 patient-days (95%CI -1.88 to -0.84; p < 0.001) in urology wards. In addition, restricted use of carbapenems was initiated in ICUs (January 2007), and their administration decreased from 190-170 DDD/1,000 patient-days (October-December 2006) to 40-60 DDD/1,000 patient-days (January-April 2007), with a reduction from 3.1 cases/1,000 patient-days in December 2006 to 2.0 cases/1,000 patient-days in May 2007. The level of initial carbapenem use rose again during 2008, and the incidence of MRPA increased progressively once more.
Conclusions
In the setting of sustained MRPA outbreaks, epidemiological findings suggest that patients may be a reservoir for further environmental contamination and cross-transmission. Although our control program was not successful in ending the outbreak, we think that our experience provides useful guidance for future approaches to this problem.
Background
Classically, P.aeruginosa nosocomial infections have been considered to be polyclonal endemic infections that follow secondary endogenous intestinal and primary respiratory tract colonization in patients admitted to ICUs or other hospital wards who have previously received antibiotic therapy [1-3]. However, in this setting, outbreaks caused by some particularly transmissible strains which often show multiresistance to antibiotics have also been reported. The capacity to control these outbreaks varies greatly and dependes on the reservoir. Outbreaks are usually circumscribed in time and attributable to a point source of infection which can be identified in the environment [4-8].
Over the past decade, an increase in hospital outbreaks caused by multiresistant P.aeruginosa (MRPA) has been reported in a new epidemiological setting. Studies have often failed to identify a specific environmental reservoir [9-15], and point toward patients as an additional potential reservoir, as in the case of a large sustained epidemic/endemic caused by multiresistant Acinetobacter baumannii [16]. While several studies of the risk factors and clinical features of patients colonized or infected with multiresistant P.aeruginosa have been conducted [17,18], the management strategies for these prolonged MRPA outbreaks and the contribution of antibiotic restriction [9] to their control have not been analyzed in depth.
Since 2003 there has been an increase in the number of carbapenem-resistant P.aeruginosa isolates in our hospital. A retrospective microbiological revision in 2005 showed a progressive increase in P.aeruginosa strains exhibiting the same multiresistant antibiotype, and molecular typing of selected strains demonstrated the presence of an outbreak in several hospital wards. Despite strict barrier precautions for controlling infection, the incidence of MRPA continued to rise throughout the hospital.
Here, we describe a large-scale sustained outbreak of a multiresistant strain of P.aeruginosa in our hospital and evaluate the effectiveness of the control measures applied.
Methods
Setting and Definitions
The study was performed at the Hospital Universitari de Bellvitge, a 900-bed public tertiary-care institution for adult patients. The MRPA surveillance programme was initiated in January 2006 and all patients colonized or infected with MRPA were prospectively identified.
MRPA was defined as strains resistant to ≥ 3 of the following classes of antibiotics: antipseudomonal penicillins, antipseudomonal oxyimino-β-lactams, fluoroquinolones, aminoglycosides, and carbapenems [19]. A clinical sample positive for MRPA was considered to have been acquired in a specific ward if it appeared either during a stay in this ward or within a week of discharge from it. The remaining patients with clinical samples positive for P.aeruginosa and without the MRPA phenotype isolate from the time of admission to the time of discharge from hospital were considered non-MRPA.
Clinical assessment was determined in accordance with the definitions of the Centers for Disease Control and Prevention for nosocomial infections [20]. Patients with positive clinical samples for MRPA but without related signs or symptoms of infection were considered to be colonized.
Microbiological studies
P. aeruginosa strains were identified and tested for antimicrobial susceptibility by a MicroScan automated microdilution system using CN1S and CO1S panels (Dade International, West Sacramento, CA, USA). CLSI (Clinical and Laboratory Standards Institute) criteria [21] were used to define susceptibility or resistance to these antimicrobial agents.
We selected 284 MRPA phenotype strains isolated throughout the whole period (2006 [132], 2007 [101], 2008 [51]) for pulsed-field gel electrophoresis (PFGE) analysis using a method described previously [13]. DNA was digested with SpeI and DNA fragments were separated with a CHEF DR III apparatus (Bio-Rad Laboratories, Hercules, CA, USA). Electrophoresis was run at 6 v/cm, 14°C for 20 hours with pulses ranging from 0,5 to 25 seconds. DNA restriction patterns generated by PFGE were interpreted according to the guidelines [22]. Isolates with PFGE patterns differing in more than four fragments were ascribed to distinct genotypes; differences in four restriction fragments or fewer were considered to be subtypes of a single genotype [23].
The mechanisms responsible for the multidrug-resistant phenotype detected were studied in three representative isolates from the epidemic clone. The potential presence of horizontally acquired β-lactamases was explored by phenotypic tests, which included Etest MBL strips (AB Biodisk, Solna, Sweden) for the detection of class B carbapenemases and double disk synergy tests (DDST) for the detection of ESBLs, using amoxicillin-clavulanate and ceftazidime, cefepime, aztreonam, imipenem, or meropenem disks (distance from 5 to 30 mm). To examine the involvement of mutation-driven resistance mechanisms in the multidrug-resistant phenotype, the relative mRNA expression of the genes encoding the major P. aeruginosa efflux pumps (mexB, mexD, mexF and mexY) and the chromosomal cephalosporinase (ampC) was determined by real-time PCR as described elsewhere [24,25]. Isolates were considered to be hyperproducers if the relative expression of the corresponding gene was at least three times (mexB) or ten times (mexD, mexF mexY, and ampC) greater than the figure documented for the reference PAO1 strain. In addition, in order to detect inactivating mutations causing carbapenem resistance, oprD genes from these isolates were amplified by PCR and fully sequenced, using previously described primers and conditions [26]. The sequences obtained were compared with those available in the GenBank.
Environmental survey
To determine the possible route of transmission or reservoir in the hospital's physical environment, a total of 78 samples, 30 from room equipment (bedside tables, bed rails, phones, plug holes, tap handles, door handles, call buttons), 36 from sink surfaces, and 12 from water samples were taken from the urology and ICU wards, the wards which initially had the highest incidence. Twenty samples, (13 from urology wards and seven from the ICU), were obtained in February 2006 using moistened swabs, another 16 samples were obtained in September 2006 from the urology wards, and the remaining 42 samples were collected from the ICU during 2007. In addition, to improve our capacity to detect contamination, in 2008 we modified the swab technique by introducing the use of moistened sterile gauze pads [27]. Another 82 samples were collected (63 from ten ICU rooms and 19 from four non-ICU rooms). All these samples were taken from room equipment. During the surveillance procedure, all the non-ICU rooms had admitted patients with known MRPA colonization, compared with only three ICU rooms. The genetic similarity of MRPA strains isolated in the environmental survey was investigated by PFGE.
Active surveillance program
To determine the strength of the human reservoir, we carried out longitudinal active surveillance of rectal swabs in an ICU unit over a one-month period (March 2006). Weekly rectal swab samples were obtained on admission in order to identify digestive tract carriage of MRPA between ICU admission and discharge or the time to MRPA clinical sample detection. Patients admitted to the unit for less than 48 hours and those with MRPA digestive tract colonization at ICU admission were excluded.
Infection control interventions
Infection control measures: Intervention 1: Disposable aprons and gloves were used while caring for MRPA colonized/infected patients, and cleaning procedures were strictly supervised by infection control nurses (rooms were routinely cleaned once a day using water and liquid soap with 500 parts per million [ppm] hypochlorite, and rooms with colonized patients were cleaned twice a day in the same way but with a hypochlorite concentration of 1,000 ppm, and using a specific checklist for high-touch surfaces). ICU patients were in single rooms and patients in non-ICU wards were spatially segregated inside a single room. These infection control measures were introduced in February 2006. Intervention 2: Environmental cleanliness was reinforced, particularly in the ICU (which was closed for extensive decontamination, consisting in rigorous cleaning of the medical equipment and painting of the wards, in July-August 2006 and July-August 2007) and the urology ward (which was decontaminated in August 2007).
Restriction of carbapenem use in ICUs: In January 2007 the use of carbapenem for ICU patients was restricted. Compliance with this policy was monitored. Consumption was expressed as defined daily doses (DDD) [28].
Statistical analysis
An interrupted time series analysis was performed using the segmented linear regression procedure [29], evaluating the impact of the intervention on the level (abrupt change immediately following the intervention) and the secular trend of the series (change in the slope of the incidence rate following the intervention). The level and trend of the pre-intervention segments served as the control for the post-intervention segment.
MRPA patients included those who presented at least one MRPA specimen obtained during admission; previously known cases who tested positive on readmission were excluded, and newly identified cases attending the outpatient clinic were included from the date of prior admission. Colonization pressure was calculated monthly in the ICUs and the urology ward as follows: number of MRPA patient-days × 100/total number of patient-days [30].
To evaluate the effect of the interventions performed in the urology ward, four time segments were defined (Figure 1). Segments 1 and 3 served as the pre-intervention (baseline) periods. Time points before May 2006 were excluded from the analysis, since there were fewer than three observations before and after an intervention performed in February 2006 [31]. The interventions studied were: Intervention 1 "reinforcement of control measures and cleaning procedures" (October 2006) and Intervention 2 "extensive decontamination" (August 2007). The analysis was adjusted for the colonization pressure of the immediately preceding month and for the level and trend of the pre-intervention periods. Statistical analysis was performed with SPSS software, version 14.0.
Figure 1. Reduction in the rate of MRPA cases in urology ward: Intervention 1: reinforcement cleaning procedures; Intervention 2: extensive decontamination.
Ethical considerations
The study formed part of the medical care provided in the setting of the surveillance and control of nosocomial outbreaks, and the physician had good reason to believe that participation in the research study would not adversely affect the health of the patients. All the samples and exams performed on patients were part of standard care procedures for epidemiological control. The study was approved by the local ethic committee of the hospital Universitari de Bellvitge and no informed consent was requested.
Results
Description of the outbreak
From January 2006 to May 2008, 390 consecutive patients were colonized or infected by a main phenotype of MRPA; 288 (74%) patients were males with a mean age of 62.4 (SD ± 19.7) years; 85 (22%) were ICU patients and 305 patients acquired the MRPA strain in a non-ICU setting. The urology service [54 patients/305 (18%)] was the non-ICU ward with the highest rates of MRPA acquisition; the remaining patients were recorded in 30 other wards.
Among the 390 patients with clinical samples for MRPA, 165 (42%) patients were infected, with urinary tract infection [45 episodes (28%)] being the most frequent MRPA infection. Forty-six episodes (28%) were intra-abdominal infections: 36 (22%) surgical site infections, and 10 (6%) biliary infections. All 37 (23%) episodes of lower respiratory tract infection were observed in ICU patients. The remaining infections were: eleven (7%) primary bloodstream infections, 12 (7%) bone and joint infections, nine (6%) soft-tissue infections and five miscellaneous infections. Seventy-two (44%) out of 165 infected patients died. Of these deaths, 14 (19%) were attributed to MRPA infection.
Microbiological and genotypic analysis
The antibiotic susceptibility patterns of MRPA strains isolated during the study period are summarized in Table 1. Genotypic analysis showed that the selected MRPA strains belonged to a single clone responsible for a large sustained outbreak and no relationship was found between this majority clone and the MRPA strains detected in our hospital in an earlier MRPA outbreak [5].
Table 1. Antibiotic susceptibility of multiresistant P.aeruginosa clone strains
All phenotypic tests for the detection of acquired β-lactamases yielded negative results. In contrast, the pan-β-lactam resistance phenotype was found to be driven by the interplay of the hyperproduction of the chromosomal cephalosporinase AmpC (238 ± 34-fold increased expression of ampC respect to wild-type reference strain PAO1) and the inactivation of OprD porin, caused by a C to T mutation in nucleotide 424 of oprD, which leads to a premature stop codon at amino acid 142. Furthermore, no significant modification of the expression of the efflux pump encoding genes mexB, mexD, mexF, or mexY was detected.
Environmental survey results
Three of 36 (8%) initial samples were positive for clonal MRPA strain. All three strains were isolated in wet areas in the urology ward. None of the remaining 42 samples (moistened surfaces, room equipment and water samples) from the ICU wards showed MRPA growth. Thirteen samples, five of them from water, showed growth for carbapenem-susceptible P.aeruginosa.
In addition, with the modified gauze technique [27], three of 19 (16%) samples from non-ICU rooms were positive for the MRPA clonal strain, while only four of 63 (6%) ICU samples were positive.
Active surveillance program
During the study period, 42 patients were admitted to the ICU; of these, six patients with an ICU stay of less than 48 hours and five carriers of digestive tract MRPA at ICU admission were excluded. Thirty-one patients were included, and six digestive tract carriers were colonized by MRPA (19%). The mean time to MRPA acquisition between ICU admission and digestive tract carriage was 22 days. The mean time between carriage of MRPA in the digestive tract and the detection of positive clinical samples was 11 days (range 1 to 21 days).
Response to infection control intervention programs
Before the interventions, there was a significant month to month increase in MRPA cases in the urology ward: segment 1: increase of 0.67 cases/1,000 patient-days (95%CI 0.21 to 1.12; p = 0.006) and segment 3: increase of 1.53 cases/1,000 patient-days (95%CI 0.84 to 2.23; p < 0.001). However, significant changes in the trends of incidence rates were noted after both interventions: intervention 1: -1.16 cases/1,000 patient-days (95%CI -1.86 to -0.46; p = 0.003) and intervention 2: -1.36 cases/1,000 patient-days (95%CI -1.88 to -0.84; p < 0.001). This represents a mean decrease of 5.4 MRPA cases/1,000 patient-days after intervention 1, and an even more marked reduction, 8.4 cases/1,000 patient-days, after intervention 2. Concomitantly, after intervention 2, an immediate effect was found in the first month: -1.58 cases/1,000 patient-days (95%CI -3.50 to 0.33; p = 0.09) (Figure 1).
In addition, restricted use of carbapenems was initiated in January 2007 (Figure 2). Carbapenem use decreased from 190-170 DDD/1,000 patient-days in the period immediately before the implementation of this restriction (October-December 2006) to 40-60 DDD/1,000 patient-days after its initiation (January-April 2007), with a reduction from 3.1 cases/1,000 patient-days in December 2006 to 2.0 cases/1,000 patient-days in May 2007. However, after this strong initial decrease, we observed a moderate increase in carbapenem consumption, which rose to around 100 DDD/1,000 patient-days from May to December 2007 and a progressive increase in incidence. Segmented linear regression analysis could not easily be applied in ICU intervention (carbapenem use restriction) since no evident linear patterns were found following the intervention.
Figure 2. MRPA incidence rates in ICUs during and after the implementation of the restricted use of carbapenems. Bars: incidence rates (n° MRPA cases/1,000 patient-days). Line: DDD carbapenem use/1,000 patient-days.
Discussion
We provide epidemiological information on a large-scale sustained MRPA outbreak occurring at our hospital over a period of more than three years. An epidemic clone prevailing in the urology and ICU wards spread rapidly throughout many other wards despite an epidemiological surveillance program; no specific source of outbreak could be identified and the clone became endemic. This is a new epidemiological scenario with serious consequences.
Our observations contrast strongly with the classical view of the epidemiology of nosocomial P.aeruginosa infections. These nosocomial P.aeruginosa infections were considered to be polyclonal, endemic and opportunistic, affecting patients with underlying diseases undergoing multiple manipulations and receiving broad spectrum antibiotic therapy [3].
Several hospital outbreaks caused by multiresistant P.aeruginosa have been reported [9-15]. Although colonization by P.aeruginosa frequently precedes overt infection, the original source of the organism and the precise mode of transmission are often unclear.
In our large clonal endemic setting, we were unable to identify a particular reservoir responsible for the outbreak. We investigated all the wet areas that were likely to be sources of contamination. Environmental surveillance was performed in the urology and ICU wards, the wards with the highest incidence, but in neither case did the study provide a great deal of information. The level of detection improved when a moistened gauze was used (0% in ICU and 8% in non-ICU using swabs versus 6% in ICU and 18% in non-ICU with gauze), although no parallel analysis of the two techniques was performed. In addition, the sites that tested positive for the epidemic clone were near colonized patients, and the presence of this clone in the environment may have been a simple consequence of the outbreak rather than a source for transmission of the strain [32]. Nevertheless, two specific interventions involving the reinforcement of cleaning procedures for extensive decontamination were applied in the urology wards and MRPA rates decreased significantly during the months following the interventions, a finding that supports the transitory efficacy of these epidemiological control measures
The results of our active surveillance program and environmental cultures in the ICU setting suggest that patients may indeed be a reservoir in the maintenance of the monoclonal MRPA outbreak. Our capacity of detection of MRPA intestinal carriers in ICUs was lower than the rates found in the screening programs of two other outbreaks at our hospital [33,34]; however, in those two settings the capacity of the outbreak to spread was higher. These findings are borne out by the longer period of time recorded between ICU admission and digestive colonization: 22 days in the present study versus < 7 days in those outbreaks.
No doubt, cross-transmission plays a relevant epidemiological role in our MRPA ICU patients [35], but as no hand-print cultures of health care workers were performed in our study, we cannot provide direct evidence that strains were transmitted patient to patient by the health care workers.
Moreover, our data suggest that antibiotic pressure plays a decisive role, by altering the ecological niche in these patients and providing a selective growth advantage for MRPA organisms. Thus, given that the carbapenem consumption in our ICUs in 2006 was high, we assumed that carbapenem restriction might make a significant contribution to controlling the outbreak. The carbapenem restriction program carried out during 2007 was initially followed by a marked reduction; however, a moderate increase was recorded in the second semester and the levels rose again during 2008. Concomitantly, an increase in the level of piperacillin-tazobactam use was observed and a new cluster, particularly in the ICU, was found [36]; the dominant use of this antibiotic could have favored the emergence and spread of this different clone, and as a result, the carbapenem restriction was relaxed. Carbapenem restriction did not significantly reduce the number of MRPA cases, although this antibiotic program may not have been sufficiently rigorous or prolonged. Furthermore, we cannot rule the possibility that the use of fluoroquinolones may also have contributed to promoting these MRPA strains [17]. Although fluoroquinolone consumption did not increase during the period analyzed (data not shown), overall fluoroquinolone use was considerably higher than that of other antipseudomonal antibiotics. Thus, the limitation of fluoroquinolone use should have been included in our antibiotic restriction program.
Finally, our epidemiological control program had several limitations. First, we did not perform decontamination in other hospital wards; second, it is possible that the evaluation of MRPA digestive tract carriers in certain non-ICU wards would have allowed earlier spatial segregation and would thus have prevented a substantial number of possible cross-transmissions; and third, an extensive antibiotic program restricted to non-ICU wards might have contributed to lower incidence rates.
Conclusion
In conclusion, we faced a large and sustained MRPA outbreak that became endemic in our hospital after three years. In the absence of a recognized source of contamination, the reasons for the wide dissemination of the clone remain unclear. However, considering the general epidemiology of nosocomial P.aeruginosa infections, we speculate that more efficient nosocomial clones are more likely to acquire resistance determinants and that antibiotic pressure in the hospital environment favors their further spread [37]. Epidemiological findings suggest that patients may be a reservoir for further environmental contamination and cross-transmission. However, although unlikely, we cannot completely rule out the possibility of an environmental reservoir. Further studies should try to determine the extent to which realistic and prolonged restriction antibiotic programs can contribute to fighting this epidemiological challenge.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
CS participated in recruitment of subjects, performed the statistical analysis and drafted the first version of the manuscript. CP was the study's principal investigator, and participated in its design and coordination, supervision of analyses, and drafting. OA participated in the collection of epidemiologic data. MAD performed the PFGE analysis. FT performed the phenotype analysis. CJ studied the mechanisms responsible for the multidrug-resistant phenotype. LG participated in the design and statistical analysis. MS calculated antimicrobial consumption. AO helped to interpret the results of mechanisms of resistance. MP participated in the design and in the analysis of results. JA participated in design and provided expert oversight. All authors read and approved the final manuscript.
Acknowledgements
This work was supported by National Health Service grant FIS 08/0276 from the Fondo de Investigación Sanitarias and Supported by Ministerio de Ciencia e Innovación, Instituto de Salud Carlos III-co-financed by European Regional Development Fund "A way to achieve Europe" ERDF, Spanish Network for the Research in Infectious Diseases (REIPI RD06/0008), and by the Ciber de Enfermedades Respiratorias (CB06/06/0037).
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Study protocol
Modified constraint-induced movement therapy or bimanual occupational therapy following injection of Botulinum toxin-A to improve bimanual performance in young children with hemiplegic cerebral palsy: a randomised controlled trial methods paper
Brian J Hoare1,2*, Christine Imms1,3,4, Hyam B Rawicki2 and Leeanne Carey1,5
Author Affiliations
1 School of Occupational Therapy, La Trobe University, Bundoora, 3086, Australia
2 Victorian Paediatric Rehabilitation Service, Monash Medical Centre, 246 Clayton Road, Clayton, 3168, Australia
3 Murdoch Children's Research Institute, Melbourne, Australia
4 Royal Children's Hospital, Melbourne, Australia
5 National Stroke Research Institute, Florey Neuroscience Institutes, Melbourne, Australia
For all author emails, please log on.
BMC Neurology 2010, 10:58 doi:10.1186/1471-2377-10-58
The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1471-2377/10/58
Received:23 December 2009
Accepted:5 July 2010
Published:5 July 2010
© 2010 Hoare 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
Use of Botulinum toxin-A (BoNT-A) for treatment of upper limb spasticity in children with cerebral palsy has become routine clinical practice in many paediatric treatment centres worldwide. There is now high-level evidence that upper limb BoNT-A injection, in combination with occupational therapy, improves outcomes in children with cerebral palsy at both the body function/structure and activity level domains of the International Classification of Functioning, Disability and Health. Investigation is now required to establish what amount and specific type of occupational therapy will further enhance functional outcomes and prolong the beneficial effects of BoNT-A.
Methods/Design
A randomised, controlled, evaluator blinded, prospective parallel-group trial. Eligible participants were children aged 18 months to 6 years, diagnosed with spastic hemiplegic cerebral palsy and who were able to demonstrate selective motor control of the affected upper limb. Both groups received upper limb injections of BoNT-A. Children were randomised to either the modified constraint-induced movement therapy group (experimental) or bimanual occupational therapy group (control). Outcome assessments were undertaken at pre-injection and 1, 3 and 6 months following injection of BoNT-A. The primary outcome measure was the Assisting Hand Assessment. Secondary outcomes included: the Quality of Upper Extremity Skills Test; Pediatric Evaluation of Disability Inventory; Canadian Occupational Performance Measure; Goal Attainment Scaling; Pediatric Motor Activity Log; modified Ashworth Scale and; the modified Tardieu Scale.
Discussion
The aim of this paper is to describe the methodology of a randomised controlled trial comparing the effects of modified constraint-induced movement therapy (a uni-manual therapy) versus bimanual occupational therapy (a bimanual therapy) on improving bimanual upper limb performance of children with hemiplegic cerebral palsy following upper limb injection of BoNT-A. The paper outlines the background to the study, the study hypotheses, outcome measures and trial methodology. It also provides a comprehensive description of the interventions provided.
Trial Registration
ACTRN12605000002684
Background
Cerebral palsy is "a group of permanent disorders of the development of movement and posture causing activity limitation(s) that are attributed to non-progressive disturbances that occurred in the developing fetal or infant brain" [[1], p. 9]. Secondary to the brain disturbance, children with cerebral palsy often experience neurological symptoms including dystonia, ataxia, athetosis and particularly spasticity [2]. Spasticity occurs as a result of a loss of upper motor neuron inhibition on the lower motor neurons which results in increased or impaired motor unit firing and altered muscle tone [2]. Muscle spasticity is characterised by a velocity-dependent increase in tonic stretch reflexes (muscle tone) with exaggerated tendon jerks (phasic stretch reflex) resulting from hyperexcitability of the stretch reflex [3]. Adding to these neurological symptoms, skeletal muscle morphology in children with cerebral palsy is also altered due to abnormally long muscle sarcomere lengths and muscle tissue containing a hypertrophic extracellular matrix of poor quality [2,4]. This results in muscle stiffness affecting posture and movement and can be described as hypertonia or increased muscle tone [3].
Hemiplegic cerebral palsy, characterised by a clinical pattern of unilateral motor impairment, accounts for 35.1% of all cerebral palsy types in Victoria, Australia [5], 15.3% in Ontario, Canada, 40% in Sweden [6] and 31.2% in North England, United Kingdom [7]. Along with muscle spasticity and hypertonia, children with hemiplegic cerebral palsy experience a loss of upper motor neurone excitation that is typically associated with poor selective motor control and weakness, and in some instances, sensory deficits. These additional impairments significantly impact on a child's ability to perform daily tasks [8-11].
The spastic motor type of cerebral palsy is the most common, comprising about 80% of all reported cases [12]. Although the mechanism is unknown, spastic muscle often shortens to create muscle contractures, which often leads to fixed deformity and further functional complications [13]. Therefore, management of the upper limb in children with cerebral palsy usually involves a variety of interventions targeting the musculoskeletal system. These may include splinting and casting, passive stretching, the facilitation of posture and movement (e.g. occupational therapy and physiotherapy) or systemic spasticity-reducing medication and surgery [14]. Botulinum toxin-A (BoNT-A) is now commonly used as an adjunct to these interventions.
Botulinum toxin-A in the treatment of the upper limb in children with cerebral palsy
BoNT-A is a powerful neuromuscular paralysing agent that is produced by the anaerobic bacterium clostridium botulinum [15]. BoNT-A acts at the neuromuscular junction by inhibiting the release of the neurotransmitter acetylcholine. Injection of BoNT-A into selected muscles produces dose-dependent chemical denervation resulting in reduced muscle activity. The denervation is temporary as sprouting of new nerve terminals from the treated nerves leads to re-innervation. The function of the original terminal is eventually restored leading to the recovery of the affected muscles [16]. The period of clinically useful relaxation appears to be 12-16 weeks [12]. The aim of BoNT-A in the treatment of the upper limb in children with cerebral palsy is to produce selective reduction in muscle spasticity using the smallest possible dose. The reduction in spasticity is intended to provide an opportunity to optimise the effects of motor training by reducing the negative interference of spasticity. It can also serve to improve tolerance and compliment the effects of splinting and casting potentially delaying the need for soft tissue surgery [9].
Use of BoNT-A for the treatment of upper limb spasticity in children with cerebral palsy has become routine clinical practice in many paediatric treatment centres worldwide. In Australia, injection of BoNT-A (Botox(r) only) is now an approved and government funded treatment for moderate to severe spasticity of the upper limbs children with cerebral palsy, 2 to 17 years of age inclusive [17]. A recent Cochrane review, including ten randomised controlled trials predominantly of high quality, provided high-level evidence that BoNT-A in the upper limb, in combination with occupational therapy, improved outcomes at both the body function/structure and activity level domains of the International Classification of Functioning, Disability and Health (ICF) [18] when compared to occupational therapy alone, BoNT-A alone or no treatment [19]. The review authors concluded that injection of BoNT-A in the upper limb in children with cerebral palsy should always be accompanied by planned post-injection therapeutic intervention. This conclusion is consistent with a recently developed international collaboration documenting upper limb BoNT-A evidence-based guidelines for intervention and follow-up for children with cerebral palsy [20].
Intramuscular injection of BoNT-A results in relaxation of the affected muscle providing a window of opportunity for maximising therapy outcomes. For children with hemiplegia however, injection of BoNT-A alone does not improve the ability of the child to use the affected limb and clinical experience suggests attempts to use the limb immediately following injection often remain effortful, inefficient and result in clumsy movement. Persistent verbal and physical encouragement by therapists and parents may frequently lead to further frustration and negative or defiant behavior. Therapy must, therefore, create the opportunity, motivation, experience and environment in which a child can learn how to use their affected limb to maximize the effectiveness of the BoNT-A.
A recent systematic review of all upper limb interventions in children with cerebral palsy found that occupational therapy in combination with BoNT-A produced the largest treatment effect of all upper limb interventions on activity level outcomes [21]. Despite high-level evidence supporting occupational therapy post-injection, no clinical trial has specifically investigated the optimal type and amount of therapy following BoNT-A. Approaches undertaken in recent trials however, all point to the effectiveness of bursts of therapy focused on specific skill acquisition or goal achievement [22-26]. Further investigation is required to establish what intensity and/or specific type of therapy will enhance functional outcomes and prolong the beneficial effects of BoNT-A [14].
Movement-based therapies in the upper limb
For occupational therapists, bimanual occupational therapy training (BOT) in children with hemiplegia is a commonly applied intervention. Theories of practice have previously been described in the literature [27-29], although clinical service delivery is eclectic and potentially covers a range of interventions. An essential element of the bimanual approach to upper limb training includes the repetitive practice of motivating, meaningful and purposeful bimanual activities (i.e. occupations). This has been well supported by recent advances in the areas of neuroscience, basic mechanisms of hand function and more specifically, motor control and motor learning theories [30]. Although this approach is commonly used and has strong theoretical foundations, it has not been systematically defined and empirically investigated.
In 2007, Gordon and colleagues comprehensively described and evaluated a bimanual approach to upper limb training in children with hemiplegic cerebral palsy called Hand Arm Bimanual Intensive Training (HABIT) [31]. Despite similarities with BOT, specific differences exist between the therapies. HABIT includes: high intensity of treatment (6 hours per day for 2 weeks); the use of behavioural shaping theory [32]; and relies solely on environmental adaptation for grading of activities, rather than physical assistance or handling of the child (Andrew Gordon, personal communication). Children participating in HABIT demonstrated improved bimanual upper limb function compared with a group receiving customary care [31]. However, the promising evidence provided by this small trial of moderate methodological quality suggests that further investigation of bimanual intervention in children with hemiplegia is both justified and warranted [28].
Constraint-induced movement therapy (CIMT) has been adopted as a method of teaching a child to use his/her affected upper limb through use of a restraint on the non-affected limb and massed practice of movements of the affected limb [33]. It has been proposed as an effective way of improving outcomes following upper limb BoNT-A injection, particularly as it removes the need for persistent prompting [34]. It may, on the other hand, lead to the opposite effect if a child demonstrates adverse behavioral responses to the restraint [33].
Developmental disregard is a term used to describe a child with hemiplegia who may disregard, or learn not to use, the affected limb during the development of motor function [35]. Children with even mild impairment in the affected limb learn effective strategies to manage daily tasks (e.g. play) with one hand, discovering that performance is more efficient using only the non-affected hand [10]. Constraint-Induced Movement Therapy (CIMT), aims to reverse the behavioral suppression of movement in the affected upper limb by constraining use of the non-affected limb and providing massed practice of activities with the affected limb [36]. Studies in adults following stroke have provided evidence of adaptation in the brain involving the motor cortical areas controlling movement of the more affected limb following CIMT [37-39]. Since the potential for central nervous system plasticity in young children is increased relative to adults [40-42], it is postulated that this approach might prove to be especially effective in children [43].
CIMT is a multi-faceted intervention and studies describing its use in children with cerebral palsy present wide variation in its application in relation to: method of restraint; length of restraint (per day, number of weeks); type and duration of therapy; intervention environment (that is home, school, or clinic) and intervention provider (therapist, parent, or teacher). Children included in studies have also varied in age, diagnosis, severity of motor and sensory impairment, cognitive abilities and behavior. Despite the emerging popularity of CIMT in children with hemiplegic cerebral palsy, a Cochrane review [44] identified a significant treatment effect in only a single trial which adopted a less intensive modified form of CIMT [8]. The modified CIMT (mCIMT) involved the application of a restraint on the unaffected upper limb and less than three hours per day of therapy provided to the affected limb [44]. While a positive trend was found favoring CIMT [35] and Forced Use [45], no significant treatment effect was demonstrated for these interventions when compared with traditional services.
Studies published to date have provided limited evidence of factors that may impact on the effectiveness of CIMT. Response to CIMT does not appear to be age-dependent and children with more impaired bimanual hand function demonstrate greater improvement than less impaired children [8]. Although not statistically significant, trends have also been reported as favoring children with cortical/subcortical lesions compared to those with periventricular white matter lesions [8]. More recently, Kuhnke et al., [46] reported that adolescents and young adults with ipsilateral corticospinal projections responded differently to CIMT intervention in comparison to those with contralateral projections. Although these findings must be interpreted cautiously due to methodological limitations [47], the study by Kuhnke et al., [46] adds information about children's potential responses to CIMT.
There is currently no published trial directly comparing a BOT protocol with a CIMT protocol. However, in 2008 Gordon and colleagues [48] published data from a quasi-randomised trial of 16 children with hemiplegic cerebral palsy that compared a one-on-one uni-manual approach to treatment (CIMT) versus a one-on-one bimanual approach to treatment (HABIT) [48]. This small trial reported similar changes for both groups on outcomes including the Jebsen-Taylor Test of Hand Function, Assisting Hand Assessment and accelerometry. The finding, measured using the Assisting Hand Assessment, indicated immediate post-treatment improvements in upper limb bimanual performance using either a uni-manual or bimanual approach to training. However, retention of gains was not examined following either intervention.
Methods/Design
The aim of this current paper is to describe the methodology of a randomised controlled trial comparing the effects of modified constraint-induced movement therapy (a uni-manual therapy) versus conventional bimanual occupational therapy on improving bimanual upper limb performance of children with hemiplegic cerebral palsy following upper limb injection of BoNT-A. Bimanual upper limb performance, assessed using the Assisting Hand Assessment, was selected as the primary measure as the central aim of all upper limb motor-based interventions in children with hemiplegia is to improve a child's actual use of their affected upper limb in a range of daily tasks, including those requiring bimanual performance; rather than what they can do in a clinical setting or how normal their movement appears. This RCT builds on the existing evidence supporting the use of BoNT-A in the upper limb of children with cerebral palsy. It aims to identify the most effective intervention following injection of BoNT-A. A further aim is to explore the individual characteristics of children that impact on response to treatment. Finally, it will also provide additional evidence regarding dose response to treatment, specificity of training effects, and the retention of treatment effects.
Primary objective
To compare the effects of modified CIMT versus bimanual occupational therapy (BOT) in improving the bimanual performance of children with hemiplegic cerebral palsy following upper limb BoNT-A injection.
Hypothesis 1
Modified CIMT is more effective than conventional BOT in improving the bimanual performance of children with hemiplegic cerebral palsy following upper limb BoNT-A injection.
Hypothesis 2
Relative to baseline performance on the Assisting Hand Assessment, modified CIMT results in greater retention of gains in bimanual performance compared with BOT.
Secondary objectives
A. To compare the effect of modified CIMT versus bimanual occupational therapy (BOT) on secondary outcomes relating to: the performance of activities of daily living; the quality of upper limb movement; occupational performance; individual goal achievement; and frequency of use of the affected upper limb of children with hemiplegic cerebral palsy following upper limb BoNT-A injection.
Hypothesis 3
Modified CIMT is more effective than conventional BOT in improving performance of activities of daily living, quality of upper limb movement, occupational performance, individual goal achievement and frequency of use of the affected upper limb in children with hemiplegic cerebral palsy following upper limb BoNT-A injection.
B. To evaluate the clinical significance of change identified on the primary outcome, the Assisting Hand Assessment.
Hypothesis 4
That changes on the Assisting Hand Assessment associated with intervention are of a clinically significant magnitude. Clinical significance was defined as a change score of more than 5 raw score points (2.6 logits) on the AHA (Lena Krumlinde-Sundholm, personal communication).
C. To evaluate the impact of age and actual treatment dosage on the primary outcome, the Assisting Hand Assessment.
Hypothesis 5
Younger children (irrespective of treatment type) will show greater improvement in bimanual performance following uni-manual or bimanual therapy and upper limb BoNT-A injection.
Hypothesis 6
Children who undertake treatment of greatest intensity (irrespective of treatment type) will show greater improvement in bimanual performance.
Study design
Randomised, controlled, evaluator-blinded, prospective parallel-group trial based on Consolidated Standards of Reporting Trials (CONSORT) Statement for Randomised Trials of Non-Pharmacologic Treatment [49].
Participants
Children were eligible to participate if they met the following criteria:
Inclusion Criteria
• Diagnosis of congenital spastic hemiplegic cerebral palsy as diagnosed and reported in the medical history by a medical specialist (i.e. neurologist, paediatrician).
• Aged 18 months to 6 years at time of recruitment.
• Active movement of the shoulder, elbow, wrist, digits and thumb of the affected upper limb, such that the:
• child is able to reach forward to an elevated position in front with mid range shoulder flexion.
• child is able to grasp a 2.5 cm cube from a table top and release it in a large container (20 cm × 14 cm).
• Able to attend to tasks and follow simple one stage commands.
• child is able to actively perform reach and grasp/release activities with verbal prompting.
• Moderate levels of muscle tone (i.e. 1-2, modified Ashworth scale) and spasticity (i.e. 1-2, Tardieu scale) and no fixed contracture in target group of muscles to be injected with BoNT-A.
• Parents able to commit to an intensive therapy program and agree to cease all other upper limb therapeutic interventions for the 6-month period of the trial.
• Assessed as appropriate for upper limb BoNT-A at neuromuscular clinic by rehabilitation specialist (BR).
Exclusion Criteria
Children who otherwise met the inclusion criteria were excluded if they had:
• Congenital quadriplegic or diplegic cerebral palsy.
• Previous BoNT-A injections in the upper limb in the past 12 months.
• Prior upper limb surgery (i.e. tendon transfer/tendon lengthening).
• Existing treatments that are not compatible with those those included in the study treatment package.
There were no criteria relating to exclusion of children with mother tongue other than English, presence of co-morbidity or socio-economic status.
Recruitment
Potential participants were identified from the Physical Rehabilitation Clinic of a major paediatric metropolitan hospital. In addition, postal and email advertising were sent to local medical practitioners and paediatric therapy networks in metropolitan Melbourne. Potential participants were screened by the chief investigator (BJH) to determine eligibility. Children eligible for inclusion were then assessed by an experienced rehabilitation specialist for suitability for upper limb BoNT-A injection (HBR). Suitable children were then invited to participate in the RCT and informed consent obtained for the participation of the child and the child's parent prior to enrolment in the RCT.
Procedure
The RCT was approved by the Ethics Committee of both Southern Health and La Trobe University. The trial is registered with the Australian New Zealand Clinical Trials Registry (ACTRN12605000002684) [50]. Data was collected from August 2003 until May 2009.
Reliability training for assessment and scoring of outcome measures
Prior to commencement of the RCT, two highly experienced paediatric therapists (one occupational therapist, one physiotherapist) were trained in the administration of all outcome measures. A manual was developed for both therapists to ensure consistency in measurement over the recruitment period. The same occupational therapist collected data for the Assisting Hand Assessment, Quality of Upper Extremity Skills Test, Pediatric Evaluation of Disability Inventory, Pediatric Motor Activity Log, Canadian Occupational Performance Measure and Goal Attainment Scaling for the entire recruitment period. Two physiotherapists collected data for the modified Ashworth and modified Tardieu Scales. Scoring of the Assisting Hand Assessment was undertaken by the assessment developers. The Quality of Upper Extremity Skills Test was scored by a highly experienced paediatric occupational therapist who had significant experience in the assessment and treatment of children following upper limb BoNT-A injections.
Randomisation
Sequence generation
Children were block randomised into pairs matched by age (± 6 months) using a computer generated set of random numbers.
Allocation Concealment
A set of random numbers was used to create an allocation sequence which was contained in individual opaque envelopes for use by the chief investigator (BJH). As children were recruited, the next envelope in the sequence was opened and the child assigned to the stated group.
Implementation
All randomisation, sequence generation, and preparation of group allocation materials were performed by a third party (the Monash Institute of Health Services Research) who had no direct contact with the clinical aspects of the trial. The master list of random numbers was located in locked cabinets at the Institute and only accessible at completion of the RCT for analysis.
Blinding
Due to the overt nature of the interventions, children, their parents and the treating therapist were not blinded to group assignment, however, children and parents were blind to the RCT hypotheses. Outcome measures were administered by a therapist blind to group assignment and scored by different assessors who were blind to group allocation. The Assisting Hand Assessment and Quality of Upper Extremity Skills Test was videotaped, randomised and scored by assessors blind to group allocation and order of assessment.
Sample Size
Sample size estimates were based on projected treatment effect on the primary outcome measure, the Assisting Hand Assessment. In 2005, the authors suggested that a change of more than 5 raw score points may represent a clinically meaningful difference on the Assisting Hand Assessment (Lena Krumlinde-Sundholm, personal communication). This was the equivalent to a change in 2.6 logits. Calculation for fractions of logits undertaken by a statistician indicated that in order to detect a change of 2.6 logits a minimum sample size of 17 per group was required. As the RCT design involves a two-group design, a total sample size of 34 was required to provide 80% power to detect clinically meaningful change between groups. Therefore, the study aimed to recruit 40 children to allow for a 10-12% drop-out rate.
Outcome measures
Outcomes measures were completed on four occasions: baseline (1 to 2 weeks prior to injection), and at 1, 3 and 6 months after injection. Outcomes included measures from across the International Classification of Functioning, Disability and Health (ICF) [18] spectrum and involved a combination of investigator observed and parent report measures (See Table 1). This enabled analysis of the impact of intervention across the body function and activity domains.
Table 1. Outcomes classified according to International Classification of Functioning, Disability and Health (ICF)
The effects of upper limb BoNT-A injection in both groups were monitored using measures of muscle spasticity and muscle tone including the Modified Ashworth Scale (MAS) and Modified Tardieu Scale (MTS).
Primary Outcome
The primary outcome measure was the Assisting Hand Assessment (AHA) [51]. The administration of the AHA (Small Kids English version 4.4) was videotaped. The AHA is a standardized, criterion-referenced test for use with children aged 18 months to 12 years, who have a unilateral upper limb impairment [51]. It aims to measure how effectively a child uses their affected hand in bimanual play activities using 22 items. Unlike most other upper limb assessments for children with cerebral palsy, the AHA attempts to capture a child's typical performance when performing tasks rather than their best effort or "capacity" [51]. Change on the AHA is therefore more likely to reflect change in the child's upper limb use across multiple environments.
The AHA is conducted by video observation of the child involved in a 10-15 minute play session using the AHA test kit with specific toys. The twenty-two items defining different actions are then scored on a 4-point scale rating the quality of the performance. Four being effective, 3 somewhat effective, 2 ineffective and 1 indicating the child does not perform the action. The sum of raw scores (sum score) varies between 22 (low ability) to 88 points (high ability). Raw scores are converted to scaled scores ranging from 0 to 100. Using a computer generated logarithmic transformation, ordinal data are then converted to equal intervals in the unit logits [51]. The range of the AHA scale is -10.18 to +8.70 logits. Logit scores will be used in the analysis of data for this RCT.
The psychometric properties of the AHA have been described in several studies [51-54]. Using Rasch measurement analysis, validity and aspects of reliability were evaluated, with excellent results. There is strong evidence that AHA items measure a uni-dimensional construct with 95% of items fitting Rasch assumptions. Person response validity demonstrated that 97% of person's responses fitted the model. A person separation index of 6.16 demonstrated a very good ability to distinguish children of different ability levels [51,52]. A standard error mean of 0.28 (range 0.26-0.32) revealed the precision and reliability of the item measures [51]. More recently, reliability of the Small Kids AHA was found to be excellent with Intraclass Coefficients (ICC's) of 0.97 (20 raters) to 0.98 (2 raters) for interrater and for the intrarater 0.99 [53]. The same version also demonstrated excellent test-retest reliability (ICC 0.99). The Smallest Detectable Difference (SDD) over time indicated that a change in AHA scores from one test session to the next must be 3.89 sum scores (0.97 logits) or more to be considered a true change with 95% probability [54]. The AHA was initially designed for evaluation within a trial of CIMT [8] where it demonstrated responsiveness to change. Since then, studies evaluating HABIT [31,48] have also shown some change in the treatment group compared to the control group, providing evidence that the AHA was also responsive to change following bimanual intervention.
Monitoring responses to Botulinum toxin-A injection
The Modified Ashworth Scale (MAS) is a 6-point, criterion referenced ordinal scale deigned to measure the resistance that is encountered when a limb is moved passively [55]. Scores range from 0 (no increase in muscle tone) to 4 (rigid). Movements assessed included: shoulder abduction and flexion; elbow extension and flexion; forearm supination and pronation; wrist extension and flexion; ulnar and radial deviation and thumb abduction.
The MAS has often been described in the literature as a measure of spasticity [55,56], however since muscle resistance in children with cerebral palsy is due to a combination of factors, only one of which is spasticity, the MAS is considered to have poor validity for spasticity evaluation [57]. Further, as there is no reference to the velocity of limb movement during examination, a central criterion in defining spasticity [3], the MAS is considered a measure of muscle tone. Following a review of papers across a broad range of diagnostic groups, Morris [58] estimated intra-rater reliability to be between 0.55 to 0.83 and 0.45 to 0.84 for inter-rater reliability. These estimations have been supported in recent studies in children with cerebral palsy where inter-rater reliability has been described as low [59,60]. To improve the reliability of using the MAS in this RCT, two physiotherapists, independent to the intervention component of the trial and experienced in administration and scoring of the MAS performed all assessments at a slow velocity. The MAS has demonstrated change in children following upper limb injection of BoNT-A combined with occupational therapy [24,25,61].
The modified Tardieu scale (MTS) was adapted from the original scale developed by Tardieu and colleagues [62]. The MTS grades the quality of the reaction of the muscle to passive stretch and measures the dynamic component of muscle spasticity [63]. This measure of spasticity is obtained when a joint is moved as fast as possible through its range of movement (V3 velocity) and the angle of "catch" elicited is measured using a goniometer. This is called R1. The difference between the angle of "catch" (R1) and the full passive range of movement (R2) reflects the potential range available in the joint if spasticity is eliminated [9,63]. The quality of the muscle reaction (resistance) is also rated when obtaining the R2 measurement, from 0 (no resistance through the course of the passive movement) to 5 (joint immovable).
In this RCT, administration of the MTS was jointly performed by an occupational therapist and physiotherapist both blinded to group assignment. Movements measured included: shoulder abduction and flexion; elbow extension and flexion; forearm supination and pronation; wrist extension and flexion; ulnar and radial deviation and thumb abduction. The velocity used to determine the angle of catch was V3 (as fast as possible), as recommended by Morris [58]. The MTS scale has demonstrated sensitivity to change when measuring spasticity following BoNT-A injection in the upper limb [24] and lower limb [63] in children with cerebral palsy. Despite this reported sensitivity, the MTS has demonstrated poor reliability as a measure of elbow flexor spasticity in children with cerebral palsy with large inter-sessional variation and difficulty in applying standardised velocities [64]. The MTS however, is still considered the only clinically valid measure of spasticity currently available [57,65]. To improve reliability of the MTS in this RCT, the same blinded raters (one occupational therapist, two physiotherapists) were used for each assessment occasion for each child.
Secondary Outcomes
The secondary outcomes included measures of: quality of upper limb movement (Quality of Upper Extremity Skills Test (QUEST)); caregiver questionnaires relating to functional status of the child (Pediatric Evaluation of Disability Inventory (PEDI)) and perceived use of the affected upper limb (Pediatric Motor Activity Log (PMAL)); individual occupational performance (Canadian Occupational Performance Measure (COPM)); individual goal attainment (Goal Attainment Scaling (GAS)); muscle tone (the modified Ashworth Scale (MAS)); and spasticity (the modified Tardieu Scale (MTS)).
The Quality of Upper Extremity Skills Test (QUEST) is a descriptive, impairment-based measure designed to evaluate movement patterns and hand function in children with cerebral palsy [66]. The QUEST involves evaluation of 36 items of upper extremity function in four domains: dissociated movement, grasp, protective extension, and weight-bearing. For this RCT, both the affected and unaffected upper limbs were tested. Each item is scored as either a pass, fail or not-tested. A standardized score is obtained for each domain using the formula detailed in the manual [66] ranging from 0 (low ability) to 100 (high ability). A total score can be obtained by summing scores for each domain tested and dividing by the total number of domains tested. In this RCT, despite data for all domains being collected, only data from domains targeted by the interventions and therefore likely to change following mCIMT or BOT were analysed. These include grasp and dissociated movement. This is consistent with a more recent study evaluating the effects of BoNT-A and occupational therapy [23].
The QUEST is a reliable and valid measure for evaluating the quality of movement in children with cerebral palsy. Inter-observer ICC's range from 0.90 to 0.96 with test-retest correlations of 0.95 [67]. Currently, there is no evidence of the magnitude of change on individual QUEST domains required to determine clinical significance. The QUEST was originally designed to evaluate Neuro-developmental Therapy (NDT) [68]. It has since been widely adopted for use in trials evaluating BoNT-A and occupational therapy [23,24,61,69] and CIMT [35].
The Pediatric Evaluation of Disability Inventory (PEDI) [70] is a standardized assessment of how a child functions with an impairment in the context of their daily life. It has been standardized for children without impairment aged 6 months to 7.5 years and has established reliability and validity to detect the presence, extent and area of a functional delay in children with physical impairment or combined physical and cognitive impairment [71]. The PEDI is designed to measure a child's ability across 3 measurement scales: functional skills, caregiver assistance, and modifications used. Each scale is divided into 3 domains of self-care, mobility and social function, each of which can be administered separately.
Due to the nature of interventions provided in this trial being targeted at upper limb function rather than mobility and social function, only the self-care domain of the PEDI was administered. This is consistent with the use of the PEDI in many other trials evaluating BoNT-A and occupational therapy [23,25,26,72,73]. The 73 items in the functional skills scale are rated on a 2-point scale with 0 indicating inability to perform a tasks and 1 indicating a child is capable to perform the task. Eight items in the caregiver assistance scale are rated on a 6-point scale indicating the amount of assistance required to complete a task (0 = total assistance, 1 = maximal assistance, 2 = moderate assistance, 3 = minimal assistance, 4 = supervision and 5 = independent). Total raw scores are calculated by summing items from both the functional skills and caregiver assistance scales. Higher scores for functional skills level and caregiver assistance indicate better performance and increased independence. Normative standard scores and scaled scores are generated from the raw scores. Scaled scores range from 0 to 100, with increasing numbers representing increasing degrees of functional performance. Scaled scores were used in the data analysis for this trial.
PEDI scale construction was developed using Rasch measurement model and analytic techniques to evaluate construct validity and develop scaled scores. The PEDI has established validity [71,74-76]. High intra-rater reliability has been reported for self-care functional skills (ICC's 0.97 - 0.99) and self-care caregiver assistance (ICC's 0.94 - 0.99) [77,78]. Inter-rater reliability has been reported for self-care functional skills when administered as a parent interview [74,77]. In 2003, Iyer and colleagues reported that change scores of about 11% on the total scale appeared to represent clinically meaningful change [79]. Although the PEDI has been reported to show responsiveness to change over 6 months [80] more recently reported trials evaluating upper limb injection of BoNT-A in children with cerebral palsy, with or without occupational therapy, did not demonstrate a significant difference between groups [23,24,26,69,72]. As recommended by Berg et al. [78] to improve reliability, a single blinded assessor administered the PEDI in this RCT with the same parent across all time periods.
The Canadian Occupational Performance Measure (COPM) [81] is a client-centered measure designed to detect change in a persons' perception of their occupational performance in self-care abilities, productivity (i.e. for children school, pre-school activities) and leisure activities. It has adequate validity, adequate test-retest reliability (ICC of 0.63 for performance and 0.84 for satisfaction) and responsiveness to change [81-85]. The COPM has been used widely in intervention studies [85-88] and previously demonstrated responsiveness to change in studies evaluating BoNT-A and occupational therapy in children with cerebral palsy [23,24,61,72].
Due to the age of the children in this RCT, parental responses to the COPM were obtained rather than the child's. This adaptation has been supported in findings by Cusick et al. [89] who demonstrated acceptable internal consistency reliability for performance (mean alpha = 0.73) and satisfaction (mean alpha = 0.82), content and construct validity and responsiveness using this approach. The five most important occupational performance problem areas were selected using a 10-point scale, where 1 equals "not important at all" and 10 equals "extremely important". Performance in these areas was then rated by a parent on two scales: perception of their child's current performance and satisfaction with their child's performance. Ratings were again on a 10-point scale where scores closer to 10 indicated perceived better performances and increased satisfaction. At baseline, a total performance score was generated by summing the performance scores and dividing by the number of identified problems. Similarly, a total satisfaction score was generated by summing the satisfaction scores and dividing by the number of problems. These scores range from 1 to 10 [81]. Upon re-assessment, performance and satisfaction for each identified problem were again evaluated, scored from 1 to 10. Change in performance and change in satisfaction were calculated by subtracting Time 1 values from Time 2, 3 or 4 values. There is evidence that a change in summary scores (i.e. between initial and subsequent scores) of two or more is clinically significant [85].
The Goal Attainment Scale (GAS) is an individualized, criterion-referenced measure of treatment-induced change [90]. The GAS aims to measure an individual's success in achieving functional goals that have been determined prior to a treatment intervention. For this RCT, the three most important areas of occupational performance identified by parents using the COPM were used as the three nominated goals for scaling the GAS. During the baseline assessment session each goal was rated on a 5-point scale from -2 (current level of performance), -1 (less than expected outcome), 0 (expected outcome), +1 (more than expected outcome) to +2 (much greater than expected outcome). In the assessment periods during and after treatment (i.e. 1, 3 & 6 months post BoNT-A) parents were again asked by the blinded assessor to rate their child's performance in the three identified goals using the 5-point scale. Goals were not weighted and were therefore assessed as being of equal importance.
Using mathematical formulae, Kiresuk et al. [90] provide a method for summing the goals and converting them to a T-score. A T-score of 50 indicates that the goals were, on average, achieved [90]. Despite violating mathematical principles, most intervention trials using the GAS have adopted this methodology for analysis of GAS data. The T-score calculation implies data obtained from GAS are interval level and uni-dimensional in nature. This however, may not be the case and using these calculations when the assumptions are violated has been found to compromise the interpretation of change scores and confound the interpretation of parametric statistical tests [91]. In this RCT, the proportion of achieved goals was analysed. Achieved goals, that is those that change from -2 (baseline level of performance) to 0 (expected level of performance) will represent a clinically significant change.
The GAS has been used in five out of ten RCT's evaluating the use of BoNT-A and occupational therapy in children with cerebral palsy making it the most commonly used outcome measure across these trials. Despite its popularity and reported sensitivity to detect change, the validity and reliability of GAS is largely unknown [92]. Validity has been questioned, due to dependence on the skill of the therapists who scale the goals, their objectivity, and ability to select realistic goals and anticipate outcomes following a specific intervention [93]. With regard to sensitivity to change, Steenbeek et al. [92] reports that the responsiveness of GAS "depends on whether therapists and parents select goals and levels of attainment for each goal that represent clinically important changes in future performance" [[92], p. 553]. In this RCT, a single blinded assessor developed goals for each child and each goal was re-rated at each time point with the same parent.
The Pediatric Motor Activity Log [94] Version 1, is a parental rating on the frequency of use and quality of movement of the affected upper limb in 22 tasks.
Despite its use in studies evaluating CIMT [35,94], and a decision in 2003 to include the original version of the PMAL in this RCT, the measure has since been found to have inadequate construct validity and reliability [95]. Recent Rasch measurement modeling undertaken by Wallen and colleagues [95] found that the original scales of the PMAL had disordered rating structure. A revised version of the scale has been recommended which demonstrates strong test-retest reliability and adequate sensitivity to change. The new, collapsed scale however, may not adequately detect change in children at the extremes of ability and requires further exploration before it can be used as an outcome measure [95]. Unfortunately discussions with Wallen [95] indicated that transformation of the data from the original PMAL version obtained in this RCT into the revised PMAL would not be valid. This is because significant rewording of items and scoring criteria between versions has been required to validate the new tool. Based on Wallen and colleagues [95] findings of inadequate psychometrics of the version used in this RCT, PMAL outcomes will not be analysed or reported.
Intervention
Botulinum toxin-A
Each child in the study received injections of BoNT-A (Allergan Australia P/L, Gordon, NSW, Australia) by a single, highly experienced rehabilitation specialist (HBR). Muscles injected were determined by the rehabilitation specialist in consultation with the chief investigator (BJH) at the Physical Rehabilitation clinic based on whether they appear to contribute to abnormal limb position and impair functional use of the limb. Injected muscles included biceps brachii, brachialis, brachioradialis, pronator teres, pronator quadratus, flexor carpi ulnaris, flexor carpi radialis, flexor digitorum profundus, flexor digitorum superficialis, flexor pollicis longus, adductor pollicis, and opponens pollicis. If indicated, children also received injections of BoNT-A into lower limb muscles during the same injection session. This was considered ethically appropriate for the child's overall management and was considered unlikely to interfere with upper limb outcomes. Injections were performed in theatre under a light general anaesthetic and all children were discharged on the same day. Muscle localisation was undertaken by the use of Teflon coated Botox injection needles (37 mm, 27 gauge) allowing electrical stimulation. Doses of BoNT-A were administered at a maximum dose of 15 U/kg (or 400 U). Dilution was 100 U/1 ml. During the first month following injection, the chief investigator (BJH) reviewed all children on two occasions to monitor the effect of the BoNT-A, identify potential adverse events and provide splinting intervention as required.
Splinting
Children with increased resistance to passive stretch or who exhibit early signs of muscle shortening were provided with a thermoplastic stretching splint designed for use overnight for a minimum of 6 hours per night. This protocol has been based on scientific support from a small number of animal studies reporting that muscles increase in length when immobilised in a lengthened position [96,97] and a few studies in adult lower limb literature that suggest a prolonged low load stretch is more effective than brief stretches in preventing contracture [98,99]. For children with cerebral palsy, evidence that static splinting maintains the mechanical-elastic properties of muscle remains weak [100]. Because passively positioning a joint during active movement and covering the skin is considered to limit the potential to strengthen antagonist muscles and impede sensory feedback from the hand, no day splints (i.e. neoprene, Second Skin(r), thermoplastic wrist cock-up) were used during enrolment in the RCT.
Movement-based therapy: General Considerations
One-month following injection of BoNT-A, children randomised to the experimental group received modified constraint-induced movement therapy (mCIMT). Children randomised to the control group received conventional bimanual occupational therapy (BOT). The individual-based treatment sessions of 45 to 60 minutes were conducted by the same occupational therapist (BJH) twice weekly for 8 weeks in an outpatient paediatric treatment room. In addition, children in the mCIMT experimental group were required to complete 3 hours of home program (with the mitt on), 7 days a week for the 8 week treatment period. Children in the BOT control group were also encouraged to undertake a home program but no time requirements were specified. A checklist was completed after each treatment session, identifying the activities used and general observations. Important general considerations for both groups related to establishing and maintaining rapport, equipment, preparing and implementing the sessions are described below.
Building rapport and establishing the therapist-child/therapist-parent relationship was a primary focus for initial treatment sessions. Along with identifying individual movement, hand skill and motor planning abilities of each child, this time allowed for selection of toys to match the demands of the task with the child's developmental level and specific hand skill goals. It was also necessary to establish expectations for future sessions, create an understanding of acceptable behaviour and to establish patterns of on-task behaviour for the child.
Most treatment was undertaken with the child sitting at a height adjustable table (See Figure 1). The therapist sat on a wheeled chair usually positioned on the child's affected side or behind the child. The child's chair, with footrest, armrest and pommel was adjusted so that the table was at waist height. This prevented the child from leaving the table to wander around the room or slip under the table. The therapist and child's position also: allowed the treatment to focus on hand skill development; provided adequate freedom of movement of upper limbs for reach and grasp; allowed visual monitoring of the hands during tasks; assisted in maintaining attention on the task; provided control of the child's immediate environment and; appropriately positioned the therapist to provide modelling of tasks and verbal and physical assistance with an emphasis on the required hand placement and movements, the task sequence and general demands of the task. Parents were seated opposite the child or on the child's unaffected side and beyond the child's reach.
Figure 1. Table and chair used in therapy sessions
Prior to each session a carefully selected range of fun and motivating toys were placed in the treatment room on an uncovered bench behind or to the side of the child's chair. To improve motivation the child was actively encouraged to choose the toys with which they wished to play. Periods without the child actively engaged in play were avoided to maintain motivation, attention and concentration. If the child became distracted, techniques such as using noise and sensory input (e.g. tapping the table with a toy, tapping the child's affected hand, raising volume of voice) were used to redirect their attention to the task. Because task repetition and practice are key components of training, any attempt by the child to prematurely stop task performance (i.e. by focussing on something else in the room) was ignored by the therapist and parent and the child's attention redirected to the task. The child was required to indicate when an activity had been finished through verbalisation or physical signs. If this occurred after only a few repetitions, further repetitions were negotiated before the activity is stopped. This was seen as important for compliance and motivation as the child felt included in the decision making process whilst further task repetition was achieved. No new activities were chosen until all parts of the activity had been packed up. This was crucial as repetitions of movement/hand skills achieved through packing up were often greater than in the activity itself.
With novel and/or challenging tasks, an inability to independently and successfully complete the demands of the task often leads to a child becoming disinterested or frustrated after only a few trials or repetitions. To facilitate learning, improve skills and task performance, develop resilience, and for the therapist to assist the child to establish patterns of on-task behaviour it was important not to allow the child to "give up" or avoid tasks. A variety of task-avoidant behaviours can be successfully used by children from a very early age. Initial and ongoing parent/therapist reinforcement can inadvertently condition task-avoidant behaviour, impeding the participation and engagement of the child and therefore the effectiveness of treatment. It was crucial for the therapist to establish a collaborative partnership with the parents to ensure consistent responses to these behaviours. On occasions where task avoidant behaviours became evident, the therapist provided advice and strategies to the parent(s) on appropriate responses to avoid reinforcement. Examples of typical task-avoidant behaviours displayed by children included:
• Throwing objects - this often occurs when a child does not understand the requirement of the task (poor motor planning) or the task is too challenging for the child's abilities. In this RCT the therapist always modelled novel task performance to demonstrate task demands. During early attempts at a task, significant physical assistance and/or verbal cuing were provided to ensure successful performance. The amount of assistance was then gradually decreased with future attempts. Any object thrown by the child was not collected by the therapist/parent and the behaviour was ignored. The object remained on the floor to ensure the child did not receive a response that reinforced the behaviour (i.e. stopping of activity, collection by parent). If the behaviour persisted the therapist pre-empted the throw preventing it from happening. The child's attention was redirected to the task and, if required, additional modelling of task performance or increased physical assistance was provided.
• Mouthing objects - young children can learn that mouthing objects leads to a quick and alarmed reaction from the parent/therapist that leads to diversion of attention away from the task. During treatment if a child demonstrated this behaviour, objects small enough for ingestion were avoided in all sessions until the behaviour ceased. To avoid encouraging this behaviour, larger objects were used (to ensure ingestion was not possible) and the behaviour was ignored when displayed. Any direct eye contact with the child from the parent and therapist was also avoided and directed to the task, so the child did not receive a response (i.e. facial expression from parent/therapist, verbal feedback).
• Crying can be an extremely effective strategy for task-avoidance by young children. The reinforcement of this behaviour occurs when the therapist immediately stops the task or parents intervene to provide comfort and reassurance through physical contact or removal of the child from the activity. The child can associate crying with successful task avoidance with resultant disruption to the session. During treatment, it was important for the therapist to quickly evaluate the situation. If the child was obviously distressed, parental comfort and reassurance was used to settle the child. Removal from the seat was avoided if at all possible. If the child was not obviously distressed and crying was deemed by the therapist/parent as task-avoidant behaviour the task was continued. A graded level of response was initiated: 1) child was not removed from position; 2) therapist negotiated with the child for one or two more repetitions prior to task completion; 3) therapist verbalised when task was completed 4) child was required to pack up the activity before choosing next activity; 5) if the child remained uncooperative the therapist and parent engaged in conversation or played the task/game whilst ignoring the child's behaviour; 6) if the child remained uncooperative the therapist asked the child if they would like the parent to leave the room; 7) as a last resort, if the child remained uncooperative the parent left the room until the child settled. As soon as the child settled the parent was invited back into the room. Great care was taken with this final response as it had the potential to cause significant anxiety and stress to the child. Over time, with consistent responses to crying from the parent and therapist, it was expected that the child would stop using crying as a technique for task avoidance.
Constraint-induced movement therapy
The modified constraint-induced movement therapy (mCIMT) protocol incorporated the two fundamental components of CIMT as described by Taub et al. [36]: the use of a restraint device (glove) and; the provision of massed practice to the affected upper limb (3 hours of home program). A comfortable neoprene (wet suit material) glove was worn on the hand of the non-affected upper limb to facilitate intensive practice of the affected upper limb. The neoprene glove, with a palmar thermoplastic insert over the fingers and thumb to prevent grasp, allowed the child to use the hand as an effective assist in bilateral activities, but did not allow active grasp of objects (see Figure 2). Importantly, the glove allowed the child to use the unaffected limb for breaking a fall, if needed. The intervention period was 2 months and involved wearing the glove 7 days per week. Children were expected to wear the glove for 3 hours per day, including therapy time and the home program, which could be split into different sessions of no less than 30 minutes duration. Families were expected to undertake an intensive home program of 3 hours per day. This could occur in the child's usual environment including home, crèche, preschool or school. Caregivers completed a log-book detailing the total period the restraint device was worn per day and any issues arising from use of the glove. The intention of the home program was to facilitate an intensive period of practice with the affected limb and to educate, empower and include families and caregivers in the treatment process. Families were provided with written and specific goals by the treating therapist after each session. These were based around development of specific hand skills such as grasp, hold, release, reach, in-hand manipulation. Families were encouraged to focus on these goals during the home program. Families were discouraged from placing the glove on the child without supervision to avoid frustration.
Figure 2. Neoprene glove used in modified constraint-induced movement therapy.
The implementation of mCIMT was based on the principles of motor-learning theory [30,101]. Eliasson [30] comprehensively describes the core principles of motor-learning including "learning to perform a task by developing strategies, learning a task by practicing skills and learning to use the hemiplegic hand through task practicing" (p. 56). Consideration of these principles highlights the important differences between adopting a motor learning approach within a CIMT protocol compared with a BOT protocol. Although both approaches use similar principles for development of motor skills (i.e. motivation and learning through repetitive practice), the uni-manual nature of CIMT only allows for practice of general aspects of hand function rather than the activity itself [30]. For the mCIMT group in this RCT, the uni-manual treatment focussed on repetitive practice of movements and skills with the affected limb (e.g. grasp, release, holding and transporting of balls into a ball tower, holding paint stamps and using adapted brushes, stacking blocks and other objects, holding a magnetic wands to catch a ball, magnetic fishing games, hammering games, Jenga). Using constraint of the unaffected upper limb and utilising games and play, uni-manual tasks were carefully selected to provide sufficient challenge and successful outcomes when using the affected upper limb. In doing so, learning was facilitated by practicing skills and the experience of using the hand through massed practice. It was not possible to target learning of bimanual strategies to achieve task performance.
Activities deemed too difficult by the therapist were avoided to prevent experiences of failure and frustration. This was particularly important in initial sessions where simple cause and effect activities were used (e.g. battery operated switch activated toys). Parents were educated that use of the glove must be perceived by the child as fun and enjoyable. Rewards, such as playing with favourite toys when the glove was used, was highly encouraged. Negative experiences, such as verbal threats and withdrawal of activities, were discouraged. The child was always kept busy and periods without the child actively engaging in play was avoided. If the child became distracted by the glove their attention was immediately redirected to the task by using noise and sensory input (e.g. tapping the table with a toy, tapping the child's affected hand, raising volume of voice). Any discussion regarding the glove was avoided. At the conclusion of each session the child was praised and informed the session had finished and the child was encouraged to independently remove the glove using his or her affected hand.
Bimanual Occupational Therapy
The bimanual occupational therapy (BOT) was underpinned by components of motor learning [102] and cognitive-based motor intervention [103,104]. This eclectic approach to treatment is commonly adopted by occupational therapists in the training of upper limb motor skills in children with cerebral palsy [30]. Details of the components adopted from motor skill acquisition, motor learning and motor control theory, the Assisting Hand Assessment hierarchy and cognitive based approaches are described below.
Motor skill acquisition, motor learning and motor control theory
Practical application of a motor learning framework requires implementation of a motor-teaching model whereby the therapist acts as a teacher and the child, a learner. Factors required to facilitate a child's learning of motor skills include: giving attention to the context; motivation and prior knowledge; instructions; modelling; taxonomy and sequencing of tasks; anticipation skills; mental and physical practice; repetition; facilitation-guidance; and feedback [102]. Similar core components have been outlined for improving motor skills in children using a motor skills acquisition frame of reference [27]. These frameworks [27,102], along with more recent advances in the knowledge of motor planning difficulties experienced by children with cerebral palsy [30,105], formed the core components of the BOT provided in this RCT. Examples of the practical implementation of these principles included:
• Initial and ongoing task analysis to identify if the child's performance was limited by execution of movement or motor planning difficulties (i.e. sequencing of movements) [30,105,106]. Motor planning impairment was observed by presenting a child with a novel task without prior modelling or task demonstration. It is evident when you know the child has the underlying physical capacity to complete the task but they simply cannot organise or plan the sequence of actions or required movements of the hands to successfully perform the task.
• Repetitive whole-task practice of challenging, motivating and purposeful bimanual activities (i.e. toys and games), carefully selected to facilitate learning and development of goal-based skills and independence with task completion [101].
• Use of modelling, physical assistance, verbal cues or environmental adaptation to enable the child to understand the critical features of the task and the environment.
• Facilitation of the child's learning and understanding of the role of their assisting hand (i.e. hemiplegic hand) using active problem solving.
• Grading of physical and/or verbal assistance provided to complete tasks.
• Provision of feedback focusing on the outcome, task and environment rather than specific movement performance.
• Provision of opportunities for the child to repetitively practice tasks in a range of contexts and environments.
Understanding and grading task difficulty in bimanual intervention
The Assisting Hand Assessment (AHA), designed specifically for children with unilateral impairment, was developed using a Rasch measurement model. This model allowed identification and ordering of the AHA test items on a scale from easiest to hardest. For example, simply approaching an item using the affected hand (easier item) to using in-hand manipulation skills to move objects in the affected hand (more difficult item). Based on the Rasch model, easier items are more likely to be easier to perform for all children than more difficult items. More able children are also more likely to perform better on more difficult items than less able children. The unique construct allows children's bimanual upper limb ability levels to be placed along a continuum of low ability to high ability. This knowledge can be used to design intervention and guide graded task performance [107]. For example, attempting to improve the bimanual performance of a child with low ability to place objects directly onto a table (most difficult item) using the assisting hand is inappropriate. Treatment for this child should focus on the development and consolidation of easier items such as holding objects or stabilising by grip. Conversely, continually focussing on easier items for a child with high abilities will not serve to improve their abilities on more difficult items. In this RCT, although specific details of the AHA scores remained unknown to the treating therapist, knowledge of the AHA hierarchy served as an important guide for selecting specific activities during the implementation of BOT. Treatment was tailored to the individual child based on their typical performance in the initial treatment sessions (see Figure 3).
Figure 3. Using the Assisting Hand Assessment hierarchy to grade treatment. DH = Dominant Hand; AH = Assisting Hand
Cognitive approach
Children with hemiplegic cerebral palsy have varied abilities to perform bimanual activities. Often children have the underlying motor abilities (i.e. adequate range of movement, ability to grasp, hold and release) to execute the required movements for task performance, however they are unable to independently identify the specific role for each hand, appropriately position the hands and object or sequence the movements or direction of force required to complete the task. These motor planning difficulties may be just as limiting for the performance of activities of daily living as is movement execution in children with cerebral palsy [105].
Cognitive-based approaches [103,104] have evolved and been adapted for use in approaches such as Cognitive Orientation to daily Occupational Performance (CO-OP). Developed in the 1990's for the treatment of motor disorders in children with DCD [88,108], CO-OP is a task oriented problem-solving approach that utilises cognitive skills to improve occupational performance [109]. The child is guided to develop his/her own strategies based on problems encountered during a task. In this RCT, the bimanual approach to training was grounded in cognitive-based intervention theory. Much of the treatment targeted the motor planning abilities of children with both low and high bimanual abilities. Children engaged in a self-instructional training program that was carefully facilitated by the treating therapist [110]. In the context of this RCT, the treating therapist reinforced to children that the difficulties they often experienced with task performance was because they were not taking advantage of certain strategies or tricks that they could learn [110]. Novel activities were chosen which the child could only solve by carefully looking and listening, and for which a plan or strategy was required before any movement or action took place (e.g. pushing plastic links together). Attempts were made to encourage the child to plan ahead and reduce impulsive tendencies to quickly grab the object using their dominant hand before they thought about the role of affected hand in the task. Before handing the toy to the child, the therapist demonstrated exactly what was involved to complete the task. The therapist modelled the required movements and emphasised important sequences and strategies whilst verbalising these aloud using simple, key words. Deliberately, the therapist occasionally performed components of the task incorrectly and talked through how they could be corrected. The child was then handed the activity and encouraged to slowly perform the task using the sequences modelled by the therapist whilst using the same verbalisations, or verbalisations the child had developed. These verbalisations during task performance have been referred to as verbal mediation. The mediators serve to teach the child how to comprehend a task, direct motor movement through self commands and importantly, to guide, monitor and control their own performance [111]. If the child had difficulty performing a previously practiced task, the therapist prompted the child using recall of verbal mediators (see Figures 4 and 5). Importantly, this technique was demonstrated and reinforced to parents to ensure a similar process is undertaken in the home environment.
Figure 4. Development of grasp, hold and release using verbal mediators
Figure 5. Development of stability of grip using a cognitive-motor approach to treatment.
Identifying and breaking down the specific sequences of a task allowed the therapist to assist the child to discover deficient sequences and to prompt them to consider the error before the end of the whole task. This inhibits failure of the task at an incipient stage thereby reducing the likelihood that the child will become frustrated or non-compliant [111]. Typically, children become more motivated if they have been active and successful participants in the problem solving process. Over time, this approach aims to promote a child's resilience by independently prompting themselves to identify incorrect sequences before becoming angry or frustrated. With proficiency in performance the need for self-rehearsal diminishes [111]. Importantly, the child learns how to learn. In an attempt to facilitate generalisation of the learning, task practice in this RCT was encouraged in various environments (i.e. using home programs) and with different activities that required similar strategies [110].
Statistical analysis
Data were managed and analysed using the Statistical Package for Social Sciences (SPSS version 16.0). Descriptive statistics were calculated to summarise the data set for both groups and to identify potential baseline differences between the groups; p values were used to indicate the strength of the evidence and will be interpreted according to Sterne and Davey Smith [112] Distributions of data from each group and for each occasion were assessed to determine if they met the assumptions for the various inferential analyses.
Testing the effectiveness of therapy (Hypotheses 1 and 2)
Using continuous, interval level data from the AHA, differences between the two groups were assessed using a linear regression approach to analysis of covariance (ANCOVA). This controlled for the covariates of baseline AHA scores and the child's age. These variables were included as covariates as they might provide alternative explanations for any observed changes in scores. The size of treatment effect was estimated by comparing differences in group means and their 95% confidence intervals. Regression analyses and scatter-plots were used to investigate the relationship of post-treatment outcome with initial deficit.
Testing the effect of therapy on secondary outcomes (Hypothesis 3)
Between group differences were assessed using ANOVA for continuous data (following tests of normality). Outcomes that had non-continuous data or did not meet usual assumptions of linear regression were investigated using non-parametric statistics.
Testing the clinical significance of the effect of therapy on primary outcome (Hypothesis 4)
The magnitude of treatment effect was evaluated relative to defined criterion of clinical significance.
Testing the effect of age and intensity of therapy (Hypotheses 5 & 6)
Data related to the number of minutes spent by all children undergoing treatment was extracted from the log book for each child. Associations between the intensity of therapy and outcome and age of children and outcome were determined, while controlling for the covariate of group, using ANCOVA.
Safety evaluation
Each child was monitored throughout the trial period by the chief investigator. Following trial completion, all medical and research records were retrospectively audited. Any adverse events were recorded and classified according to whether they could be attributed to BoNT-A injection, general anaesthesia or movement-based treatment.
Current study status
The study commenced recruitment in May 2003 and achieved target recruitment in September 2008. Participant follow-up was completed in March 2009. Data analysis is currently being undertaken.
Amendments to the study since commencement (2003)
(1) Extension for completion date
Approval was granted by Southern Health and La Trobe University Ethics Committees to extend the completion date of the project to June 2010. Approval was requested due to the trial being behind schedule due to slow recruitment rates.
(2) Inclusion criteria
In March 2005 approval was granted by Southern Health and La Trobe University Ethics Committees to extend the upper age limit of children included in the trial from 4 to 6 years of age. Modification was requested due to the development of the upper age limit for the AHA, allowing the effective/valid measurement of children aged 4 to 6 years old. Children in this age range were also considered to potentially benefit from the treatments provided in the trial and therefore would be a clinical population these treatments would be offered to in the future.
Discussion
This paper describes the methodology of a randomised controlled trial comparing the effects of modified constraint-induced movement therapy (a uni-manual therapy) versus conventional occupational therapy (a bimanual therapy) on improving bimanual upper limb performance of young children with hemiplegic cerebral palsy following upper limb injection of BoNT-A. The results of the paper will be disseminated through peer reviewed journal publications.
Abbreviations
AHA: Assisting Hand Assessment; BoNT-A: Botulinum toxin-A; BOT: Bimanual occupational therapy; CIMT: Constraint-induced movement therapy; COPM: Occupational Performance Measure: GAS: Goal Attainment Scaling; HABIT: Hand arm bimanual intensive training; ICF: International Classification of Functioning, Disability and Health; MAS: Modified Ashworth Scale; mCIMT: Modified constraint-induced movement therapy; MTS: Modified Tardieu Scale; PEDI: Pediatric Evaluation of Disability Inventory; PMAL: Pediatric Motor Activity Log; QUEST: Quality of Upper Extremity Skills Test; RCT: Randomised controlled trial.
Competing interests
Allergan Australia provided partial support by providing the BoNT-A (Botox(r)) used in the study, by payment of research assistants blind to group allocation, a blinded rater for the QUEST, and video editing services. The authors have no pecuniary interest in Allergan.
BJH is an occupational therapist and has received sponsorship from Allergan Australia to attend and teach at conferences and meetings but has no personal financial interest in Botox(r) or any related product.
CI is co-investigator of a randomised controlled trial investigating the effect of repeat injections of BoNT-A and occupational therapy in the upper limbs of children with hemiplegic cerebral palsy that has received support from Allergan Australia. In 2008, CI received a grant from Allergan Australia to present results of this trial at the American Academy of Cerebral Palsy and Developmental Medicine in Atlanta but has no personal financial interest in Botox® or any related product.
HBR has received sponsorship from Allergan Australia to attend and teach at conferences and meetings but has no personal financial interest in Botox(r) or any related product.
LC - No competing interests
Authors' contributions
BJH: PhD candidate, designed the RCT and wrote the paper. CI: PhD supervisor, designed the RCT and monitored progress revising the paper critically for intellectual content. HBR: monitored progress revising the paper critically for intellectual content. LC: PhD supervisor, designed the RCT and monitored progress revising the paper critically for intellectual content. All authors have read and approved the final manuscript.
Acknowledgements
The authors wish to express their gratitude to the children and their families who participated in the trial. They would also like to sincerely thank Trisnawati Tanumihardjo, Melanie Toy-Laing and Cate Clancy for administering the assessments and Lena Krumlinde-Sundholm, Marie Holmefur and Sue Greaves for scoring all the videos. Finally they would like to acknowledge the Allergan Australia and Southern Health and La Trobe University for supporting this research
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Research article
Effects of Greek orthodox christian church fasting on serum lipids and obesity
Katerina O Sarri*, Nikolaos E Tzanakis, Manolis K Linardakis, George D Mamalakis and Anthony G Kafatos
Author Affiliations
Department of Social Medicine, University of Crete, School of Medicine, P.O Box 1393, Iraklion 71110, Crete, Greece
For all author emails, please log on.
BMC Public Health 2003, 3:16 doi:10.1186/1471-2458-3-16
The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1471-2458/3/16
Received:30 October 2002
Accepted:16 May 2003
Published:16 May 2003
© 2003 Sarri et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.
Abstract
Background
No study to date has focused on the impact of Greek Orthodox Christian fasting on serum lipoproteins and obesity yet.
Methods
120 Greek adults were followed longitudinally for one year. Sixty fasted regularly in all fasting periods (fasters) and 60 did not fast at all (controls). The three major fasting periods under study were: Christmas (40 days), Lent (48 days) and Assumption (August, 15 days). A total of 6 measurements were made during one year including pre- and end-fasting blood collection, serum lipoprotein analyses and anthropometric measurements.
Results
Statistically significant end-fasting total and LDL cholesterol differences were found in fasters. Fasters compared to controls presented 12.5% lower end-total cholesterol (p < 0.001), 15.9% lower end-LDL cholesterol (p < 0.001) and 1.5% lower end-BMI (p < 0.001). The end- LDL/HDL ratio was lower in fasters (6.5%, p < 0.05) while the change in end- HDL cholesterol in fasters (4.6% decline) was not significant. Similar results were found when the pre- and end-fasting values of fasters were compared. No change was found in control subjects.
Conclusions
Adherence to Greek Orthodox fasting periods contributes to a reduction in the blood lipid profile including a non-significant reduction in HDL cholesterol and possible impact on obesity.
Keywords:
Greek Orthodox Christian; religion; fasting; effect; serum lipids
Background
Fasting, the voluntary abstention from all restricted foods, is a feature of many religions, and the putative health benefits have attracted both scientific and popular interest. Commonly, religious doctrines proscribe foods from animal sources permanently or for particular periods.
There are several religions, such as Hinduism, Buddhism, Judaism, Islam, Seventh-Day-Adventism that have often been studied regarding their relation to health [1-14]. However, the studies on Orthodox Christianity are very limited.
Orthodox Christian holy books recommend a total of 180–200 days of fasting per year. The faithful are advised to avoid olive oil, meat, fish, milk and dairy products every Wednesday and Friday throughout the year. Additionally, there are three principal fasting periods per year: i) a total of 40 days preceding Christmas (meat, dairy products and eggs are not allowed, while fish and olive oil are allowed except on Wednesdays and Fridays), ii) a period of 48 days preceding Easter (Lent). During Lent fish is allowed only two days whereas meat, dairy products and eggs are not allowed. Olive oil consumption is allowed only at weekends, iii) a total of 15 days in August (the Assumption) when the same dietary rules apply as for Lent with the exception of fish consumption which is allowed only on August 6th. Seafood such as shrimps, squid, cuttlefish, octopus, lobsters, crabs as well as snails are allowed on all fasting days throughout the year. The Greek Orthodox fasting practices can therefore be characterized as requiring a periodic vegetarian diet including fish and seafood.
The variant of vegetarianism followed during fasting periods by Orthodox Christians, with a diet of vegetables, legumes, nuts, fruits, olives, bread, snails and seafood, is a type of the so-called Mediterranean diet [15,16]. To date little is known as to the effects of this 'hidden' element of the traditional Orthodox Christian diet on health and no data exist on the effect of Orthodox Christianity's dietary rules on blood lipid levels and obesity. The objective of this study was therefore to assess the effects of intermittent short-term religious fasting, according to the dietary rules of the Orthodox Christian Church, on blood lipoprotein profile and the prevalence of obesity.
Methods
Subjects
The subjects of this study were selected from an adult population in the region of Heraklion, Crete. One hundred-twenty Orthodox Christians were asked to participate in this study. Sixty individuals (31 males, 29 females), mean age (x ± SD) 41 ± 12 years, fasted regularly according to the dietary rules and the fasting periods of the Christian Orthodox Church. Fasters had been practicing the fasting rituals for a mean of 20 ± 14 years. Another group of sixty subjects (24 males, 36 females), mean age (x ± SD) 38 ± 9 years, were control subjects that did not fast. Among the fasting group, 20 were lay persons (fasted for 13 ± 10 years) and 40 were under religious order (fasted for 23 ± 15 years): 19 nuns living in a convent and 21 priests living with their families in community parishes. The family history of each subject was recorded with regard to diabetes, CHD, smoking, hormonal disturbances and drug intake.
Study design
Three pairs of measurements were made over a 1-year period (2000–2001), coinciding with the beginning and end of each of the three major fasting periods of the Christian Orthodox Church: Christmas, Lent and the Assumption. All measurements were made between 8.00–10.00 am and they included fasting blood collection, anthropometric measurements and the completion of questionnaires.
Questionnaires
All subjects signed informed consent forms and completed questionnaires on fasting, health habits (coffee and alcohol consumption, smoking), certain items of personal data (marital status, educational level), physical activity, dietary habits (24 h recall, 3-day dietary record). Fasters were defined as those who fasted regularly during all three principal fasting periods, while current smokers as those who smoked at least one cigarette per day.
Anthropometric variables
Body weight was measured all six times by a digital scale (Seca, Hamburg, Germany, Model 770) with an accuracy of ± 100 g. Subjects were weighed barefoot in very light clothing. Standing height was measured once without shoes to the nearest 0.5 cm with the use of a stadiometer with the shoulders in relaxed position and arms hanging freely. Body Mass Index (BMI) was calculated by dividing weight (kg) by height squared (m2). Waist and hip circumferences were measured twice, at the first and sixth measurement of the study [17]. Blood pressure (BP) was measured all six times in the right arm with a traditional sphygmomanometer. Three seated BP measurements were taken for each subject spaced two minutes apart.
Biochemical assays
Serum lipoprotein concentrations were always determined after 12 h of fasting. Blood samples were transferred to the University hospital of Crete in tanks containing ice packs that maintained the temperature at 3–4°C. Total cholesterol was determined by Allain's method [18], HDL-C was measured by the heparin-manganese precipitation method [19] and triacylglycerols were determined using Fossati's method [20], while LDL-C was calculated as follows: LDL-C = TC - (HDL -C + TG/5) [21]. During the period October 2000 – September 2001 the coefficient of variation for the biochemical analysis of total cholesterol was 2,85%, for HDL was 5,40% and for triacylglycerols was 3,92%. DNA extraction was performed according to the method of Miller et al [22]. Apo E genotype was determined by PCR amplification and subsequent digestion with the restriction enzyme Hha I (New England Biolabs) as described by Reymer et al [23] in Harokopio University of Athens.
Statistical methods
Differences in gender, tobacco use, educational level and apolipoprotein E distribution were compared using χ2 analysis, while differences in age were compared by ANOVA analysis. Regression analysis was used to compare end-fasting lipid concentrations and BMI with age, sex, smoking, educational level, BMI, WHR, fasting and the pre-fasting values. The influence of fasting on end-fasting values was examined using ANCOVA analysis. Paired samples T-test and Mann-Whitney test were used to compare pre and end-fasting values in fasters.
Pre-fasting values comprise the mean of the three measurements that were made before the beginning of the Christmas, Lent and Assumption fasting periods, while end-fasting values are the mean of the three measurements that were made at the end of each one of the fasting periods.
Results
Demographic data on a hundred and twenty subjects are presented in Table 1. Sixty of the subjects were fasters (26% male, 24% female) with a mean age of 42 ± 12; the other sixty were control subjects (20% male, 30% female) with a mean age of 38 ± 9. There was no statistically significant difference in the age of the two groups (ANOVA). The subjects in the fasters group had been observing the fasting rituals for a mean of 20 ± 14 years. The rate of compliance with the fasting rules was 100%. All subjects in both groups did not suffer from any disease like thyroid, diabetes, cancer, cardiovascular diseases, and did not take any medication.
Table 1. Sociodemographic characteristics of the population.
The levels of serum lipids, blood pressure and body measurements of all three periods for fasters and controls are presented in table 2.
Table 2. Levels of serum lipids, blood pressure and body measurements.
Effect of fasting on end-fasting values
Multiple Linear Stepwise Regression Analysis indicated that fasting is a significant determinant for end-total cholesterol, end-LDL cholesterol, end-LDL/HDL ratio and end-BMI (Table 3), showing that fasters have lower levels of these variables.
Table 3. Effect of various variables including fasting on serum lipids and BMI.a
Females have higher levels of end-HDL cholesterol while men have higher levels of end-TC/HDL and end-LDL/HDL ratios. Waist-to-hip ratio was positively related to end-total cholesterol and end-LDL cholesterol.
Comparisons of end-fasting values between the two groups
Comparisons of mean end-fasting values between fasters and controls are shown in Table 4. Mean end-TC, end-LDL and end-BMI were statistically lower (p < 0.001) in fasters compared to controls. Fasters presented 12.5% lower end-TC, 15.9% lower end-LDL cholesterol and 1.5% lower end-BMI compared to controls. Moreover, fasters had significantly lower LDL/HDL ratios (p < 0.05). All results were adjusted for age, sex, BMI and smoking.
Table 4. ANCOVA analysis. Effect of fasting on end-fasting mean ratios with covariates the respective pre-fasting mean ratios between fasters and control subjects.
Comparisons of pre and end-fasting values in the fasters' group
The fasters who had 3 complete pairs of measurements were included in this analysis (Table 5). Paired samples T-test showed that fasters presented 9.1% decline in end- total cholesterol, 12.4% decline in end- LDL, 8.5% decline in end- HDL and 1.4% decline in end- BMI compared to their respective pre-values. All these differences were significant (p < 0.001). As for the ratios end- TC/HDL and end- LDL/HDL although they declined the changes were not significant. The same analysis was done in controls that presented no significant changes over the year. A further step was to categorize fasters in two subgroups: 1) nun-priests and 2) lay people and to compare their pre- and end- fasting values. Mann-Whitney test showed that the changes seen in fasters' group remained when each subgroup was analyzed separately though they were not significant. It was observed that between the major fasting periods studied (between the end of Christmas and the beginning of Lent; and between the end of Lent and the beginning of the Assumption fasting period) when fasters returned to their usual dietary habits (non-fasting periods) total cholesterol and LDL cholesterol were increased by 6% and 9% respectively.
Table 5. Pared samples T-test. Mean pre-fasting values compared to mean end-fasting values in the group of fasters (n = 43).
Dietary data
Table 6 shows that at end-fasting periods fasters had 10% reduction in energy intake (EI), 17% reduction in total fat (%EI), 23% increase in carbohydrates (%EI) and 43.5% increase in fiber consumption, whereas the respective percentages for the controls are +7%, +1%, +1.7% and +3.3%. All the differences found between the two groups are significant.
Table 6. Ancova analysis. Dietary differences between fasters and controls among pre and end-fasting periods based on the 24 h dietary record.
Distribution of Apo E polymorphism
Subjects in this study were screened for the common apolipoprotein E (apoE) polymorphism, as genetic variation at the apoE locus has been shown to influence serum lipid responsiveness to dietary interventions and account for much of the interindividual variability in dietary response [24,25] Several studies, for example, support the concept that the ε4 allele is associated with an increased cholesterol response to dietary manipulation, and that subjects carrying the ε4 allele are the most responsive to diets restricted in saturated fat and cholesterol [24-27].
Fasters and control subjects were classified in three groups according to their apoE genotype: subjects homozygous for the common ε3 allele (apoE3/3 genotype, (38 fasters and 40 controls); subjects with the apoE2/3 genotype (nine fasters and four controls); carriers of the ε4 allele (apoE3/4 and apoE4/4 genotypes; four fasters and six controls, respectively). Chi-square analysis showed that apoE genotype distribution did not differ between fasters and controls (data not shown).
Discussion
The most important finding of this study is that most serum lipid variables decreased significantly over the fasting periods. Fasters, as compared to controls, had decreased levels of mean end- total cholesterol, LDL-C, LDL/HDL-C ratio and BMI. Several genetic factors account for the variation in cholesterol levels and obesity indices, however, we believe that the possibilities of genetic differences between the two groups are minimal since the population of Crete is stable with a long history over 4000 years. In addition to this, the ApoE genotype distribution found no differences between the two groups (fasters vs controls). In the fasters' group the mean decrease within all three fasting periods was 9% for total cholesterol and 12% for LDL-C. However, it was observed that during non-fasting periods when fasters returned to their usual dietary habits, total cholesterol and LDL-C increased by 6% and 9% respectively. This shows that the reduced end-total and LDL cholesterol concentrations that were observed within the fasting periods were not sustained when the subjects returned to their usual dietary habits even though the increase did not reach the initial pre- levels. The reduction in HDL that occurred in fasters is a common finding with low-fat and vegetarian diets [28-31]. The findings above are in agreement with the results reported by Barnard et al who conducted a strict vegetarian-diet intervention study for 5 weeks on 35 women [30]. The intervention diet consisted of grains, legumes, vegetables and fruit. After the intervention diet phase total cholesterol, LDL and HDL were decreased by 13.2%, 16.9% and 16.5% respectively [30]. BMI was also significantly reduced (p < 0.001) while, in agreement with our findings, the TC/HDL and LDL/HDL ratios remained unchanged (table 5)[30]. Similar were the findings in another 6-week vegetarian-diet intervention study by Masarei et al [28] and in a 12-week low-fat-vegan-diet intervention study by Nicholson et al [32]. Lee [33] and Hoffman [34], who compared omnivores with lacto-ovo-vegetarians, found no difference in LDL/HDL ratio between the two groups. The contrasting results on LDL/HDL ratio could be attributed to differences in the population samples studied.
Nieman et al [14] and Toohey et al [35] investigated Seventh-Day Adventists with similar demographic and life-style factors and with comparable diets and dietary habits to our cohort. They found that lacto-ovo-vegetarians and lifetime strict vegetarians had lower concentrations of total and LDL cholesterol when compared with non-vegetarians and lacto-ovo-vegetarians respectively (p < 0.05) [14]. Toohey et al found also found lower levels of BMI, triacylglycerols and TC/HDL ratio [35]. The present study showed that women had lower levels of LDL/HDL ratio and TC/HDL ratio, which is also a better predictor for CHD in women [36-38]. This is explained by the higher concentrations of HDL that women have compared to men [39].
The positive association of waist-to-hip ratio with total and LDL cholesterol is in agreement with other studies that correlate waist-to-hip ratio with coronary risk factors and CHD prevalence [40-42]. Waist-to-hip ratio measurement is a simple and cost-effective measure that contributes in predicting abnormal lipoprotein levels and increased risk of cardiovascular disease.
Both the fasting and control groups had mean BMI in the overweight category. Fasting had a small but statistically significant impact on fasters' BMI at the end of the fasting periods that was not sustained in non-fasting periods. In accordance to the results in this study, Haddad et al studying a group of vegans and nonvegetarians found significantly lower BMI levels in the vegan group [43]. Moreover, others found that vegetarians have lower BMI than meat eaters [44-46]. At the same time following a Mediterranean-style diet has also been proven to be beneficial to weight loss [47]. As regards religious fasting some studies associate it with weight loss and decline in BMI [2,3] while others do not [4,5,48].
Educational level was not found to influence any of the blood lipid variables in this study (Table 3). This was an unexpected result since higher education is associated with better health care and awareness whereas low educational level has been related to unfavorable lipid profile [49], all-cause and CAD mortality [50] and hypertension [51].
The beneficial changes seen in fasters diet during the fasting periods, especially regarding energy intake, total fat and fiber consumption, can also explain the reductions in the biochemical and obesity indices. A recent study of the University of Crete showed that the Christian Orthodox nuns' diet was very low in cholesterol and in saturated fat intake (6% of total energy intake), and high in fiber and antioxidant vitamins [16]. This could be attributed to nuns' high consumption of fruit, vegetables, cereals and legumes. In another study Haddad et al found that vegans consume more grains, vegetables, fruit, legumes and seeds and as a result their diet consists of more dietary fiber and less dietary cholesterol [43]. It is well known that reduced intakes of dietary SFA and cholesterol lower total and LDL cholesterol concentration and are associated with low risk of cardiovascular diseases [52,53]. The Orthodox Christians' diet, which is based on vegetables, legumes, fruit, cereals, bread and olive oil, is a Mediterranean-type of diet with periodic abstinence from meat and other animal products during the fasting periods. Numerous investigators [54-56] have recognized the beneficial role of the Mediterranean diet in cardiovascular diseases, and the protective effect in terms of cancer and longevity have also been noted [57,58]. In addition, supplementary studies have associated religiosity with good health [10]. This has been confirmed in a recent study by Chliaoutakis et al [59], which is the only published work to date which investigates the association between the Orthodox Christian lifestyle and health. Chliaoutakis et al found that devout Orthodox Christians adopt healthier life-styles and that religion has a substantial impact on mental and physical health-related behaviors [59]. In the present study, contrary to Chliaoutakis' findings, the physical activity of the two groups (fasters vs controls) did not differ in any of the testing periods.
Our study attempts to provide an understanding of the impact of Christian Orthodox fasting on serum blood lipids and obesity indices before and at the end of the three major fasting periods. Compared to controls, fasters presented decreased lipoproteins and BMI levels. These results support our hypothesis by highlighting the beneficial influence of Christian Orthodox fasting on lipoprotein profile and prevalence of obesity.
Competing interests
None declared.
Authors' contributions
Author K.S mainly organized and performed the study, and drafted the manuscript. Author N.T participated in the design of the study and supervised the manuscript. Author M.L performed the statistical analyses. Author G.M performed part of the statistical analysis. Author A.K conceived of the study, participated in its design, and supervised the study and the manuscript.
Acknowledgments
We appreciate the assistance of Bishop Nektarios of Crete in supporting the study and the Monasteries of Sabbathiana, Isodia Theotokou and Kremaston for their participation. We are also grateful to Dr N.Yiannakouris, Mrs C.Codrington, Dr C.Hatzis, Ms F.Bervanaki, Mr M.Kiriakakis and Mr G.Tsibinos.
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Pre-publication history
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2458/3/16/prepub
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Pre-publication history
Microbicides Development Programme: Engaging the community in the standard of care debate in a vaginal microbicide trial in Mwanza, Tanzania
Andrew Vallely*, Charles Shagi, Shelley Lees, Katherine Shapiro, Joseph Masanja, Lawi Nikolau, Johari Kazimoto, Selephina Soteli, Claire Moffat, John Changalucha, Sheena McCormack and Richard J Hayes
BMC Medical Ethics 2009, 10:17 doi:10.1186/1472-6939-10-17
Pre-publication versions of this article and reviewers' reports
Original Submission - Version 1 Manuscript 18 Jul 2009
Reviewer's Report Gita Ramjee 13 Aug 2009
Reviewer's Report Thomas Moench 10 Sep 2009
Resubmission - Version 2 Manuscript Author's comment 23 Sep 2009
Editorial acceptance 23 Sep 2009
Published 09 Oct 2009
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Case Report
Capnocytophaga canimorsus infection presenting with complete splenic infarction and thrombotic thrombocytopenic purpura: a case report
Michal Brichacek1, Peter Blake2 and Raymond Kao1,3*
Author affiliations
1 Critical Care Trauma Centre, London Health Sciences Center, 800 Commissioners Road East, London, Ontario N6A 5W9, Canada
2 Department of Nephrology, London Health Sciences Center, 800 Commissioners Road East, London, Ontario N6A 5W9, Canada
3 Department of National Defense, Canadian Forces Health Services, 1745 Alta Vista Drive, Ottawa, Ontario K1A 0K6, Canada
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Citation and License
BMC Research Notes 2012, 5:695 doi:10.1186/1756-0500-5-695
Published: 26 December 2012
Abstract
Background
Animal bites are typically harmless, but in rare cases infections introduced by such bites can be fatal. Capnocytophaga canimorsus, found in the normal oral flora of dogs, has the potential to cause conditions ranging from minor cellulitis to fatal sepsis. The tendency of C. canimorsus infections to present with varied symptoms, the organism’s fastidious nature, and difficulty of culturing make this a challenging diagnosis. Rarely, bacterial cytotoxins such as those produced by C. canimorsus may act as causative agents of TTP, further complicating the diagnosis. Early recognition is crucial for survival, and the variability of presentation must be appreciated. We present the first known case of C. canimorsus infection resulting in TTP that initially presented as splenic infarction.
Case presentation
72-year-old Caucasian male presented with a four-day history of abdominal pain, nausea, vomiting, diarrhea, and intermittent confusion. On presentation, vital signs were stable and the patient was afebrile. Physical examination was unremarkable apart from petechiae on the inner left thigh, and extreme diffuse abdominal pain to palpation and percussion along with positive rebound tenderness. Initial investigations revealed leukocytosis with left shift and thrombocytopenia, but normal liver enzymes, cardiac enzymes, lipase, INR and PTT. Abdominal CT demonstrated a non-enhancing spleen and hemoperitoneum, suggesting complete splenic infarction. Although the patient remained afebrile, he continued deteriorating over the next two days with worsening thrombocytopenia. After becoming febrile, he developed microangiopathic hemolytic anemia and hemodynamic instability, and soon after was intubated due to hypoxic respiratory failure and decreased consciousness. Plasma exchange was initiated but subsequently stopped when positive blood cultures grew a gram-negative organism. The patient progressively improved following therapy with piperacillin-tazobactam, which was switched to imipenem, then meropenem when Capnocytophaga was identified.
Conclusions
There is a common misconception amongst practitioners that the presence of systemic infection excludes the possibility of TTP and vice versa. This case emphasizes that TTP may occur secondary to a systemic infection, thereby allowing the two processes to coexist. It is important to maintain a wide differential when considering the diagnosis of either TTP or C. canimorsus infection since delays in treatment may have fatal consequences.
Keywords:
Capnocytophaga canimorsus; Thrombotic thrombocytopenic purpura; Splenic infarction; Dog bite
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Another photo for future stock photo use.
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Alleghany County, North CarolinaEdit This Page
From FamilySearch Wiki
Revision as of 20:20, 21 November 2012 by Lsgc (Talk | contribs)
This article is about a northwestern North Carolina county. For other uses, see Alleghany.
United States North Carolina Alleghany County
North Carolina
Online Records
Alleghany County, North Carolina
Map
Location in the state of North Carolina
Location of North Carolina in the U.S.
Facts
Founded 1776
County Seat Sparta
Courthouse
Adopt-a-wiki page
This page adopted by:
NCGenWeb Project
who welcome you to contribute.
County Coordinator
Alleghany Co. NCGenWeb
Adopt a page today
Contents
County Courthouse
Alleghany County Courthouse
Main Street PO Box 186
Sparta, NC 28675
Phone: 336-372-4342
Clerk Superior Court has birth death records from 1914
Court records from 1869 & land records from 1860
Alleghany County was fromed in 1859 from Ashe[1]
History
In 1776 settlers in what would eventually become Tennessee successfully petitioned North Carolina to recognize the Washington District. The District included all of modern Tennessee except two small settlements (North-of-Holston, Fincastle County, and Pendleton, Washington County) in the far northeast that were considered part of Virginia at the time. Washington (old) County was created from Washington District by North Carolina in 1777 as the western county of North Carolina.[2]
In August 1784 delegates from Washington and two other western North Carolina counties which had split off from Washington (all now in Tennessee), declared their Independence from North Carolina because of perceived neglect, and misuse by North Carolina’s legislature. By May 1785 they had petitioned to be admitted to the United States as the new State of Franklin. The Franklin statehood request was denied. By 1789 the hopes for a State of Franklin faded. North Carolina refused to recognize several counties created by Franklin out of Washington County.[3]
North Carolina was admitted to the Union in 1789 and ceded her western counties to the United States. The United States made these western counties into the Southwest Territory. In 1792 North Carolina divided Washington (old) County and annexed some of its land that would later become Alleghany, Ashe, and Watauga counties in North Carolina to Wilkes County, North Carolina.[4] In 1796 the remainder of Washington County became part of the new State of Tennessee.
North Carolina created Ashe County out of Wilkes County in 1799, and in 1859 erected Alleghany County out of Ashe County.[5]
For a detailed assessment of Alleghany records and their availability, see:
Parent County
1859--Alleghany County was created in 1859 from the eastern part of Ashe County. County seat: Sparta[5]
Boundary Changes
Record Loss
Some records were lost in a 1932 courthouse fire. For more information on extant records, see the following:
Places/Localities
Townships
Alleghany County currently has seven townships:
• Cherry Lane
• Cranberry
• Gap Civil
• Glade Creek
• Piney Creek
• Prathers Creek
• Whitehead
Populated Places
Neighboring Counties
Resources
Cemeteries
Census
For tips on accessing Alleghany County, North Carolina census records online, see: North Carolina Census.
Church
Court
• Court (U.S. GenWeb Archives)
Land
Local Histories
Maps
Military
Civil War
Civil War Confederate units - Brief history, counties where recruited, etc.
- 4th Regiment, Virginia State Line (Cavalry and Infantry) (Confederate). Company B.[6]
Newspapers
Probate
Taxation
Vital Records
Births
Marriages
Deaths
Yearbooks
Societies and Libraries
Family History Centers
Web Sites
References
1. Handybook for Genealogists: United States of America, 10th ed. (Draper, Utah: Everton Pub., 2002), Allegheny County, North Carolina p. 506. At various libraries (WorldCat); FHL Book 973 D27e 2002.
2. Joyce Cox, and W. Eugene Cox, History of Washington County Tennessee (Jonesborough, Tenn.: Washington County Historical Assoc., 2001), 54.
3. “State of Franklin” in North Carolina History Project at http://www.northcarolinahistory.org/encyclopedia/99/entry (accessed 27 June 2010).
4. Arthur L. Fletcher, Ashe County: A History (Jefferson, N.C.: Ashe County Research Assoc., 1963), 33-34.
5. 5.0 5.1 The Handybook for Genealogists: United States of America,10th ed. (Draper, UT:Everton Publishers, 2002), 506.
6. The Virginia State Line: Organizational Structure of the Virginia State Line, Ranger95.com, accessed 11 June 2012.
Need additional research help? Contact our research help specialists.
Need wiki, indexing, or website help? Contact our product teams.
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Compiler Error & problems
Newbie Member
27May2010,23:26 #1
HeLLo
i M a basic C programmer, just started to learn it.
So till now i was using Turbo C++ 3.0 but i had fEwW problems with it.
So i shifted toCode::Blocks using MinGW Compiler
But 1 thing i cant understand.
As from when i have learnt C, i have learnt to write clrscr ()
i Have leanrt using Turbo C++ 3.0
But whenevr i compile the programe in Code::Blocks & try to write clrscr (), it rejects the programme, giving clrscr () as error.
& whenever i compile a programme, i get this msg after successful compilation
So can u pls. explain me why are this errors happening???
Newbie Member
27May2010,23:27 #2
The error is this :
Go4Expert Founder
28May2010,09:55 #3
This error can be result of lot of things.
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About this Journal Submit a Manuscript Table of Contents
Case Reports in Dermatological Medicine
Volume 2012 (2012), Article ID 486724, 2 pages
doi:10.1155/2012/486724
Case Report
Rhabdomyolysis due to Multiple Wasp Stings
1Department of Dermatology, Almeida Hospital, 1509-2 Miyazaki, 2-Chome, Oaza, Oita 870-1195, Japan
2Department of Dermatology, Fukuoka University Hospital, 6-45 Momochihama, 3-Chome, Sawara-ku, Fukuoka, Japan
Received 2 August 2012; Accepted 26 August 2012
Academic Editors: I. D. Bassukas and M. Hide
Copyright © 2012 K. Ito 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.
How to Cite this Article
K. Ito, S. Imafuku, and J. Nakayama, “Rhabdomyolysis due to Multiple Wasp Stings,” Case Reports in Dermatological Medicine, vol. 2012, Article ID 486724, 2 pages, 2012. doi:10.1155/2012/486724
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About this Journal Submit a Manuscript Table of Contents
Abstract and Applied Analysis
Volume 2012 (2012), Article ID 329702, 2 pages
doi:10.1155/2012/329702
Editorial
Ulam's Type Stability
1Department of Mathematics, Pedagogical University, Podchorążych 2, 30-084 Krakow, Poland
2Département d'Informatique et de Mathématiques, Ecole Centrale de Nantes, 1 rue de la Noë, B.P. 92101, 44321 Nantes Cedex 3, France
3Department of Mathematics, Sichuan University, Sichuan, Chengdu 610064, China
Received 13 November 2012; Accepted 13 November 2012
Copyright © 2012 Janusz Brzdęk 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.
The original stability problem was posed by S. M. Ulam in 1940 and concerned approximate homomorphisms. The pursuit of solutions to this problem, but also to its generalizations and modifications for various classes of (difference, functional, differential, and integral) equations and inequalities, is an expanding area of research and has led to the development of what is now quite often called Ulam’s type stability theory or the Hyers-Ulam stability theory. This theory has been the subject of many papers as well as talks presented at various conferences, especially at the series of ICFEI conferences (International Conference on Functional Equations and Inequalities) organized by the Department of Mathematics of the Pedagogical University in Cracow (Poland) since 1984.
This special issue on Ulam’s type stability is focused on the recent achievements in that type of stability for various objects. It contains 16 articles (a survey and 15 regular research papers) which have been written by 29 authors from 11 countries.
As usual, most of the authors use in their investigations direct and fixed point methods. Some open problems are also formulated.
The issue covers a wide variety of problems for different classes of functional equations both in a single variable and in several variables. Their stability is traditionally investigated in classical Banach spaces, but also in complete (probabilistic) metric spaces, complete probabilistic quasimetric spaces, -Banach spaces, -Banach spaces, and fuzzy Banach spaces.
Several papers deal with the stability of several kinds of derivations, and, thus, derivations in Riesz algebras, -derivations in normed algebras, cubic -derivations in Banach -algebras, and some higher ring derivations in intuitionistic fuzzy Banach algebras are studied.
The issue contains a few papers on the phenomenon of superstability, an article on the stability of a functional inequality in -Banach spaces, and a paper on the Cauchy fractional differential equation in the unit disk.
Moreover, general solutions of two conditional quadratic functional equations of Pexider type and the structure of the set of all regular points and the set of all irregular points for a Brouwer homeomorphism which is embeddable in a flow are also considered.
Finally, the survey presents some selected recent developments (results and methods) in the theory of Ulam’s type stability. In particular, some aspects of stability and nonstability of functional equations in a single variable, the effect "stability implies completeness," some methods of proofs applied in that theory (the Forti method and the methods of fixed points), stability in non-Archimedean spaces, selected results on functional congruences, the notion of hyperstability, and stability of composite functional equations (e.g., of the Gołąb-Schinzel equation and its generalizations) are discussed there.
We believe that this volume will have some influence on the further research in that area of mathematics.
Janusz Brzdęk
Nicole Brillouët-Belluot
Krzysztof Ciepliński
Bing Xu
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Bibliography: Lady Robotica
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Title: Lady Robotica
Author: G. O. Clark
Year: 2004
Type: POEM
ISFDB Record Number: 1030785
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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Image:Kuliah 7 tambahan MEDIA KULTUR.pdf
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User:David Johnston Monje/Notebook/Maize Endophyte Biofertilizers/2013/01/03
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Applied Soil Microbial Ecology Main project page
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Remains of the Day
• Streaked out LGI enrichment cultures of potato endophytes onto R2A media and incubated at 25 degrees.
• Photographed plates of microbes from tomato vine decline growth room study and potatoe endophyte growth room study
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User:Mary Mendoza/Notebook/CHEM572 Exp. Biological Chemistry II/2013/02/20
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ADA Kinetic Assay for obtaining the zero point
• The objective of this laboratory period is to conduct adenosine deaminase (ADA) kinetic assay runs for the new calculated concentrations.
• UV 2550 Shimadzu spectrophotometer was baseline with 0.05 M sodium phosphate buffer.
• The assays were prepared according to the data below.
• After running the first trial, it was observed that the absorbance for 12.34 μM adenosine of trial 2 was superimposed over the 10.52 μM adenosine of trial 1.
• It was suggested by Dr. Hartings to use Beer's Law for accurate measurement of the concentration of adenosine in solution.
• An article was provided by Dr. Hartings with the molar extinction coefficient, 1.53 x 10-4 of adenosine at 260 nm.
• Thus, it was decided to run a full spectrum of adenosine at each given concentration specified above for accurate measurement of the concentration of adenosine.
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CMD sent two reporters to track ALEC in Oklahoma
Click here to help support our future investigations.
Edwardsport Plant
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This article is part of the Coal Issues portal on SourceWatch, a project of CoalSwarm and the Center for Media and Democracy. See here for help on adding material to CoalSwarm.
This article is part of the CoalSwarm coverage of coal plants
Sub-articles:
Edwardsport Plant is a power generation plant in Edwardsport, Indiana. [1] As of late 2012, the plant was expected to begin commercial operation in mid-2013.[2]
Contents
Timeline
In 2006, Duke Energy and Vectren proposed the Edwardsport coal plant to be built in Knox County, Indiana, intended to replace two existing oil and coal plants built in the 1940’s, the Edwardsport Generating Station (existing). There is significant opposition to the plant, both because the concentration of coal generators in this area is one of the highest in the world, and because the local power needs have already been met.
May 2007 update: Environmental and consumer groups – including Citizens Action Coalition of Indiana, Save the Valley, the Sierra Club and Valley Watch – testified that the proposed plant is not the best available option. These groups have urged the Indian Utility Regulatory Commission (IURC) to direct Duke Energy/Vectren towards energy efficiency and renewable power. Among the many objections to the plant is the significant potential for wind generation in Indiana, which could meet or exceed the energy capacity of the new Edwardsport plant. The IURC approved the plant on Nov. 20, 2007. An air permit from the Indiana Dept. of Environmental Management (IDEM) is still pending; the comment period for the IDEM hearing closes Dec. 31, 2007, but the Citizen’s Action Coalition is trying to get that deadline extended until February. [3]
April 2008 update: The Sierra Club filed suit in federal court in Indiana seeking to hold Duke Energy accountable for failing to install modern pollution controls at the existing Edwardsport plant. According to the Clean Air Act, coal-fired power plants are required to install modern pollution controls when they upgrade the rest of the facility, something the Sierra Club contends Duke has not done.[4]
May 2008 update: Duke Energy announced that the new IGCC plant would cost $2.35 billion, an increase of $365 million from earlier estimates. If approved, the overage would be paid for by a 2 percent rate increase between 2008 and 2013.[5] Duke has been given a grant of $1 million from the U.S. Department of Energy's Regional Carbon Sequestration Partnership Program "to study the permanent storage of carbon dioxide from the plant near the site."[6]
October 2008 update: Three citizen and environmental groups filed a request for public record disclosure to obtain records detailing Governor Daniels' actions in promoting Duke Energy's proposed Edwardsport power plant. The Hoosier Chapter of the Sierra Club, Citizens Action Coalition, and Valley Watch are seeking to determine whether the Daniels administration and the Indiana Utility Regulatory Commission (IURC) violated ratepayer protections in an effort to fasttrack the new plant. The citizen groups want to reveal the level of financial support received from Duke Energy in support of the Governor's energy summit, which was held in Indianapolis two months before the election. The groups are also looking for any evidence of improper communications between Duke and the IURC.[7]
January 2009 update: Indiana Utility Regulatory Commission approved Duke's request to pass increased construction costs for the plant on to its Indiana customers. The ruling clears the way for Duke to raise rates by 18 percent over the next five years.[8]
May 2009 update: Under the proposed Waxman-Markey Climate Bill, two Duke Energy plants - Cliffside and Edwardsport - are exempted from new pollution regulations. The bill has been criticized because Duke Energy helped draft the US-CAP blueprint that provided the basis for bill, and CEO Jim Rogers testified before the House Energy and Commerce Committee hearings on the bill.[9]
July 2009 update: Duke filed a proposal for a carbon dioxide storage project with the Indiana Utility Regulatory Commission. The company would invest over $120 million to store a portion of the CO2 emissions from the Edwardsport plant in saline aquifers and in depleted oil and gas fields. The experiment would result in an average 1 percent rate increase for customers between 2010 and 2013. Duke is also applying for a federal grant to cover about half of the project's costs. If the project is successful, Duke will apply to capture and store the emissions on a permanent basis.[10]
October 2009 update: Construction on the plant is about 30 percent complete. Duke expects to have the plant in service in 2012.[11]
November 2009 update: Duke announced that the construction costs for Edwardsport have increased at least six percent. The company said building the plant would cost at least an extra $150 million. Originally the company estimated a 17 percent rate increase to cover construction costs.[12]
April 2010 Update: Duke Energy Indiana filed testimony and exhibits with the Indiana Utility Regulatory Commission increasing the cost estimate for the Edwardsport coal gasification plant under construction, adding approximately $530 million to the previously approved $2.35 billion cost. Indiana state utility regulators must review and approve Duke Energy Indiana’s filing before any new costs can be phased into customer electric rates. If approved, the increase in costs will add about 3 percent to the project’s customer rate impact, and an overall average 19 percent rate increase by 2013.[13]
July 2010 Update: Duke is marketing $500 million of bonds to help fund the power facility’s construction and to repay bank debt. The 10-year first-mortgage bonds may be sold as soon as July 6, 2010, and pay about 80 basis points more than similar-maturity Treasuries, according to a person familiar with the transaction. A portion of proceeds from the debt sale will be used to fund the coal plant’s construction, said Tom Shiel, a spokesman for Duke Energy Corp. The Edwardsport plant is more than 55 percent complete, and is expected to be operational in 2012.[14]
October 2010: Update: Following an alleged ethics violation, Indiana Governor Mitch Daniels fired the state regulatory chairman Dave Lott Hardy and Duke Energy placed two employees on paid leave. The firing was made after it was discovered that the regulatory chairman did not removes himself from reviewing Duke Energy matters despite the fact that he was in discussions with the company about working for them.[15]
November 2010 Update: State regulators met the first week of November to consider Duke Energy's rate hike request. The plant's cost is now estimated at nearly three billion dollars, twice its original estimate. The Indiana Utility Regulatory Commission (URC) will hold a hearing on Nov. 3 in Indianapolis.[16] Governor Mitch Daniels later fired the URC’s chairman David Lott Hardy for allowing agency official Scott Storms to continue presiding over the case even after talking with Duke about a job opportunity, which Storms took in September 2010. Additionally, later Duke Energy fired Indiana president Michael Reed and staff attorney Scott Storms, following the ethics scandal.[17]
December 2010 Update: Duke puts former URC official Scott Storms and Mike Reed, the Duke Energy Indiana president who hired him, on administrative leave. Afterward, e-mails obtained by the Indianapolis Star showed that Jim Turner, who headed Duke’s regulated utilities, discussed Storms and Reed with former commission chairman David Hardy, both of whom were dismissed over the ethics issues. Turner and Reed were part of the team that negotiated the settlement with the Indiana Utility Customer Counsel, the Duke Energy Indiana Industrial Group and Nucor Steel Indiana, so Duke Energy withdrew a proposed settlement worked out with with the Indiana customer groups. The company and groups hope to negotiate a new settlement that will not be tainted by the ethics inquiries.[18] On December 6, 2010, James Turner resigned.[19]
January 2011 Update: In a confidential letter to Duke dated Oct. 5, 2010, project manager Brian Hartman of engineering contractor Bechtel Corp., wrote that Duke had taken "significant risks" with the project, in violation of its contract with Bechtel. The risks included limiting oversight and monthly reports, consolidating functions, reducing documentation, and taking personnel action against Bechtel management's recommendations, in the name of expediency. Duke, in turn, had rated Bechtel's performance on the project as "very low" in an internal e-mail. In another email, top Duke executive Richard W. Haviland had said of the many accidents at the project: "We need an exorcist on this job." The letters and emails were among dozens between the companies released after a filing was made by a collection of consumer and environmental groups, who want the state to investigate whether Duke mismanaged the project, committed fraud, or concealed vital facts. If the groups can prove that, Duke would have to absorb a large portion of the construction costs, without passing them along to ratepayers.[20]
Meanwhile, some are questioning a timetable for approving the plant, saying it leaves too little time for public input. The Indiana Utility Regulatory Commission's schedule for the plant would conclude hearings by early May 2011, which attorneys say appears expedited. The Indianapolis Star reported that the timetable was largely put together part by Randolph Seger, an Indianapolis lawyer representing the Indiana Finance Authority, which has a 30-year deal to buy the plant's synthetic natural gas. Seger has said the project is a "good one for Indiana and the benefits should be realized as quickly as possible."[21]
March 2011 Update: On March 11, 2011, Duke asked state regulators to approve the company's newly drafted plan to cap at $2.72 billion, and Duke Indiana President Doug Easamann said the company remains committed to completing the 618-megawatt Edwardsport plant despite growing protests about the overruns and ethics issues involving the regulatory approval process.[22]
July 2011 Update: The Indiana Office of Utility Consumer Counselor rejects Duke Energy's request for an extra $530 million to recover more of the cost overruns of the Edwardsport plant: "Duke has not demonstrated any budgetary constraints on this project," the office's Barbara Smith wrote in pre-filed testimony to the commission. "There appears to be a lack of responsibility or accountability on the part of those causing these multi-million dollar cost overruns. In addition, there has been no evidence presented to indicate that Duke management, or any other entity, has conducted any kind of timely prudency review regarding these cost overruns."[23] On July 15, Indianapolis-based uitility watchdog Citizens Action Coalition (CAC) filed copies of e-mails pointing to numerous meetings between top Duke executives and former IURC chairman David Hardy, and said the emails show the Duke executives secretly informed Hardy that costs for the coal-gasification plant, initially projected at $1.9 billion, had escalated to $2.88 billion. Such back-channel, “ex parte” communications with the head of regulatory agency are considered highly inappropriate, if not illegal, CAC officials said. The CAC wants Duke to cancel the Edwardsport project. Duke did not yet file testimony in response.[24]
October 2011 Update: On October 20, 2011, Duke Energy said the price tag of the plant will climb $200 million, to $2.98 billion, but that the utility will pay for the latest overruns out of its own pocket, rather than seek to pass them along to customers in the form of higher electricity bills.[25] State regulators are scheduled to begin hearings in late October 2011 on whether Duke Energy or Indiana ratepayers will cover the more than $1 billion in cost overruns. The state ruled that former IURC counsel Scott Storms violated Indiana ethics laws by making judicial decisions regarding Duke and Edwardsport while making plans to work for the company. The ethics commission fined Storms and stripped him of his law license. Storms has since appealed the rulings.[26]
March 2012 Update: In March 2012 the Indiana Utility Regulatory Commission considered opening up additional documents about Duke Energy. Citizens Action Coalition urged the commission to open the documents since, as they claimed, Duke had not been consistent about classifying some material, and some designated as confidential has shown up on websites and in newspapers.[27]
December 2012: Indiana Utility Regulatory Commission capped the amount of construction costs Duke Energy Corp. can pass on to consumers at $2.6 billion.[28]
Project Details
Sponsor: Duke Energy/Vectren
Location: Edwardsport, Knox County, Indiana
Size: 630 MW
Type: IGCC
Projected in service: 2012
Status: Permit application pending (November 2007)
Financing
Citizen Groups
Resources
References
1. “Tracking New Coal-Fired Power Plants,” National Energy Tech Lab, May 1, 2007, page 12. (Pdf)
2. Bruce Henderson, "Troubled plants to cost Duke Energy $280 million," Charlotte Observer, October 31, 2012
3. "Stopping the Coal Rush", Sierra Club, accessed December 2007. (This is a Sierra Club list of new coal plant proposals.)
4. "Sierra Club Challenges Duke Energy", Sierra Club Indiana, March 31, 2008.
5. "Duke Energy Revises Cost Estimate For Edwardsport", Inside Indiana Business, May 5, 2008.
6. Duke Energy, " Duke Energy's Edwardsport Coal Gasification Project to Receive Federal Funds for Carbon Study", Media Release, May 9, 2008.
7. "Coal plant opponents seek Daniels e-mail records," Chicago Tribune, October 29, 2008.
8. "Ind. panel approves cost increase for coal plant," WTHR, January 8, 2009.
9. “Waxman-Markey climate bill a wish list for Duke Energy?”, Matt Leonard, It’s Getting Hot in Here, May 5, 2009.
10. "Duke Energy files plans for carbon-storage study," Charlotte Business Journal, July 7, 2009.
11. "Coal plant construction continues in earnest at some sites," SNL Interactive, October 5, 2009. (Subscription required.)
12. "Power Plant Cost Going Up," Tristatehomepage.com, November 25, 2009.
13. "Duke Energy Indiana Files Cost Update for Clean Coal Gasification Power Plant" Duke Energy, April 16, 2010.
14. Sapna Maheshwari, "Duke Energy Unit Markets Debt to Help Fund Coal Plant (Update1)" Bloomberg BusinessWeek, July 6, 2010.
15. [ http://policyintegrity.org/documents/Binder_Edwardsport%20IGCC%20Scandal.pdf "Ethics controversy erupts"] Jeff Swiatek and Tim Evans, The Indianapolis Star, October 6, 2010.
16. "Duke Energy Hearing Preview" mywabashvalley, Nov. 2, 2010.
17. Chris O'Malley, "Duke Energy puts local CEO on leave amid state probe" Indiana Business Journal, Oct. 5, 2010.
18. John Downey, "Ethics issue kills Duke Energy deal on Indiana plant costs" Charlotte Business Journal, Dec. 9, 2010.
19. Julie Rose, "Duke Energy Exec James Turner Resigns Over Ethics Flap" WFAE 90.7FM, Dec. 6, 2010.
20. John Russell, "Contractor says Duke took risks at plant" Indystar.com, Jan. 28, 2011.
21. "Utility lawyers question coal-gas plant timetable" Bloomberg, Jan. 28, 2011.
22. "Duke proposes new cap on coal-gasification plant" Fox News, March 11, 2011.
23. Bruce Henderson, "Consumer advocates slam Duke's Indiana costs" Charlotte Observer, July 11, 2011.
24. Chris O'Malley, "Consumer group accuses Duke of 'gross mismanagement'" IBJ, July 15, 2011.
25. "Duke says Edwardsport project cost increases more than $200M" Indy Star, Oct. 20, 2011.
26. "Ethics Scandal Zaps Former Duke Energy Attorney" ABC, Oct. 20, 2011.
27. "Secrets Can Strain Credibility" Indystar.com, March 6, 2012.
28. "Ind. regulators OK deal involving cost overruns at Duke Energy's coal-gasification plant," The Republic, Dec. 27, 2012.
Related SourceWatch Articles
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Australian Bureau of Statistics
Celebrating the International Year of Statistics 2013
ABS Home > Statistics > By Catalogue Number
4196.0.00.010 - National Aboriginal and Torres Strait Islander Survey: Regional Statistics - Cairns, 1994
Latest ISSUE Released at 11:30 AM (CANBERRA TIME) 27/02/1996
Page tools: Print Page Print All RSS Search this Product
• About this Release
Irregular; Contains a range of statistics from the 1994 National Aboriginal and Torres Strait Islander Survey plus some data from the 1991 Census of Population and Housing and the 1992 ATSIC Housing and Community Infrastructure Needs Survey. A range of charts and dot point commentary on major statistics is presented. The introductory chapter describes the region, its size, major towns and localities and the demographics of the indigenous population of the region. The six chapters are, family and culture, health, housing, education and training, employment and income, and law and justice.
This publication has been converted from older electronic formats and does not necessarily have the same appearance and functionality as later releases.
© 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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}
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Australian Bureau of Statistics
Celebrating the International Year of Statistics 2013
ABS Home > News & Media
ABS Response to 'The 'R' word's already here, and things will get worse' (The Age, 4 June 2009, pg 8, Business Day)
ABS Response to 'The 'R' word's already here, and things will get worse' (The Age, 4 June 2009, pg 8, Business Day)
Jiggery-Pokery in Age Article
I write to express my disappointment that The Age published an inaccurate and misleading article about the Australian Bureau of Statistics and the statistics it publishes ('The 'R' word's already here, and things will get worse', The Age, 4 June 2009, with authorship attributed to Gerard Minack). The author did not check the claims made in the article with the ABS, nor apparently with any other authority familiar with macro-economic statistics.
Many of your experienced readers who are familiar with macro-economic statistics will realise that the claims made in the article cannot be correct. Unfortunately, queries received by the ABS about the inaccurate article indicate that some people have been mislead by it.
The methodology to estimate the volume of bulk commodity exports described in The Age article attributed to Mr Minack is not used by the ABS .
The statement in the article that "Yesterday it (the ABS) announced that it was factoring in lower prices in the March quarter' is not correct. No such announcement was made. Furthermore, given the methodology that the ABS uses to measure the volume of bulk commodity exports, such a claim could not possibly have been correct.
The volume measures of exports of bulk commodities recorded for the latest quarters in the ABS Balance of Payments and National Accounts Statistics are calculated by multiplying the quantities of such exports, as reported by exporters in tonnes or some other unit of quantity, by the average price of such commodities, as reported by exporters in the reference year. For the March quarter 2009 volume estimates, 2006-07 is the reference year for prices. The reference year prices are updated annually, in the September quarter accounts. For example, the September quarter 2009 accounts will use average prices reported in 2007-08. This methodology assures that movements in the volume of bulk exports from one quarter to the next reflect only changes in actual volumes and are not influenced by changes in prices.
The ABS methodology for compiling the volume estimates of bulk commodity exports has been in place for many decades, is very well documented on the ABS website, and well understood by most analysts. In 1997 the ABS moved to update the historic reference year from once every 5 years or so, to once every year -- apart from this the methodology has been essentially unchanged.
The ABS has tried, without success, to contact Mr Minack about the erroneous claims in his article. If Mr Minack had bothered to contact the ABS to gain an understanding of the relevant macro-economic statistics methodology, your readers may have been spared the misleading reporting that appeared in The Age.
I would encourage Mr Minack in future to either contact the ABS or visit our website if he is seeking to understand the statistical methods that are used by the ABS.
Peter Harper
Acting Australia Statistician
© 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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Ask Your Question
2
Cannot access built-in help
asked 2012-08-02 21:19:56 +0200
Schalk
23 4
updated 2012-08-03 12:41:01 +0200
manj_k
5622 4 31 48
I installed Libre Office 3.5.5, running windows7. I cannot access built-in help by either pressing F1 or selecting Help on the taskbar. The help button only directs me to the Libre office webpage. Is help available?
delete close flag offensive retag edit
1 Answer
Sort by » oldest newest most voted
4
answered 2012-08-02 22:42:26 +0200
hyogapag
821 7 19
http://www.insolit.org/
This is because the built-in help is not included in the main download. You have to download it apart. Here's a page you can download it :
https://www.libreoffice.org/download/
Once downloaded, just install it as a usual program. You're done.
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Stats
Asked: 2012-08-02 21:19:56 +0200
Seen: 115 times
Last updated: Aug 02 '12
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LS-Pro - chroot Arm Build Environment
From NAS-Central Buffalo - The Linkstation Wiki
(Difference between revisions)
Jump to: navigation, search
m (New page: ==Get Started== by armstation Why: To not fill up LinkStation's system partition & To test toolchains and building programs without risk of breaking anything ===Create a chroot somew...)
Line 1: Line 1:
-
-
==Get Started==
==Get Started==
Revision as of 10:08, 11 September 2007
Contents
Get Started
by armstation
Why: To not fill up LinkStation's system partition & To test toolchains and building programs without risk of breaking anything
Create a chroot somewhere outside of the LinkStation system partition
mkdir /mnt/disk1/share/arm-tools
Transfer entire Linkstation system to build directory
cd /
for d in `ls |grep -v dev |grep -v proc|grep -v mnt`
do
tar -cvf /mnt/disk1/share/arm-tools/$d.tar $d
done
Unpack the system directories
cd /mnt/disk1/share/arm-tools
for f in *.tar
do
tar -xvf $f
done
Download jonli447's excellent arm-tools and copy them into /mnt/disk1/share/arm-tools/
then Gunzip & Untar the arm tool
tar -xvzf arm-tools-0_16-3.tgz
Mount special files in chroot environment (thanks Zoolook)
mount -t proc none /mnt/disk1/share/arm-tools/proc
mount -o bind /dev /mnt/disk1/share/arm-tools/dev
Create /opt directory in chroot
mkdir /mnt/disk1/share/arm-tools/opt
Create symbolic link from chrooted /opt to system /opt
ln -s /mnt/disk1/share/arm-tools/opt /opt
chroot into development environment.
chroot /mnt/disk1/share/arm-tools /bin/sh
That's it
That's it, now everyting compiled and installed to /opt from chroot will be available from the main Linkstation system without adding any files to the system partition besides 1 symbolic link.
This article is currently a stub. You can help this Wiki by expanding it
. This template will categorize articles that include it into Category:Stubs.
Personal tools
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Roku and Linkstation - setup and customization tips
From NAS-Central Buffalo - The Linkstation Wiki
(Difference between revisions)
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m
Line 145: Line 145:
Thanks to Bauldrick for advice on model & setup, and digging up the Weather & MOTD hacks at [http://www.last-outpost.com/~malakai/roku/index.html Jeff's Roku Soundbridge page]
Thanks to Bauldrick for advice on model & setup, and digging up the Weather & MOTD hacks at [http://www.last-outpost.com/~malakai/roku/index.html Jeff's Roku Soundbridge page]
-
Nas-central.org Forum Thread on Roku & Other Soundbridges w/ Firefly/mt-daapd - http://forum.nas-central.org/index.php?action=vthread&forum=4&topic=1892
+
'''Nas-central.org Forum Thread''' on Roku & Other Soundbridges w/ Firefly/mt-daapd - http://buffalo.nas-central.org/forums/viewtopic.php?f=4&t=1892
Support for SoundBridge products at Roku Labs - http://www.rokulabs.com/support_sb.php
Support for SoundBridge products at Roku Labs - http://www.rokulabs.com/support_sb.php
Latest revision as of 23:34, 26 February 2008
Contents
Background
The term Roku can refer to any device produced by Roku Labs. These devices seem to play well with Firefly , Firefly-equipped Linkstations and other hacked NAS's. Below are some tips and tricks that may provide a more positive experience for Roku & Linkstation users.
Hardware/Software Requirements
• Any Roku soundbridge device (M500, M1000, M1001, M2000)
• Any Linkstation w/ OpenLink or FreeLink installed on it
• A wired or wireless (b or g standard) network - some Roku devices are WEP & WPA compatible
• regular computer w/ web browser & terminal/command line interface program
Directory setup
On the share/data partition (hda3 usually) of your Linkstation, it is a good idea to set up some sort of directory structure for organizing your music files. Here is one possible example:
/share/freelinktunes/e_Music
/share/freelinktunes/i_Tunes
/share/freelinktunes/hpodderdownloads
/share/freelinktunes/mediarippertunes
/share/freelinktunes/lossless
/share/freelinktunes/internet_streams
Managing these files is easy if you have flexible access to them with Samba or NFS, so one can modify ownership and permissions to these directory with chown and/or chmod.
Firefly/mt-daapd Tweaks
Making Firefly's web interface configuration page "configurable"
If you notice that your Configuration page on the web interface (available by web browser at <linkstation-ip>:3689/config.html) won't allow you to make changes, then you may need to adjust permissions on the configuration file. The very first entry on the Configuration page lists the location of this file, usually at /etc/mt-daapd.conf . To make the web configuration work, cd to the directory where this configuration file is and modify the permissions of the file:
cd /etc
chown nobody mt-daapd.conf
chmod 600 mt-daapd.conf
Reload the web Configuration page and it should work now. This is a method suggested by an expert at Firefly Forums. If you have concerns about security, you may want to read this thread for details on keeping it secure.
Automatically refresh the database
Also on the Configuration page, there is an option for periodic updating of the database. To make your Firefly server update every hour, go to the Database area on the Configuration page, choose or enter the following:
• Scan Type 0 - Normal,
• Rescan Interval 3600 (seconds, ie. 1 hour),
• Always Scan Yes.
Add to Recognized File Extensions
Also on the Configuration page, there is an option for getting Firefly to scan for more types of files. Normally it will only scan for .mp3 and a few others. In the section Music Files, you can add other types, such as .url files (for internet/stream play lists).
If you want to enable scanning for .url internet playlist files for instance, go to the section
• Music Files, and in Extentions add in
• .url so that the field looks like
.mp3,.m4a,.m4p,.url
• and press Save. Don't forget to restart Firefly for this change to take effect.
See Firefly's documentation for more details on types of files supported.
Restart Firefly from the command line
You may have to restart your mt-daapd server from the command line for some of these configuration changes to take effect.
/etc/init.d/mt-daapd restart
It takes a minute or so for it to come on line after a (re)start.
Playlists
Static Playlists
Smart Playlists
URL/Internet/Stream Playlists
Although Roku's firmware has built-in presets, these can be cumbersome to use, as they require the user to Change Configuration. Some people find it easier to put the internet/radio streams in a .url file and access them through the Roku's Playlist feature - it is much faster that way. To create and use an internet stream playlist for Firefly and Roku, do the following:
1. Get the name, bit rate and URL of a stream that you want to listen to through your Roku SB. For example: Public Radio WNYC-AM from New York City, at 32 kilobit per second, with URL ,http://wnycam.streamguys.com/ (no link arrow, though).
2. Start your favorite editor, e.g. nano, and create a file called (again, for example): nano wnyc-nypr.url.
3. Enter the following information on one line, in the format and order of <bit rate in kbps>,<desired display title>,<URL> . For example it would look like the following, all on one line:32,WNYC-AM Public Radio,http://wnycam.streamguys.com/ (no link arrow, though)
4. Save it to a directory for streams, such as /share/freelinktunes/internet_streams, so they are easy to find/organize/edit.
5. Add as many as you like. They are easier and faster to access from your own Playlist than they are from the Presets. In fact, you may want to copy the presets that you like to your own internet_streams directory.
6. In Firefly's Configuration page, add .url as a recognized extension.
7. In Firefly's Smart Playlist page, create a smart playlist called Internet Radio Stations with criteria description = "Playlist URL"
8. Update Firefly's database.
When you turn on your Roku and go to Playlists, you should see Internet Radio Stations, and when you select it, you should see WNYC-AM Public Radio. Once you have it working, create as many as you want.
Hpodder
Hpodder is a podcatcher that is available as a Debian package, so it is a natural choice for those running Freelink. At the time this article was written, it is available in testing and instable, so remember to (temporarily) adjust your /etc/apt/sources.list as needed - that is, you must have either the testing or unstable branch of Debian enabled.
Once you install hpodder, check its man page. It gives details on configuring it and the first run of hpodder.
You will have to declare a download directory for hpodder (like /share/freelinktunes/hpodderdownloads) , as listed above in Directory setup.
One nice implementation would be to run hpodder as a daily or periodic cron job.
Customizing the Roku
CAUTION: Customizing your Roku, like any device, could void its warranty or turn it into a Brick, or in the case of a M500/M1000/M1001/M2000, a Baton, or an Expensive Aluminum Pipe. Proceed at your own risk. See your Roku manual for details.
It is relatively easy to gain access to a Roku M1001, for instance, via telnet at your Roku's own IP address, through port 4444. A list of command available is given below. :
telnet <roku IP address> 4444
Trying <roku IP address>...
Connected to <roku IP address>.
Escape character is '^]'.
Welcome to the SoundBridge Shell version 2.7.38 Release
Type '?' for help or 'help <command>' for help on <command>.
SoundBridge> ?
Valid commands are:
? - displays this list
help - provides help on a command
clear - clears the terminal window
stty - sets shell tty parameters
consoleprint - sets consoleprint on or off
version - prints out the software version
exit - exits the shell
cycles - displays a count of processor cycles
uptime - reports system uptime
reboot - reboots the system
ps - lists threads
ifconfig - lists network interface configurations
ping - pings an ip address
ipset - sets up a manual config
mfg - performs mfg tests
sketch - draw on the display
irman - capture, monitor or send IR commands
memstat - prints out memory statistics
romcheck - print rom sizes
log - dump the persistent log
logclear - clear the log
logadd - add string to log
attract - attract eyeballs
irwait - wait for a specific IR command
displaytype - print type of currently connected DISplay
time - displays the current time
clearsettings - clears CascadeSettings and reboots
softwareupgrade - perform software upgrades from network or local storage
srbmod - modify srb for oem test
rcp - enter the RCP shell
Many accounts of hacking Roku devices can be found on the web:
References, etc
Thanks to Bauldrick for advice on model & setup, and digging up the Weather & MOTD hacks at Jeff's Roku Soundbridge page
Nas-central.org Forum Thread on Roku & Other Soundbridges w/ Firefly/mt-daapd - http://buffalo.nas-central.org/forums/viewtopic.php?f=4&t=1892
Support for SoundBridge products at Roku Labs - http://www.rokulabs.com/support_sb.php
Personal tools
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Age Effects in Foreign Language Learning for Children in China
Zhiliang Liu, Guanying Chen
Abstract
To know the age effects in foreign language learning for children in China, we made both qualitative survey on the English teachers, the students and their parents by means of questionnaire; and quantitative survey on the students’ scores from junior 1 to senior 2 (5 years) in the secondary school by analyses between those (over 30 students) who had learned English for 3 years in the elementary school and those (over 400 students) who hadn’t. Finally conclusions that “‘the younger, the better’ in FLL is doubted, FLES shouldn’t be done in lower grades of elementary school in China, qualified English teacher is the decisive factor on FLES” were drawn from the surveys on FLES in China.
Full Text: PDF
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English Language Teaching ISSN 1916-4742 (Print) ISSN 1916-4750 (Online)
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Texas IntroductionEdit This Page
From FamilySearch Wiki
United States Texas Introduction
Adopt-a-wiki page
This page adopted by:
TXGenWeb Project
who welcome you to contribute.
Adopt a page today
This set of Wiki pages describes major sources of information about families from Texas. As you read this outline, study the United States set of Wiki pages, which will help you understand terminology and the contents and uses of genealogical records.
Records of the Family History Library
The Family History Library has many of the records described in these Texas Wiki pages. The library's major holdings include vital, probate, land, court, history, military, church, cemetery, and naturalization records. Most of these have been microfilmed at the county courthouses.
Some of the sources described in this set of Wiki pages list the Family History Library's book, microfilm, and microfiche. These are preceded by FHL, the abbreviation for Family History Library. You can use these numbers to locate materials in the library and to order microfilm and microfiche at Family History Centers
Family History Library Catalog
The library's records are listed in the Family History Library Catalog found at the library and at each Family History Center. The catalog is available in a online or microfiche version. To find a record, look in the Place Search of the catalog for:
• The place where your ancestor lived, such as:
UNITED STATES - CENSUS
TEXAS - HISTORY
TEXAS, BEXAR - VITAL RECORDS
TEXAS, BEXAR, SAN ANTONIO - CHURCH RECORDS
• The record type you want to search, such as:
UNITED STATES - CENSUS
TEXAS - HISTORY
TEXAS, BEXAR - VITAL RECORDS
TEXAS, BEXAR, SAN ANTONIO - CHURCH RECORDS
The page titles in the Texas Wiki pages match the names of record types used in the catalog.
Need additional research help? Contact our research help specialists.
Need wiki, indexing, or website help? Contact our product teams.
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• This page was last modified on 23 September 2011, at 15:44.
• This page has been accessed 581 times.
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Leichhardt Municipal Council
Leichhardt Municipal Council is located in inner west Sydney in New South Wales. The Council covers an area of 11 km² and has a population of 51,127.
Locality Plan
How to embed a Google Map in a page
Software Systems
Asset Management System: JRA Lifecycle Asset Knowledge Management System
Document/Records Management System: Unknown
Financial System: FinanceOne
Geographical Information System: ArcGIS and Latitude
Contact Information
Street Address: 7-15 Wetherill Street, Leichhardt NSW
Postal Address: P.O Box 45, Leichhardt NSW 2040
Phone: (02) 9367 9222
Fax: (02) 9367 9111
E-mail: ua.vog.wsn.cml|tdrahhciel#ua.vog.wsn.cml|tdrahhciel
Additional Contacts
Name: Peter Cormican
Position: Manager Assets
E-mail: ua.vog.wsn.cml|cretep#ua.vog.wsn.cml|cretep
Site Members
Neighbouring Councils
About This Page
If you are looking at this page because you work for this Council, please consider signing up to the site and contributing some information or circulating this link to other Council employees who might be interested. Some information about what software systems the Council uses & some contact details would be a good start, but any information that you can contribute to the site, on any topic will be greatly appreciated. There are a lot of good reasons to contribute, and it is pretty easy. Click here for an explanation about how to edit pages.
External Links & References
1. Council Website
2. Wikipedia
Unless otherwise stated, the content of this page is licensed under Creative Commons Attribution-ShareAlike 3.0 License
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Affinity purification of antibodies
From OpenWetWare
Revision as of 06:41, 15 December 2008 by Torsten Waldminghaus (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
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Contents
Curators
Abstract
This protocol describes purification of a polyclonal antibody that might be needed for immuno fluorescens microscopy or co-immuno precipitations. A comman way to do this is to couple the antigen to activated beads on a column, run the serum over it and than after some washing elute the antibody that was bound to the antigen. However this protocol describes a kind of 'affinity substraction'. Instead of the antigen a protein extract of an antigen deletion strain is coupled to the beads. The advantages are that one does not need purified antigen and that one can simply use the flow through instead of having to elute and dialyze. The disadvantage off course is that one needs a deletion strain which might be difficult if the respective protein is essential.
Note:This protocol is currently under developement and testing in the lab. So please watch changes over the next weeks and edit and comment all that you can.
Materials
List everything necessary to perform the protocol here. Include all information about suppliers, ordering details, etc. Links to the suppliers' page on that material are also appropriate and encouraged. Please be aware that users of this protocol may not be working in the same country as you.
Reagents
Biological resources e.g. cell lines, buffers (link to a method for making them), enzymes, chemicals, kits, etc.
Equipment
Any equipment used to perform the protocol (link to a method for using them).
Procedure
Coupling to activated beads
The first step is to couple the antigen to activated beads. In the following procedure CNBr-activated beads from Sigma are used and the procedure is modified from their product information (sigma-aldrich)
1. Dissolve protein to be coupled in 0.1 M NaHCO3 buffer containing 0.5 M NaCl, pH 8.3-8.5 (about 5-10 mg protein per mL of gel). Note: Other buffers can be used, but avoid amine-containing buffers such as Trizma or other nucleophiles (buffers with amino groups) which will react with the binding sites.
2. Wash and swell cyanogen-bromide activated resin in cold 1 mM HCl for at least 30 minutes. A total of 200 ml per gram of dry gel is added in several aliquots. Remove the supernatant (contains lactose) by gentle suction in a Büchner or suction funnel between successive additions. Note: Lactose is necessary to stabilize the beads during freeze-drying, but it will interfere with binding if present during coupling. The use of HCl preserves the activity of the reactive groups which hydrolyze at high pH.
3. Wash the resin with distilled water, 5 - 10 column volumes, then wash the resin with the NaHCO3/NaCl coupling buffer (5 ml per gram dry gel) and immediately transfer to a solution of the ligand in coupling buffer. Note: The reactive groups hydrolyze in basic solution!
4. Mix protein with gel for 2 hours at room temperature or overnight at 4°C. Use a paddle stirrer or end-over-end mixer, but not a magnetic stir bar (which may grind beads).
5. Wash away unreacted ligand using NaHCO3/NaCl coupling buffer described above.
6. Block unreacted groups with either 1 M ethanolamine or 0.2 M glycine, pH 8.0 for 2 hours at room temperature or 16 hours at 4°C.
7. Wash extensively to remove the blocking solution, first with basic coupling buffer at pH ≈ 8.5, then with acetate buffer (0.1 M, pH 4) containing NaCl (0.5 M).
8. Complete this wash cycle of high and low pH buffer solutions four or five times.
9. If the resin is to be used immediately, equilibrate it in buffer. If not, store the resin in 1.0 M NaCl at 2-8°C with a suitable bacteriostat.
Critical steps
Referenced from the main protocol, a more thorough explanation of particularly important steps in the protocol.
Troubleshooting
Referenced from the main protocol, an explanation of what can cause things to go wrong with the protocol.
Notes
Referenced from the main protocol, any comments about the protocol should be made here; i.e. how it was developed. Any comments added should be signed (by adding *'''~~~~''': in front) and explained. Links to FAQs/tips provided by other sources such as the manufacturer or other websites would be best made here.
Anecdotal observations that might be of use to others can also be posted here; e.g. 'my cells were still floating'.
It might also be good to add an image to show the workflow and timescales for experiment planning.
Acknowledgments
Acnkowledge any help you had in development, testing, writing this protocol.
References
See OpenWetWare:Biblio for information on how to reference within a wiki.
Specific Protocols
Add links to all the OWW protocols that have been used in making the consensus.
Discussion
You can discuss this protocol.
Tag this page with categories to allow easier indexing and searching. See Categories for information on existing categories.
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Synthetic Biology:Conferences
From OpenWetWare
Revision as of 11:29, 27 October 2009 by Anthony C. Forster (Talk | contribs)
Jump to: navigation, search
Go to http://syntheticbiology.org/
Synthetic Biology is
A) the design and construction of new biological parts, devices, and systems, and
B) the re-design of existing, natural biological systems for useful purposes.
Home About Conferences Labs Courses Resources FAQ
Upcoming Events
Previous events
2008
2007
• BioSysBio 2007: Computational Systems Biology, Bioinformatics, Synthetic Biology
January 11-13, 2007 in Manchester, United Kingdom
2006
2005
This site is hosted on OpenWetWare and can be edited by all members of the Synthetic Biology community.
Making life better, one part at a time.
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Quotation added by staff
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Anger is the most impotent of passions. It effects nothing it goes about, and hurts the one who is possessed by it more than the one against whom it is directed. Clarendon
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In politics, nothing is contemptible. Disraeli, Benjamin
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Quotation added by staff
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We are enslaved by anything we do not consciously see. We are freed by conscious perception. Howard, Vernon
This quote is about conflict · Search on Google Books to find all references and sources for this quotation.
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Vernon Howard saw through the illusion of suffering and fear and loneliness. From 1965 until his death in 1992 he wrote books and conducted classes which reflect a degree of skill and understanding that may be unsurpassed in modern history. His warmth and refreshing sense of humor made him a delightful subject for interviews, talk shows and articles. Today more than 7 million readers worldwide enjoy his exceptionally clear and inspiring presentations of the great truths of the ages. Libraries, bookstores, health food stores and church bookshops all over the US sell Vernon Howard books, booklets, cassette tapes and videotapes. His material is widely used by doctors, psychiatrists, psychologists, clergymen, counselors, educators and people from all walks of life. Many of Vernon Howard's class talks are available on cassette tape. All his teachings center around one grand theme: "There is a way out of the human problem and anyone can find it."
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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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When you pray for anyone you tend to modify your personal attitude toward him. Peale, Norman Vincent
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glen davis
Glen Davis to Magic for Brandon Bass
UPDATE: Sherrod Blakely says it's a done deal. UPDATE: Marc Spears reports the trade will happen. Dan Duggan of the Herald has the scoop on this rumor: The Celtics have apparently decided that Glen Davis’ future is better elsewhere. According to a league source, the Cs are in discussions with Orlando to send Big Baby, [...]
December 9, 2011 Chuck - Red's Army Uncategorized 8
Your Morning Dump… Hornets with lukewarm interest in Celtics offers
Every morning, we compile the links of the day and dump them here… highlighting the big storyline. Because there's nothing quite as satisfying as a good morning dump. The Celtics have reportedly changed their stance on whether they'd require a long-term commitment before trading for Paul. It was believed to be a requisite to a [...]
December 7, 2011 Chuck - Red's Army Uncategorized 13
Danny Ainge dials up Glen Davis
CSNNE's Sherrod Blakely reports that Danny Ainge called Glen Davis today – the first day teams were able to contact free agents: "It's very important," John Hamilton, Davis' agent, told CSNNE.com. "It shows Danny and the Celtics appreciate the contributions he has made to the club, and are open to seeing if they can continue [...]
December 5, 2011 Chuck - Red's Army Uncategorized 3
Glen Davis channels his inner Junkyard Dog
Glen Davis tweeted out a couple of photos (there's this one too) of himself at the gym. Not only does Baby look like he's in decent shape… we also appreciate the homage (intentional or otherwise) to one of our favorite wrestlers of all time: Junkyard Dog! One thing JYD had that Baby doesn't is a [...]
December 5, 2011 RedsArmyAdmin Uncategorized Comments Off
In the GreenLab: Defeating the Under Screen
Welcome to the new series here at RedsArmy called "GreenLab" where I will delve into the film room and examine some of the Celtics plays. If you've been a regular reader of RedsArmy then you'll remember a couple of my previous posts from last season, going over the excellent "2-3 Arc" set (my term, not [...]
December 2, 2011 Jay Uncategorized 2
Your Morning Dump… Is Rondo’s attitude wearing on his teammates?
Every morning, we compile the links of the day and dump them here… highlighting the big storyline. Because there's nothing quite as satisfying as a good morning dump. CSN New England’s Donny Marshall — the former NBA player who covers the Celtics and does some color commentary for the team’s broadcasts — was on NBC’s SportsTalk [...]
December 2, 2011 Chuck - Red's Army Uncategorized 4
Danny Ainge: I’m not trying to trade Rajon Rondo
Danny Ainge and Doc Rivers just wrapped up their first news conference of the upcoming season, and the big topic, of course, was all the trade rumors swirling around the team right away. The highlights: When asked "are you actively trying to trade Rajon Rondo?" Danny replied "I'm not." Danny called his core group of [...]
December 1, 2011 RedsArmyAdmin Uncategorized 2
Your Morning Dump… Where the C’s are concerned about Chris Paul’s knee
Every morning, we compile the links of the day and dump them here… highlighting the big storyline. Because there's nothing quite as satisfying as a good morning dump. Slowly but surely, the talk of Chris Paul coming to Boston is beginning to die a slow death. [...] One of the reasons that's giving the C's some [...]
December 1, 2011 RedsArmyAdmin Uncategorized 5
The new CBA hurts more than helps this year
How the new CBA affects every NBA team – ESPN via kwout I guess this is the kind of effect small market teams wanted out of the new deal. ESPN put together how the new CBA helps and hurts both short and long term. The biggest impact on the Celtics is in the short term. [...]
November 30, 2011 RedsArmyAdmin Uncategorized Comments Off
Jackie Mac on Baby: “He drove [the Celtics] crazy”
It gets no clearer than what Jackie MacMullan said about Glen Davis on the WEEI today. “You have wonder what was going on inside his head. He had a weight clause in his contract and he had to keep a certain weight. You take a picture of Big Baby from November to April and it’s [...]
November 29, 2011 RedsArmyAdmin Uncategorized 2
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Thread Tools Search this Thread
Posts: 93 | Thanked: 198 times | Joined on Feb 2012
#1
I build the newest xu4 1.0beta4 package (http://xu4.sourceforge.net/) for the n900.
The original Ultima IV is freeware. In the package ultima4-data the data-file is downloaded in the postinst script from http://www.ultima-universe.com/downl...ltima4v101.zip and saved in /opt/xu4/share/games/ultima4/ultima4.zip. This means you have to be online, if you install ultima4-data.
The package xu4 contains the game interface. I uploaded also ultima4-graphics-upgrade to Extras-devel non-free. This package upgrades Ultima IV to 256 color graphics with MIDI support.
I changed the key mapping from alt + key to FN (blue arrow) + key.
In the Menu you have to press the starting letter
• j - continue playing
• i - new game
• c - configure
• a -about
In the Game you can press FN+h for a list key mapping.
You can enable fullscreen in video options.
I am playing at the moment with the following settings:
At the moment i have set the game cycles to 15.
I disabled the mouse.
I set the keyboard repeat Interval to 10 msec.
If you press G and you are not over a chest, then the game crashes.
You have to kill the game with
killall -9 u4
Have Fun!
Last edited by HolgerN; 05-09-2012 at 11:26 AM.
The Following 8 Users Say Thank You to HolgerN For This Useful Post:
Posts: 782 | Thanked: 315 times | Joined on May 2010
#2
just tried this and it has no sound...
Posts: 23 | Thanked: 2 times | Joined on Nov 2010
#3
I've also noticed no sound, but that isnt a big problem.
What is causing me problems is that I cant seem to type the number "1".
Therefore I can't ready a weapon for "1" or buy 10 packets of food!
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Australian Bureau of Statistics
Celebrating the International Year of Statistics 2013
ABS Home > Statistics > By Release Date
6354.0 - Job Vacancies and Overtime, Australia, Feb 1999
Previous ISSUE Released at 11:30 AM (CANBERRA TIME) 01/04/1999
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Australian Bureau of Statistics
Celebrating the International Year of Statistics 2013
ABS Home > Statistics > By Release Date
8731.2 - Building Approvals, Victoria, Sep 1994
Previous ISSUE Released at 11:30 AM (CANBERRA TIME) 11/08/1994
Past Releases
First Release
• First Issue: Apr 1959
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Australian Bureau of Statistics
Celebrating the International Year of Statistics 2013
ABS Home > Statistics > By Catalogue Number
4430.0 - Disability, Ageing and Carers, Australia: Summary of Findings, 2009 Quality Declaration
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MEDIA RELEASE
16 December 2010
Embargoed: 11.30 am (Canberra time)
163/2010
Percentage of Australians disabled due to physical health conditions decreased
Four million Australians (18.5%) had a disability in 2009, according to findings from the 2009 Survey of Disability, Ageing and Carers released today by the Australian Bureau of Statistics (ABS).
Since 2003, the prevalence of disability in Australia has dropped by 1.5 percentage points. The proportion of Australians disabled because of physical health conditions such as asthma and back problems declined. In 2003, 6.8% of Australians were disabled because of musculoskeletal disorders like arthritis and back problems, while only 6.5% reported such a disability in 2009. Asthma-related disability also declined, from 0.8% in 2003 to 0.5% in 2009. In particular, the proportion of children (aged 0 to 17 years) disabled by asthma almost halved since 2003, from 0.9% to 0.5%.
Older people were more likely to have a disability, affecting almost nine out of ten (88%) of those aged 90 years and over, compared to only 3.4% of those aged four years and under. Males and females were similarly affected by disability.
The percentage of Australians involved in caring for a person with a disability or an older person also declined since 2003, in line with the decrease in disability prevalence. In 2009, 12% of people (living in households) were carers who provided assistance to someone requiring help because of disability or old age; down from 13% in 2003.
Just under one third of carers (29%) were primary carers; that is, they provided the majority of the informal help needed by a person with a disability or an older person. In 2009, just over two thirds of primary carers were women (68%).
Further details are in Disability, Ageing and Carers, Australia: Summary of Findings, 2009 (cat. no. 4430.0).
Media note: When reporting ABS data you must attribute the Australian Bureau of Statistics (or the ABS) as the source.
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Health effects of 1,1-dichloroethene
Health effects of 1,1-dichloroethene
This article has been reviewed by the following Topic Editor: Sidney Draggan Ph.D.
Introduction
1,1-Dichloroethene—also known as vinylidene chloride—is a chemical used to make certain plastics (for example, packaging materials, flexible films like SARAN wrap, and flame-retardant coatings for fiber and carpet backing. It is a colorless liquid that evaporates quickly at room temperature. It has a mild sweet smell and burns quickly.
1,1-Dichloroethene is a man-made chemical and is not found naturally in the environment. Although 1,1-dichloroethene is manufactured in large quantities, most of it is used to make other substances or products such as polyvinylidene chloride
Pathways for 1,1-dichloroethene in the environment
1,1-Dichloroethene can enter the environment when it is released to the air during its production or released to surface water or soil as a result of waste disposal. Most 1,1-dichloroethene evaporates quickly and mainly enters the environment through the air, although some enters into rivers or lakes. 1,1-Dichloroethene can enter soil, water, and air in large amounts during an accidental spill. 1,1-Dichloroethene can also enter the environment as a breakdown product of other chemicals in the environment.
1,1-Dichloroethene behaves differently in air, water, and soil. 1,1-Dichloroethene evaporates to the air very quickly from soil and water. In the air, 1,1-dichloroethene is broken down by reactive compounds formed by sunlight. 1,1-Dichloroethene remains in the air for about 4 days.
From water, 1,1-dichloroethene evaporates into the air; it breaks down very slowly in water. We do not know exactly how long 1,1-dichloroethene stays in water. It is not readily transferred to fish or birds, and only very small amounts enter the food chain.
In soil, 1,1-dichloroethene either evaporates to the air or percolates down through soil with rainwater and enters underground water. Small living organisms in soil and groundwater may transform it into other less harmful substances, although this happens slowly.
Exposure to 1,1-dichloroethene
You may be exposed to 1,1-dichloroethene by breathing it when it is in the air or eating food or water that contains it. You may also be exposed to 1,1-dichloroethene if it touches your skin. 1,1-Dichloroethene is found at very low levels in indoor and outdoor air (estimated as less than 1 part per trillion parts of air [ppt]). Therefore, the potential for exposure in the environment is extremely low. The amounts are somewhat higher near some factories that make or use 1,1-dichloroethene (those that make food-packaging films, adhesives, flame-retardant coatings for fiber and carpet backing, piping, and coating for steel pipes), hazardous waste sites, and areas near accidental spills. The exact amount of 1,1-dichloroethene in the air near these factories is not known. In air around waste sites where it has been identified, the amount of 1,1-dichloroethene ranges from 0.39 to 97 parts 1,1-dichloroethene per billion parts of air (ppb, 1 ppb is 1,000 times more than 1 ppt). The levels of 1,1-dichloroethene in air around waste sites are usually much lower than those that have caused health effects in animals. We estimate that 1,1-dichloroethene contaminates the air around 97 chemical factories in the United States. Factories that make 1,1-dichloroethene are mainly located in Texas and Louisiana. Measured air levels inside manufacturing plants range from less than 5 to 1,900 parts 1,1-dichloroethene per million parts of air (ppm, 1 ppm is 1,000 times more than 1 ppb).
A small percentage (3%) of the drinking water sources in the United States contain low amounts of 1,1-dichloroethene (0.2-0.5 ppb with an estimated average of 0.3 ppb). The amounts are very low compared with levels that are expected to affect human health. The concentration of 1,1-dichloroethene in groundwater samples from hazardous waste sites ranged from 0.001 to 0.09 ppm.
Since 1,1-dichloroethene is used to make some consumer products, exposure might occur while these products are made or used. For example, the estimated average amount of 1,1-dichloroethene in plastic food-packaging
Health effects of 1,1-dichloroethene
You should know that one way to learn whether a chemical will harm people is to determine how the body absorbs, uses, and releases the chemical. For some chemicals, animal testing may be necessary. Animal testing may also help identify such health effects as cancer or birth defects. Without laboratory animals, scientists would lose a basic method for getting information needed to make wise decisions that protect public health. Scientists have the responsibility to treat research animals with care and compassion. Scientists must comply with strict animal care guidelines because laws today protect the welfare of research animals.
Additionally, there are vigorous national and international efforts to develop alternatives to animal testing. The efforts focus on both in vitro and in silico approaches and methods. For example, the National Toxicology Program (NTP) at the National Institute of Environmental Health Sciences (NIEHS) created the NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) in 1998. The role of NICEATM is to serve the needs of high quality, credible science by facilitating development and validation—and regulatory and public acceptance—of innovative, revised test methods that reduce, refine, and replace the use of animals in testing while strengthening protection of human health, animal health and welfare, and the environment. In Europe, similar efforts at developing alternatives to animal based testing are taking place under the aegis of the European Centre for the Validation of Alternative Methods (ECVAM).
How a chemical affects your health depends on how much you are exposed to and for how long. As the level and length of your exposure increase, the effects are likely to become more severe. Information on the health effects in humans after breathing 1,1-dichloroethene is insufficient. People who breathed high amounts of 1,1-dichloroethene in a closed space lost their breath and fainted. Some people who breathed 1,1-dichloroethene at work for several years had abnormal liver function. However, exposure to other chemicals may have also contributed to this effect. Available information indicates that prolonged inhalation of 1,1-dichloroethene can induce adverse neurological effects and is possibly associated with liver and kidney damage in humans. Studies in animals indicate that 1,1-dichloroethene can affect the normal functions of the liver, kidneys, and lungs. However, the amount of 1,1-dichloroethene in the air to which the animals were exposed was much higher than the amounts in the air that the general public usually breathes. Some animals that breathed large amounts of 1,1-dichloroethene died within a few days. The liver and kidneys of animals were affected after breathing air that contained 1,1-dichloroethene for days, months, or years. After pregnant rats breathed 1,1-dichloroethene in air, some of the newborn rats had birth defects.
We have no information on health effects in humans who ate food or drank water that contained 1,1-dichloroethene. Animals fed food that contained 1,1-dichloroethene or that had 1,1-dichloroethene placed experimentally in their stomachs developed liver and kidney disease, and some even died. These amounts, however, were very much higher than those which occur in drinking water supplies. Birth defects did not occur in the newborn of female rats that drank 1,1-dichloroethene.
Spilling 1,1-dichloroethene on your skin or in your eyes can cause irritation. We do not know what other health effects might occur if 1,1-dichloroethene comes into contact with your skin for long periods. However, no serious effects or deaths occurred in mice after small amounts of 1,1-dichloroethene were put on their skin over a period of months. We do not know whether spilling 1,1-dichloroethene on your skin can cause birth defects or affect fertility.
We do not know whether coming into contact with 1,1-dichloroethene increases the risk of cancer in humans. Evidence from epidemiology studies of workers exposed to 1,1-dichloroethene is inconclusive. Several studies examined the possibility that 1,1-dichloroethene may increase the risk of cancer in animals. Only one of these studies indicated that mice breathing 1,1-dichloroethene for 1 year developed kidney cancer, but the particular type of mouse used may be especially sensitive to 1,1-dichloroethene.
The U.S. Department of Health and Human Services has not classified 1,1-dichloroethene with respect to carcinogenicity. The International Agency for Research on Cancer (IARC) has determined that 1,1-dichloroethene is not classifiable as to its carcinogenicity in humans. The EPA has determined that 1,1-dichloroethene is a possible human carcinogen. NTP does not include it in its list of substances expected to be human carcinogens.
Medical tests for exposure to 1,1-dichloroethene
1,1-Dichloroethene can be measured in the breath, blood, urine, and body tissues of individuals who come in contact with the chemical. However, only relatively high levels of 1,1-dichloroethene in body tissues and fluids can be measured. Because breath samples are easily collected, tests of exhaled air are now the most common way to tell whether a person has been exposed to high levels of 1,1-dichloroethene. One of the breakdown products of 1,1-dichloroethene, dithioglycolic acid, can also be measured in urine. None of these tests are regularly available at a doctor's office because they require special equipment, but your doctor can tell you where you can get the tests done. Although these tests can prove that a person has been exposed to 1,1-dichloroethene, they cannot tell if any health effects will occur. Since most of the 1,1-dichloroethene leaves the body within a few days, these methods are best for determining whether exposures have occurred within the last several days. Detection of 1,1-dichloroethene or its breakdown products in the body may not necessarily mean that exposure to 1,1-dichloroethene alone has occurred. People exposed to 1,1-dichloroethene at hazardous waste sites were probably also exposed to other organic compounds, that produce breakdown products similar to those of 1,1-dichloroethene. Other methods for measuring the effects associated with exposure to 1,1-dichloroethene (such as reduced enzyme levels) are not specific enough to detect effects caused by exposure to 1,1-dichloroethene alone.
Further Reading
Disclaimer: This article is taken wholly from, or contains information that was originally published by, the Agency for Toxic Substances and Disease Registry. Topic editors and authors for the Encyclopedia of Earth may have edited its content or added new information. The use of information from the Agency for Toxic Substances and Disease Registry 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
ATSDR (Content Source);Sidney Draggan Ph.D. (Topic Editor) "Health effects of 1,1-dichloroethene". 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 26, 2008; Last revised Date March 26, 2008; Retrieved May 18, 2013 <http://www.eoearth.org/article/Health_effects_of_1,1-dichloroethene>
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Alexandra Park St Andrew, MiddlesexEdit This Page
From FamilySearch Wiki
England Middlesex Middlesex Parishes Alexandra Park St Andrew
Alexandra Park St Andrew, Middlesex family history and genealogy research page. Guide to parish registers (baptisms, christenings, marriages, and burials), civil registration (births, marriages, and deaths), census records, history, wills, cemetery, online transcriptions and indexes, an interactive map and website resources.
Contents
Church History
Alexandra Park St Andrew was a district church built in 1900 and stood within the civil parish boundaries of both Clerkenwell and Tottenham All Hallows parishes.
Resources
Civil Registration
Birth, marriages and deaths were kept by the government, from July 1837 to the present day. The civil registration article tells more about these records. There are several Internet sites with name lists or indexes. A popular site is FreeBMD.
Church records
To find the names of the neighbouring parishes, use England Jurisdictions 1851. In this site, search for the name of the parish, click on the location "pin", click Options and click List contiguous parishes.
Include here information for parish registers, Bishop’s Transcripts and other types of church records, such as parish chest records. Add the contact information for the office holding the original records. Add links to the Family History Library Catalog showing the film numbers in their collection
Census records
Census records from 1841-1891 are available on film through a Family History Center or at the Family History Library. The first film number is 438773. To view these census images online, they are available through the following websites for a fee ($) or free:
• FamilySearch has some of the British Censuses available.
• FindMyPast ($) has all available census records including images, and is free at Family History Centers and the Family History Library and some public and academic libraries.
• Ancestry.co.uk ($) has now all available census records but free at Family History Centers and the Family History Library and at numerous public and academic libraries. The library versions are known as AncestryInstitution.com.
• The Genealogist.co.uk ($) has all available censuses and is free at Family History Centers and the Family History Library and various other libraries.
• FreeCen is a UK census searches. It is not complete and individuals are always asked to consider helping out with transcriptions.
Probate records
Records of wills, administrations, inventories, indexes, etc. were filed by the court with jurisdiction over this parish. Go to Middlesex Probate Records to find the name of the court having primary jurisdiction. Scroll down in the article to the section Court Jurisdictions by Parish.
Maps and Gazetteers
Maps are a visual look at the locations in England. Gazetteers contain brief summaries about a place.
Web sites
References
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 9 November 2012, at 18:56.
• This page has been accessed 296 times.
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Gaston County, North Carolina
From FamilySearch Wiki
(Difference between revisions)
(cw units)
(added gaston county students in college)
Line 82: Line 82:
==== Schools ====
==== Schools ====
*[http://www.lib.unc.edu/ncc/gbnf/shc.html Sacred Heart College] - founded in 1892 by the order of the Roman Catholic Sisters of Mercy. Closed in 1987.
+
*[http://www.lib.unc.edu/ncc/gbnf/shc.html Sacred Heart College] - founded in 1892 by the order of the Roman Catholic Sisters of Mercy. Closed in 1987.
+
*[http://www.ncgenweb-data.com/ybook/resultlist.php?county=gaston Gaston County College Students] - a name list of students at various NC colleges with identified hometowns in the county.
==== Taxation ====
==== Taxation ====
Revision as of 20:46, 12 February 2012
Gaston County, North Carolina
Map
Location in the state of North Carolina
Location of North Carolina in the U.S.
Facts
Founded December 21, 1846
County Seat Gastonia
Courthouse
Adopt-a-wiki page
This page adopted by:
NCGenWeb Project
who welcome you to contribute.
County Coordinator
Gaston Co. NCGenWeb
Adopt a page today
United States North Carolina Gaston County
Contents
County Courthouse
History
Parent County
1846--Gaston County was created 21 December 1846 from Lincoln County.
County seat: Gastonia [1]
Boundary Changes
Record Loss
1874--Courthouse fire destroyed many court records.
Places/Localities
Populated Places
Neighboring Counties
Resources
Cemeteries
Church
Court
Land
Local Histories
Maps
Migration
Early migration routes to and from Gaston County for European settlers included:[2]
Military
Civil War
Civil War Confederate units - Brief history, counties where recruited, etc.
-11th Regiment, North Carolina Infantry
Newspapers
Probate
Schools
Taxation
Vital Records
Societies and Libraries
Family History Centers
Web Sites
References
1. The Handybook for Genealogists: United States of America,10th ed. (Draper, UT:Everton Publishers, 2002).
2. Handybook for Genealogists: United States of America, 10th ed. (Draper, Utah: Everton Pub., 2002), 847-61. (FHL Book 973 D27e 2002) WorldCat entry., and William E. Myer, Indian Trails of the Southeast. (Nashville, Tenn.: Blue and Gray Press, 1971), 12-14, and the book's pocket map "The Trail System of the Southeastern United States in the Early Colonial Period" (1923). (FHL Book 970.1 M992i) WorldCat entry.
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Help with finding maya converter to C code
Newbie Member
22Dec2009,17:34 #1
Hello,
I tried to look for program that can convert 3D image in maya to C code.
I have to find a program that can do that as fast as possible because I have to finish my thesis (which is a 3D paccman game in C language) in 5 mounths from now.
Please, if anyone knows any programs that can do that, please give me a download link.
It's really important.
Thank you very much for all the helpers and your time,
Ofir.
Skilled contributor
22Dec2009,17:49 #2
very interesting question..i have worked on 3D effects in C. As far as my knowledge is concerned 3D effects in C in not so usual nd easy. The max knowledge i have in is making " A star field effect which u see in screensavers" in C. But the complete paccman game is a big deal. I have to try that out.
Newbie Member
22Dec2009,18:04 #3
thank you very much for your help, I really appreciate it.
If you find something that could help me, send me a message.
and thank you again.
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Publication Listing
You are not logged in. If you create a free account and sign in, you will be able to customize what is displayed.
Contents (view Concise Listing)
Verification Status
Reference Status
Primary Verified by Mhhutchins on 2010-09-07 12:19:23
Clute/Nicholls Not Verified
Clute/Grant Not Verified
Contento1 (anth/coll) Not Verified
Locus1 Not Verified
Reginald1 Not Verified
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Copyright (c) 1995-2011 Al von Ruff.
ISFDB Engine - Version 4.00 (04/24/06)
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Pharmaceuticals 2012, 5(12), 1265-1281; doi:10.3390/ph5121265
Article
Cell Penetrating Peptoids (CPPos): Synthesis of a Small Combinatorial Library by Using IRORI MiniKans
1 Institute of Organic Chemistry, Karlsruhe Institute of Technology (KIT), Fritz Haber Weg 6, 76131 Karlsruhe, Germany 2 Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany
* Author to whom correspondence should be addressed.
Received: 10 October 2012; in revised form: 13 November 2012 / Accepted: 14 November 2012 / Published: 23 November 2012
(This article belongs to the Special Issue Cell-penetrating Peptides)
Download PDF Full-Text [1121 KB, Updated Version, uploaded 26 November 2012 14:39 CET]
The original version is still available [1092 KB, uploaded 23 November 2012 09:07 CET]
Abstract: Cell penetrating peptoids (CPPos) are potent mimics of the corresponding cell penetrating peptides (CPPs). The synthesis of diverse oligomeric libraries that display a variety of backbone scaffolds and side-chain appendages are a very promising source of novel CPPos, which can be used to either target different cellular organelles or even different tissues and organs. In this study we established the submonomer-based solid phase synthesis of a “proof of principle” peptoid library in IRORI MiniKans to expand the amount for phenotypic high throughput screens of CPPos. The library consisting of tetrameric peptoids [oligo(N-alkylglycines)] was established on Rink amide resin in a split and mix approach with hydrophilic and hydrophobic peptoid side chains. All CPPos of the presented library were labeled with rhodamine B to allow for the monitoring of cellular uptake by fluorescent confocal microscopy. Eventually, all the purified peptoids were subjected to live cell imaging to screen for CPPos with organelle specificity. While highly charged CPPos enter the cells by endocytosis with subsequent endosomal release, critical levels of lipophilicity allow other CPPos to specifically localize to mitochondria once a certain lipophilicity threshold is reached.
Keywords: combinatorial chemistry; split and mix; library; peptoid; IRORI; sub-monomer; cell penetrating
Article Statistics
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Cite This Article
MDPI and ACS Style
Kölmel, D.K.; Fürniss, D.; Susanto, S.; Lauer, A.; Grabher, C.; Bräse, S.; Schepers, U. Cell Penetrating Peptoids (CPPos): Synthesis of a Small Combinatorial Library by Using IRORI MiniKans. Pharmaceuticals 2012, 5, 1265-1281.
AMA Style
Kölmel DK, Fürniss D, Susanto S, Lauer A, Grabher C, Bräse S, Schepers U. Cell Penetrating Peptoids (CPPos): Synthesis of a Small Combinatorial Library by Using IRORI MiniKans. Pharmaceuticals. 2012; 5(12):1265-1281.
Chicago/Turabian Style
Kölmel, Dominik K.; Fürniss, Daniel; Susanto, Steven; Lauer, Andrea; Grabher, Clemens; Bräse, Stefan; Schepers, Ute. 2012. " Cell Penetrating Peptoids (CPPos): Synthesis of a Small Combinatorial Library by Using IRORI MiniKans." Pharmaceuticals 5, no. 12: 1265-1281.
Pharmaceuticals EISSN 1424-8247 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
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Activity Not Available
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AltiMario
CTO at OpenPRJ
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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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Very Low Activity
Estimated Cost
Analyzed 1 day ago based on code collected 1 day ago.
Project Cost Calculator
$ .00
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*Using the Basic COCOMO Model
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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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Very Low Activity
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Default
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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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Human-friendly or Search-Engine-Optimized-Friendly Title Tags?
Jun 20, 2006 • 4:06 pm | (1) by | Filed Under SEO - Search Engine Optimization
Just when you may have thought that writing a good, optimized page title tag was elementary SEO, someone suggests that maybe they should be written for humans, too. And, as this Cre8asiteforums discussion suggests, even that is not enough.
Thought provoking discussion in How to write SEO friendly TITLE tag? Ron Carnell rocks the boat with:
"Yes, the Title should focus on the visitor, not just the search engine, but I doubt many visitors specifically go looking for Calls to Action or long, grammatically correct sentences in the web page Title. Just as with a book, just as with a movie, just as with a song on the radio, the web page Title should capture interest succinctly, even at times, tersely. Your call to action, I think, should be in the SERP description, not the SERP Title."
Previous story: MSN Reporting Less Links For Web Sites
blog comments powered by Disqus
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Sunday, July 3, 2011
Our Supreme Corporate Court: 3 Strikes and We’re Out!
Activist Post
by Gary Corseri, Contributing Writer
Supreme Court or Corruption?
First, some aphoristic opals:
“Eternal vigilance is the price of liberty.” -- Wendell Phillips (1811-1884), abolitionist, orator and columnist for The Liberator.
“Truth can never be told so as to be understood, and not be believ’d.” -- William Blake
“Morality is the custom of one’s country and the current feeling of one’s peers. Cannibalism is moral in a cannibal country.” -- Samuel Butler
What is Truth? Is often asked, as though it were harder to say what truth is than what anything else is. But what is Justice? What is anything? An eternal contradiction in terms meets us at the end of every enquiry. We are not required to know what truth is, but to speak the truth, and so with justice.” – Samuel Butler
“Sin is not hurtful because it is forbidden, but it is forbidden because it is hurtful.” – Benjamin Franklin
“You want to be very careful about lying; otherwise you are nearly sure to get caught.”--Mark Twain
Is there a common thread to these statements? Each writer/thinker/orator is training a highly honed mind upon some of the profoundest concepts of our frail human intellect and imagination: liberty; truth and lying; morality and sin. Each brief statement is a flourishing note—the memorable, essential solo arpeggio in the midst of the orchestral performance. But… beyond the particular insight or theme, each author shares a certain quality of mind—the ability to probe deeper, to turn the mundane or jejune or vapid idea on its head: to look within the essence of the question and oneself… to rotate the squares of the Rubik’s Cube till one gets just the right fit.
Now consider this statement by Justice Antonin Scalia on the Supreme Court’s recent decision to nullify the state of California’s ban on selling “gory” videos to minors:
“Grimm’s Fairy Tales, for example, are grim indeed. As her just desserts for trying to poison Snow White, the wicked queen is made to dance in red hot slippers ‘till she fell dead on the floor.”
What’s missing?
Well, as Wordswoth once responded to a noisome fellow who claimed he could write as well as he—if only he had a mind to: “It is clear that the only thing missing is the mind.”
It is not just that Justice Scalia is making a false analogy, comparing apples and eggs—two very different media—the interactive, sensory-flooding world of “Mortal Kombat,” for example, with the word-by-word, progressive-sequential approach of the literate world… but, also, he seems to have missed a key point. Snow White—and not even an “avatar” of Snow White—is not the agent of the wicked queen’s demise. The queen’s wretched end is a consequence of her violation of higher moral codes—and the ultimate “enforcer” is not some kid with a joystick, but… fate.
Perhaps it is wrong to expect a higher level of thought from our Supreme Court justices? After all, they are not charged with upholding wisdom; merely with the far-easier task of upholding our Constitution—with all its faults.
And just what is this “Constitution,” this “living” document? Reading it, we wander around labyrinths of legalese with various elite interests—slave state vs. commercial; agrarian vs nascent manufacturing—until we come to the fairly clear Bill of Rights.
Except… we’re still trying to figure out “Freedom of Speech”… and, God knows, the Second Amendment is as wide open as Jared Loughner’s surreal gaze. The Constitution is not exactly William Blake’s territory: “Truth can never be told so as to be understood, and not be believed.” More like Butler’s: “We are not required to know the truth, but to speak it, and so with justice.” And in this murky world of truth, half-truths, falsehoods and confusion, the “eternal vigilance” of which Phillips reminds us is the “price of liberty.” And, that vigilance, that review and interpretation is not, ultimately, the province of Supreme Court justices, but is, inviolably, ours—i.e., We the People’s.
Three times in the past 18 months our Supreme Corporate Court has expressed contempt for We the People and elevated the rights and privileges of a select few above the increasingly disenfranchised many. The “prejudice” of these Supremes was clearly manifested in January, 2010 when, according to the New York Times, the Court “ruled that the government may not ban political spending by corporations in candidate elections. … The 5-4 decision was a vindication, the majority said, of the First Amendment’s most basic free speech principle—that the government has no business regulating political speech.” On the other hand, “the dissenters said that allowing corporate money to flood the political marketplace would corrupt democracy.”
Now here’s where things get murky. Nowhere in the Constitution are corporations mentioned. Not until 1819 does the Supreme Court recognize corporations as having some of the “contractual rights” of “persons.” But, while the “rights” of corporations have expanded exponentially in the past couple of centuries, the rights of the People have been abridged. Money, after all, is a marvelous lubricator of “political speech.” While the Court has been telling the wealthy “Full speed ahead,” some 45 million Americans have been getting by on food stamps, and several million more are too worried about their jobs &/or foreclosures to help bankroll local or national candidates. The First Amendment is about Freedom of Speech, Freedom of Expression. It has nothing to do with permitting corporate financial power to overwhelm the free speech of the people—to drown out their voices. Here we are in Mark Twain territory: “You want to be very careful about lying; otherwise, you are nearly sure to get caught.”
The two other instances of Supreme Court-Constitutional perfidies came lickety-split in June, 2011. First the Court decided that 1.5 million female employees of Walmart could not exercise their First Amendment right of Free Speech by uniting in a class-action suit against their alleged gender-biased employer—that global corporation that has helped to finance thousands of factories and sweatshops around the world and driven down wages in the homeland. Again, one thinks of Samuel Butler: “Cannibalism is moral in a cannibal country.” Cannibalizing the working class is fine and dandy, the fat cats caterwaul, but the tasty morsels would be gauche to complain!
Perhaps these salivating cats have not read the First Amendment carefully or they would have understood that the right to petition for governmental redress of grievances also comes within its purview. And, one wonders: if corporations have expanded their rights as persons and have increasingly assumed quasi-governmental powers—often writing legislation through their lobbyists—haven’t We the People the right to petition corporations and our government for a redress of grievances?
The third wave of these judicial outrages came just in time for the 4th of July celebrations of our freedoms! In another 5-4 decision, with Don Scalia writing the majority opinion, the Court effectively told California’s parents they could go screw themselves. (But not in public!)
On John Stewart’s show the other night, I caught a sample of the kind of videos California’s parents did not want sold to their children: an attractive blond in a skin-tight wet-suit was literally being torn apart by two hulking males on either side of her, pulling on her limbs like a chicken’s wishing bone. Guts, blood and gore spill out of the cracked carcass.
Perhaps we should not be surprised that the Court honored the First Amendment Right of Expression of the multi-billion dollar video-“game” industry over the First Amendment Right of millions of Californians to express their opprobrium. (And these citizens, one should note, were not insisting on censorship—they wanted regulation: under the same principles that we regulate the sale of alcohol, tobacco or firearms to minors, or restrict their access to potentially dangerous motor vehicles.) Wise justices might have recalled Ben Franklin: “Sin is not hurtful because it is forbidden, it is forbidden because it is hurtful.”
Probably it is too much to hope, in the majority of these “Justices,” for the quality of mind that can penetrate the great mysteries of life, truth, and morality—not to mention justice and law!. We hope for wisdom and the understanding of great hearts… and we are met with the Wall of the Law. About one hundred and fifty years ago, Chief Seattle of the Duwampo tribe, perceived our fatal dichotomies all too well:
“He gave you laws. … Your religion was written upon tables of stone by the iron finger of your God. … Our religion is the traditions of our ancestors—the dreams of our old men… and it is written in the hearts of our people. … Tribe follows tribe, and nation follows nation, like the waves of the sea. It is the order of nature, and regret is useless. Your time of decay may be distant, but it will surely come, for even the White Man whose God walked and talked with him as friend with friend cannot be exempt from the common destiny.”
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MySource:Pkeegstra/Presidents of Dordt College
Watchers
MySource Presidents of Dordt College
Coverage
Place Sioux Center, Sioux, Iowa, United States
Year range 1957 -
Citation
Presidents of Dordt College.
Repository
URL http://www.dordt.edu/
What would become Dordt College was founded in 1953. Its first President was appointed in 1957.
Presidents of Dordt College
Date Person
1957-1982 Rev. B. J. Haan
1982-1996 Dr. J. B. Hulst
(living)
1996-2012 Dr. Carl E. Zylstra
(living)
2012- Dr. Erik Hoekstra
(living)
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Place:Varginha, Minas Gerais, Brazil
Watchers
NameVarginha
TypeCity
Coordinates21.55°S 45.417°W
Located inMinas Gerais, Brazil
source: Getty Thesaurus of Geographic Names
source: Family History Library Catalog
the text in this section is copied from an article in Wikipedia
Varginha is a city and municipality in southwest Minas Gerais state, Brazil. The estimated population is approximately 123,000 inhabitants and the area of the municipality is 396.39 km². The elevation is 950 mts.
Known throughout Brazil for the supposed appearance of alien creatures in 1996, an episode known as the "ET Varginha."
Varginha stands out as one of the major centers of commerce and coffee production in Brazil and the world, producing excellent quality coffee (Gourmet Coffee), the city is a center for export of coffee draining most of the production of the South mines, making the grain trade with several countries.
The city has a strategic location and, with the shores of Lake Furnas, while equidistant from the three main capitals of Brazil, São Paulo, Rio de Janeiro and Belo Horizonte, was considered by Veja magazine in 2011 as one of the best medium-sized cities in Brazil to live and invest.
The city has an excellent and is nearby one of the country's most important and busiest highways, the BR-381, or Rodovia Fernão Dias, which has recently been widened to four lanes. The city is served by Maj. Brig. Trompowsky Airport .
Ranked among the top cities in the country in standard of living (life expectancy: 73.6; average years of study: 6.15) the city has fifteen health centers in the neighborhoods, three hospitals—one specializing in heart surgery—and two emergency units to attend the population. There is also a regional center for treatment of cancer, which serves a population of three million people in the south of Minas Gerais.
Varginha achieved a certain fame in UFO circles due to the so-called Varginha UFO incident in 1996 [1], in which two extraterrestrial beings were allegedly spotted by locals and later captured by Brazilian Army, along with the local police and fire department. Three girls who saw one of the creatures became quite famous all over Brazil. One of them later converted to an evangelical church. Brazilian UFO researchers fear she can deny having seen the creature because of her new faith.
After this episode known as the "visit from the ET", the city began to invest in "UFO tourism". Today there are bus stops with the shape of spaceships, a huge water tower downtown also in the shape of a spaceship; all with lights and considered quite attractive. In August 2004, UFO researchers from all over Brazil came together at the I UFO Congress of Varginha, organized with the support of the City Hall.
Varginha is located by the Furnas lake where people can practice Aquatic sports, fishing and relaxing.
Research Tips
This page uses content from the English Wikipedia. The original content was at Varginha. 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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Tell me more ×
Answers OnStartups is a question and answer site for entrepreneurs looking to start or run a new business. It's 100% free, no registration required.
I was told by a lawyer that I can file a wage claim against the startup I've been working with for the last 3 years. I am in the middle of equity talks with the owners of the startup. I want to use the wage claim as leverage during equity talks because the owners do not believe my argument that I was underpaid during my first 3 years at the startup; thus, their equity offer is below what I believe it should be.
Background:
I helped start an online startup 3 years ago. I have run the operations at the startup since inception. We're an online educational startup selling training programs in a particular field. The startup is fully owned by two people (not me). I was working for these two people at another company they owned 4 years ago. A year in we started the online startup. Basically, the two owners developed content for a training program used for employees at the original company I worked for, and the online startup idea was to put the content online and sell it to people outside of the original company. My job in the startup was to turn the training program into something that would work for people online, sell it, market it, and train and manage trainees and developers (most everything).
At first, I never worked out equity with the owners, because I was still focusing on my job at the original company (a job in which I never made a dime), but now I only work on the startup. In my first year I agreed to take a small commission, 5% gross, from the sale of training programs I sold to people online, with no base salary. Several months later the owners agreed to give me 1k of the first training program I sold along with 5% commission; which moved to 7% commission if I sold 5 training programs or more. I made around 18k my first year and 24k my second year off commission in the startup (since I made no money off the original job, I was struggling to get by those first 2 years).
In the 3rd year the commission moved to 10%. I sell training programs every month now so they moved me to a base salary structure instead of giving me 1k of the first training program sold. Now I get a base salary of 1.5K every month along with my 10% commission.
The lawyer I talked to claims that I was supposed to receive at least 24k a year as a base salary for the startup to be exempt from minimum wage or overtime and other state & federal law. I never really had a base salary my first 2 years. Even now my base is only 18k. The lawyer claims that the commission I made on sales does not count towards the base salary as well. The only exception to the 24k base salary minimum would be if I owned 20% or more of the company (or if I was in the service industry).
Is this correct? I want to be sure before using it in negotiations. thx!
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1 Answer
I have not heard the 24k figure exactly, but I think I know where it came from. If an employee is making commission on sales while working full time there are laws to handle the minimum wages for the salesperson.
My disclaimer is this applies to me in the US, in the state of Virginia. I believe it applies across the US but I am not sure about that.
So if you do not have a guaranteed constant supply of sales leads, then you must be paid at least the equivalent of minimum wage for your hours worked. This includes time-and-a-half for overtime worked, which is why I don't think the 24k figure is exact but is instead the average for 40 hour work weeks.
So if a salesperson is expected to find leads and close sales for commission, and does not manage to do so, the company must pay minimum wages.
Again, not a lawyer or a CPA, but had to do this with my own salespeople at a small web company.
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81 reputation
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location Farmville, VA
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Estwhile software entrepreneur thinking about the next big thaing.
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answered Dealing with a string of business Failures? Is there a time to get a job and drop the dream?
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Research article
The tumour-associated glycoprotein podoplanin is expressed in fibroblast-like synoviocytes of the hyperplastic synovial lining layer in rheumatoid arthritis
Anna-Karin H Ekwall1*, Thomas Eisler2, Christian Anderberg3, Chunsheng Jin4, Niclas Karlsson4, Mikael Brisslert1 and Maria I Bokarewa1
Author Affiliations
1 Department of Rheumatology and Inflammation Research, Institute of Medicine, Sahlgrenska Academy, Göteborg University, Box 480, 405 30 Göteborg, Sweden
2 Department of Orthopaedics, Institute of Clinical Sciences, Sahlgrenska Academy, Göteborg University, Bruna Stråket 11, 314 45 Göteborg, Sweden
3 Division of Orthopaedics, Spenshult Hospital, 313 92 Oskarström, Sweden
4 Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska Academy, Göteborg University, Box 440, 405 30 Göteborg, Sweden
For all author emails, please log on.
Arthritis Research & Therapy 2011, 13:R40 doi:10.1186/ar3274
The electronic version of this article is the complete one and can be found online at: http://arthritis-research.com/content/13/2/R40
Received:10 September 2010
Revisions received:11 January 2011
Accepted:7 March 2011
Published:7 March 2011
© 2011 Ekwall 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
Introduction
Activated fibroblast-like synoviocytes (FLSs) in rheumatoid arthritis (RA) share many characteristics with tumour cells and are key mediators of synovial tissue transformation and joint destruction. The glycoprotein podoplanin is upregulated in the invasive front of several human cancers and has been associated with epithelial-mesenchymal transition, increased cell migration and tissue invasion. The aim of this study was to investigate whether podoplanin is expressed in areas of synovial transformation in RA and especially in promigratory RA-FLS.
Methods
Podoplanin expression in human synovial tissue from 18 RA patients and nine osteoarthritis (OA) patients was assessed by immunohistochemistry and confirmed by Western blot analysis. The expression was related to markers of synoviocytes and myofibroblasts detected by using confocal immunofluoresence microscopy. Expression of podoplanin, with or without the addition of proinflammatory cytokines and growth factors, in primary human FLS was evaluated by using flow cytometry.
Results
Podoplanin was highly expressed in cadherin-11-positive cells throughout the synovial lining layer in RA. The expression was most pronounced in areas with lining layer hyperplasia and high matrix metalloproteinase 9 expression, where it coincided with upregulation of α-smooth muscle actin (α-sma). The synovium in OA was predominantly podoplanin-negative. Podoplanin was expressed in 50% of cultured primary FLSs, and the expression was increased by interleukin 1β, tumour necrosis factor α and transforming growth factor β receptor 1.
Conclusions
Here we show that podoplanin is highly expressed in FLSs of the invading synovial tissue in RA. The concomitant upregulation of α-sma and podoplanin in a subpopulation of FLSs indicates a myofibroblast phenotype. Proinflammatory mediators increased the podoplanin expression in cultured RA-FLS. We conclude that podoplanin might be involved in the synovial tissue transformation and increased migratory potential of activated FLSs in RA.
Introduction
Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease predominantly affecting joints, leading to tissue destruction and functional disability [1,2]. Both genetic and environmental factors are believed to contribute to the dysregulated immune responses seen in this heterogeneous autoimmune disease [3]. Today, treatment strategies involve traditional disease-modifying antirheumatic drugs as well as biologic agents targeting proinflammatory cytokines (tumour necrosis factor α (TNFα), interleukin (IL)-1 and IL-6), B cells or the activation of T cells [4]. Despite this arsenal of drugs, at least 30% of the patients are resistant to the available therapies, suggesting that yet other mediators must be important.
The most prominent feature of RA is the progressive destruction of articular cartilage and bone, which is orchestrated by activated RA fibroblast-like synoviocytes (RA-FLSs) [5,6]. RA-FLSs not only mediate tissue destruction but also are considered to play a major role in initiating and driving RA in concert with inflammatory cells [7]. In the healthy synovium, one to three layers of synoviocytes, the macrophage-like type A and the more abundant fibroblast-like type B (also referred to as synovial fibroblast), form the synovial lining layer separating the synovial sublining layer of loose connective tissue from the joint cavity [8,9]. The synoviocytes are interconnected with adherens junctions containing cadherin-11 [10,11] and E-cadherin [12,13] and are embedded in a lattice of extracellular matrix (ECM) resembling an epithelium but lacking a discrete basal membrane as well as gap junctions and desmosomes. Apart from being a marker of FLSs, cadherin-11 has been shown to be essential for the formation of synovial lining structures in vitro and for the development of inflammatory arthritis in mice [14,15].
The morphological hallmarks of RA include activation of FLSs; infiltration of inflammatory cells such as T cells, B cells and macrophages in the sublining; hyperplasia of the synovial lining layer; fibrotic deposition; and subsequent formation of the "pannus" [16]. This tissue mass expands and attaches to and invades the adjacent cartilage and subchondral bone [17]. The major cell type accounting for the thickened lining layer as well as for pannus formation is believed to be activated FLSs [18,19]. These aggressive cells share many characteristics with tumour cells, with upregulated expression of proto-oncogenes and promigratory adhesion molecules, increased production of proinflammatory cytokines and matrix-degrading enzymes [7], as well as increased resistance to apoptosis [20,21]. There are data indicating that the transformed phenotype of RA-FLS is stable and maintained even in the absence of stimulus from inflammatory cells [22]. In high-inflammation synovial tissue, RA-FLSs show a gene expression profile characteristic of myofibroblasts, and cells of the synovial lining in RA have been found to express α-smooth muscle actin (α-sma) and type IV collagen [13,23]. Thus, it has been suggested that RA-FLSs can undergo a process resembling epithelial-mesenchymal transition (EMT), a phenomenon known from early developmental processes, tissue repair, fibrosis and carcinogenesis [24,25]. Recently, it was also suggested that migrating RA-FLSs might be responsible for spreading the disease to distant joints [26].
Podoplanin (identical to human PA2.26, aggrus and T1α-2), is a small, 38- to 40-kDa, mucin-type transmembrane glycoprotein normally expressed on human lymphatic endothelia, basal epithelial keratinocytes, myoepithelial cells and myofibroblasts of certain glandular tissues, follicular dendritic cells and fibroblastic reticular cells of lymphoid organs and alveolar type I cells [27,28]. We demonstrated strong podoplanin expression on subepithelial interstitial cells in human endolymphatic tissue of the inner ear [29]. The physiologic function of podoplanin is to a large extent unknown, but knockout (KO) studies showed that it is crucial for the development of the lung and deep lymphatics in mice [28]. The podoplanin-KO mice died at birth as a result of respiratory failure and generalised lymphoedema. Overexpression of this glycoprotein in epithelial cells induced a dentritic cell morphology and increased cell adhesion and migration [27]. Interestingly, increasing data show that podoplanin is upregulated on the invasive front of human cancers [27,30]. The expression of podoplanin is correlated with metastasis and a bad prognosis. In addition, podoplanin (or aggrus) induces platelet aggregation of tumour cells [31] and has been associated with both EMT-dependent and EMT-independent tumour cell invasion [32]. There are a few studies indicating increased podoplanin expression in fibroblasts in reactive tissues, such as in chronic pleuritis, in cancer-associated fibroblasts [33] and in cultured fibroblasts [34]. However, little is known about the potential role of podoplanin in inflammation and tissue repair. In this study, we were interested to see whether podoplanin is expressed in FLSs in RA and could be associated with the fibrotic transformation of the synovium in this disease.
Materials and methods
Human synovial tissue and cells
Synovial tissue specimens and fluid were obtained from patients with RA (n = 18) or OA (n = 9) during joint replacement surgery or therapeutic joint aspiration at Sahlgrenska University Hospital and Spenshult Hospital in Sweden. Both weight-bearing (knee and hip) and non-weight-bearing (shoulder and elbow) joint specimens were included. All RA patients fulfilled the American College of Rheumatology 1987 revised criteria for RA [35]. Preoperative radiographs were scored according to Larsen index (1 to 5) [36]: 0 = normal; 1 = slight abnormality, soft tissue swelling, periarticular osteoporosis and slight joint space narrowing; 2 = early abnormality, erosions (obligatory in non-weight-bearing joints) and joint space narrowing; 3 = medium destructive abnormality, erosions and joint space narrowing; 4 = severe destructive abnormality, erosions, joint space narrowing and bone deformation; and 5 = mutilating abnormality. The patient characteristics are outlined in Table 1. All patients gave informed consent, and the procedure was approved by the Ethics Committee of Gothenburg in Sweden. Human primary FLS cultures were established as follows: representative tissue pieces were minced, treated with 1 mg/ml collagenase/dispase (Roche, Mannheim, Germany) for 1 hour at 37°C and passaged through a cell strainer. The cell suspension was rinsed twice in phosphate-buffered saline (PBS), resuspended in Dulbecco's modified Eagle's medium (DMEM) GlutaMAX (Invitrogen, Camarillo, CA, USA) supplemented with 10% heat-inactivated foetal bovine serum (HIFBS) (Sigma, St. Louis, MO, USA), 50 μg/ml gentamicin (Sanofi-Aventis, Paris, France) and 100 μg/ml normocin (Invivogen, San Diego, CA, USA) and incubated at 5% CO2 at 37°C. Cells in passages 3 through 6 were used.
Table 1. Characteristics of patientsa
Immunohistochemistry
Paraformaldehyde (PFA)-fixed (Histolab, Göteborg, Sweden), paraffin-embedded (4 μm) or acetone-fixed (Histolab) frozen sections (6 μm) were rehydrated in Tris-buffered saline for 10 minutes. Antigen retrieval was performed when required in a pressure chamber (2100 Retriever; Histolab). Unspecific binding was blocked using serum-free protein block or normal rabbit serum (Dako, Glostrup, Denmark). After incubation with mouse monoclonal antihuman podoplanin (clone D2-40; AbD Serotec, Oxford, UK), mouse monoclonal antihuman cadherin-11 (clone 5B2H5; Invitrogen) or mouse monoclonal antihuman CD90 antibodies (clone AS02; Dianova, Hamburg, Germany), respectively, the specimens were incubated with a biotinylated rabbit antimouse immunoglobulin G F(ab')2 fragment (Dako) followed by streptavidin-conjugated alkaline phosphatase (Dako). Fast Red Naphthol (Sigma) was used as a substrate, and the specimens were counterstained with Mayer's haematoxylin (Histolab) and mounted in Aqua-Mount mounting medium (VWR International Ltd, Leicestershire, UK). The same staining protocol was used for immunocytochemistry of primary FLS seeded onto chamber slides (Lab-Tek; Nunc, Rochester, NY, USA) and fixed in PFA. Normal mouse IgG1 (Dako) was used as a negative control. The podoplanin staining was scored by two independent observers blinded to the procedure according to the following scoring method: 0 = negative staining, 1 = positive staining of single or limited groups of cells in the lining layer, 2 = continuous positive staining of the cells of the synovial lining layer and 3 = same as 2, but with the addition of positive staining of cells in the sublining layer.
Immunofluorescence and confocal microscopy
Paraffin-embedded synovial sections were subjected to a double-staining procedure: incubation with rabbit antihuman cadherin-11 (Invitrogen), rabbit anti-matrix metalloproteinase (MMP)-9 (AB805; Millipore, Billerica, MA, USA), rabbit antihuman E-cadherin (clone H-108; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or rabbit anti-α-sma (PA1-37024; Thermo Scientific, Rockford, IL, USA) antibodies followed by addition of Alexa Fluor 555-conjugated goat antirabbit IgG (Invitrogen) or, in one step, Alexa Fluor 647-conjugated mouse antihuman CD68 (clone KP1; Santa Cruz Biotechnology). Second, mouse antihuman podoplanin (clone D2-40) incubation was followed by Alexa Fluor 488-conjugated goat antimouse IgG (Invitrogen). Alternatively, biotinylated mouse antihuman podoplanin (Acris Antibodies GmbH, Herford, Germany) and Alexa Fluor 488-conjugated streptavidin were added prior to mouse antihuman cadherin-11 (clone 5B2H5) and Alexa Fluor 555-conjugated goat antimouse IgG (Invitrogen). Slides were placed in ProLong Gold antifade reagent mounting medium with 4',6-diamidino-2-phenylindole (Invitrogen). Normal mouse IgG1 or normal rabbit serum (Dako) was used as negative controls. Images were collected using a confocal microscope (LSM700; Zeiss, Oberkochen, Germany). The background fluorescence level was set with the negative controls, and images were analysed using Zen image analysis software 2009 (Zeiss).
Western blot analysis
Membrane proteins from tissue and cell pellets were prepared by sodium carbonate treatment [37]. In brief, lyophilized material was resuspended in 0.1 M sodium carbonate before sonication. After removal of cell debris, the membrane fraction was collected by ultracentrifugation at 115,000 g for 75 minutes. The membrane proteins were solubilised with 7 M urea, 2 M thiourea, 40 mM Tris, 1% C7 detergent (wt/vol) and 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate buffer (wt/vol) and kept at -80°C before use.
Samples, together with recombinant unglycosylated human podoplanin core protein (ProSpec, Ness-Ziona, Israel), were separated by 20% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions with 10 mM dithiothreitol. After being transferred onto polyvinylidene fluoride membrane, the blots were probed with mouse antihuman podoplanin (1:50; D2-40) and detected with a horseradish peroxidise-conjugated rabbit antimouse antibody (1:2,000; DakoCytomation) and chemiluminescence (SuperSignal West Femto Maximum Sensitivity Substrate; Thermo Scientific).
Flow cytometry
Primary synovial cell cultures from patients with RA (n = 6) and patients with OA (n = 5) were trypsinised, resuspended in fluorescence-activated cell sorting buffer (5% HIFBS, 0.09% sodium azide and 0.5% ethylenediaminetetraacetic acid in PBS) and transferred onto a 96-well plate. For intracellular staining (CD68; α-sma), cells were PFA-fixed and permeabilised with 0.1% Triton X-100 in PBS. Unspecific binding was blocked using 1% HIFBS in PBS or Beriglobin P (human IgG; Apoteket, Sweden). Staining was performed with allophycocyanin (APC)-conjugated mouse antihuman CD90, phycoerythrin (PE)-conjugated mouse antihuman CD68, PE-conjugated mouse antihuman CD29 (BD Biosciences, San Jose, CA, USA), mouse antihuman podoplanin (clone D2-40), mouse antihuman cadherin-11 (clone 5B2H5), rabbit antihuman α-sma (PA1-37024) and isotype controls (BD Biosciences). The unconjugated antibodies were incubated with secondary PE-conjugated rat antimouse IgG1 (BD Biosciences) or APC-conjugated goat antirabbit IgG (Santa Cruz Biotechnology) in a second step. Fluorescence was measured using the FACSCanto II system (BD Biosciences) equipped with DIVA 6.2 software (BD Biosciences), and data were analyzed using FlowJo 8.7.3 software (Tree Star Inc., Ashland, OR, USA). The isotype controls were used to set the gates for positive and negative populations.
Stimulation experiments
Primary FLSs from one OA patient were seeded into complete DMEM in triplicates in six-well plates (100,000 cells/well) and incubated until confluence. The cells were serum-starved in DMEM supplemented with 2% heat-inactivated foetal calf serum for 6 hours before the different human recombinant cytokines were added: 10 ng/ml TNFα (Sigma), 1 ng/ml IL-1β and 1 ng/ml TGF-β1 (R&D Systems, Minneapolis, MN, USA). The cells were harvested by trypsinisation after 12, 24 and 48 hours, and podoplanin expression was measured using flow cytometry with antipodoplanin antibody (clone D2-40). The experiment was repeated four times with different primary cell cultures, including RA-FLSs, with similar results.
Statistical analysis
Differences in protein expression between the patient groups detected by immunohistochemistry (IHC) and flow cytometry were evaluated using the Mann-Whitney nonparametric test.
Results
Podoplanin is expressed in the human synovial lining layer in RA
By carrying out IHC on paraffin sections of human synovia, we found that podoplanin was highly expressed in rounded cells of the epithelium-like synovial lining layer in 17 of the 18 RA specimens (Figures 1A-D and 1L-M). In most cases, the podoplanin staining covered the whole cell surface and was continuous along and throughout the lining layer. Podoplanin expression was most pronounced in areas with strong hyperplasia and disrupted synovial architecture (Figures 1C, 1D and 1L), staining not only the surface of all the lining layer cells with high intensity but also adjacent interstitial cells of the sublining layer (Figure 1D). The podoplanin expression was prominent in long cytoplasmatic processes and was maintained on rounded, dispersed and disaggregated cells in "invasive" areas (Figure 1C). Podoplanin stained lymph vessels in all tissues (Figure 1J). The synovium in OA was predominantly negative (Figures 1F and 1N), but single positive cells or a limited group of them were occasionally found in the lining layer (Figures 1E and 1H). Discrete staining was sometimes detected on the apical surface of the outermost lining layer (Figure 1G, arrowhead). The mean score of podoplanin expression in the synovium of the RA specimens was 2.61 (SEM, 0.18) versus 0.33 (SEM, 0.17) for OA specimens (P < 0.0001) (Figure 1K). The subsynovial connective tissue in OA was negative in all cases.
Figure 1. Podoplanin is expressed in human synovial tissue in RA. Immunohistochemistry (IHC) of human synovial tissue from (A-D, I) rheumatoid arthritis (RA), (E-H, J) osteoarthritis (OA) (A-D, E-H, J) using antipodoplanin antibody (D2-40) or (I) mouse immunoglobulin G1. (J) Positive control showing lymph vessels (arrow). (K) IHC staining score of podoplanin on human synovial tissue from 18 RA and 9 OA patients. Double immunofluorescence staining of (L and M) RA and (N) OA synovium using antipodoplanin (green) and anti-cadherin-11 (red) antibodies. Note the extensive hyperplasia of the podoplanin-positive lining layer cells (C and D, L) and the podoplanin-positive lymph vessel (arrowhead in N) but negative lining layer in OA (N). L, lumen. ***statistical significance P < 0,0001.
To verify that podoplanin is expressed in human synovial tissue in RA and to evaluate the specificity of the antipodoplanin antibody, extracted membrane proteins from synovial tissue samples from two RA patients were subjected to SDS-PAGE and Western blot analysis using D2-40 monoclonal antibody. The Western blot analysis showed one distinct band of about 45 kDa (Figure 2) in both samples. The antibody also recognised the recombinant immature podoplanin core protein (13.4 kDa according to the manufacturer) as a band of estimated molecular weight of about 18 kDa. The lung fibroblast cell line MRC-5, shown by us not to express podoplanin by flow cytometry, was used as a negative control.
Figure 2. Anti-podoplanin antibody D2-40 recognizes 45kD band in Western blot of synovial protein extracts. Western blot of extracted membrane proteins from human synovial tissue from a patient with rheumatoid arthritis (RA) (lane 1), cell pellet of human MRC-5 lung fibroblast cell line (lane 2) and recombinant immature podoplanin core protein (lane 3) separated on a 20% sodium dodecyl sulphate polyacrylamide gel electrophoresis gel probed with the antipodoplanin antibody (D2-40).
Podoplanin is expressed on cadherin-11-positive synoviocytes of the lining layer in RA
To identify which type of synoviocyte express podoplanin, we performed IHC and double-immunofluorescence (double-IF) on human RA synovium using different cellular markers. We found that the fibroblast marker CD90 was expressed by interstitial cells, typically forming sheet structures around capillaries, of the synovial sublining in frozen sections of human synovium (Figure 3B). However, the lining layer was CD90-negative (in contrast to podoplanin) (Figure 3A). Both podoplanin and anti-cadherin-11 were present in the lining layer of serial sections of RA synovia (Figure 3C and 3D). Double-IF staining and confocal microscopy confirmed a colocalization of cadherin-11 and podoplanin on the cellular level of lining cells (Figure 3E). The cadherin-11 expression in RA, compared with OA, was increased both in the lining and in the sublining layers, especially in areas with hyperplasia (Figure 1L). Double-staining for podoplanin and the macrophage marker CD68 clearly did not show any colocalization (Figure 3F). CD68-positive cells were dispersed in the lining layer and in the sublining tissue in both RA and OA.
Figure 3. Podoplanin is expressed in FLS in areas of synovial transformation. Immunohistochemistry of (A and B) frozen and (C-J) paraffin-embedded human synovial tissue from patients with rheumatoid arthritis (RA) using antibodies against (A and C) podoplanin (D2-40), (B) CD90 (AS02) and (D) cadherin-11 (3B2H5) (arrowhead). Double immunofluorescence staining analysed by confocal microscopy showing, in green, (E) podoplanin 18H5 and (F-J) podoplanin D2-40, and in red, (E) cadherin-11 (3B2H5), (F) CD68 (KP1), (G and overview in H) E-cadherin (H-108), (I) α-smooth muscle actin (α-sma) (PA1-37024) and (J) matrix metalloproteinase 9 (MMP-9). (E) Note colocalization of podoplanin and cadherin-11 (arrowhead). L, lumen.
α-sma is upregulated in podoplanin-expressing synovial lining layer cells
Next, we were interested to see whether podoplanin could be involved in EMT-like transdifferentiation of RA-FLSs. We therefore investigated the expression of α-sma and E-cadherin in relation to podoplanin in RA synovia. We found that α-sma was expressed in the cytoplasm of podoplanin-positive synovial lining cells in hyperplastic areas (Figure 3I). In addition, α-sma was expressed in vessel walls and on a few dispersed cells in the sublining. E-cadherin could be detected in some areas of the synovial lining layer in both RA and OA specimens. Interestingly, the expression of E-cadherin was very low or absent in podoplanin-expressing lining layer cells (Figure 3G and overview in Figure 3H).
Different MMPs (especially MMP-1, MMP-3, MMP-9 and MMP-13) are upregulated in the RA synovium and are responsible for the degradation of ECM and cartilage [5,38]. We used MMP-9 as an indicator of inflamed synovium and of the presence of matrix degradation. MMP-9 is reportedly expressed in synovial lining cells, in leukocytes and in endothelia of the RA synovium [39]. In agreement with this, we found high expression of MMP-9 in the synovial lining, in sublining ectopic lymphoid structures and in vessels in RA synovial tissue (Figure 3J). Double-staining of podoplanin and MMP-9 showed that the podoplanin-positive lining layer cells expressed MMP-9 (Figure 3J).
Podoplanin is expressed in cultured CD90-positive FLSs
To characterise podoplanin expression on the cellular level, we established primary FLS cultures from both RA (RA-FLSs) and OA (OA-FLSs) synovial specimens. At passage 3, the cultures were homogeneous. Using IF and confocal microscopy, we found that the primary FLSs had a typical cultured fibroblast phenotype with prominent stress fibres and that approximately 50% of the FLS cells expressed podoplanin (Figure 4A and 4B). The podoplanin expression was most pronounced in areas of focal attachment and in small membrane protrusions (microspikes) (Figure 4A, arrowheads).
Figure 4. Podoplanin is expressed in cultured primary FLS and the expression is increased by pro-inflammatory cytokines. (A) Immunofluorescence staining of primary rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) showing podoplanin (red) and actin stress fibres (green). Note accumulated podoplanin staining in membrane protrusions (arrowheads). (B) Magnification of podoplanin-positive RA-FLS. (C and D) Flow cytometry (FACS) of primary FLS cultures from patients with RA (filled bars) and patients with osteoarthritis (OA) (striped bars) showing the percentage of positive cells of viable cell populations using (C) antipodoplanin and phenotype markers (CD90, CD68 and CD29) and (D) cadherin-11 antibodies. (E) Immunocytochemistry of an aggressively growing RA-FLS culture using antipodoplanin antibody. Note the dendritic phenotype with long cytoplasmatic protrusions. (F) Representative flow cytometry plot of primary RA-FLS stained for podoplanin and α-smooth muscle actin (α-sma) showing the double-positive population (podoplanin+ and α-sma+) of 58.2% in the upper right quadrant. (G) Graph showing the percentage of podoplanin-positive primary FLS by flow cytometry at baseline, 24 and 48 hours of stimulation with control (complete medium) (open circles), 10 ng/ml tumour necrosis factor (TNF)-α (filled squares), 1 ng/ml interleukin (IL)-1β (open diamond) and 1 ng/ml transforming growth factor β receptor 1 (TGF-β1) (filled triangles), respectively, of a representative culture. The experiment was run in triplicate and repeated four times using different OA-FLS and RA-FLS cultures, which showed similar results but starting at different baseline levels of podoplanin expression.
The podoplanin expression was further evaluated in six RA-FLS and five OA-FLS primary cell cultures using flow cytometry, showing an average expression of 52 ± 24% and 64 ± 6%, respectively. The variation in RA-FLS compared to OA-FLS was evident. Furthermore, 99 ± 6% of RA-FLSs expressed the fibroblast marker CD90, 97 ± 1% of RA-FLSs expressed CD29 (β1-integrin) and less than 0.6 ± 0.5% of RA-FLSs expressed the macrophage marker CD68. A percentage of 95 ± 0.7% OA-FLS expressed CD90, 98 ± 1% expressed CD29 and less than 0.8 ± 0.4% expressed CD68 (Figure 4C). The mean expression of cadherin-11 was low in OA-FLSs (2.3%) and slightly increased in RA-FLSs (up to 21%) (Figure 4D). Interestingly, one RA-FLS cell line had a more dendritic phenotype (Figure 4E) and was growing without contact inhibition. This cell line had a 100% expression of podoplanin on the basis of flow cytometry.
Cultured fibroblasts upregulate α-sma (up to 100% by passage 5) in culture [40,41]. We were interested to see whether this is true also for primary FLSs. Using flow cytometry, we found that nearly 100% of both OA-FLSs and RA-FLSs express α-sma by passage 6. About 60% (58.2% for RA-FLSs and 61.7% for OA-FLSs) were double-positive for podoplanin and α-sma. All podoplanin-positive cells expressed α-sma (Figure 4F).
Podoplanin expression in vitro is increased by proinflammatory cytokines
IL-1β and TNFα are known to activate RA-FLSs. TGF-β1 is a key mediator of EMT and promotes the differentiation of fibroblasts into myofibroblasts in wound healing and fibrosis.
In this study, we investigated the effects of IL-1β, TNFα and TGF-β1 stimulation on podoplanin expression in primary FLSs. We found a more than twofold increase in podoplanin expression in OA-FLS culture after stimulation with 1 ng/ml IL-1β and 10 ng/ml TNFα. This culture had a low baseline expression of podoplanin (30%), which increased to 72% after 48 hours of IL-1β stimulation (Figure 4G). Using two different primary RA-FLS cultures, we found an early increase in podoplanin expression from, on average, 50% at baseline to about 90% after 12 hours of IL-1β stimulation. These effects were maintained after 36 hours of stimulation (data not shown). TGF-β1 (1 ng/ml) had a moderate effect (+1.7-fold) on podoplanin expression evident at late time points (48 hours) (Figure 4G).
Discussion
In this study, we have shown that the tumour-associated proinvasive glycoprotein podoplanin is highly expressed in synovial lining layer cells in RA but is rarely found in OA synovial specimens. The expression of podoplanin was most pronounced in areas with signs of inflammation (that is, the presence of leukocyte infiltrates and ectopic lymphoid structures) and synovial transformation (indicated by lining layer hyperplasia, MMP-9 expression and upregulation of cadherin-11 and α-sma). Furthermore, the podoplanin-expressing lining layer cells expressed cadherin-11 but not the macrophage marker CD68, suggesting that these synoviocytes were FLSs rather than synovial macrophages.
All included RA patients had progressed to erosive disease (Larsen index score >1), and all except one had a high podoplanin expression score (IHC score >1) of the synovial tissue from the replaced joint (Table 1). However, without the rarely available tissue specimens from nonerosive and early RA joints to compare these tissues with, we could not analyze whether there is a correlation between erosive disease and podoplanin expression.
The function of podoplanin is far from elucidated. On one hand, this small glycoprotein is constitutively expressed on the apical surface of lymph endothelia as well as on specialised epithelia (for example, podocytes) facing fluid compartments [28,42,43]. On the other hand, podoplanin is crucial for processes involving cell migration, such as the specific embryologic development of deep lymphatics [28] and the invasion and metastasis of certain tumour cells or tissues [32]. Podoplanin has been shown to bind ezrin, an actin filament membrane linker protein, on the inside of the cell in vitro [30,44]. It has therefore been suggested that podoplanin is involved in directing actin polymerisation, thereby forming the cellular protrusions needed for migration.
In our study, the marked and widespread expression of podoplanin in lining layer cells in RA was not restricted to the apical cell surface. Instead, it resembled the strong whole cell surface-staining pattern of podoplanin in tumour tissues [30]. It has been shown that RA-FLSs of highly inflammatory synovial tissue show a gene expression profile characteristic of myofibroblasts [23]. We detected coexpression of podoplanin and α-sma of FLSs in areas of synovial transformation and found that the expression of E-cadherin was low or absent in the podoplanin-expressing lining layer cells. We know from earlier studies that podoplanin can promote EMT of epithelial Madin-Darby canine kidney cells in vitro [44]. EMT is a biologic process in which polarised epithelial cells undergo sequential changes into a mesenchymal cell phenotype with increased migratory potential and the production of ECM components [24]. Loss of E-cadherin and gain of α-sma expression constitute examples of such changes. We therefore hypothesise that podoplanin is involved in an EMT-like transdifferentiation of RA-FLSs into myofibroblasts.
Podoplanin has been observed in interstitial fibroblasts in different inflammatory environments in vivo and in vitro [33,34]. In agreement with this observation, we found a locally increased expression of podoplanin in interstitial cells of the sublining connective tissue in specimens from patients with RA. However, it is difficult to determine whether upregulated podoplanin expression in the sublining in some RA specimens was a result of general inflammation or whether this phenomenon was part of a specific activation and transdifferentiation of FLSs in RA.
To confirm the specificity of the D2-40 antibody and the expression of podoplanin in RA synovial tissue, we performed SDS-PAGE and Western blot analysis of protein extracts showing a distinct band of about 45 kDa. The mature glycosylated form of podoplanin has been estimated to be about 38 to 40 kDa [27]. The difference in approximated molecular weight could be explained by the reported heterogeneity of podoplanin in SDS-PAGE, which arises as a result of heavily O-linked glycosylation of the core protein [27] as well as a slightly unspecific migration of the used molecular weight markers.
Characteristics of RA are the phenotypic changes and hyperplasia of FLSs of the lining layer. Conventional isolation of FLSs from synovial tissue yields homogeneous fibroblast cultures [45], but the interindividual morphological variation is large, and cultures presumably arise from both the synovial lining and sublining layers.
We established primary cultures of FLSs from human synovial tissues by enzyme digestion and found that the cells had typical fibroblast morphology. Nearly all of the primary FLSs stained positive for the fibroblast marker CD90/Thy-1 and most expressed β1 integrins. However, the IHC staining of human synovia using the anti-CD90 antibody revealed positive expression in the sublining, but not in the lining layer cells (Figures 3A and 3B). Fibroblasts possess a remarkable phenotypic plasticity [41] as well as a positional identity [46]. The synovium (lining layer versus sublining layer) of both healthy and RA patients harbour phenotypically different (by morphology and expression of surface markers) populations of fibroblasts. CD90 might therefore be a good marker for interstitial tissue fibroblasts, but not for the FLSs forming the epithelium-like lining of the synovium. In addition, fibroblasts change the expression of several surface molecules in vitro and acquire an "active" phenotype with prominent stress fibres and focal adhesions [47] when cultured on plastic. We therefore concluded that most of the established primary FLS cultures in this study originated from the sublining connective tissue or acquired a sublining fibroblast phenotype (with respect to CD90 expression) in culture. Using IHC, we found that cadherin-11 was expressed both in the lining layer and in cells of the sublining tissue in reactive areas, but when using flow cytometry, we found it on average in only 10% of the isolated primary FLSs. These data support the assumption that the isolated primary FLSs in these experiments originated from the sublining rather than from the lining layer.
Fibroblasts have been shown to upregulate podoplanin in culture [34]. In this study, we did not observe any significant difference in mean podoplanin expression between the RA-FLS and OA-FLS cultures. Only one culture, derived from an RA patient, was growing without contact inhibition, a characteristic of activated FLSs in RA. All cells of this culture were expressing podoplanin. Taken together, our results suggest that cultures of the lining layer FLS phenotype are hard to establish by using this technique and that primary FLSs, like other fibroblasts, probably upregulate podoplanin in culture. The observed upregulated expression of α-sma of the primary FLSs constitutes another example of an acquired feature of cultured fibroblasts.
Finally, we found a more than twofold increase in podoplanin expression in primary FLSs after stimulation with IL-1β and TNFα compared with controls. Interestingly, we also detected an increase in podoplanin expression in response to TGF-β1 stimulation. TGF-β1 is a key mediator of EMT and promotes the differentiation of fibroblasts into myofibroblasts in wound healing and fibrosis. Furthermore, TGF-β1-induced podoplanin in human fibrosarcomas [48] was found to be increased in arthritic joints in RA [49] and promoted EMT of FLS in vitro [13]. The fact that proinflammatory cytokines and growth factors, known to be present in high concentrations in the RA joint, stimulate podoplanin expression in primary FLSs in vitro supports our finding that podoplanin is upregulated in the synovium of RA patients and might be involved in the transdifferentiation of FLSs in RA.
Conclusions
We can now add podoplanin expression as a shared characteristic of activated RA-FLSs and tumour cells that possibly affects common features of RA and carcinoma-like fibrotic tissue transformation and tissue invasion. Podoplanin might therefore be an important target not only in cancer therapy but also in the treatment of RA.
Abbreviations
α-sma: α-smooth muscle actin; DMARDs: disease-modifying antirheumatic drugs; EMT: epithelial-mesenchymal transition; FACS: fluorescence-activated cell sorting; FLS: fibroblast-like synoviocyte; IF: immunofluorescence; IHC: immunohistochemistry; IL-1β: interleukin-1β; OA: osteoarthritis; RA: rheumatoid arthritis; TGF-β1: transforming growth factor-1β; TNF-α: tumour necrosis factor-α.
Competing interests
MIB has received consulting fees from Schering-Plough, UCB, Pfizer, Roche and GlaxoSmithKline (less than US$10,000 each). NK was working for Bristol-Myers Squibb from 2006 to 2009 on an unrelated project. The authors declare no other competing interests.
Authors' contributions
AKHE did the major design of the study and the coordination and establishment of the biobank, carried out the experiments, performed the imaging and FACS analysis and drafted the manuscript. TE participated in the design of the study and performed the Larsen scoring of radiographs. TE and CA included the patients in the study and collected patient data and tissue samples. CJ performed the SDS-PAGE and Western blot analyses. MB participated in the analysis of the FACS data and helped to draft the manuscript. MIB participated in the design of the study and analysis of the data and helped to draft the manuscript. All authors read and approved the final manuscript.
Acknowledgements
We thank Ing-Marie Jonsson, Sofia Andersson and the late Berit Ertmann-Ericsson for skillful technical assistance with paraffin embedding and sectioning. This work was supported by grants from The Göteborg Medical Society, The Swedish Rheumatism Association and The Rune and Ulla Amlöv Foundation.
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Proc Natl Acad Sci USA 2002, 99:12877-12882. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
47. Grinnell F: Fibroblast biology in three-dimensional collagen matrices.
Trends Cell Biol 2003, 13:264-269. PubMed Abstract | Publisher Full Text
48. Suzuki H, Kato Y, Kaneko MK, Okita Y, Narimatsu H, Kato M: Induction of podoplanin by transforming growth factor-β in human fibrosarcoma.
FEBS Lett 2008, 582:341-345. PubMed Abstract | Publisher Full Text
49. Pohlers D, Beyer A, Koczan D, Wilhelm T, Thiesen HJ, Kinne RW: Constitutive upregulation of the transforming growth factor-β pathway in rheumatoid arthritis synovial fibroblasts.
Arthritis Res Ther 2007, 9:R59. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
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Connexions
Sections
You are here: Home » Content » Music Inquiry
About: Music Inquiry
Collection type: Course
Course by: Catherine Schmidt-Jones. E-mail the author
View the content: Music Inquiry
Metadata
Name: Music Inquiry
ID: col11455
Language: English (en)
Summary: An inquiry based approach to learning about music provides flexible lessons that deepen and broaden learners' understanding of music while engaging with their current musical interests, preferences, and goals. This hands-on experience with inquiry in a subject area that appeals to most learners also provides practice in self-directed learning that can be applied in any area of life-long learning.
Collection Subtype: Course
Subject: Arts
Keywords: active learning, ethnomusicology, IBL, inquiry, inquiry based learning, learner-centered education, life-long learning, music, music education, music theory, self-directed learning
License: Creative Commons Attribution License CC-BY 3.0
Authors: Catherine Schmidt-Jones (caschmidtjones@gmail.com)
Copyright Holders: Catherine Schmidt-Jones (caschmidtjones@gmail.com)
Maintainers: Catherine Schmidt-Jones (caschmidtjones@gmail.com)
Latest version: 1.1 (history)
First publication date: Nov 7, 2012 10:40 am US/Central
Last revision to collection: Nov 7, 2012 11:11 am US/Central
Version History
Version: 1.4 Mar 18, 2013 5:50 pm GMT-5 by Catherine Schmidt-Jones
Changes:
added one new module
Version: 1.3 Feb 5, 2013 11:58 am US/Central by Catherine Schmidt-Jones
Changes:
added new modules
Version: 1.2 Jan 16, 2013 10:03 am US/Central by Catherine Schmidt-Jones
Changes:
added new modules
Version: 1.1 Nov 7, 2012 11:11 am US/Central by Catherine Schmidt-Jones Currently viewing this version.
Changes:
new collection
How to Reuse and Attribute This Content
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American Chemical Society (ACS) Style Guide:
Schmidt-Jones, C. Music Inquiry, Connexions Web site. http://cnx.org/content/col11455/1.1/, Nov 7, 2012.
American Medical Assocation (AMA) Manual of Style:
Schmidt-Jones C. Music Inquiry [Connexions Web site]. November 7, 2012. Available at: http://cnx.org/content/col11455/1.1/.
American Psychological Assocation (APA) Publication Manual:
Schmidt-Jones, C. (2012, November 7). Music Inquiry. Retrieved from the Connexions Web site: http://cnx.org/content/col11455/1.1/
Chicago Manual of Style (Bibliography):
Schmidt-Jones, Catherine. "Music Inquiry." Connexions. November 7, 2012. http://cnx.org/content/col11455/1.1/.
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Catherine Schmidt-Jones, "Music Inquiry," Connexions, November 7, 2012, http://cnx.org/content/col11455/1.1/.
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Schmidt-Jones, C. 2012. Music Inquiry. Connexions, November 7, 2012. http://cnx.org/content/col11455/1.1/.
Modern Languages Association (MLA) Style Manual:
Schmidt-Jones, Catherine. Music Inquiry. Connexions. 7 Nov. 2012 <http://cnx.org/content/col11455/1.1/>.
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{
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"url": "dotnetkicks.com/stories/43827/How_To_Debug_GC_Issues_Using_PerfView",
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"warc_filename": "<urn:uuid:6ffa0ce8-479f-45b8-9fe9-926a8877b6c4>",
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Error!
Success!
How To Debug GC Issues Using PerfView
0
kicks
How To Debug GC Issues Using PerfView (Unpublished)
In my previous artlcle, I discussed 4 ways to optimize your server application for good garbage collection performance. An essential part of that process is being able to analyze your GC performance to know where to focus your efforts. One of the first tools I always turn to is a little utility that has been publically released by Microsoft.
Kicked By:
Drop Kicked By:
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{
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"uncompressed_offset": 78136887,
"url": "elinux.org/index.php?oldid=80858&title=A10_tablets",
"warc_date": "2013-11-22T14:52:14.000Z",
"warc_filename": "<urn:uuid:6ffa0ce8-479f-45b8-9fe9-926a8877b6c4>",
"warc_url": "http://elinux.org/index.php?title=A10_tablets&oldid=80858"
}
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A10 tablets
From eLinux.org
Revision as of 04:50, 23 December 2011 by Hipboi (Talk | contribs)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
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Tablets powered by Allwinner A10
Currently there are many tablets on the Chinese market with Allwinner A10 inside. These tablets usually are not very expensive. Below lists some of them:
• Teclast 76
• Teclast P76Ti
• Ainol Novo7 Advanced
• Onda Vi20W
• Bmorn V11
• WoPad A7 (upcoming)
• Onda Vi20W deluxe (the original Vi20W is RK2918-based)
• Onda Vi30W deluxe
• Onda Vx610w
• Ployer MOMO9
• Sanei N70
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{
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"url": "josm.openstreetmap.de/ticket/2340",
"warc_date": "2013-11-22T14:52:14.000Z",
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"warc_url": "http://josm.openstreetmap.de/ticket/2340"
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Modify
Opened 4 years ago
Closed 4 years ago
Last modified 4 years ago
#2340 closed defect (duplicate)
Error occured whilst entering points - first use of this version of JOSM
Reported by: stevecarter@… Owned by: team
Priority: major Component: Core
Version: Keywords:
Cc:
Description
Path: trunk
URL: http://josm.openstreetmap.de/svn/trunk
Repository Root: http://josm.openstreetmap.de/svn
Repository UUID: 0c6e7542-c601-0410-84e7-c038aed88b3b
Revision: 1497
Node Kind: directory
Last Changed Author: stoecker
Last Changed Rev: 1497
Last Changed Date: 2009-03-17 19:28:03 +0100 (Tue, 17 Mar 2009)
Java version: 1.6.0_12
Plugins: osmarender;utilsplugin;validator;wmsplugin
Plugin osmarender Version: 14015
Plugin utilsplugin Version: 14015
Plugin validator Version: 14015
Plugin wmsplugin Version: 14110
java.lang.NullPointerException
at org.openstreetmap.josm.gui.NavigatableComponent.getNearestNode(NavigatableComponent.java:167)
at org.openstreetmap.josm.actions.mapmode.DrawAction.computeHelperLine(DrawAction.java:626)
at org.openstreetmap.josm.actions.mapmode.DrawAction.mouseMoved(DrawAction.java:580)
at java.awt.AWTEventMulticaster.mouseMoved(Unknown Source)
at java.awt.Component.processMouseMotionEvent(Unknown Source)
at javax.swing.JComponent.processMouseMotionEvent(Unknown Source)
at java.awt.Component.processEvent(Unknown Source)
at java.awt.Container.processEvent(Unknown Source)
at java.awt.Component.dispatchEventImpl(Unknown Source)
at java.awt.Container.dispatchEventImpl(Unknown Source)
at java.awt.Component.dispatchEvent(Unknown Source)
at java.awt.LightweightDispatcher.retargetMouseEvent(Unknown Source)
at java.awt.LightweightDispatcher.processMouseEvent(Unknown Source)
at java.awt.LightweightDispatcher.dispatchEvent(Unknown Source)
at java.awt.Container.dispatchEventImpl(Unknown Source)
at java.awt.Window.dispatchEventImpl(Unknown Source)
at java.awt.Component.dispatchEvent(Unknown Source)
at java.awt.EventQueue.dispatchEvent(Unknown Source)
at java.awt.EventDispatchThread.pumpOneEventForFilters(Unknown Source)
at java.awt.EventDispatchThread.pumpEventsForFilter(Unknown Source)
at java.awt.EventDispatchThread.pumpEventsForHierarchy(Unknown Source)
at java.awt.EventDispatchThread.pumpEvents(Unknown Source)
at java.awt.EventDispatchThread.pumpEvents(Unknown Source)
at java.awt.EventDispatchThread.run(Unknown Source)
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Change History (2)
comment:1 Changed 4 years ago by stoecker
• Resolution set to duplicate
• Status changed from new to closed
comment:2 Changed 4 years ago by stoecker
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as The resolution will be set. Next status will be 'closed'.
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Lippard Lab
From OpenWetWare
Revision as of 12:03, 6 April 2007 by Mateo (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search
Home Contact Internal Lab Members Publications Research Talks
The Lippard lab studies biological interactions involving metal ions, focusing on reactions and physical and structural properties of metal complexes. Such complexes can be useful as cancer drugs and as models for the active sites of metalloproteins. Metal ions also promote key biological reactions in enzymes and metal complexes can be employed to sense biological signaling agents.
Lippard is affiliated with MIT’s Center for Cancer Research and is well known for his work on the mechanism of the anti-cancer drug cisplatin, which contains platinum and is primarily used to treat testicular cancer and ovarian cancer. His lab is currently working on designing more effective platinum anti-tumor agents.
The Lippard group also determined the structure of the component proteins of methane monooxygenase, an enzyme from aerobic bacteria that convert methane (natural gas) and oxygen to liquid methanol and water in the first step of their life process. They elucidated several key steps in the activation of oxygen and methane at a closely spaced pair of iron atoms in the enzyme. This chemistry is closely related to that used in bioremediation, processes by which microorganisms are employed to clean the environment. Examples include removal of trichloroethylene from drinking water and the cleanup of oil spills from the land. Subsequently, structures of the related hydroxylase enzymes from toluene/o-xylene monooxygenase and phenol hydroxylase, the latter in complex with its regulatory protein, were determined. Oxygenated intermediates in the catalytic cycles of the latter two enzymes have been identified.
Lippard recently developed a fluorescent sensor that monitors nitric oxide, a molecule that plays critical roles in the human body, from destroying invading microorganisms to relaying neuronal signals. The molecule had long eluded scientists because it often exists in minute concentrations and for only short periods of time. The sensor allows scientists to view nitric oxide in living cells. In related work, Lippard has also developed fluorescent and MRI sensors to detect and understand the roles of mobile zinc in the brain.
Personal tools
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{
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photodawg's bookmarks
"I give the fight up: let there be an end, a privacy, an obscure nook for me. I want to be forgotten even by God."
Browning, Robert on defeat
5 fans of this quote
"So free we seem, so fettered we are!"
Browning, Robert on freedom
11 fans of this quote
"Ah, but a man's reach should exceed his grasp, or what's a heaven for?"
Browning, Robert on ambition
"Stung by the splendor of a sudden thought."
Browning, Robert on inspiration
Robert Langenfeld's quote collection
I'm male and made my book on 4th July 2009.
My book as a pdf
My feed
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}
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Help Wikitravel grow by contributing to an article! Learn how.
Discount airlines in Europe
From Wikitravel
(Redirected from European Discount Airlines)
Jump to: navigation, search
Boarding at Ryanair, no assigned seats
This article is a travel topic
This is one of several Wikitravel articles about Discount airlines.
Europe has a number of low cost airlines, the largest and most established being easyJet [1], Ryanair [2], germanwings [3] and Air Berlin [4]. These airlines have stirred up air travel within Europe by dramatically cutting fares.
The European Open-Skies Treaty of 1992 blew the lid off the system in place before, where national government would restrict access to their airspace to expensive 'flag-carriers', such as British Airways [5] or Lufthansa [6]. This enabled airlines to fly anywhere they wished in the European Union without government approval.
Ryanair was the first airline in Europe to try this model, and now has many followers offering low fares across the continent. These are boom times for cheap air travel in the European Union, with fares on some routes as low as €10 (£7, US$12) one-way including tax (though average fares for international flights are around €80 one-way).
But do not overlook other European airlines; for example, depending on the destination, dates and time, the usually expensive Swiss airline can be cheaper than a so-called discount airline when all costs and times are included from source to destination. Another example is OLT Express[7], which offers business flights that can be cheaper than any alternative airline for some legs.
[edit] Tickets and pricing
Most discount airlines in Europe sell their tickets exclusively over their website or over the phone, and tickets are not available via travel agents. Most are ticketless; you simply turn up at the check-in desk or even just at the departure gate with your passport and confirmation number (and print-out of your e-ticket). A credit or debit card is a very good idea for booking tickets. Most discount airlines sell their tickets as single journeys only.
The pricing structure is complex, with fares fluctuating strongly according to demand, often on an hourly basis, and the same rule "get as much money as a traveler is ready to pay" that was invented by traditional carriers applies. There are no hard and fast rules for obtaining the cheapest fares. In fact, fares can vary from as little as £1 or £2 on special promotions, right up to £500 - such as a London-Geneva return flight, during the February half-term weekend (winter holidays in most of the schools).
The following will however increase your probability of obtaining very inexpensive fares:
• Do fly mid-week
• Do fly early in the morning or late at night
• Do fly in low season (Spring and Autumn)
• Do make use of sales. These sometimes appear 3-5 weeks prior to departure, however this is by no means guaranteed.
• Don't fly during public holidays.
• Don't book your ticket less than two weeks in advance
• Opt for return tickets, but keep in mind, in most cases airlines will charge extra fees for changes of date or time.
[edit] Connecting tickets
For most traditional airlines it is possible to book a flight from A to B with a connection at C on a single ticket. For many low-cost carriers, this is not possible as they only offer single "point to point" flights. To make a connection with a low-cost carrier, you need to purchase two separate tickets, one from A to C, and another from C to B, and these count as separate contracts. Connecting low-cost flights can save on cost but it has a few disadvantages:
• You are not guaranteed to make the connection to your final destination. If your first flight is delayed, so that you miss the connection, it is your responsibility. The airline fulfilled the first contract by bringing you to the connection point albeit delayed, and it is your problem that you failed to arrive at the airport in time to get your second flight. Travel insurance may sometimes cover an event like this, paying for another ticket on a later flight, but only if you have put up a safe connection time.
• All checked luggage will need to be picked up at your connection point as if that were your final destination. It then needs to be checked in again as if you are departing from that airport.
However, some low-cost carriers want a share of the market with transporting connecting passengers, and have policies which allow for connecting tickets. Note that connecting flights here refers to connections to another flight on the same airline. Planning low-cost flight connections can be complicated and requires access to vast amounts of data.
Very few low-cost carriers offer connecting tickets to different airlines. (Many traditional carriers don't offer such tickets either although interline agreements often exist.)
• Ryanair does not offer connecting tickets, and discourages people from flying with them if they need to connect.[8]
• EasyJet do not offer connecting tickets, and advise passengers who need to connect to calculate a two hour connection time.[9]
• Air Berlin offers connecting flights on their website and codeshares with some members of the oneworld Alliance
• Norwegian generally operate point-to-point and recommend a connection time of 2 hours. If you have calculated two hours but still miss the connection, Norwegian will rebook you to a later flight subject to available space. Connecting flights can sometimes be booked on a single ticket online ("onlining") for a surcharge of 40 NOK, and in those cases they are responsible for bringing you to your destination.[10]
• Wizzair do not offer connecting flights, and accept no liability for missed connections. Passengers are advised to calculate "sufficient time".
• German Wings offer connecting flights, and can often check your luggage through to your destination.[11]
• bmibaby do not offer connecting flights, and do not accept responsibility for missed connections.[12]
• Flybe offer connecting tickets, and will try to reaccommodate passengers onto the nearest available flight if a connection is missed.
• Jet2 do not offer connecting flights, and accept no responsibility for missed connections.[13]
• Monarch Airlines do not offer connecting flights, and accept no responsibility for missed connections.[14]
• Smart Wings do not offer connecting flights unless expressly stated otherwise. They recommed a two hour connecting time, but do not guarantee the connection.[15]
• Transavia do not offer connecting flights on their website. Their general conditions of carriage say "If a passenger is prevented from traveling within the period of validity of the ticket because Carrier: ... (5) causes the passenger to miss a connection; ... the validity of such passenger’s ticket will be extended until Carrier’s first flight on which space is available for that passenger in the class of service for which the fare has been paid."[16]
• Meridiana do not offer connecting flights on the website; the conditions of carriage do not cover the issue.[17]
• Iceland Express do not offer connecting flights per se, though they have a service which sets up connecting routes. The contract of carriage accepts no responsibility for missed connections.[18]
[edit] Searching Flights
• Fly Low-Cost Airlines You can search flights from most of low cost airlines.[19]
• The Low-Cost Airlines Guide Has useful pages on low cost carriers sorted by country. [20]
• Viagens low cost You can search flights from every European low cost airlines.[21]
[edit] Restrictions
• Discount airlines are often much more strict about their fares. For example, you have to pay an extra fee for your baggage, e.g. 15-20 € one-way at Ryanair or 8 € at Germanwings. While in traditional airlines they usually allow some baggage over the weight limit, WizzAir will charge you €6 for each kg over the limit. Also, some airlines have lower limits than the usual 20 kg. A few kilograms of weight can double your ticket price. Check your terms carefully and weigh your luggage before a journey. You should weigh and measure your hand luggage too; some discount airlines have lattice boxes at the gate to measure your hand luggage.
• Food is usually not served during the flight, or it is available for a fee. It's best to bring your own food and water. Liquids are allowed through security only in bottles of 100 mL or less (and contained in a clear, 1-liter bag), but you can bring an empty bottle and fill it up at a drinking fountain or restroom tap. Alternatively, buy bottled water after security check.
• In-flight entertainment isn't normally provided either. Again, bring your own (laptop, music player, book or magazine)--although electronic devices are not allowed during landing and takeoff, as they are not with traditional carriers.
• Most discount airlines try to lower airport fees, so they often use smaller and more distant airports, sometimes quite far away from the city they state they fly to. For example Paris Beauvais Airport is some 90 km from Paris, bus costs about €14 one-way and it takes about 1h15 to get to Paris (taxi would be €130-150 one-way), "Frankfurt-Hahn" near the hamlet "Hahn", is actually nearer to the Netherlands (about 100 km) than to Frankfurt (about 125km). Especially Ryanair (the biggest discount airline) uses nearly exclusively such airports.
• Discount airlines do not wait for late running passengers, since an idle plane waiting for a passenger costs money. Check in desks shut promptly at the advertised time. If you are one minute late, they will not let you check in. Also, if you do not get to the boarding gate in time, you may find the plane gone and your luggage sitting on the ground. In these circumstances you will not get a refund, but you may get a transfer to a later flight if there is room.
• Many airlines have changed their schedule with as little as week before departure, so the flight is up to 10 hours earlier/later than in the original reservation. Options they typically give are: accept the change; re-book on a different flight (normally you still have to pay the difference in ticket price but no fee); or accept a refund. Note that purchasing another ticket with either that airline or another at a week's notice may be very expensive relative to your original purchase. When flying low cost it is always better to have good cancellation policy from the supplier connecting with the flight (next flight in your itinerary; hotel at your destination; car rental at the destination airport etc).
• Many discount airliners are "point-to-point" airlines, and do not sell connecting tickets if you need to take two planes to reach your destination. This means you might need to collect your luggage and check it in again for the next leg of the journey, and they do not take responsibility if you miss your connection, even if your connecting flight is with the same airline. This could force you to purchase a new ticket for the next flight. Some low cost carriers (notably Air Berlin) do offer end-to-end tickets, but normally only if you book the entire journey as a single ticket.
• Especially Ryanair desires a internet-check-in. Checking in at the airport costs a surcharge of about 40 € one-way per person.
[edit] Other points to consider
• Do check out deals from the traditional carriers as well, especially on return trips they may have offers rivalling those of the discount carriers. Following competition from discount airlines, traditional carriers such as BA have also cut their fares on competing routes, and are often only about 20% more expensive than discount airlines, a price worth paying if the journey to the airport is cheaper and faster. Sometimes they can even be cheaper than discount airlines, especially during public holidays.
• Also check high speed rail connections in countries where they are available (France, Spain, Germany, Italy, UK). Railway companies have started to offer discounted advance fares (as low as €20 one-way) in response to competition from budget airlines. Travelling by high speed train can often be faster and cheaper than by discount airline, once you take into account the cost and time needed to get to the airport, as well as lenghty embarking/disembarking procedures.
• Contrary to public perceptions, most budget airlines have an excellent safety record.
• The flight frequency and departure/arrival times are usually worse on discount carriers compared to traditional ones.
• Traditional carriers will rebook you on the next available flight for free if something happens. Low cost carriers often charge for this or force you to buy a new ticket.
[edit] easyJet
easyJet plane taking off
easyJet [22] carried 30.3 million in 2005 making them just smaller than Ryanair and the 7th busiest airline in Europe. Fares are priced as single segment one way trips. Their website allows you to book multiple flights simultaneously however, and even allows you to exchange a flight you have purchased for a different flight of your choice on their website providing a partial refund (e.g. changing to a flight on a different date and/or with different passenger names). If you change planes at an Easyjet hub you must collect your luggage and check it in again at the hub. You can book a return at the same time as the outbound but you get no discount for doing so. Some of the advanced features on the Easyjet website are only available if you create an account for yourself on the website.
Following Ryanair, easyJet has no free weight allowance for luggage, and charge for all checked luggage. However, they do not charge extra for printing a boarding pass at the airport.
EasyJet has hubs in 3 London airports (Gatwick, Stansted, Luton) as well as Basel, Berlin, Bristol, Madrid, Dortmund, Edinburgh, Geneva, Paris (CDG and Orly), Liverpool and various other UK and European airports. As well as these hubs they serve 70 other airports throughout Europe, Morocco, Egypt and Israel with over 260 routes.
EasyJet operate an ever-expanding network, keep your eyes peeled to their site. Unlike Ryanair, easyJet tend to operate out of principal airports, such as Barcelona rather than Barcelona Girona, in Spain.
Tickets can range from €20 to €420, all inclusive one-way.
There are no advance seat assignments. However, Easyjet offers the option for pre-boarding for a small surcharge. This could be well worth the money for some travellers. However, this only works where gates lead directly to the plane: if you have to take a bus to the plane, your pre-boarding will only get you on the bus early. After that, it's a scramble with everyone else, pre-boarding or not. Requests for refunds for Speedy Boarding where planes are boarded by bus are generally met with refusal via form emails.
[edit] Ryanair
Ryanair plane
Ryanair [23], with 65 Million passengers in 2009 is Europe's largest low-cost carrier, the 3rd largest airline in Europe in terms of passenger numbers and the largest in the world in terms of international passenger numbers. Ryanair carries more international passengers than any other airline. Fares are priced as single segment one-way trips. If you wish to change planes at one of Ryanair's hubs, then you must book the two segments separately. Luggage is not transferred and must be collected and checked in again. You can book a return at the same time as the outbound but there is no discount for doing so. If you miss a second flight due to a delay in the earlier Ryanair flight, you will not get a refund for the missed flight and will be forced to buy a new ticket at the applicable price, which may be higher than you originally paid.
Tickets start from €0.01 all inclusive one-way during promotions, however always check the full final cost of the fare including all "taxes" and "fees" before booking. Most Ryanair flights that are advertised for €0.01 end up costing at least €10 after such fees, even before airport tax. When booking online (the only reasonable method and possibly the only one available at all), the final cost of your reservation may be difficult to find out until you have confirmed everything, including the payment details. Ryanair charges a credit card fee of €6 per person and segment, which can only be avoided with a prepaid Mastercard credit card. The average price one-way is about €40-50 inc. baggage and all fees and taxes.
Ryanair has a lot of add-on fees such as €15-20 per bag per segment. The fee for overweight luggage is €40 per kilo. There is no free checked baggage allowance on Ryanair, plus they have strict carry-on rules about cabin baggage, only one piece which may weigh no more than 10 kilograms. The cabin baggage rules are not always seem to be enforced though. Airports which are heavily dependent upon Ryanair flights tend to be stricter about enforcing cabin baggage rules and will often check that cabin baggage conforms to Ryanairs limit on dimensions, which is currently 55cm x 40cm x 20cm. Other airports, such as Düsseldorf Weeze, will ask passengers to put their cabin luggage on a set of scales, if they suspect that the luggage may be overweight.
Ryanair operate a large network in Europe, and are generally (but not always) the cheapest airline on the routes where they compete with another airline. They have 41 hubs: in Shannon, Dublin, Glasgow (Prestwick), Liverpool, London (Stansted & Luton), Madrid, Brussels (Charleroi), Düsseldorf (Weeze), Frankfurt (Hahn), Stockholm (Skavsta), Milan Bergamo, Rome, Kaunas, Barcelona (Girona). They serve 142 European destinations as well as Morocco, with over 1,000 routes.
Ryanair uses small airports that can be quite far from the the city they purport to serve so check your travelling time and cost estimates carefully. Coach tickets from airport to city centre may be available through Ryanair, but there may be local competition with more favourable schedules and/or fares. Check through the airport's website, or do an online search for "airport coach" plus the name of the airport.
Ryanair keeps extremely low prices by setting a standard customer behaviour (typically an airport to airport travel without on flight meal and hand luggage only) and placing additional fees for every addition you need. So although you may be able to pick up a €20 fare for a London - Milan flight, in-flight meals and snacks are charged at a premium and there are other charges for things like items of checked-in luggage. Ryanair's "no-frills" approach is aimed at travellers requiring a basic cheap transport service. Beware of getting on last when the plane is fully booked. Because everyone takes the maximum size hand baggage to avoid paying the suitcase fee the containers for hand baggage rapidly get filled up. If there is no room for yours it will be put underneath with the checked-in baggage and you will have to wait for it to arrive at the carousel.
In order to offer faster check-in to passengers with hand luggage only, Ryanair allows passengers to check-in in advance via their website. If booked in advance, there the fixed fare €15.00 per single trip per checked bag is €15 (€20 in July and August and for international flights to and from the Canary Islands). The cost if paid at the airport is €35. Luggage weight limits are 10kg for hand luggage and 15kg for checked luggage or 20kg for an increased charge of €25. It is possible to check in a second piece of luggage, but this is charged at €35 and limited to 15kg (€40 in July and August and for international flights to and from the Canary Islands).
Compared to most other budget airlines, Ryanair provides very limited compensation in the event of flight cancellations, despite the EU regulations. Typically, Ryanair will only provide a replacement seat on a later Ryanair flight (which can depart up to 3 days later than the original flight), or a full refund of the single journey price. Alternative travel arrangements and accommodation are not normally provided by Ryanair. Passengers wishing to return on the same day are normally forced to purchase a new non-advance ticket with a different airline, which can far exceed the price of the original ticket. Therefore, it is advisable to obtain insurance against flight cancellation when travelling with Ryanair.
Also bear in mind that since October 2009, Ryanair is very strict about checking in online and printing your boarding pass at home. You will be charged with a £40/€40 issuing fee if you do not have your boarding pass with you. You must have deposited any checked-in luggage no later than 40 minutes prior to the scheduled departure time. When flying with Ryanair it is advisable to get to the airport early as bag drop desks (as Ryanair terms them) can often have long queues.
[edit] airberlin
airberlin is Europe's third largest discount airline and the second largest German carrier. It offers a huge network between Germany, Austria, Spain and other regions around the Mediterranean Sea like Greece, Tunisia and Egypt as well as throughout most European countries like France, Italy, Russia and Scandinavia. airberlin also offers some long-haul flights, for example to New York, Bangkok and Dubai.
Tickets start from €44,99 one-way including all taxes and fees, free beverages, snacks, sweets and newspapers aboard and up to 20 kg checked baggage. They can be booked one-way at no penalty. Connecting flights via their hubs in Düsseldorf, Berlin, Nuremberg (all in Germany) and Palma de Mallorca (Spain) or Munich are also available.
Some flights, especially from and to Austria, are carried out by their partner NIKI. airberlin is in the preparations to join the oneworld Alliance in spring 2012. Therefore other codeshare partners are American Airlines or S7 Airlines.
[edit] Other low cost airlines
There are 62 low cost airlines in Europe, and this number is rapidly changing. Here are a few of the biggest, grouped by their base country.
[edit] Baltic states
[edit] Latvia
• airBaltic [24] have a wide variety of cheap fares from Riga, which can be used as a transit point. E.g. it is cheaper to travel Odessa-Riga-Kiev with airBaltic than Odessa-Kiev directly with regular-fare airlines.
Be careful when booking from Latvia as the charge in LVL (Latvian Lats) and it is roughly 1.5 euros to a Lat, but the number looks a lot smaller.
[edit] Lithuania
• Ryanair [25] has 18 cheap flights [26] from Kaunas (second biggest city in Lithuania).
[edit] Balkans
[edit] Bulgaria
• Wizzair [27] offers regular flights to Sofia, Bourgas and seasonal (summer) flights to Varna
• Easyjet [28] offers regular flights to Sofia
• Ryanair [29] offers regular flights from Plovdiv to Bergamo-Orio al Serio, Hahn , London-Stansted
[edit] Romania
• Blue Air [30] operates 24 routes
• Ryanair [31] has cheap flights from Constanta to Pisa or Milan Bergamo. Ryanair pulled out the Constanta-Bologna route.
• Wizzair [32] is currently the cheapest operator out of Bucharest, and has many routes to major European cities
[edit] Serbia
• Germanwings [33] offer flights from Belgrade to Cologne-Bonn and Stuttgart.
• Norwegian Air Shuttle [34] operates routes from Belgrade to Oslo and Stockholm.
• Wizzair [35] offers regular flights from Belgrade to Dortmund and London-Luton.
• AirBaltic [36] offers regular flights from Belgrade to Riga
[edit] Kosovo
• Easyjet [37] offers regular flights to Pristina from Basel/Freiburg and Geneva.
[edit] Western and Central Europe
[edit] Ireland
• Aer Lingus [38] operate many routes to and from the Republic of Ireland.
• Ryanair (see above)
[edit] United Kingdom
• EasyJet (see above)
• FlyBe [39] operates out of the UK to many European destinations
• Jet2 [40] operate out of UK airports Belfast, Blackpool, Edinburgh, Leeds/Bradford, Manchester and Newcastle to destinations throughout continental Europe.
• Monarch [41] Operates scheduled low-cost flights from London Luton, London Gatwick, Birmingham and Manchester airports to various destinations in mainland Spain, the Balearics, Portugal, Gibraltar, the Canaries and Cyprus.
• Thomsonfly [42] Operates from many UK airports to destinations across Europe and Northern Africa, as well as to Tel Aviv.
• Thomas Cook [43] Offering flights from 21 UK airports to 75 destinations.
• bmibaby [44], a subsidiary of bmi, had bases in Cardiff, Manchester and East Midlands. They operated domestic flights to Scotland and Northern Ireland and international flights all over Europe. bmibaby ceased flying in September 2012 following the takeover of its parent, bmi, by British Airways.
[edit] Austria
• NIKI operates out of Austria to many European destinations and is a partner of airberlin (see above). Beverages and snacks as well as up to 20 kg checked luggage are included in the fares.
[edit] Czech Republic
• Smartwings [45] operates out of Prague.
[edit] Germany
• airberlin (see above)
• Condor [46] operates out of major airports in Germany mainly to holiday destinations and sells tickets starting at 29,00 € one-way within Europe, putting it into the discount airline bracket as well. Offers free food and beverages catered by Lufthansa parent.
• germanwings [47] operate a large network out of German airports Berlin, Cologne-Bonn, Hamburg, and Stuttgart. The airline also offers guaranteed connection flights between some of its destinations. (from €20 all inclusive, one-way, to all destinations.)
• TUIfly [48] former Hapag-Lloyd Express operate a large network out of Stuttgart, Cologne/Bonn, Hanover, Hamburg and Berlin. Flies as far as Greece and Israel. (from €20 all inclusive, one-way.)
[edit] Hungary
• Wizzair [49] is a Hungarian airline which operates out of Poland and Hungary (fares are from €20 all inclusive, one-way.) At the beginning they charged for all checked luggage, but in the last years they allow weights up to 15 kg free. Please check in advance as the policy on baggage can change without any notice
[edit] Slovakia
• Danube Wings [50] is a Slovakian airline based out of Bratislava which flies to numerous destinations across Ireland, the United Kingdom, Italy, Croatia, Greece, Spain and France just to name a few. Some of the flights are summer only destinations. They have become infamous for cancelling flights with little notice and then not providing a refund. Book with them at your peril.
[edit] Poland
• Wizzair [51] (see 'Hungary' above)
• Norwegian [52] operates direct flights from Warsaw to Oslo-Gardermoen, Bergen, Stavanger, Stockholm-Arlanda, Athens, Dubrovnik, Split, Rome, Salzburg, Paris-Orly and Malaga; from Krakow to Oslo-Gardermoen, Oslo-Rygge, Bergen, Stavanger, Copenhagen-Kastrup and Stockholm-Arlanda; from Wroclaw, Szczecin and Gdansk to Oslo-Gardermoen.
[edit] Switzerland
• Helvetic [53] operates out of Zürich to mainly destinations around the Mediterranean.
• FlyBaboo [54] operates out of Geneva to mainly destinations around the Mediterranean and East Europe.
[edit] France and the Benelux
[edit] Belgium
• Brussels Airlines [55], the successor of SN Brussels Airlines and Virgin Express, operates from Brussels to destinations in Italy, Spain, Greece, Germany, Switzerland, the UK and other countries. With b.light, fares start at €99 all inclusive, return. Brussels Airlines also has an extensive network of destinations in Africa.
• Jetairfly [56] has very cheap flights to far off cities in Europe, America, the Caribbean and even Asia.
• Air Pegasus is a turkish airline that flies from Paris, Frankfurt, Brussels into Asia, with connections in Istanbul. [57]
[edit] France
[edit] Netherlands
• easyJet [58] flies to serveral destinations from Amsterdam Schiphol.
• Martinair [59], a subsidary of KLM, operates out of Amsterdam, offers worldwide destinations. They no longer provide passenger flights since November 2011.
• Ryanair [60] has many cheap flights from Eindhoven Airport
• transavia.com [61] (former BasiqAir), operates out of Amsterdam, Rotterdam, Eindhoven and Groningen to many European destinations.
[edit] Iberia
[edit] Spain
• Air Europa [62] Based in Madrid, travels around Spain, Europe and South America.
• Vueling [63] operates out of Barcelona (Spain) to many Spanish and European destinations,
[edit] Italy
• Meridianafly [64] operates out of Italy to many European & Italian destinations.
• Wind-Jet [65] operates out of Italy to many European & Italian destinations.
• Blu-Express [66] operates within Italy and to Thailand & Mediterranean destinations
[edit] Greece, Turkey and Cyprus
[edit] Greece
• Aegean Airlines [67] operates from Athens to the Greek islands and the biggest cities of Europe.
[edit] Turkey
• Corendon [68] operates out of Turkey to Germany, Belgium and the Netherlands
• Easyjet [69] operates from Istanbul to Basel/Mulhouse.
• Onur Air [70] operates a Turkish domestic network.
• Pegasus Airlines [71] operates from Istanbul to Germany, Netherlands, Switzerland, France, Denmark, Great Britain, Austria, Greece, Ukraine and a domestic network.
• Atlasjet [72] operates a domestic network and flies to northern Iraq, Egypt, and Greece.
• Anadolujet [73], the discount wing of national carrier Turkish Air, operates a domestic network and flies to London, Stockholm, Copenhagen, Dusseldorf, Brussels, and Vienna.
[edit] Scandinavia
[edit] Finland
• Blue1 [74], a Scandinavian Airlines subsidiary and a Star Alliance member, operate routes within Europe (mainly from Helsinki) and eight different cities in Finland. Not really low-cost, but often offers very competitive prices.
[edit] Iceland
• Iceland Express [75] operates out of Reykjavik to 13 airports in Europe including London, Berlin, Paris and the Nordic capitals Copenhagen, Stockholm & Oslo.
[edit] Norway, Sweden, Denmark
• Norwegian [76] operates out of Norway, Sweden, Denmark and Poland. Has very cheap flights. Took over Sweden's FlyNordic in 2008. Main hubs: Oslo (Gardermoen and Rygge), Stockholm, Copenhagen and Warsaw. Domestic flights within Norway, Sweden and Denmark, and international flights to destinations in Europe (primarly from the four hubs, but also from some smaller cities in Norway). Norwegian operates in 25 european countries (plus Morocco, Egypt and the United Arab Emirates), and has flights to more than 70 destinations.
• Cimber Sterling [77] was formed when Danish airline Cimber, bought some of the assets of the now bankrupt low cost airline Sterling. Operates out of Copenhagen and Billund to several destinations in southern Europe and Britain as well as Danish domestic and intra Scandinavian flights. Cimber Sterling filed for bankruptcy in February 2012 and has ceased operations.
[edit] Based outside Europe
[edit] Canada
• AirTransat [78] is a Canadian airline which operates from several Canadian cities (Calgary, Edmonton, Halifax, Montreal, Ottawa, Vancouver and Québec City) to the UK and Europe (fares are from $99 all inclusive, one-way.)
[edit] Asia & Australia
• Air Asia [79] is a low cost airline based in Kuala Lumpur. It provides network services to Australia.
Since of 2013 there is no connection with europe by Air Asia
[edit] Low cost airline hubs
If you cannot find a direct flight with a low cost carrier, it may be necessary to change flights at a low cost airline hub. Make sure you leave plenty of time for connections, as you will not be refunded if you miss a flight. It may be sensible to stay overnight in a city near the hub to be sure you won't miss the flight.
[edit] List of low cost airline hubs
Airport name (airport code) Low cost airlines Local cities
Alicante (ALC) Monarch, Easyjet, FlyBe, Ryanair, Thomas Cook, Jet2, Thomson Airways, transavia Alicante, Barcelona
Amsterdam Schiphol (AMS) Easyjet, transavia, Blue1 Amsterdam, Haarlem, Leiden
Barcelona (BCN) Monarch, Vueling, Easyjet, Thomson Airways, tuiFly, Germanwings, Jet2 Barcelona, Alicante
Orio al Serio Airport (Bergamo - BGY) Ryanair, My Air, Alitalia, BMI Baby, and many others flying Europe-wide, see [80] Milan
Berlin Schoenefeld (Brandenburg - SXF) Easyjet, Ryanair, Germanwings, tuiFly, Condor Berlin, Potsdam
Berlin Tegel (TXL) Air Berlin, HapagLloydExpress, tuiFly, transavia Berlin
Bucharest Baneasa (BBU) Blue Air, Wizz Air, Germanwings, MyAir, EasyJet Bucharest
Bratislava Ivánka (BTS) Danube Wings, Ryanair, Sun D´Or Bratislava, Vienna
Brussels (BRU) Brussels Airlines, Sterling, Blue1, Condor, Jetairfly, Thomas Cook, Brussels
Brussels South (CRL) Ryanair, Jetairfly, Wizz Air Charleroi
Budapest (BUD) Wizz Air, EasyJet, Ryanair, Jet2.com, AerLingus, Germanwings, Norwegian Air, Transavia, Volotea Budapest
Cologne/Bonn (CGN) Easyjet, Germanwings, tuiFly, Wizz Air Cologne, Bonn
Dublin (DUB) Ryanair, Blue1, FlyBe, Germanwings Dublin
Düsseldorf International Airport (DUS) Air Berlin, Easyjet Düsseldorf
East Midlands (EMA) Ryanair, bmi Baby, Jet2, Thomson Airways Nottingham, Leicester, Derby, Peak District
Hamburg (HAM) airbaltic, Blue Wings, Brussels Airlines, Easyjet, FlyBE, Germanwings, Hamburg International, Intersky, norwegian.no, Pegasus, TUIfly, Hamburg
Leeds Bradford (LBA) Jet2.com, Ryanair Leeds, Yorkshire Dales,
Liverpool (LPL) Easyjet, Ryanair, Flybe, Wizz Air Liverpool, Manchester, Preston
London Luton (LTN) Monarch, Easyjet, Ryanair, Flybe, Thomas Cook, Thomson Airways, Wizz Air Luton, London
London Stansted (STN) Easyjet, Ryanair, Kibris Turkish Airlines, Norwegian.No, Germanwings, Atlantic Airways, Blue1, AtlasJet, Transavia, Fly Niki Cambridge, London
Malaga (AGP) Monarch, easyJet, Ryanair, Thomsonfly, Flybe Malaga
Munich (MUC) Easyjet, Brussels Airlines, tuiFly, HapagLloydExpress, German Wings, Blue1 Munich
Paris Beauvais (BVA) Blue Air, Ryanair, Wizz Air Paris
Paris Charles de Gaulle (CDG) Easyjet Paris
Pisa (PSA) Ryanair, Easyjet, tuiFly, Wizzair Pisa
Reykjavik (KEF) Iceland Express Reykjavik, Keflavic
Stuttgart (STR) tuiFly, Condor, Germanwings Stuttgart
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Togo
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[[File:|250px|frameless|Togo]]
Location
[[File:|250px|frameless]]
Flag
[[File:|108px|frameless]]
Quick Facts
Capital Lomé
Government Republic under transition to multiparty democratic rule
Currency Communaute Financiere Africaine franc (XOF)
Area 56,785 km2
Population 7,154,237 (July 2013 est.)
Language French (official and the language of commerce), Ewe and Mina (the two major African languages in the south), Kabye (sometimes spelled Kabiye) and Dagomba (the two major African languages in the north)
Religion Indigenous beliefs 51%, Christian 29%, Muslim 20%
Electricity 127-220/50 Hz (European plug)
Country code +228
Internet TLD .tg
Time Zone UTC
Togo [1] is a narrow country in West Africa, sandwiched between Ghana on the west and Benin on the east, with a small border with Burkina Faso to the north, and a 56km coastline on the Atlantic Ocean to the south.
[edit] Understand
Togo is probably one of the nicest places in Western Africa. Roads are pretty good, distances small, beaches sandy and white, people friendly, hills and mountains waiting to be explored. What else do you need? The capital city, Lomé is an excellent place to start your trip. Lots of daytrips can be made to Togoville on the borders of Lake Togo or Aneho.
When you go north Kpalime and its beautiful hilly surroundings deserve a visit; trekking and hiking in the area is wonderful. Continue further north if you are into hiking. Kara is the place to go. Nearby is Tamberma Valley which has intriguing castle like structures known as Tatas. The national parks of Fazao and Keran offer good opportunities to view wildlife.
A good place to stay in Lomé is Chez Alice in Avepozo, a few miles east of the city along the main coastal road to Benin. You can rent affordable rooms or pitch up your tent in the yard. Good places to eat can be found nearby on the roadside.
Another place to stay is Villa Suedoisé in Aného. It is not big and is definitely affordable. It has a nice view over te Atlantic Ocean.
[edit] History
In an 1884 treaty signed at Togoville, Germany declared a protectorate over a stretch of territory along the coast and gradually extended its control inland. This became the German colony Togoland in 1905. After the German defeat during World War I in August 1914 at the hands of British troops (coming from the Gold Coast) and the French troops (coming from Dahomey), Togoland became two League of Nations mandates, administered by the United Kingdom and France. After World War II, these mandates became UN Trust Territories. The residents of British Togoland voted to join the Gold Coast as part of the new independent nation of Ghana, and French Togoland became an autonomous republic within the French Union.
Togo's size is just less than 57,000 square kilometres (22,000 sq mi). It has a population of more than 7 million people, which is dependent mainly on agriculture. The mild weather makes for good growing seasons. Togo is a tropical, sub-Saharan nation.
Togo gained its independence from France in 1960. In 1967, Gnassingbé Eyadéma, the former leader of the country, led a successful military coup, after which he became President. Eyadéma was the longest-serving leader in African history (after being president for 38 years) at the time of his death in 2005.[4] In 2005, his son Faure Gnassingbé was elected president. About a third of the population live below the international poverty line of US$1.25 a day.
[edit] People
In Togo, there are about 40 different ethnic groups, the most numerous of which are the Ewe in the south (46%) (Although along the south coastline they account for 21% of the population), Kotokoli and Tchamba in the center, Kabyé in the north (22%). Another classification lists Uaci or Ouatchis (14%) as a separate ethnic group from the Ewe which brings the proportion of Ewe down to (32%). However, there are no historic or ethnic facts that justify the separation between Ewes and Ouatchis. On the contrary, the term Ouatchi relates to a subgroup of Ewes which migrated south during the 16th century from Notse the ancient Ewe Kingdom capital. This classification is inaccurate and has been contested for being politically biased; Mina, Mossi, and Aja (about 8%) are the remainder; and under 1% are European expatriates who live in Togo as diplomats and for economic reasons. The Ouatchis are a sub-group of the Ewe just as the Anlo in the Republic of Ghana are a subgroup of the Ewe ethnic group.
More than 60% of Togo's population is under 25. Life expectancy in Togo is somewhere between 60-65. AIDS is a big problem in the country and to this day continues to spread at a very high pace.
[edit] Climate
The climate is generally tropical with average temperatures ranging from 27°C on the coast to about 30°C in the northernmost regions, with a dry climate and characteristics of a tropical savanna. To the south there are two seasons of rain (the first between April and July and the second between October and November).
[edit] Landscape
Highly variable stretching from north to south. Gently rolling savanna in north; central hills; southern plateau; low coastal plain with extensive lagoons and marshes.
Map of Togo with regions colour-coded
[edit] Regions
Maritime Togo (Lomé)
the Atlantic coast and the region that the vast majority of visitors see
Central Togo
rolling hills and forests, seldom visited
Northern Togo (Kara)
land of the Kabye people
[edit] Cities
[edit] Get in
A week long visa will cost you 15,000 CFA at the border. An extension costs 30,000 for a month. American citizens can extend their visa for one year, for 300CFA.
[edit] By plane
Several airlines offer regular flights to Lomé. But flying directly to Togo is often more expensive than flying to Accra in neighboring Ghana. Comfortable, air-conditioned, and reasonably priced buses leave Accra for the border at Aflao. At Aflao, travellers must walk across the border into Lomé and find their own transport inside Togo.
[edit] By car
There are bush taxis everywhere. These are basically four door cars, with four people in the back, and two sharing the front. From either Accra or Benin, you can take bush taxis for $5 to Lomé. From there, you can take them out to more rural areas. You can also offer to pay for the entire car, so that you're not cramped. For this, calculate the price of six people, and then bargain down from there. The Trans-West African Coastal Highway crosses Togo, connecting it to Benin and Nigeria to the east, and Ghana and Côte d'Ivoire to the west. When construction in Liberia and Sierra Leone is finished, the highway will continue west to 7 other ECOWAS nations. A paved highway also connects Togo northwards to Burkina Faso and from there north-west to Mali and north-east to Niger.
[edit] By bus
There are overland buses from Burkina Faso, Ghana, and Benin.
[edit] Get around
A taxi-moto (motorcycle taxi) will cost 150-500 CFA to get you around. You can tell who the taxi-moto drivers are--they will honk or hiss at you as they drive by and usually wear baseball caps and sunglasses. A cab will usually cost about 500 CFA for a one-way short trip inside the city, for trips to the northern parts of the city expect to pay up to 2,500 CFA. Taxis will have yellow license plates and their registration number painted on the car. Always negotiate before you get on/in, the quoted price will include tip!
Sometimes, when you are on a side street, it might be helpful if you ask a security guard to wave down a cab for you. Tipping at around 300 to 600 CFA is expected.
[edit] By train
The train service in Togo is not currently available.
[edit] Talk
French is the national language and the lingua franca. Virtually no English is spoken in the whole of the country, aside from business offices and major banks in the capital.
Ewe is far and away the most widely spoken native language, with the Ewe people populating the southern half of the country. You may also come across the related Mina language in the area around Aneho. Kabiyè is the predominant language of the north.
[edit][add listing] See
Togo is a charming country, but most of the charm comes from the charming people; this is a small country with a small number of small attractions. Lomé's markets, both general and voodoo, are the most popular stop in the country along the road between Ghana and Benin. The smaller towns of Togoville on Lake Togo and Aneho on the ocean are also popular stops for the former's voodoo shrines and historic sights and the latter's beaches.
Lately, the coffee growing region around Kpalimé has become popular with the errant tourist in Togo, with a good number of nice hikes, cooler weather, and pleasant views.
Perhaps the most alluring part of the country is the hardest to get to—the hilly and sparsely populated north. The best known destination is Tamberma Valley—the Koutammakou UNESCO World Heritage site, to the north of Kara. The local Batammariba people (known by colonists as the Tamberma) constructed and live in unique Takienta (a.k.a. Tata) "tower-houses" of mud and straw, which arguably have become the Togolese national symbol. It's a surreal dreamland of a place, and easily a highlight of a trip to Togo, although it is a journey to get there.
Togo's few parks/reserves are relatively rarely visited, but if you manage to make it out there on a safari, Fazao Mafakassa National Park in the center-west of the country is quite beautiful. In the far north of the country is Kéran National Park, with one of the larger elephant populations in West Africa. Aside from Kéran, the north also offers a ton of potential outdoor excursions, with nice hikes up mountains, out to waterfalls, etc.
[edit][add listing] Do
Sports, especially football, are the main entertaining activity in Togo. You can watch the football (soccer) league games played in the weekends (check listings). Apart from football, there are several night clubs that can keep you awake at night, and the capital is full of them; the Chess BSBG is among the most popular. TV programs are not the best in the world, with movies and sitcoms that have been played for years. Plus, the beach offers another type of fun. Many activities and parties are organized there, with people coming from all over Lomé to enjoy the beautiful weather in the weekends. Despite those great things at the beach, you really have to choose a good spot, to avoid stepping or sitting on the unwanted.
[edit][add listing] Buy
Most goods are not supposed to have negotiable prices. Don't haggle with the poor woman trying to sell you a banana. If you are worried about Yovo (white person) surcharges, just ask anyone other than the person you intend to buy from what the price should be. You should, however, haggle over taxi rides, some items, such as clothes, in open markets, and always curios. Keep things in perspective, though. If you are being overcharged 50¢ for being white, that amount is not a big deal to you, but really would help the poor person sitting in the dust all day every day, trying to make ends meet.
[edit] Costs
A liter of gasoline will cost you around 600 CFA, a liter of water around 300 CFA. A baguette is around 175 CFA and half a pound of local coffee will cost 1,200 CFA. A beer in the supermarket will cost your around 350 CFA, at an expat restaurant this will be around 1,000 CFA. A coca-cola will cost you between 200 and 400 CFA in the supermarket. "Western food", mostly imported from France, can be found in supermarkets, but is more pricey than in Europe.
[edit] Markets
The most popular souvenirs from Togo tend to be something voodoo related, like a charm or mask. The obvious place to shop for these curios is Lomé's voodoo market, although you will be paying tourist trap-premium prices.
[edit][add listing] Eat
Pâte is made from corn flour. The "national" dish of West-Africa is Fufu. In Togo, it consists of white yams pounded into a doughy consistency. You will find plenty of Fufu Restaurants in the cities as well as roadside stands. Pâte and Fufu are usually eaten with your hands and come with different sauces (from smoked fish to spicy tomato to peanut). Plantains can also be found in various forms; grilled, cooked, mashed or fried. In the season, Mangos, Avocados, Papayas, and Pineapples are for sale everywhere.
[edit][add listing] Drink
Lemonade and Bissap juice are the most popular drinks. There are many bars almost around all corners in Lomé where you will be able to have a beer.
[edit][add listing] Sleep
If you are interested in staying somewhere different there is the Villa Suédoise, just by the ocean in Aneho. It is an Hostel owned by a swedish woman.
It is far from any tourist streaks and offers a good place to relax. It has a small pool on the roof and offers a nice beach to enjoy. Prices per night streatches from 5000 - 20 000 CFA.
[edit] Work
[edit] Stay safe
As a rule, stay away from public beaches, where tourists find themselves mugged any time of day or night. Most of the country has little crime, but Lomé is a clear exception, and is a good deal more dangerous than any city in Ghana or Benin. If going somewhere at night, take a car taxi, and get the numbers of a few trusted taxi drivers if you plan to stay for a while.
Driving is obscenity in Togo, with fatalistic overloaded speed demons chancing it on curves and hills, capital streets swarming with motorcycles throughout the black of night, and worrisome accident scenes along the main roads. The hilly north-south road north of Kara is particularly dangerous. If you are skeptical, take a day trip, and marvel at all the husks of buses and trucks that weren't there on the way out! Traffic is the single biggest danger to travelers in Togo.
[edit] Stay healthy
Drink bottled water such as Volta or sachets of "Pure Water". Bissop juice is also fairly safe as it is boiled, and avoid the lemonade "citron" despite its delicious aspect. Stay away from road-side meals if possible. People relieve themselves in the streets in Lomé, so be aware of that.
[edit] Respect
Greetings are a little more elaborate in Togo. Say hello to everyone when coming and going. Handshakes are key. Also, maybe if you try to get to know them, you will fit in. Make sure you make yourself feel like you are at home. Don't make it too homey, though, because you don't want to get on their bad side.
[edit] Contact
Lome has Internet cafes, and they are cheap. You buy time by the hour (something like a couple dollars an hour), but most of the cafes feature very slow computers and internet connection speeds. You can buy calling cards along the street. It is, however, much cheaper for people in the United States to call with their calling cards to a Togo cell phone.
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Australian Bureau of Statistics
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ABS Home > Statistics > By Release Date
5368.0 - International Trade in Goods and Services, Australia, July 2012 Quality Declaration
Previous ISSUE Released at 11:30 AM (CANBERRA TIME) 07/09/2012
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EXPLANATORY NOTES
INTRODUCTION
1 This publication presents preliminary estimates of Australia's international trade in goods and services on a balance of payments basis (tables 1–11 and 17) and merchandise import and export statistics on an international merchandise trade basis (tables 12–15). In addition, table 16 Exchange rates and trade–weighted indexes (TWI) are derived by using exchange rates and indexes provided by the Reserve Bank of Australia (RBA). As of December 2011 the RBA has changed the methodology for compiling the TWI to include both merchandise and services trade, rather than merchandise trade only. This takes advantage of country–level services trade data that have been published by the Australian Bureau of Statistics (ABS).
2 Merchandise trade statistics on an international merchandise trade basis are compiled from information submitted by exporters and importers or their agents to the Australian Customs and Border Protection Service (Customs and Border Protection). Adjustments for coverage, timing and valuation are made to international merchandise trade data to convert them to a balance of payments basis. The services data are sourced from the quarterly Survey of International Trade in Services and a range of administrative data and indicator series.
3 More comprehensive quarterly estimates of Australia’s trade in goods and services, together with comprehensive details of Australia’s balance of payments are included in the quarterly publication, Balance of Payments and International Investment Position, Australia (cat. no. 5302.0). Detailed monthly statistics on merchandise trade are available in Time Series Spreadsheets on the ABS website or by subscription to tailored services. More information on the International Trade in Services by Country, by State and by Detailed Services Category are available on a financial year basis (cat. no. 5368.0.55.003) following the September issue of this publication and calendar year basis (cat. no. 5368.0.55.004) following the March issue of this publication.
CONCEPTS, SOURCES AND METHODS
4 The conceptual framework used in compiling Australia's merchandise trade statistics can be found in International Merchandise Trade, Australia: Concepts, Sources and Methods, 2001 (cat. no. 5489.0).
5 The conceptual framework used in compiling Australia's balance of payments statistics is based on the International Monetary Fund's Balance of Payments and International Investment Position Manual, Sixth Edition (BPM6). Descriptions of the underlying concepts and structure of the balance of payments and the sources, methods and terms used in compiling estimates are presented in the publication Balance of Payments and International Investment Position, Australia: Concepts, Sources and Methods, 1998 (cat. no. 5331.0). This version reflects the international standards prior to BPM6 and is currently being revised. The first part of the revised document was released on 8 March 2011, featuring only the Goods Account. Other components will be released as they become available. Further information on the key changes introduced with BPM6 can be found in the Information paper: Implementation of new international statistical standards in the ABS National and International Accounts, September 2009 (cat. no. 5310.0.55.002).
6 To bring merchandise trade statistics on an international merchandise trade basis to a balance of payments basis, timing adjustments are made to ensure that the transaction is recorded in the period in which ownership changed, rather than in the period in which the transaction was recorded by Customs and Border Protection or the period in which the goods arrived in/departed from an Australian port. Adjustments are also made to account for the change of ownership of goods not included in merchandise trade statistics. Chapter 6 of cat. no. 5331.0 provides more detail on the relationship between statistics on an international merchandise trade basis and on a balance of payments basis.
7 International merchandise trade exports data presented for recent months in tables 12–15 of this publication are based on information initially reported by exporters to Customs and Border Protection. At the time of initial reporting to Customs and Border Protection the final prices may not be known for some commodities. Therefore, the information recorded for recent months for commodities like iron ore and coal can include a variety of prices including previous or current contract prices and the prevailing spot prices. Newly negotiated contract prices may not be fully reflected in the data first reported to Customs and Border Protection, and to compensate for this, the balance of payments series may reflect adjusted price levels.
8 The Concepts, Sources and Methods publications and information papers are available to download for free from the ABS website. Select Statistics, then select By Catalogue Number, and then the catalogue numbers as above.
CLASSIFICATIONS
9 The merchandise trade data on an international merchandise trade basis are presented using the following commodity classifications:
• the Harmonized System 2012 (available on request)
• the Standard International Trade Classification (SITC Revision 4)
• the Classification by Broad Economic Categories (BEC)
• the industry classification: Australian and New Zealand Standard Industrial Classification, 2006 (ANZSIC 06) from July 2005.
10 The balance of payments 'goods and services' series are presented according to three classifications with the goods classifications derived from SITC Revision 4 and BEC. The classifications are:
• for goods:
• the Balance of Payments Commodities for Exports (BoPCE)
• the Balance of Payments Broad Economic Categories (BoPBEC) for Imports
• for services:
• the Extended Balance of Payments Services Classification (EBOPS).
ACCURACY, RELIABILITY AND REVISIONS
11 While every effort is made to ensure the accuracy and reliability of estimates, most series are subject to revision as more complete and accurate information becomes available. Care should be exercised in the use and interpretation of estimates in this publication. The transactions occurring in international trade in goods and services are of many different kinds, and therefore the compilation of trade estimates entails the use of a very wide range of statistical data of varying degrees of accuracy and timeliness. For further information on the monthly services series, see paragraph 25.
12 The revisions are applied differently for merchandise trade and balance of payments series. Each month, merchandise trade data are revised for the previous six months to incorporate latest available data.
13 For the balance of payments 'goods and services' series, in original terms, revisions are not usually applied in the July, October, January or April issues. This is to ensure the monthly series align with the comparable series in the Balance of Payments and International Investment Position, Australia (cat. no. 5302.0). In the September issue revisions can be applied to the four previous financial years. In other issues revisions can be applied to the previous and current financial years to incorporate the latest available survey and administrative data.
14 In seasonally adjusted and trend terms, revisions may occur at any time point but tend to focus on the most recent estimates. Please also refer to paragraphs 17 (seasonal adjustment) and 19 (trend estimates) below.
SIGN CONVENTION
15 In keeping with BPM6 conventions, balance of payments basis credit entries are shown with an implied positive sign and debit items are shown as negative entries. For statistics on an international merchandise trade basis, both imports and exports are shown without sign. The calculation of percentage changes on balance of payments debit items are made without regard to sign. References to balance of payments debit items in Key Figures, Key Points, and Analysis and Comments are also made without regard to sign.
SEASONALLY ADJUSTED AND TREND ESTIMATES
16 The estimates of international trade in goods and services on a balance of payments basis are seasonally adjusted, however the merchandise exports and imports statistics on an international merchandise trade basis are not. Monthly original estimates are volatile, being subject to calendar–related and large irregular influences. Seasonally adjusted estimates are derived by estimating and removing from the original series systematic calendar related effects, such as seasonal (e.g. Christmas), trading day and moving holiday (e.g. Easter) influences. Seasonal adjustment does not aim to remove the irregular or non–seasonal influences which may be present in any particular month. These irregular influences may reflect both random economic events and difficulties of statistical recording.
17 The seasonally adjusted statistics in this publication use the concurrent seasonal adjustment technique and Autoregressive Integrated Moving Average (ARIMA) modelling to estimate factors for the current and previous months. Under concurrent seasonal adjustment, the estimates of seasonal factors are fine tuned as new or revised original estimates become available each period. The seasonally adjusted estimates are subject to revisions at each reference month as the estimates of seasonal factors are improved.
18 ARIMA modelling relies on the characteristics of the series being analysed to project future period data. ARIMA modelling is used on a case–by–case basis where it results in reduced revisions to seasonally adjusted series when subsequent data becomes available. The projected values are temporary, intermediate values, that are only used internally to improve the estimation of the seasonal factors. The projected data do not affect the original estimates and are discarded at the end of the seasonal adjustment process. The ARIMA model is assessed as part of the annual review and, following the 2011 annual review, the majority of directly seasonally adjusted trade in goods and services time series use an ARIMA model.
19 The month–to–month movements of the seasonally adjusted estimates may not be reliable indicators of underlying behaviour because they include irregular or non–seasonal movements. Trend estimates reduce the effect of these movements as they are derived by applying a 13–term Henderson moving average to the seasonally adjusted series. The 13–term Henderson moving average (like all Henderson averages) is symmetric, but as the end of a time series is approached, asymmetric forms of the average are applied. While the asymmetric weights enable trend estimates for recent months to be produced, it does result in revisions to the estimates for the most recent six months as additional observations become available. Revisions to trend estimates can also occur because of revisions to the original data and as a result of the re–estimation of the seasonal factors. Trend estimates should be used with caution, especially around the time of unusual influences, until these have been appropriately taken into account.
20 Information papers and articles on time series methods are available on the ABS website:
ECONOMIC TERRITORY
21 In accordance with BPM6 definitions, Australia's economic territory, on a balance of payments basis, is the area under the effective control of the Australian government. It includes the land area, airspace, territorial waters, including jurisdiction over fishing rights and rights to fuels and minerals. Australian economic territory also includes territorial enclaves in the rest of the world. These are clearly demarcated areas of land, located in other countries and which are owned or rented by the Australian government for diplomatic, military, scientific or other purposes. Specifically, the economic territory of Australia consists of:
• Geographic Australia which includes Cocos (Keeling) Islands and Christmas Island;
• Norfolk Island;
• Australian Antarctic Territory;
• Heard Island and McDonald Islands;
• Territory of Ashmore Reef and Cartier Island;
• Coral Sea Islands;
• Australia's territorial enclaves overseas; and
• the Joint Petroleum Development Area (joint territory between Australia and East Timor (Timor–Leste)).
22 Because of administrative complexities and measurement difficulties, Norfolk Island transactions with the rest of the world will not always be captured in all relevant balance of payments statistics. Most of the transactions involving Norfolk Island are not material to Australia's trade performance and not capturing these transactions will not distort these statistics. However, any significant transactions will be identified and included in the relevant statistics.
COMMODITY BREAKDOWN OF GOODS
23 For details of the classifications used to present goods exports (credits) and imports (debits) on a balance of payments basis, see tables 6.6 and 6.7, respectively, in Balance of Payments and International Investment Position, Australia: Concepts, Sources and Methods (cat. no. 5331.0).
24 The international merchandise trade statistics shown in tables 12 and 13 are classified by the Standard International Trade Classification (SITC). Imports and exports of goods at a more detailed level are available from the time series spreadsheets on the ABS website. All data from July 2005 are presented according to SITC Rev 4 and data prior to July 2005 are presented according to SITC Rev 3. For details refer to the 'Classifications and Standards Update' in the July 2008 issue of this publication.
MONTHLY SERVICES SERIES
25 Monthly indicators for many of the services components that are only surveyed quarterly are not available. Monthly estimates for the freight and other transportation components of services credits in table 9 are derived by dividing the quarterly estimate by three. Freight debits are derived directly from imports data for the reference month as a difference between total merchandise cost, insurance and freight (CIF) and free on board (FOB), adjusted to reflect timing and processing adjustments. For freight credits and other transportation credits and debits, estimates are derived by extrapolating the last quarter's data by an average of movements for the same quarter over the three previous years, and then dividing the estimate by three. Similarly other services estimates are derived by extrapolating the last quarter's data by an average of movements for the same quarter over the previous three years, and then dividing the estimate by three.
CONFIDENTIALITY OF MERCHANDISE TRADE STATISTICS
26 The release of statistics for certain merchandise trade commodities is restricted in order to prevent the identification of the activities of an individual business, where this is requested by the business concerned. These restrictions do not affect the total value of exports and imports, but they can affect statistics at the country, state and commodity levels. Imports data that have the confidentiality restrictions 'No commodity details' or 'No value details' are aggregated into a single confidential commodity code. For data prior to September 2008, these data are added back into the appropriate state total and country total (i.e. these totals show the correct level of trade). From September 2008 these confidential data are not added back. Instead the confidential data are published as 'No country details' in the country totals and 'State not available for publication' in the state totals. Therefore, country and state totals from September 2008 may not represent the actual amount of trade in each country/state but only the trade in commodities without a 'No commodity details' or 'No value details' restriction. For information on the confidentiality restrictions applied to the merchandise trade statistics in this publication, refer to the current issue of International Merchandise Trade: Confidential Commodities List (CCL) (cat. no. 5372.0.55.001).
TOURISM RELATED SERVICES
27 The tourism related services memorandum items provide timely indicators of the movements in tourism related activities, not an absolute measure of the level of these activities. The tourism related indicator has been derived by combining total travel services (business, education–related and other personal travel) and passenger transportation services (which includes agency fees and commissions for air transport).
SERVICES BY STATE AND BY PARTNER COUNTRY
28 Annual services data by state, by country and detailed services are released twice yearly. Calendar year data are usually released following the March issue of this publication and financial year data are usually released following the September issue. Refer to time series data under trade in services data cubes for details.
29 Services credits are classified by the state of provision, while services debits are classified by the state of consumption. The state allocations for transportation, travel, postal and courier, and telecommunication services are based on a number of indicators including merchandise trade statistics by state, overseas passenger arrivals and departures by state of clearance and data provided by the Department of Immigration and Citizenship. The allocation for other services (about 25% of all trade in services) is primarily based on the location of the business reporting the information, which serves as a proxy for the state of provision/consumption of that service. The data should be used with care but are considered suitable for analysis over time.
30 A comprehensive explanation of the data sources used and the methodology applied in the compilation of partner country statistics is provided in Chapter 17 of Balance of Payments and International Investment Position, Australia: Concepts, Sources and Methods (cat. no. 5331.0). Approximately 5% of total services credits and debits for both 2009–10 and 2010–11 were either confidential, or unable to be allocated to individual countries.
ABS DATA AVAILABLE
31 More detailed balance of payments and international merchandise trade data, including forward seasonal factors are available as time series spreadsheets or data cubes from the ABS website. Merchandise trade data by commodity, country and state that are not on the ABS website may be available on request. Inquiries should be made to the National Information and Referral Service as shown on the back of this publication.
RELATED PRODUCTS AND PUBLICATIONS
32 Users may also wish to refer to the following publications which can be downloaded free of charge from the ABS website by using the 'Statistics' tab and selecting the relevant catalogue number:
33 Current publications and other products released by the ABS are available from the 'Statistics' tab on the ABS website. The ABS also issues a daily Release Advice on the website which details products to be released in the week ahead. A foreign trade theme page and a balance of payments theme page are available on the ABS website. Select Topics @ a GlanceEconomyForeign Trade or Balance of Payments. This page provides direct links to all foreign trade and balance of payments related data and publications, recent changes and forthcoming events, links to relevant websites and a range of other information about the Australian International Accounts.
ROUNDING
34 Where figures have been rounded, discrepancies may occur between sums of the component items and totals. Percentage movements are calculated from data at the level of precision presented in this publication (i.e. $m) except for international merchandise trade tables.
© 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
5625.0 - Private New Capital Expenditure and Expected Expenditure, Australia, Dec 1996
Previous ISSUE Released at 11:30 AM (CANBERRA TIME) 27/02/1997
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• About this Release
Preliminary estimates derived from a sample survey of business enterprises. Contains estimates of actual and expected new capital expenditure by type of asset (new buildings and structures/other new capital equipment) and by selected ASIC subdivisions. These statistics are expressed in current and constant prices and in original, and seasonally adjusted terms.
This publication has been converted from older electronic formats and does not necessarily have the same appearance and functionality as later releases.
© 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
4390.0.40.002 - Private Health Establishments: Free Standing Day Hospital Facilities Data Report on Hardcopy, 2002-03
Previous ISSUE Released at 11:30 AM (CANBERRA TIME) 22/11/2004
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NOTES
ABOUT THE PUBLICATION
This Special Data Series publication presents details on Private Free-standing Day Hospital Facilities from the 2002-2003 national census of private hospitals.
The 2002-2003 national census of private hospitals used two forms to collect data: PHE1 for private acute and psychiatric hospitals and PHE2 for free-standing day hospital facilities. A copy of the relevant collection form can be provided on request. The form contains definitions which may help you with the interpretation of the data in the tables.
Please note that in each table title, a reference to the relevant question number on the collection form is shown in brackets.
State level data has been provided, where possible, for New South Wales, Victoria, Queensland and Balance.
In most tables, data has been rounded. This rounding can cause totals to be different to the combine values of the data shown in the table.
The associated publications, Private Hospitals, Australia, 2002-2003 (cat. no. 4390.0), released 21 Septemer 2004 ($23.00) and Private Health Establishments, Private Acute and Psychiatric Hospitals Data Report 2002-2003 (cat. no. 4390.0.40.001) released 22 November 2004 ($505.00) can be obtained by contacting any ABS office.
Please contact Andrew Cumpsty (1800 806 415) if you would like to find out more about the tables provided herewith or the associated publications.
INQUIRIES
For further information about these and related statistics, contact the National Information and Referral Service on 1300 135 070 or Andrew Cumpsty on Brisbane (07) 3222 6374.
© 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 Catalogue Number
4651.0 - Land Management: Fitzroy and Livingstone Shires Queensland, 2004-2005
Latest ISSUE Released at 11:30 AM (CANBERRA TIME) 01/02/2006
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• Main Features
• Natural resource management survey puts a spotlight on Fitzroy and Livingstone Shires in Queensland (Media Release)
MEDIA RELEASE
February 1, 2006
Embargoed: 11:30 AM (AEST)
09/2006
Natural resource management survey puts a spotlight on Fitzroy and Livingstone Shires in Queensland
A new survey approach by the Australian Bureau of Statistics (ABS) presents Queensland's Fitzroy and Livingstone Shires' natural resource management (NRM) practices across previously unavailable boundaries.
The results of the survey, released today, use information from about 1,300 land owners and provide an important insight into land management in both shires. The survey covered 1.2 million rural hectares.
The data is presented for five different geographical boundaries - by shire, for a riparian zone 5km either side of the Fitzroy River, for a 20km wide coastal zone, for radial zones from the Rockhampton City Centre and by neighbourhood catchments.
Of the 4,025 rural landholders in both shires, 3,289 reported that their holdings had NRM issues during the year ended 30 June 2005. Residential landholders in the Fitzroy Shire spend an average of $40 per hectare addressing NRM issues compared to $54 per hectare in the Livingstone Shire. In the Fitzroy Shire, non-residential landholders spent an average of $12 per hectare addressing NRM issues compared to $10 per hectare in the Livingstone Shire.
Age or ill health was reported as a barrier to improving NRM practices by 63% of residential landholders within the 5km Fitzroy River riparian zone. Nearly three-quarters of the non-residential landholders within the riparian zone reported age or health issues as a barrier.
The survey showed that 14% of the non-residential landholders within the riparian zone plan to sell all or part of their holdings in the next five years.
Within the 20km coastal zone 88,075 hectares were classified as grazing land. As at 30 June 2005, a total of 28,200 cattle were being grazed on 94% of this grazing land. The stocking rate was 2.9 hectares per beast. In comparison, the stocking rate outside the coastal zone was 4.0 hectares per beast.
The full results of the survey are found in Land Management: Fitzroy and Livingstone Shires Queensland, 2004-2005 (cat. no. 4651.0) which is available free from the ABS web site.
The new survey approach could revolutionise collection of land management data in rural areas and is strongly supported by those areas which have experienced it.
It was first used in the Eurobodalla Shire (NSW) in 2004. The 2005 trial focused on the Fitzroy and Livingstone Shires in Queensland.
© 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 Catalogue Number
8164.0 - Discussion paper: The first iteration of the Business Longitudinal Database, 2004-05
Latest ISSUE Released at 11:30 AM (CANBERRA TIME) 10/05/2007 First Issue
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• About this Release
This publication provides an overview of the data contained in the Business Longitudinal Database (BLD) and examples of its analytical potential. The BLD comprises characteristics and financial data sourced from the first Business Characteristics Survey (BCS), ATO administrative data (including BAS and BIT data), as well as data on exports and imports from the Australian Customs Service.
Whilst the publication does contain some initial data from the BLD its primary purpose is to encourage feedback from BLD users as to the adequacy of the topics and financial variables contained in the BLD and the methodology by which productivity is to measured.
© 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 Catalogue Number
1301.0 - Year Book Australia, 2012
Latest ISSUE Released at 11:30 AM (CANBERRA TIME) 24/05/2012
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HEALTH STATUS
The World Health Organization (WHO) defines health as “... a state of complete physical, mental and social wellbeing, not merely the absence of disease or infirmity”. Aspects of the physical and mental health of a population can be assessed by examining the prevalence of diseases (i.e. the total number of cases of a disease in a given population at a specific time), as well as disability and mortality rates. Assessing the social and mental wellbeing of a population is more subjective, although the ABS can partly meet these information needs through instruments such as the SF-12 questions on self-assessed health and bodily pain, and the K10 questions that determine levels of psychological distress.
The SF-12 is a short-form health survey with 12 questions that provide data on physical and mental health and wellbeing. The K10 is a 10-item questionnaire that provides a global measure of distress based on questions about anxiety and depressive symptoms that a person has experienced in the most recent four-week period (the Kessler Psychological Distress Scale). Data for the SF-12 were collected from people aged 15 years and over, while the K10 was asked of people 18 years and over.
This section presents general wellbeing data from these question sets in the 2007–08 National Health Survey (NHS), along with data for selected long–term health conditions in Australia in 2007–08. Long-term conditions are defined as medical conditions or injuries that were current at the time of reporting and had lasted, or were expected to last, for at least six months. In most cases, respondents were asked about conditions that had been medically diagnosed. The section includes an overview of the most prevalent conditions and more detailed data for the Australian national health priority areas.
The ABS classifies data on health conditions according to the International Statistical Classification of Diseases and Related Health Problems, 10th Revision (ICD-10).
The data presented in this section are as reported by respondents to the 2007–08 NHS. The reported data may underestimate or overestimate actual prevalence. For example, some people may not realise that they are suffering from a condition, or may not have understood their diagnosis correctly. Biomedical data from the 2012–13 Australian Health Survey will be invaluable in measuring some of this uncertainty.
WELLBEING
Self-assessed health
In 2007–08, the majority of people aged 15 years and over considered themselves to be in good health, with 85% reporting their health status as good, very good or excellent. The proportion of people reporting fair or poor health increased with age, from 7% of those aged 15–24 years to 39% of those aged 75 years and over (graph 11.1).
Bodily pain
One in ten people aged 15 years and over in 2007–08 reported feeling severe (8%) or very severe (2%) pain in the four weeks prior to being surveyed. One in five (19%) reported moderate pain and 39% had mild or very mild bodily pain. Rates of experiencing moderate to very severe pain increased steadily with age, from 18% of people aged 15–24 years to 43% of people aged 75 years and over. Females were slightly more likely than males to report any bodily pain in the previous four weeks (69% compared with 65%). A third of people 15 years and over felt no bodily pain in the previous four weeks (33%).
Bodily pain did not necessarily correlate with feelings of poor health. Four out of five people (81%) who reported any bodily pain in the previous four weeks rated their general health as good to excellent, as did nearly half (46%) of people who reported experiencing very severe pain in this time. On the other hand, 33% of people with very severe pain rated their health as poor, compared with the average of 4%.
Levels of psychological distress
In 2007–08, around 12% of people aged 18 years and over experienced high or very high levels of psychological distress and a further 21% experienced moderate levels of psychological distress (similar to 2001 and 2004–05 rates). Women were more likely than men to experience psychological distress, with 14% of women and 10% of men experiencing high or very high levels of distress, and 23% of women and 19% of men experiencing moderate levels.
People with high or very high levels of psychological distress were much more likely to rate their general health as only fair or poor (42% compared with 9% of those with low distress levels – see graph 11.2).
LONG-TERM HEALTH CONDITIONS
In 2007–08, three-quarters of people (of all ages) reported (or had reported on their behalf) having a long-term health condition. Just over one in four people were long-sighted (26% or 5.3 million people), and a further 23% were short-sighted (4.7 million people). Arthritis and Hayfever and allergic rhinitis each affected one in seven people (15% or 3.1 million people), similar to Back pain and disc disorders (14% or 2.8 million). Around one in ten people was affected by each of the following conditions:
• Mental and behavioural problems (11% or 2.3 million people)
• Complete or partial hearing loss (10% or 2.1 million people)
• Asthma (10% or 2 million people)
• Hypertensive disease (9% or 1.9 million people).
Other commonly reported conditions were High cholesterol and Migraine (both 6% or 1.2 million people); Heart, stroke and vascular diseases (5% or 1 million); Diabetes mellitus (4% or 818,000 people); Disorders of thyroid gland (2% or 486,000 people); and Malignant neoplasms (cancer) (2% or 327,000 people). Graph 11.3 shows rates for these conditions by sex.
The prevalence of long-term health conditions rose steadily with age, from 37% of people under 15 years of age, to 99% of people aged 55 years and over. Females were slightly more likely to have one or more long-term health conditions than males (78% compared with 73%), which may be partly due to the fact that there are more women in the older age ranges (In 2007–08, 54% of people over the age of 65 years were women).
Interestingly, 83% of people with a long-term health condition felt that their general health was good, very good or excellent.
NATIONAL HEALTH PRIORITY AREAS
The Australian Government National Health Priority Areas (NHPA) initiative focuses on conditions that contribute most to the burden of disease in the community, particularly where this can be significantly reduced through health policy or programs. The burden of disease and injury is a measurement of time lost due to premature death, and years of healthy life lost due to disability; however, the NHPAs also represent a considerable economic and social burden.
This section presents data on arthritis and musculoskeletal conditions, asthma, cancer, conditions of the circulatory system, diabetes, injuries and mental health. The remaining NHPA, obesity, is discussed in HEALTH RISK FACTORS later in this chapter.
Wellbeing of people with NHPA conditions
People aged 18 years and over with NHPA conditions were more likely to report experiencing severe or very severe levels of bodily pain, general health that was only fair or poor, and higher levels of psychological distress than average, as shown in the graphs 11.4, 11.5 and 11.6.
Arthritis and other musculoskeletal diseases
Osteoarthritis, rheumatoid arthritis and osteoporosis are the most commonly occurring musculoskeletal conditions. Although they are not immediately life threatening and have low associated mortality, they have substantial influence on people's quality of life and impose a heavy economic burden on the community. In 2004–05, total health expenditure attributable to musculoskeletal diseases was $4.0 billion, which accounted for 8% of allocated health system expenditure in Australia (AIHW, 2010a).
In 2007–08, 15% of people reported that they currently had arthritis (13% of males and 18% of females). Of people with arthritis, 51% had osteoarthritis and 14% rheumatoid arthritis. The proportion of people with arthritis increased with age, from 2% of people under 35 years to 53% of people aged 75 years and over.
Around 1% of males and 6% of females had osteoporosis (3% overall). Rates of osteoporosis also increased with age, from less than 1% of people under 35 years to 16% of people aged 65 and over (graph 11.7).
Arthritis and musculoskeletal diseases were the underlying cause for 1,078 registered deaths or 0.8% of all deaths registered in Australia in 2009, and were identified as either an underlying cause or associated cause of death for 6,410 deaths registered in Australia in that year.
Of all deaths due to arthritis and musculoskeletal diseases in 2009, 761 (71%) were female, predominantly in the age group 75 to 94 years. The median age at death for deaths due to these diseases was 83 years (80.9 years for males and 84.0 years for females).
Asthma
By international standards, the prevalence of asthma is relatively high in Australia, with more than two million people (10%) reporting having the disease in 2007–08. Asthma was the most prevalent chronic illness in children under 15 years of age (10%). Boys (12%) were more likely than girls (8%) to have asthma, although this was reversed for people aged 15 years and over, with 12% of females and 8% of males having the condition.
Asthma is more likely to be reported by people living in more disadvantaged areas than those in less disadvantaged areas (graph 11.8).
People with asthma can experience reduced quality of life and require a range of health services to manage their condition. Asthma is a common cause of absenteeism from school and also affects family, work and recreation (AIHW, 2011a). In 2004–05, the condition accounted for 1.2% of allocated health system expenditure in Australia, while respiratory diseases as a whole accounted for $3.3 billion, or 6% of allocated health expenditure.
In 2007–08, just over a quarter (26%) of adults aged 18 years and over with asthma rated their health as only 'fair' or 'poor' compared with 15% of adults without asthma (graph 11.5). Asthmatics also reported higher levels of psychological distress, with 20% of asthmatics aged 18 years and over reporting high or very high levels of psychological distress in the previous four weeks, compared with 11% of people without asthma (graph 11.6).
Around 22% of people with asthma reported that their asthma had become worse or out of control in the previous year. Of those people, 42% went to hospital for treatment. Children tended to have higher rates of hospitalisation than adults – 64% of children whose asthma got worse or out of control in the previous year had been to hospital for their asthma, compared with 33% of people aged 15 years and over. One-fifth (21%) of people with asthma had a written asthma action plan.
Asthma symptoms are usually reversible with treatment, but death can sometimes result if a severe asthmatic episode is not managed properly. In 2009, asthma was the underlying cause for 411 registered deaths, or 0.3% of all deaths in Australia, and was identified as either an underlying cause or associated cause of death for 1,344 deaths in this time.
Women were twice as likely to have asthma as an underlying cause of death as men, with 100 female deaths for every 46 male deaths. The median age at death for deaths due to asthma was 73.1 years for males, 80.2 years for females and 77.9 years overall.
Cancer
In 2007–08, an estimated 1.6% of people reported that they had a medically diagnosed malignant neoplasm (cancer). Rates of cancer increased with age, from less than 1% of people aged under 15 years to around 5% of people aged 65 years and over. It should be noted that the 2007–08 National Health Survey excluded people in hospitals, nursing and convalescent homes and hospices, which may have a greater effect on cancer data than on other conditions.
Of people aged 18 years and over with cancer:
• 17% reported experiencing severe or very severe bodily pain in the previous four weeks (compared with the national average of 10%, see graph 11.4)
• 47% felt that their general health was only fair or poor (much higher than the national average of 15%, see graph 11.5) and
• 20% experienced high or very high levels of psychological distress in the previous four weeks (compared with the national average of 12%, see graph 11.6).
Cancer accounted for $3.8 billion (7%) of Australia's expenditure on health in 2004–05; however, it was responsible for nearly a fifth (19%) of premature death and disability in that year.
In 2009, cancer was the underlying cause of death for 41,952 registered deaths, accounting for 30% of all registered deaths. Cancer contributed to a total of 48,165 deaths as either an underlying or associated cause of death. The standardised death rate for cancer was 175.6 per 100,000 population in 2009, with 130 male deaths per 100 female deaths for the reference year. The median age of people dying from cancer was 75.1 (75.0 years for men and 75.4 years for women).
Conditions of the circulatory system
Although death rates for conditions of the circulatory system have declined in recent decades, this group of diseases continues to be one of the biggest health problems in Australia, and its health and economic burden continues to exceed that of any other disease (AIHW, 2011b). In 2004–05, the highest health expenditure of all disease groups was for conditions of the circulatory system at $5.9 billion, or 11% of total allocated health expenditure.
Many Australians are at increased risk of developing some form of circulatory disease due to risk factors such as cigarette smoking, high blood pressure, high cholesterol level, being overweight or leading a sedentary lifestyle. High cholesterol levels were reported by 6% of people, rising with age to 17% of people aged 65 years and over. Data for other risk factors are presented in HEALTH RISK FACTORS in this chapter.
In 2007–08, 16% of people reported one or more long-term conditions of the circulatory system, with the most common being hypertension (high blood pressure) at 9%. One in ten people aged 45–54 years reported having hypertension, increasing to 35% of people aged 65 years and over.
Circulatory conditions were mostly experienced by people in middle and older age groups. Almost one in five (19%) people aged 45–54 years had a current long-term circulatory condition, rising progressively to 62% of people aged 75 years and over. The circulatory conditions that form the most significant health and economic burden are heart, stroke and vascular diseases (including ischaemic heart disease, cerebrovascular disease, oedema, heart failure, and diseases of the arteries, arterioles and capillaries). Around 7% of people reported having a heart, stroke or vascular disease in 2007–08 (8% of males and 6% of females).
Ischaemic heart disease was the leading cause of death in 2009, with 12,047 male and 10,476 female deaths (115 male deaths per 100 female deaths). In 2007–08, around a quarter of people who had a heart, stroke or vascular disease (25%) reported experiencing severe or very severe levels of bodily pain in the last 4 weeks, compared with the national average of just under 10% (graph 11.4).
Overall, conditions of the circulatory system accounted for 46,106 deaths in 2009, or 33% of all deaths, contributing to 80,375 deaths overall as either an underlying or associated cause of death. The general decrease in deaths from these conditions since the 1960s (graph 11.9) is due in part to a reclassification of underlying causes of death (with some deaths shifting from Cerebrovascular diseases to Vascular dementia). The median age of people dying from circulatory conditions was 81.3 years for males and 87.2 years for females.
Diabetes mellitus
Diabetes is a costly disease associated with substantial morbidity and mortality, mostly from cardiovascular complications, eye and kidney diseases, and limb amputations. In 2004–05, total health expenditure attributable to diabetes was nearly $1.0 billion, accounting for 2% of allocated health system expenditure.
In the 2007–08 NHS, 4% of people reported having diabetes as a long-term condition, rising from 2.4% in 1995. More males than females had the condition (5% and 3% respectively). Diabetes rates also increased with age, particularly after the age of 45 years (graph 11.10).
Over half of all people aged 18 years and over with diabetes felt that their general health was only fair or poor (52%), compared with the national average of 15% (graph 11.5).
The majority of people with diabetes (88%) reported that they had Type 2 diabetes. Type 2 diabetes is associated with a number of risk factors, such as excess weight, poor diet, inactivity and smoking. For example, of people aged 35 years and over, those who were obese were about twice as likely to have Type 2 diabetes as those in other weight ranges (51% compared with 27% after adjusting for age). People in this age group who were sedentary or exercised at low levels were also more likely to have diabetes than people who exercised at high or moderate levels (8% and 6% after adjusting for age).
Diabetes was the underlying cause for 4,170 (3%) deaths registered in Australia in 2009, and contributed to 14,286 (10%) deaths as either an underlying or associated cause of death. The standardised death rate for this condition was 17.1 per 100,000 population in 2009 (20.6 per 100,000 males, and 14.2 per 100,000 females), an increase from 16.0 per 100,000 population in 2000.
The median age at death was 80.9 years (78.5 years for men and 83.3 years for women). Type 2 diabetes accounted for 1,772 deaths, or 42% of all diabetes deaths.
Injuries
Injuries (including poisoning) are associated with high morbidity, and are a leading cause of premature mortality in Australia. They can result in fatalities, survival with ongoing dysfunction or the onset of secondary conditions (such as osteoarthritis in injured joints). In 2009, just over 6% of people with a disability reported injury or accident as their main disabling condition.
The terms ‘injury’ and ‘poisoning’ encompass the adverse effects on the human body that result from particular events. These can be accidental such as falls, vehicle accidents and exposure to chemicals, or intentional such as suicide attempts and assaults by other people. Injury patterns vary significantly by age and sex. Near-drowning and drowning, for example, are major causes of injury and death in early childhood, while self-harm and road crashes are primary causes of injury in young adulthood. Falls are the most common cause of death caused by injury among the elderly. Incidence rates of serious injury are higher for males than females, both overall and for most types of injury (AIHW, 2010b).
In 2004–05, total health expenditure attributable to injuries was $3.4 billion, accounting for 7% of allocated health system expenditure.
In 2007–08, injuries accounted for over 1 in 20 (426,000) hospitalisations in Australia (AIHW, 2010b). About 2.4 million people had a long-term condition caused by an injury. Almost half (47%) of disc disorders and 30% of back pain/problems were caused by an injury, as were 15% of arthropathies (joint diseases) and 10% of partial deafness/hearing loss cases. Nearly 80,000 people had an amputation as a result of an injury.
Most people who had a long-term condition as the result of an injury acquired it at work (1 million people or 41%), followed by participation in exercise and sport (531,000 people or 22%). A further 410,000 people (17%) had a long-term condition as the result of a motor vehicle accident, and 334,000 people (14%) injured themselves at home. Men were more likely to acquire a long-term condition as a result of a work injury, whereas women were more likely to acquire their long-term injury at home or in a motor vehicle accident (graphs 11.11 and 11.12).
In 2009, injuries due to external causes accounted for 8,884 deaths, or 6.3% of all registered deaths. The standardised death rate for injuries was 38.6 per 100,000 of population (55.1 per 100,000 for males and 23.2 per 100,000 for females).
Over time, more men than women have died from external causes, and at younger ages. Consistent with previous years, around two-thirds of the total number of deaths resulting from external causes in 2009 were males (5,886, or 66%). Median age at death for injuries was 47.4 years for males, 66.7 years for females and 51.8 years overall.
Mental health
The designation of mental health as one of the national health priority areas recognises its enormous social and public health importance. Mental health is one of the leading contributors to the non-fatal burden of disease and injury in Australia. It is associated with increased exposure to health risk factors, greater rates of disability, poorer physical health and higher rates of death from many causes including suicide. Mental health problems incur high direct and indirect costs, result in a high number of hospitalisations and impose a heavy burden of human suffering (AIHW, 2011c).
In 2004–05, mental health accounted for 8% of allocated health system expenditure, at $4.1 billion, but by 2008–09 this figure had increased to more than $5.8 billion, or $272 per person (up from $225 per person in 2004–05) (AIHW, 2010a).
In 2009–10, the Australian Government paid $755 million in benefits for Medicare Benefits Schedule (MBS) subsidised mental health related services, around 4.9% of all MBS subsidies. Subsidies for psychologist services made up $287 million of the expenditure. Around 9.7% of subsidies for prescription medication were spent on prescriptions for mental health conditions (equivalent to $35 per Australian) (AIHW, 2011d).
In the 2007 ABS Survey of Mental Health and Wellbeing (SMHWB), mental health condition status was assessed through a diagnostic instrument (the World Health Organization Composite International Diagnostic Interview (CIDI)). Of the 16 million Australians aged 16–85 years in 2007, almost half (45% or 7.3 million) were assessed as having had a mental disorder at some time in their life. One in five (20% or 3.2 million) were assessed as having a current mental health disorder (having had symptoms of that disorder in the 12 months prior to interview).
Women were more likely to experience anxiety disorders (18% compared with 11% for men) and affective disorders (7% compared with 5% for men). Men had twice the rate of substance use disorders (7% compared with 3% for women). Prevalence varied by age, with people in younger age groups experiencing higher rates of disorder (graph 11.13)
Adolescence and young adulthood is a critical stage of transition in physical and mental development, and vulnerability to mental illness is heightened at this time. Around three-quarters (76%) of people who experience mental disorder during their lifetime will first develop a disorder before the age of 25 years. In 2007, just over a quarter of all young people aged 16–24 years had a mental disorder in the previous year (approximately 26% or 671,000 young people). Young women were more likely than young men to have had a mental disorder (30% compared with 23%).
Around 21% of young people with a mental disorder had experienced high or very high levels of psychological distress in the previous 4 weeks, compared with 32% of people over 25 with a mental disorder, and 4% of people with no mental disorder. However, over half (56%) of young people with a mental disorder were pleased or satisfied with the general quality of their life, and 16% were delighted with it.
Overall, one in ten people with a mental disorder reported being delighted with the quality of their life, compared with 20% of people without a mental disorder. However, almost half (48%) of the people who reported a mental health condition in 2007–08 also reported high or very high levels of psychological distress in the previous four weeks – around four times the national average of 12% (graph 11.6). In 2007, almost 2% of people with a 12-month mental disorder reported that they had attempted suicide, and 8% had thought about it sometime in the previous year.
In 2007, 38% of all people with a mental disorder (or 1.2 million people) had two or more mental disorders. A mix of mood and anxiety disorders was the most common combination, making up 39% of all co-morbidity cases (472,000 people), with people with more than one anxiety disorder making up a further 27% (331,000 people).
Around 59% of people with a mental disorder also had a physical condition, compared with 48% of those without a mental disorder. After adjusting for age differences in the populations with and without mental disorders, the gap between the rates of those with physical conditions further widened (from 11 to 17 percentage points). Co-morbidity with physical conditions was most common for people with mood disorders, 64% of whom also had a physical condition (Australian Social Trends, March 2009, 4102.0).
In 2007, 1.9 million Australians aged 16–85 years (12%) accessed services for mental health problems in the previous year. Around a third of people with a current, long-term mental disorder (35%) accessed these services, with more females doing so than males (41% and 28% respectively).
Mental health disorders were identified as the underlying cause of 6,522 registered deaths in 2009, representing 4.6% of all registered deaths in Australia in that year. (Dementia accounted for 89% of these deaths.) In total, 21,384 deaths were due to, or associated with, mental health disorders.
The prevalence of mental health disorders as an underlying cause has increased significantly over the last ten years. In 2009, the standardised death rate for mental health disorders was 25.2 per 100,000 of population, an increase from 16.5 per 100,000 population in 2000.
In 2009, more than half the deaths due to mental health disorders were females (4,130 or 63%). The median age at death was higher for females at 88.9 years, compared with 84.6 years for males
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Statistics contained in the Year Book are the most recent available at the time of preparation. In many cases, the ABS website and the websites of other organisations provide access to more recent data. Each Year Book table or graph and the bibliography at the end of each chapter provides hyperlinks to the most up to date data release where available.
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Research article
Patient, caregiver, health professional and researcher views and experiences of participating in research at the end of life: a critical interpretive synthesis of the literature
Marjolein H Gysels*, Catherine Evans and Irene J Higginson
Author Affiliations
King’s College London, Cicely Saunders Institute, Department of Palliative Care, Policy & Rehabilitation School of Medicine, London, UK
For all author emails, please log on.
BMC Medical Research Methodology 2012, 12:123 doi:10.1186/1471-2288-12-123
Published: 17 August 2012
Abstract
Background
The development of the evidence-base informing end of life (EoL) care is hampered by the assumption that patients at the EoL are too vulnerable to participate in research. This study aims to systematically and critically review the evidence regarding the experiences and views of patients, caregivers, professionals and researchers about participation in EoL care research, and to identify best practices in research participation.
Methods
We searched seven electronic databases, and hand searched three journals and the bibliographies of relevant papers. Inclusion criteria were original research papers on involvement in EoL care research or its impact on participants. Critical interpretive synthesis was used to integrate the whole body of empirical evidence on this topic and generate theoretical categories from the evidence.
Results
Of a total of 239 identified studies, 20 studies met the inclusion criteria, from: the US (11), the UK (6) and Australia (3). Most focused on patients with cancer (12) and were conducted in hospices (9) or hospitals (7). Studies enquired about issues related to: EoL care research in general (5), specific research methods (13), and trial research (2). The studies evaluating willingness to participate in EoL care research showed positive outcomes across the different parties involved in research. Factors influencing willingness were mainly physical and cognitive impairment. Participating in research was a positive experience for most patients and carers but a minority experienced distress. This was related to: characteristics of the participants; the type of research; or the way it was conducted. Participatory study designs were found particularly suitable for enabling the inclusion of a wide range of participants.
Conclusion
The evidence explored within this study demonstrates that the ethical concerns regarding patient participation in EoL care research are often unjustified. However, research studies in EoL care require careful design and execution that incorporates sensitivity to participants’ needs and concerns to enable their participation. An innovative conceptual model for research participation relevant for potentially vulnerable people was developed.
Keywords:
Research participation; End of life; Palliative care; Evidence; Critical interpretive synthesis
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Email this article to a friend
Evaluation of underreporting tuberculosis in Central Italy by means of record linkage
Lorenza Melosini*, Umberto Vetrano, Federico L Dente, Michele Cristofano, Mauro Giraldi, Luciano Gabbrielli, Federica Novelli, Ferruccio Aquilini, Laura Rindi, Francesco Menichetti, Giulia Freer and Pierluigi L Paggiaro
BMC Public Health 2012, 12:472 doi:10.1186/1471-2458-12-472
Fields marked * are required
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"warc_url": "http://www.biomedcentral.com/1472-6769/12/2/abstract?fmt_view=mobile"
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Methodology article
Comprehensive predictions of target proteins based on protein-chemical interaction using virtual screening and experimental verifications
Hiroki Kobayashi1, Hiroko Harada1, Masaomi Nakamura1, Yushi Futamura1, Akihiro Ito2, Minoru Yoshida2, Shun-ichiro Iemura3, Kazuo Shin-ya3, Takayuki Doi4, Takashi Takahashi5, Tohru Natsume3, Masaya Imoto1 and Yasubumi Sakakibara1*
Author affiliations
1 Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, 223-8522, Japan
2 Chemical Genetics Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan
3 National Institute of Advanced Industrial Science and Technology (AIST), 2-4-7 Aomi, Koto-ku, Tokyo, 135-0064, Japan
4 Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aza-Aoba, Aramaki, Aoba, Sendai, 980-8578, Japan
5 Department of Applied Chemistry, Tokyo Institute of Technology, 2-12-1 Ookayama, Meguro, Tokyo, 152-8552, Japan
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Citation and License
BMC Chemical Biology 2012, 12:2 doi:10.1186/1472-6769-12-2
Published: 5 April 2012
Abstract
Background
Identification of the target proteins of bioactive compounds is critical for elucidating the mode of action; however, target identification has been difficult in general, mostly due to the low sensitivity of detection using affinity chromatography followed by CBB staining and MS/MS analysis.
Results
We applied our protocol of predicting target proteins combining in silico screening and experimental verification for incednine, which inhibits the anti-apoptotic function of Bcl-xL by an unknown mechanism. One hundred eighty-two target protein candidates were computationally predicted to bind to incednine by the statistical prediction method, and the predictions were verified by in vitro binding of incednine to seven proteins, whose expression can be confirmed in our cell system.
As a result, 40% accuracy of the computational predictions was achieved successfully, and we newly found 3 incednine-binding proteins.
Conclusions
This study revealed that our proposed protocol of predicting target protein combining in silico screening and experimental verification is useful, and provides new insight into a strategy for identifying target proteins of small molecules.
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Market Delineation Study of the Fish Market in Nigeria: An Application of Cointegration Analysis
Teslim Bada, M.A.Y. Rahji
Abstract
In Nigeria aquaculture has provided an avenue to bridge the ever-widening demand and supply gap in fish. It
uses land resources that would have otherwise been a waste. This paper examined whether Catfish is in the same
market with Hake, Mackerel and Sadinnela. Unit root tests, Johansen`s bivariate and multivariate co integration
analyses were carried out. The analyses show that there is co integration among the species.. The hypothesis of
no substitution between Catfish and the imported species was rejected All the species were classified as being in
the same market and are close substitutes. The results indicate that the price of catfish is not insulated from the
prices of the imported species. The prices of the imported species are however insulated from the price of the
local species. Fish production policies designed to alter fish prices without taking into account the foreign prices
are not likely to be effective in Nigeria.
Full Text: PDF
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Journal of Agricultural Science ISSN 1916-9752 (Print) ISSN 1916-9760 (Online)
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Please disable AdBlock. CAN is an ad-supported site that takes hundreds of hours and thousands of dollars to sustain.
Jasper van Loenen
02/08/2012 / Arduino, Objects, Other
Created by Quinten Swagerman, Jasper van Loenen and Mr. Stock, the Pristitrope is a modification of the zoetrope, a pre-cinematic optical toy that shows short, looped animations. Instead of using static illustrations, Pristitrope is equipped with 18 tiny LCD screens, merging a ...
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29/07/2010 / openFrameworks
Created by Jasper van Loenen using OpenFrameworks and Arduino, Test Screen is an installation designed with a physical interface for single purpose and to allow the viewer to see inside the complexity of the code involved. Included are a total of ...
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Photos for 2000
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This document (source) is part of Crummy, the webspace of Leonard Richardson (contact information). It was last modified on Sunday, October 17 2010, 02:58:35 Nowhere Standard Time and last built on Saturday, May 18 2013, 07:00:06 Nowhere Standard Time.
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Convention on Wetlands of International Importance especially as Waterfowl Habitat
Convention on Wetlands of International Importance especially as Waterfowl Habitat
This article has been reviewed by the following Topic Editor: Kristen Hite
Introduction
(Source: Photograph by D. Tate)
The Convention on Wetlands of International Importance, Especially as Waterfowl Habitat, came into force in 1975. The treaty is commonly referred to as Ramsar or the Ramsar Convention, named after the Iranian town in which it was first signed in 1971. It is the oldest of the multilateral international conservation conventions and the only one to deal with one ecosystem type and one taxonomic group. As of December, 2006, Ramsar had 153 Parties. Moreover, 1,630 wetland sites worldwide, covering almost 1.5 million square kilometers, were designated for inclusion onto Ramsar’s List of Wetlands of International Importance, hereafter referred to as the List. The Ramsar Secretariat and Depositary are located in Gland, Switzerland and work closely with IUCN – The World Conservation Union.
Ramsar’s Preamble calls upon its Parties to recognize the interdependence of humans and their environment, and to consider the importance of the many ecological functions of wetlands, including flood control, nutrient cycling, and habitat for migratory wildlife and commercially important fish. The Preamble also suggests that wetland losses are irreparable because of their economic as well as scientific and recreational values. Parties are instructed to develop national policies to decrease wetland losses and to recognize that migratory waterfowl are important international resources because of their seasonal movements. The overall intent of Ramsar is to enhance national policies and international coordination for the conservation of both wetlands and waterfowl.
The Articles of Ramsar
The Convention is comprised of 12 articles, and several important amendments have been passed since it came into force. Article 1 defines wetlands and waterfowlThe scope of the term “wetlands” in Ramsar is relatively expansive and includes fresh, brackish, and marine wetlands (bogs, fens, marshes, swamps, etc). This clause allows for the inclusion of many near-shore areas (including some reef systems) that are permanently underwater and thus not wetlands as classically understood, but what can be very important for the conservation of waterfowl. The definition of “waterfowl” under Ramsar is also fairly broad, encompassing birds that are dependent on wetlands (as defined above) for at least part of their lifecycle. RAMSAR's waterfoul definition can encompass traditional wetland birds such as geese, swans, ducks, and their allies are included, and may also include shorebirds, herons, egrets, rails, grebes, cormorants, coots other aquatic and partly aquatic species, raptors and passerine species such as Marsh Harriers and reed warblers. This expanded definition is meant to facilitate effective conservation for the myriad avian species that may use similar flyways and stopover and staging areas during migrations regardless of taxonomic identity.
Article 2 instructs Parties to include at least one site within their jurisdiction on the List of Wetlands of International Importance, which is maintained by IUCN – The World Conservation Union. This designation is based very broadly on ecological, zoological, botanical, limnological and/or hydrological criteria as outlined in the article, but, in practice, Ramsar Parties have a good deal of leeway in proposing sites for the List. Article 3 requires Parties to promote the conservation of listed sites through national policies and to inform the Secretariat of changes in the ecological status of listed sites. Most Ramsar sites are in fact designated as some category of protected area under national law, but there are exceptions. Article 4 expands on the concept of national obligations to protect wetlands by encouraging Parties to establish nature reserves at and around important wetland sites within their respective jurisdictions, even if those sites are not listed as Internationally Important. It also asks Parties to compensate and mitigate for adverse impacts to listed sites, to share data and research with other parties, to provide training for waterfowl research, and to manage habitats to increase waterfowl populations. Article 5 instructs the Parties to consult with each other about implementation of the Convention, particularly for transboundary wetlands--an area in which Ramsar has been particularly progressive. Article 6 establishes a Conference of the Parties to convene periodically for the purposes of discussing Ramsar implementation and changes to listed areas. Conferences of the Parties have generally occurred biennially in the 30+ years since Ramsar came into force.
Bureaucratic and institutional aspects of Ramsar are considered in the remaining 5 articles. Article 7 requires that national representatives to the Conference of the Parties be wetland experts while Article 8 designates the World Conservation Union/IUCN to house the Secretariat for the purposes of maintaining the List of Internationally Important sites and convening conferences. Article 9 stipulates that the Convention remains open for signature indefinitely. Any member agency of the United Nations can become a Party to Ramsar, as can any country. As stipulated in Article 10, the Convention came into force 4 months after the accession of the seventh Party. Amendment procedures are described in Article 10 bis. Under Article 11, Parties have the right to denounce the Convention 5 years after ratification, and the Depositary is required (in Article 12) to announce new Parties, deposit all ratification or accession documents, dates of entry and any notification of denunciation, and to register the Convention with the Secretariat of the United Nations.
Discussion
Only about 60 countries became a Party to Ramsar in its first 20 years of existence, but almost twice that many have done so in the last 15 years. This is in part testament to the importance of the Convention and in part due to the proliferation of countries in the post-communist era, but it also in part due to some changes to Ramsar policies. As with the worldwide movement to create parks and equivalent reserves since the 1970s, the movement to protect wetlands for their own sake did not become feasible in many developing countries until there was a concerted and deliberate effort to make conservation programs work in coordination with, and not in opposition to, local-level development. Many individuals worldwide are dependent to some degree on subsistence harvesting of important natural products, and wetlands are among the most productive natural ecosystems. With the inclusion of more Parties, Ramsar implementation policies expanded to include concepts such as “wise use” for both conservation and local development, and the sustainable harvest of important wetland products such as reeds, thatch grasses, small-scale fisheries, etc.
Many of the policy directives issued by the Conference of Parties since 1990 have been in the spirit of community-based conservation, collaborative management, and sustainable harvest. Many developing countries have since promulgated their own national wetland policies and have thus affected Ramsar in some ways that were not originally intended by the (primarily) western and medium-developed nations that first signed the Convention. Although the concept of wise use for sustainable development was incorporated early into Ramsar, it was not until the expansion of many community-based forms of conservation (largely since the 1990s) in many nations, that these concepts began to become operational within Ramsar. Nepal, for example (a Party since 1987) drafted its own community-based wetlands conservation principles in 2000, and its national Wetlands Policy in 2003, which has still not been fully passed by the Government.
The Convention was broadly drafted to be as inclusive as possible. This has included both the definitions of wetlands and waterfowl as discussed above, and in the designation of sites within countries. For example, Canada and the United States, with vast areas and large wetland resources, have relatively few, but big, sites on the List (e.g. Everglades National Park), while Greece and Italy have many sites listed, some of which are very small. The population densities and historical land uses of course are very different in the former than in the latter, but this likely also may be attributed to cultural perceptions as well as [ecological reality about what constitutes international importance. The few large North American sites are, under any definition, classified as wetlands of international importance for migratory birds, but the many small sites along the Mediterranean could also be considered as such because they are stopover areas along the European-African flyway. Similar sites also exist in North America, but they are not listed, perhaps due to the presence of other, much larger sites, which are lacking in much of Europe.
Since many wetland areas are shallow and prone to seasonal drying, wetlands can serve as natural barometers of global warming. The Parties to Ramsar from almost the outset of the Convention have actively encouraged global change research at listed sites, contributing to efforts to both assess climatic trends and impacts. As such, the Convention was quite progressive at the time it was created in 1971.
In spite of all the interest in wetlands and associated fauna and flora, and in spite of current knowledge of the ecological and economic importance of wetland resources, implementation of the Ramsar’s mandates at the national level often still remains disappointing, both in terms of inadequate national legislation in many cases and the failure to protect wetlands on the ground. Given myriad hydro-periods, surface versus groundwater connections, and the vast array of landscapes under which wetlands can form, challenges to wetland preservation exist even with a successful and widely accepted Convention such as Ramsar.
Further Reading
Citation
Joel Heinen (Lead Author);Kristen Hite (Topic Editor) "Convention on Wetlands of International Importance especially as Waterfowl Habitat". 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 June 20, 2007; Last revised Date June 20, 2007; Retrieved May 18, 2013 <http://www.eoearth.org/article/Convention_on_Wetlands_of_International_Importance_especially_as_Waterfowl_Habitat>
The Author
Joel Heinen is Chair and Associate Professor in the Department of Environmental Studies at Florida International University in Miami, FL, USA. He received his Master’s from Virginia Tech and his PhD from the University of Michigan. Dr. Heinen's research is in the area of international biodiversity conservation policy, especially focusing on aspects of protected area management. He is particularly interested in the implementation of conservation treaties, the formulation of national conservatio ... (Full Bio)
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Lemi parish, FinlandEdit This Page
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Tyburn, MiddlesexEdit This Page
From FamilySearch Wiki
England Middlesex Middlesex Parishes Tyburn
Contents
Parish History
The ancient parish of St Marylebone, once was known as "Tyburn Church", (see St Marylebone).[1]
Resources
Civil Registration
Birth, marriages and deaths were kept by the government, from July 1837 to the present day. The civil registration article tells more about these records. There are several Internet sites with name lists or indexes. A popular site is FreeBMD.
Church records
To find the names of the neighbouring parishes, use England Jurisdictions 1851. In this site, search for the name of the parish, click on the location "pin", click Options and click List contiguous parishes.
Contributor: Include here information for parish registers, Bishop’s Transcripts, nonconformist and other types of church records, such as parish chest records. Add the contact information for the office holding the original records. Add links to the Family History Library Catalog showing the film numbers in their collection.
Census records
Census records from 1841-1891 are available on film through a Family History Center or at the Family History Library. The first film number is 438791. To view these census images online, they are available through the following websites for a fee ($) or free:
• FamilySearch has some of the British Censuses available.
• FindMyPast ($) has all available census records including images, and is free at Family History Centers and the Family History Library and some public and academic libraries.
• Ancestry.co.uk ($) has now all available census records but free at Family History Centers and the Family History Library and at numerous public and academic libraries. The library versions are known as AncestryInstitution.com.
• The Genealogist.co.uk ($) has all available censuses and is free at Family History Centers and the Family History Library and various other libraries.
• FreeCen is a UK census searches. It is not complete and individuals are always asked to consider helping out with transcriptions.
Probate records
Records of wills, administrations, inventories, indexes, etc. were filed by the court with jurisdiction over this parish. Go to Middlesex Probate Records to find the name of the court having primary jurisdiction. Scroll down in the article to the section Court Jurisdictions by Parish.
Poor Law Unions
Contributor: Add information about the pertinent poor law unions in the area.
Maps and Gazetteers
Maps are a visual look at the locations in England. Gazetteers contain brief summaries about a place.
Web sites
References
1. Cunningham, Peter. A Handbook for London: past and present, Abemarle Street, London: 1849. Volume 2. Digitised at [Google Books].
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UruguayEdit This Page
From FamilySearch Wiki
Revision as of 00:07, 27 August 2010 by Dianekay (Talk | contribs)
Contents
Intro
Montevideo, founded by the Spanish in 1726 as a military stronghold, soon took advantage of its natural harbor to become an important commercial center. Claimed by Argentina but annexed by Brazil in 1821, Uruguay declared its independence four years later and secured its freedom in 1828 after a three-year struggle.
The administrations of President Jose Batlle in the early 20th century established widespread political, social, and economic reforms that established a statist tradition. A violent Marxist urban guerrilla movement named the Tupamaros, launched in the late 1960s, led Uruguay's president to cede control of the government to the military in 1973. By yearend, the rebels had been crushed, but the military continued to expand its hold over the government. Civilian rule was not restored until 1985.
In 2004, the left-of-center Frente Amplio Coalition won national elections that effectively ended 170 years of political control previously held by the Colorado and Blanco parties. Uruguay's political and labor conditions are among the freest on the continent.
News
Events
Topics
Did you know?
Juristictions
Uruguay is divided into 19 departments (departamentos, singular - departamento):
• Artigas
• Canelones
• Cerro Largo
• Colonia
• Durazno
• Flores
• Florida
• Lavalleja
• Maldonado
• Montevideo
• Paysandu
• Rio Negro
• Rivera
• Rocha
• Salto
• San Jose
• Soriano
• Tacuarembo
• Treinta y Tres
Research Tools
Things you can do
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You are here: Home > Free Data Downloads
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keep form over the taskbar in vb plz help me vb geeks
Contributor
2Nov2009,19:23 #1
Hell dost..
Plz help me about this
How can i keep my form over the taskbar at run time in vb.
I know there are some api by that i can hide the taskbar but my problem is this that if i use that api method then when my program is terminated then task bar is hidden then also.
For showing that i have to use the api function again.
I know i can put the api code for showing the taskbar on form unload nd terminate event but then also i have one problem if i make my program's exe nd run that then if i terminate this from task manager by from process tab then my showtask bar api code is not executed,
my form show in taskbar property is false nd it does'nt have control box(min,max nd close button)
and app.TaskVisible=false
so plz help me friend
Go4Expert Founder
3Nov2009,09:03 #2
You posted this as an article and I moved to forum for discussion
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About this Journal Submit a Manuscript Table of Contents
Mediators of Inflammation
Volume 2012 (2012), Article ID 607276, 16 pages
doi:10.1155/2012/607276
Review Article
Interplay between Human Cytomegalovirus and Intrinsic/Innate Host Responses: A Complex Bidirectional Relationship
1Section of Microbiology, Department of Hematology and Oncology “L. & A. Seragnoli”, University of Bologna, 40138 Bologna, Italy
2Department of Molecular Medicine, Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy
3Department of Public Health and Microbiology, University of Turin, Turin, Italy
Received 21 December 2011; Accepted 22 March 2012
Academic Editor: Mohamed Lamkanfi
Copyright © 2012 Giada Rossini 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
The interaction between human cytomegalovirus (HCMV) and its host is a complex process that begins with viral attachment and entry into host cells, culminating in the development of a specific adaptive response that clears the acute infection but fails to eradicate HCMV. We review the viral and cellular partners that mediate early host responses to HCMV with regard to the interaction between structural components of virions (viral glycoproteins) and cellular receptors (attachment/entry receptors, toll-like receptors, and other nucleic acid sensors) or intrinsic factors (PML, hDaxx, Sp100, viperin, interferon inducible protein 16), the reactions of innate immune cells (antigen presenting cells and natural killer cells), the numerous mechanisms of viral immunoevasion, and the potential exploitation of events that are associated with early phases of virus-host interplay as a therapeutic strategy.
1. Introduction
Human cytomegalovirus (HCMV) is a ubiquitous, highly specific herpesvirus. As the other herpesviruses, after an initial primary infection HCMV establishes latency for the life of the host with periodic and spontaneous reactivation. In immunocompetent subjects, primary HCMV infection is usually asymptomatic but occasionally gives rise to a self-limited mononucleosis-like syndrome. In immunocompromised patients, HCMV is one of the most common opportunistic pathogens and causes different clinical syndromes, whose severity parallels the degree of the immunosuppression [1]; in these patients HCMV infection causes both direct effects, reflecting cell destruction by the virus, and indirect effects, such as acute and chronic rejection, cardiovascular disease, and HCMV-associated opportunistic infections [2]. During the acute phase of infection, HCMV can infect a remarkably broad cell range within its host, including endothelial cells, epithelial cells, smooth muscle cells, fibroblasts, neuronal cells, hepatocytes, trophoblasts, monocytes/macrophages (Mφs), and dendritic cells (DCs) [3].
HCMV induces many hallmarks of innate immune responses, such as the production of inflammatory cytokines and activation of the interferon (IFN) pathway in both immunocompetent and immunocompromised patients. This induction is rapid and does not require transcriptionally active viral particles [4]. The ability of the soluble forms of envelope glycoproteins B (gB) and H (gH) to effect a similar pattern of cellular responses suggests that their interactions with host cell components, such as integrin heterodimers, toll-like receptors, and entry receptors, are sensed by host cells, leading to early signaling and transcriptional events in infected cells and activating innate immune responses before the outset of viral replication [46].
Proper activation of innate immunity appears to be crucial to efficiently combat infections; in addition to the release of primary IFNs, professional antigen-presenting cells (APCs) are activated and natural killer (NK) cells are recruited and stimulated, triggering APCs and T cells. Further, unlike the innate and adaptive components of the immune system that require pathogen-induced signaling cascades for activation, intrinsic immune mechanisms are significant, forming an antiviral frontline defense that is mediated by cellular proteins, called restriction factors, that are constitutively expressed and active, even before a pathogen enters a cell [79]. Notably, interplay exists between innate and intrinsic immune mechanisms, wherein several restriction factors are upregulated by IFN, enhancing their antiviral activity [10, 11].
This paper describes the viral and cellular partners that mediate early host responses to HCMV with regard to the interaction between structural components of virions and cellular receptors and intrinsic factors, the reactions of innate immune cells, the mechanisms of viral immunoevasion, and the potential exploitation of events that are associated with these early phases of virus-host interplay as a therapeutic strategy.
2. Binding and Activation: Function of Receptors in Early Stages of HCMV Infection
Several receptors, including epidermal growth factor receptor (EGFR) [12, 13], platelet-derived growth factor receptor (PDGFR)-α [14], and integrins [15, 16], mediate HCMV attachment and entry. Virus-receptor interactions appear to be cell-type specific. For example, in the interaction between HCMV and monocyte-derived dendritic cells (Mo-DCs), viral envelope glycoprotein gB binds to the DC membrane protein DC-SIGN [17]. Polymorphisms in the promoter of DC-SIGN that enhance its expression on the surface of Mo-DCs are linked to higher levels of HCMV infection in vitro and in vivo [18], implicating DC-SIGN in viral entry into DC-SIGN-positive immune cells.
In addition to its binding to receptors, facilitating its entry, the virus is sensed by pattern recognition receptors (PRRs), such as toll-like receptors (TLRs), which initiate immune responses by recognizing pathogen-associated molecular patterns (PAMPs). TLR activation is followed by inflammatory cytokine secretion, upregulation of costimulatory molecules on APCs, and, in most cases, type I IFN production [19].
The initial evidence that HCMV activates innate immunity in a TLR-dependent manner was obtained with TLR2; stimulation of TLR2 by HCMV is replication independent and results in the activation of NF-κB and the release of inflammatory cytokines [20] without affecting the IFN pathway [21]. The envelope glycoproteins gB and gH also interact with TLR2, and neutralizing antibodies against TLR2, gB, and gH inhibit inflammatory cytokine responses to HCMV infection in permissive human fibroblasts [22]. Further, HCMV fusion inhibitors block virus-induced IFN signaling but not inflammatory cytokine secretion, suggesting that the latter is effected by surface sensing by TLR2 and does not require viral entry [21]. These findings indicate that HCMV-induced activation of cell surface TLR2 occurs at the earliest stages of infection; that is, the recognition and binding of envelope glycoproteins.
In addition to the in vitro findings, there is clinical evidence that implicates TLR2 in the pathogenesis of HCMV infection; liver transplant recipients who carry the homozygous Arg753Gln mutation of TLR2 have a higher incidence of HCMV-related disease that is associated with increased levels of HCMV DNA in the peripheral blood [23]. This clinical finding is explained by in vitro data that cells with the Arg753Gln mutation in TLR2 fail to recognize HCMV gB. Thus, impaired innate viral recognition might impede the development of a robust antiviral immune response, resulting in symptomatic disease in immunocompromised transplant recipients [24]. Chan and Guilbert have also demonstrated the significance of TRL2 in the immunopathogenesis of HCMV, reporting that UV-inactivated virions stimulate apoptosis in syncytiotrophoblast-like cells in a TLR2-dependent manner, likely contributing to chronic villitis and disruption of syncytiotrophoblasts, which often develop in placentas on delivery of newborns with congenital HCMV [25].
Intracellular TLRs, including TLR3, TLR7, TLR8, and TLR9, detect nucleic acids and are primarily involved in viral detection; TLR3, 7, and 9 recognize microbial nucleic acids in endolysosomes and trigger innate and downstream adaptive immune responses [26]. Endosomal TLR3 and TLR9 are essential components in the innate response to murine CMV (MCMV) in DCs and Mφs, and TLR9 is critical for NK cell activation and control of MCMV infection [2729]. TLR9 also functions in the early responses to HCMV in humans; HCMV induces IFN-α secretion from human plasmacytoid DCs (PDCs) by engaging the TLR7 and/or TLR9 pathways in vitro [30] and upregulates TLR9 expression in human PDCs [30] and fibroblasts [31].
Notably, the stimulation of TLR9 by its ligand, CpG-B, when added after viral entry, enhances HCMV infection in fibroblasts by an unknown mechanism, suggesting that the virus exploits TLR9 signaling to further its replication during infection of stromal cells. Moreover, the presence of T-1237C polymorphism that alters TLR9 promoter activity [32] correlates with symptomatic HCMV infection in stem cell transplants [33], implicating the TLR9 pathway in the recognition of and response to HCMV.
HCMV infection in fibroblasts is also influenced by the TLR3 and TLR4 pathways; stimulation of fibroblasts with TLR3 and TLR4 ligands inhibits viral replication through an IFN-β-dependent mechanism [31, 34]. Nevertheless, TLR3 has no function in the innate/early phases of the cellular response to HCMV in human Mo-DCs, as recently demonstrated by experiments in which TLR3 was silenced before HCMV infection [35]. HCMV also triggers TLR-independent DNA sensing mechanisms [36], as evidenced by findings that the DNA sensor ZBPI/DNA-dependent activator of IFN-regulatory factors (DAI) activates IFN regulatory factor (IRF) 3 and upregulates type I IFN on HCMV infection [37]. Further, HCMV modulates the activity of other innate immunity receptors that induce type I IFN secretion, such as retinoic acid-inducible gene I (RIG-I-) like helicases (RLHs); RIG-I is upregulated quickly in the early phase of HCMV infection in fibroblasts [38].
Other HCMV attachment/entry receptors might mediate the development of innate responses. Because they associate with TLRs [39] and HCMV glycoproteins [15, 40, 41], surface integrins have been proposed to facilitate the interactions of gB and gH with TLR2 [22, 42]. However, the ligation of gB to β1 integrin stimulates IFN signaling but not NF-κB-mediated inflammatory signalling [21], suggesting that this interaction induces a TLR-independent antiviral state before viral entry. The activation of innate mechanisms following HCMV attachment and entry and virus-induced modulation of host responses is depicted in Figure 1.
Figure 1: Activation and viral-induced modulation of early phases, HCMV attachment, entry, and intracellular phases of the viral cycle. (a) The binding of viral glycoprotein B (gB) induces the release of type I interferons (IFN) via IFN regulatory factor (IRF) 3, whereas contact between viral glycoproteins gB and gH and toll-like receptor (TLR)2 induces the activation of NF-κB and the release of proinflammatory cytokines. Expression of the intracellular receptor retinoic acid-inducible gene I (RIG-I) is also upregulated in the early phases, the DNA sensor DNA-dependent activator of IFN-regulatory factors (DAI) is activated, triggering IRF-3 activation and type I IFN production. (b) After viral entry, HCMV immunoevasion strategies are activated. Virion-associated and newly produced pp65 prevents IRF3 activation and subsequently impairs the production of type I IFN. Viral pp65 also inhibits NF-κB activation. RIG-I is downmodulated by an unknown mechanism, likely contributing to reduced IFN production. +; upregulation or activation, −; downmodulation or inhibition.
HCMV infects a variety of nonimmune cells in vivo, including fibroblasts, endothelial cells, epithelial cells, smooth muscle cells, and stromal cells; each of which expresses a unique subset of TLRs and other innate receptors, allowing them to respond specifically to HCMV infection and contribute to early antiviral defense. The activation of immune receptors on HCMV infection has significant function in fibroblasts [21, 22, 31]. HCMV-induced activation of innate receptors in other nonimmune cells might also be critical, an area that merits further study.
2.1. Viral Escape Starts at Very Early Phases
After viral entry, HCMV immunoevasion strategies are activated. The expression of HCMV pp65/UL83 blocks IRF3 signaling, which lies downstream of the RIG-I, DAI, and TLR3 pathways; pp65-mediated impairment of IRF3 signalling occurs by reducing IRF phosphorylation status and by inhibiting its nuclear accumulation [43]. pp65 also blocks IRF1 and NF-κB activation by an unknown mechanism [44], suggesting that HCMV counteracts the activation of the IFN and proinflammatory pathways at several steps. Further, RIG-I is downmodulated by an unknown mechanism starting at 48-hour postinfection [38], likely contributing to reduced IFN production.
3. Function of IFN Inducible Restriction Factors in Antiviral Defense
Intrinsic immune mechanisms were discovered as being active against retroviruses and involving the APOBEC3 class of cytidine deaminases, a large family of proteins that are collectively termed the TRIM family, and tetherin, an IFN-inducible protein whose expression blocks the release of HIV-1. Increasing evidence, however, suggests that such mechanisms also counter other viruses [45, 46]. Moreover, four proteins, promyelocytic leukemia protein (PML) [47], hDaxx [48], Sp100 [49], and viperin [50], have been identified as restriction factors that mediate intrinsic immunity against HCMV infection.
PML and hDaxx are components of subnuclear structures called nuclear domain 10 (ND10) or nuclear bodies (NBs). Direct evidence for their antiviral function comes from studies of cells that lack ND10. Primary human fibroblasts from which PML was depleted by small interfering RNA (siRNA) significantly increased the plaque-forming efficiency of HCMV due to enhanced immediate early (IE) expression. hDaxx represses HCMV IE expression and replication through histone deacetylases (HDACs), inducing transcriptionally inactive chromatin around the major IE promoter (MIEP) [51]. These findings demonstrate that the ND10 proteins PML and hDaxx are restriction factors that silence HCMV IE expression, thus controlling viral replication.
Viperin is an IFN-inducible iron-sulfur (Fe-S) cluster-binding antiviral protein that is induced in various cell types by type I, II, and III IFNs and on infection by many viruses, including HCMV. Ectopic expression of viperin in fibroblasts has no effect on the expression of HCMV IE1 or IE2, whereas the synthesis of early late (pp65), late (gB), and true late (pp28) genes is reduced significantly in viperin-expressing cells compared with control [52]. Because it interferes with the secretion of soluble proteins by disrupting lipid rafts of the plasma membrane, viperin likely exerts its antiviral effects by preventing virion assembly at a late stage of the viral life cycle.
An IFN-inducible family of proteins, previously known as the p200 family, has recently been demonstrated to suppress HCMV replication. This family, now designated PYHIN, comprises homologous human and mouse proteins that have an N-terminal Pyrin domain (PYD) and 1 or 2 partially conserved 200-residue C-terminal domains (HIN domain) [53]. These proteins are pleiotropic, based on their ability to bind to various target proteins (e.g., transcription factors, signaling proteins, and tumor suppressors) and modulate various cell functions. Increasing evidence implicates them as regulators of many processes, including proliferation, differentiation, apoptosis, senescence, inflammasome assembly, and the control of organ transplants.
Two members of the PYHIN family, AIM2, and IFN inducible protein 16 (IFI16), bind to and function as PRRs of virus-derived intracellular DNA [8]. In particular, IFI16 interacts with the adaptor molecule ASC and procaspase-1, forming a functional inflammasome during Kaposi sarcoma-associated herpesvirus (KSHV) infection [54]. Moreover, the induction of IRF3 and NF-κB-dependent genes by herpes simplex virus (HSV)-1 infection in RAW264.7 cells is impaired by siRNA that targets p204, the murine ortholog of IFI16 [55].
Using two approaches, we recently determined IFI16 to be an antiviral factor against HCMV [56]; IFI16 expression was knocked down by specific siRNA, enhancing HCMV replication, and transduction with dominant-negative IFI16 (lacking the PYD) increased HCMV replication, whereas overexpression of wild-type IFI16 impaired HCMV viral yield. In the latter set of experiments, early (E) and late (L), but not IE, mRNA and protein were downregulated, indicating that IFI16 exerts its antiviral effects by hindering viral DNA synthesis. The HCMV UL54 (also called pol) is the catalytic subunit of HCMV DNA polymerase and represents a prototypical early gene required for viral DNA replication. We have shown that IFI16 overexpression induces a significant inhibition of UL44, UL54, and UL83 mRNAs. These data were also confirmed at protein level. Moreover, transfection and electrophoretic mobility shift assay experiments performed with nuclear extracts of HCMV infected cells demonstrated that the UL54 promoter is the target of IFI16-induced viral suppression. In fact, using luciferase constructs that were driven by a site specifically mutated HCMV DNA polymerase (UL54) promoter, we noted that IFI16 suppresses UL54 transcription [56]. These data indicate that IFI16 has antiviral activity against HCMV and provide novel insights into the functions of IFI16 as a viral restriction factor.
Type I IFN-induced restriction factors, briefly described and summarized in Figure 2, constitute a potent antiviral defense mechanism against HCMV infection, rendering viral replication a true hurdle race.
Figure 2: Type I IFN restriction factors that target HCMV. Type I interferons (IFN) are effector molecules of the immune response to virus. This antiviral action is mediated by IFN-stimulated genes. ND10 proteins are induced by IFN and function as part of an intrinsic antiviral defense mechanism of the cell by suppressing viral immediate early (IE) gene expression. The IFN-inducible protein IFI16 interacts with and displaces the transcription factor Sp1 from its DNA cognate element, the IR-1 element, in the viral UL54 promoter. This interaction inhibits the UL54 promoter and decreases HCMV DNA synthesis. The IFN-inducible protein viperin exerts its antiviral effects at a late stage of the HCMV life cycle. During infection, viperin is redistributed from the endoplasmic reticulum (ER) to the Golgi apparatus (TGN, trans Golgi network) and then to cytoplasmic vacuoles that contain gB and pp28.
3.1. Strategies Adopted by HCMV to Escape Activity of IFN Restriction Factors
In response to the antiviral action of type I IFN factors, HCMV has evolved regulatory proteins and counteracting mechanisms that subvert and inactivate such factors. For example, IE1 disrupts ND10 by inducing the deSUMOylation of PML [47]. Recent evidence has demonstrated that HCMV relocates viperin from the endoplasmic reticulum to the mitochondria, where it reduces the generation of ATP, disrupting the actin cytoskeleton and enhancing viral infection [57]. Nuclear IFI16 appears to become inactivated, following its egress from the nucleus, during early gene expression by molecular mechanisms that appear to rely on protein ubiquitination (Landolfo et al. unpublished results).
4. Function of Innate Immunity Cells during HCMV Infection
HCMV infects host cells of the myeloid lineage, such as monocytes, Mφs, and myeloid DCs. Despite their resistance to HCMV infection, lymphoid lineage cells, such as NK cells and PDCs [58], are also activated rapidly by viral components, confirming the importance of early virus-host interactions in the induction of prompt host defense mechanisms. However, HCMV has developed myriad immunoevasion strategies, allowing it to subvert host cell functions for its own advantage.
4.1. HCMV Efficiently Infects APCs and Employs These Cells as Vehicle of Viral Dissemination
APCs, including monocytes and various DC and Mφ subsets, are critical in initiating specific naive and memory T-cell responses and coordinating and modulating host responses. Nevertheless, it is evident that HCMV hijacks these cells, transforming them into vehicles for viral dissemination in the first phase of infection and sheltered reservoirs in which the virus can persist, reactivate, and replicate under favorable conditions [59].
HCMV infects myeloid APCs, based on the detection of viral genome and antigens [6063]. Monocytes do not support productive viral replication, and viral gene expression is restricted to early events [64, 65], whereas infected fully differentiated Mφs and myeloid DCs undergo lytic viral cycles, express late HCMV genes, release infectious virus, and stimulate T-cell responses in vitro [62, 63, 66, 67]. Thus, the ability of HCMV to replicate in myeloid cells depends on their stage of differentiation, as shown in an experimental model of HCMV latency, which was established by infecting human monocytes with a clinical isolate in vitro, in which monocytic differentiation to Mφs or DCs induced viral reactivation [68].
During the differentiation of DC progenitors to mature DCs ex vivo, chromatin structure is altered, permitting robust IE expression and, consequently, reactivation of latent HCMV [69]. Consistent with these observations, the inhibition of viral lytic genes that occurs during latency in undifferentiated myeloid precursors, including monocytes, is attributed to their inability to sustain high IE levels; the histone modifications present on the MIEP impart on it a repressive chromatin structure preventing transcriptional activity [70]. Recent evidence implicates IL-6 signaling and activation of the ERK/MAPK pathway in HCMV reactivation from potentially permissive cells, such as interstitial DCs [71]. Thus, myeloid cell differentiation, which is driven by inflammation and proinflammatory factors, such as IL-6, contribute to reactivation of latent HCMV infection (Figure 3(a)).
Figure 3: Cells of innate immunity, activation and virus counterattack. (a) HCMV reactivates from latency in infected monocytes by inflammation or cellular differentiation, in which IL-6 and ERK/MAPK signaling are involved. Differentiated macrophages (Mφ) and dendritic cells (DC) are permissive for viral replication and, once infected, release proinflammatory factors. HCMV hampers the ability of Mφ and DC to properly differentiate from monocytes and present antigens to T lymphocytes by downregulating surface expression of CD1 and HLA class II molecules. DC-induced T-cell proliferation also decreases through mechanisms that involve virally encoded IL-10 and pUL18. IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; MIF, macrophage migration inhibitory factor; MIP-1α, macrophage inflammatory protein-1α; MIP-1β, macrophage inflammatory protein-1β. +; upregulation or activation, −; downmodulation or inhibition. (b) HCMV-encoded proteins modulate NK-cell recognition of infected cells. pUL40 binds to HLA-E and upregulates its surface expression, potentiating its interaction with the inhibitory receptor CD94/NKG2A. pUL18, an HLA-I viral homolog, binds to the inhibitory receptor LIR-1. Expression of the ligands of the activating receptor NKG2D is inhibited by pUL16 (which targets MICB, ULBP1, and ULBP2) and pUL142 (targeting MICA and ULBP3). pUL141 prevents the expression of CD112 and CD155, ligands of the activating receptors CD226 and CD96, whereas pp65 interferes with the signal transduction of the activating receptor NKp30. Solid lines: possible interactions resulting in NK-cell inhibition. Dotted lines: impairment of interactions between activating receptors and their ligands.
Conversely, the virus can enhance inflammation by acting on APCs; HCMV infection of peripheral monocytes induces a proinflammatory state, resulting in their adhesion to endothelial cells and transendothelial migration [72] and the secretion of proinflammatory cytokines and chemotactic factors [73]. Further, Mo-DCs [74, 75] and monocyte-derived Mφs [76] release proinflammatory factors on productive HCMV infection in vitro.
4.2. Immunoevasion Mechanisms Adopted by HCMV against APC Responses
In addition to enhancing inflammation for its own sake, HCMV hampers APCs in taking up and presenting the proper antigen to T lymphocytes. Several counteracting mechanisms have been evolved by HCMV to circumvent APC activity (Figure 3(a)). Immunoevasive viral transcripts, such as gpUS3 and gpUS8, that block human leukocyte antigen (HLA-) mediated antigen presentation pathways predominate during the early phases of HCMV infection of myeloid DCs [77]. HCMV inhibits the differentiation of Mφs and DCs from monocytic precursors, blocking their phagocytic, migratory, and allostimulatory activities [78, 79].
HCMV also impairs the immunophenotype and function of differentiated APCs. For example, it downmodulates integrin-like receptors, such as CD11b/CD18 (CR3) and CD11c/CD18 (CR4), on the surface of monocyte-derived Mφs, reduces their phagocytic activity [80], and impairs migration by downregulating CCR1 and CCR5, reorganizing the cytoskeleton, and inducing the secretion of soluble inhibitors [76]. Further, HCMV-infected, immature Mo-DCs have fewer surface HLA class I and class II molecules and impaired migratory and immunostimulatory capacity [74, 81, 82]. The virus also inhibits Mo-DC maturation and impedes the migration of mature DCs in response to lymphoid stimuli and induction of T-cell proliferation [75, 82, 83]. Similarly, on infection with HCMV, activation markers are downregulated in mature Langerhans DCs, decreasing their ability to stimulate T-cell proliferation [84, 85].
Many events have been implicated in the HCMV-induced impairments to immunostimulation by DCs, such as the release of soluble CD83 [86], upregulation of apoptosis-stimulating molecules [87], expression of the HCMV-encoded HLA class I-like homolog pUL18 [88], and secretion of the viral homolog of IL-10, which is expressed during the productive phase of infection (cmvIL-10) [89]. cmvIL-10 also impairs CD1-mediated antigen presentation (by reducing CD1 transcription) [90], monocyte function [91, 92], and TLR-induced transcriptional activation of IFN α/β genes in PDCs [93]. cmvIL-10 enhances HCMV infectivity by upregulating the viral entry receptor DC-SIGN [89]. Thus, secretion of cmvIL-10 during HCMV infection has many effects in hindering APC function.
4.3. APCs and HCMV: A Double-Edged Sword
Despite the subversion of APC function by the virus, specific effector and memory T cells develop during acute HCMV infection [94, 95] and robust adaptive immune responses develop to many HCMV antigens, of which IE1 is a significant target of CD4+ and CD8+ T-cell responses [94]. Whereas immunostimulation by DCs is profoundly impaired by the virus, HCMV-infected Mφs induce efficient T-cell activation through presentation of endogenous IE antigen [62]. Further, mechanisms of crosspresentation, the exogenous acquisition of antigen that is presented directly to CD8+ T cells without endogenous processing, are also initiated during HCMV infection of APCs [96]. However, the effective role of cross-presentation in inducing an efficient cellular imunity to HCMV has not yet been addressed.
4.4. NK Cell Activation during HCMV Infection
NK cells are a critical component of early innate immune responses against certain viruses, including HCMV. Individuals with NK-cell defects have increased susceptibility to herpesviruses and, in particular, HCMV [97, 98]. Moreover, the extensive mechanisms that HCMV implements to prevent NK-cell activation are indirect evidence of their importance in the innate response to HCMV.
NK cells accumulate rapidly in several organs during viral infections, taking active part in the direct elimination of injured target cells by cytotoxicity and in the activation and recruitment of other cells of the immune system by secreting cytokines and chemokines, including IFN-γ and TNF-α [99]. In secondary lymphoid organs and damaged tissues, NK cells establish a dialog with APCs, thus regulating innate and adaptive immune responses [100].
NK cells recognize virus-infected cells, using a repertoire of stimulatory and inhibitory cell surface receptors [101] that control NK-cell activation, proliferation, and effector functions; their cytotoxic function depends primarily on stimulatory receptors. Different receptors are expressed to respond to different ligands on target cells: (i) HLA class I molecules (HLA-I), frequently downmodulated in virus-infected cells are recognized by specific inhibitory receptors, including killer cell-Ig-like receptors (KIRs), leukocyte Ig-like receptor 1 LILRB1 (LIR-1), and C-type lectin receptor CD94/NKG2A; (ii) pathogen-derived molecules are recognized by activating receptors, and (iii) self-proteins that are upregulated on “stressed” or damaged cells bind to a major activating receptor, NKG2D [102].
4.5. Mechanisms of Viral Immunoevasion Employed against NK Cells
Many inhibitory receptors on NK cells, including KIRs and LIR-1, recognize HLA-I, and under normal conditions, the engagement of inhibitory receptors by self-molecules suppresses NK-cell attack. However, HCMV is able to reduce cell surface expression of HLA-I by several mechanisms (reviewed in [103]). Consequently, it was predicted that according to the missing self hypothesis, low levels of HLA-I on HCMV-infected cells render them vulnerable to NK-cell lysis [104]. Yet, NK cells fail to discriminate between normal and infected cells on the basis of virus-induced HLA-I downmodulation [105, 106]. HCMV circumvents other aspects of the NK cell-target cell interaction [107], and HCMV-infected cells become resistant to be attacked by NK cells, due to a vast array of virally encoded immunomodulatory molecules [108].
Two mechanisms describing HCMV-mediated inhibitory signalling have been proposed. In the first, HCMV encodes for pUL18, an HLA-I homolog [109] that, like HLA-I, binds β2-microglobulin [110] and peptides [111] and engages the inhibitory receptor LIR-1 with 1000-fold higher affinity compared with HLA-I [112114]. pUL18 inhibits LIR-1+ NK cells but has additional effects, because LIR-1 is expressed on other cells of the immune system, including APCs [115]. For example, the binding of pUL18 to DCs impairs cell migration and CD40 ligand-induced maturation, reducing T-cell proliferation [88]. Thus, pUL18 can be exploited by HCMV to avoid host immune responses [116]. Clinical isolates of HCMV retain UL18, underscoring its importance for viral survival in the host [117, 118].
In the second mechanism, HCMV uses the host HLA-E pathway to suppress NK cells through the inhibitory receptor complex CD94/NKG2A. A nonameric peptide that is derived from the leader sequence of the viral protein pUL40 is a canonical ligand for the nonclassical HLA-I molecule HLA-E and promotes HLA-E expression on the cell surface [119121], facilitating the interaction between HLA-E and CD94/NKG2A receptor and conferring resistance to NK-cell lysis [122125].
Because the decision by NK cells to attack relies on the sum of signals from inhibitory and activating receptors, it is important for the virus to prevent the engagement of activating receptors. HCMV encodes five genes that impede signaling by activating receptors on NK cells: UL16, UL141, UL142, UL83, and microRNA-UL112-1 (miRNA-UL112) [108]. pUL16, pUL142, and miRNA-UL112 inhibit the expression of ligands of a major activating receptor, NKG2D. In humans, the ligands for NKG2D are the human major histocompatibility complex (MHC) class I chain-related genes (MIC)A, MICB, and ULBP1-6 molecules, which are particularly expressed under stress and on stimulation by innate cytokines that are produced during viral infections (reviewed in [126]).
Because NKG2D has an important role in controlling both NK- and T-cell-mediated immunity, it is reasonable that this receptor and its ligands forced the virus to evolve specific strategies of evasion. pUL16 prevents cell surface expression of MICB, ULBP1, and ULBP2 by binding and sequestering them in the endoplasmic reticulum or Golgi [127129]. The selective pressure that is exerted by pUL16 likely contributes to drive the diversification of NKG2D ligands, eventually leading to the emergence of proteins that do not interact with UL16, such as MICA and ULBP3; the expression of which, however, is countered by the HCMV protein pUL142, which retains them in the cis-Golgi [130132]. In addition, MICB is under the control of the virally encoded miRNA-UL112 which specifically reduces its cell surface expression [133].
Another tactic that was evolved by HCMV to interfere with activating receptors relies on pUL141, which sequesters the adhesion molecules CD155 (PVR/necl-5) [134] and CD112 (nectin-2) intracellularly [135]; these proteins are ligands for the NK-cell activating receptors CD226 (DNAM-1) and CD96 (TACTILE) [136]. Notably, pUL141 is the most robust modulator of NK cells that has been tested in vitro, inhibiting a wide range of human NK-cell populations [134]. This important function explains in part the increased resistance to NK-cell lysis of low-passaged HCMV clinical isolates compared with the laboratory strain AD169 [105], from which 13–15 kbp of DNA has been deleted due to extensive passaging in vitro [137], a segment that contains UL141 [108, 134].
The pp65 tegument protein also affects NK-cell functions, dissociating the ζ-chain from the natural cytotoxicity receptor NKp30 and preventing it from transducing signals through an unknown mechanism [138]. The outcome of these disparate strategies is impaired NK-cell-mediated recognition and elimination of HCMV-infected cells, as depicted in Figure 3(b).
4.6. NK Cells and HCMV: Windows of Opportunity for Host Counterattack
Despite the many viral strategies that modulate the antiviral functions of NK cells, there is a window of opportunity during which host responses can prevail, potentially rendering infected cells detectable by the immune system. Such a circumstance could be achieved through several mechanisms, depending on genetic variations in the virus and host. For example, some allelic variants of NKG2D ligands are unaffected by known viral strategies. The MICA*008 allele, the most frequent allele in several populations, does not bind to viral pUL142. This variant has a truncated cytoplasmic tail, making it resistant to pUL142 and allowing it to persist on the surface of infected cells, where it can induce NK cells to lyse [132, 139]. This finding suggests that UL142 may be driving the selection of certain MICA alleles in humans [140, 141].
Genetic variations have also been detected in UL142 from different clinical isolates of HCMV, some of which are more efficient in downregulating MICA expression [132]. Variations have also been identified in pUL40 and pUL18 [117, 118, 124].
Despite of the wide range of strategies that are used by HCMV to modulate NK-cell function, there is still the possibility of a time interval during which host responses prevail. MICA and MICB expression appears to be regulated by IE1 and IE2 proteins, indicating that viral trans activation is largely mediated by these HCMV gene products [142]. Notably, this effect might allow NK-activating ligands to be expressed before late immunoevasion genes are expressed and exert their effects. Collectively, this evidence suggests that the cellular response to infection could be sufficiently robust in some individuals against certain viral strains and/or at a specific time after infection, allowing to achieve elevated, functionally relevant levels of activating signals.
4.7. Interplay between NK Cells and APCs during HCMV Infection
NK-DC crosstalk is bidirectional, NK cells can kill immature DCs or promote their maturation, and in turn, mature DCs can stimulate NK-cell cytotoxicity and proliferation. These processes depend primarily on the activating receptors NKp30 and DNAM-1 and on the production of cytokines, such as IL-12, IL-15, IL-18, and IFN-α/β [100, 143149].
Recent evidence has demonstrated that NK cells regulate HCMV infection through interactions with autologous APCs, such as Mo-DCs and polarized Mφs; NK cells respond vigorously against infected Mo-DCs by producing IFN-γ and becoming cytotoxic, where NKp46 and DNAM-1 have a dominant role [150]. Such a response is evident early after infection, whereas later, the virus-mediated downregulation of the DNAM-1 ligands CD155 and CD112 prevails, illustrating the significance of the course of infection with regard to the efficacy of the host response. Further, the production of IFN-γ by NK cells is influenced by the polarization of Mφs, wherein proinflammatory Mφs induce more efficient IFN-γ responses than anti-inflammatory Mφs on HCMV infection [151].
5. Early Events of HCMV Replication as Potential Targets for Therapeutic Intervention
The identification of cellular and viral components that regulate early HCMV-host cell interactions has increased our understanding of the pathogenesis of HCMV diseases and formed the rationale for the design of novel antiviral interventions that target these initial events.
The need for anti-HCMV drugs with novel mechanisms of action is underscored by the findings that conventional standard therapy is often associated with considerable adverse events and that prolonged treatment can lead to the emergence of drug-resistant strains [152]. Further, agents that target viral DNA polymerase are unable to prevent viral attachment or entry or the expression of IE proteins, which mediate proinflammatory responses and immunomodulation. Thus, blocking pre-IE events and IE expression and function may represent an alternative strategy of combating HCMV-induced immunopathological phenomena [153]. Several molecules that effect such outcomes have been identified (reviewed in [154]). However, with the sole exception of hyperimmune globulin preparations, compounds that target HCMV attachment and entry remain at the preclinical stage of development. We briefly review the properties of those experimental agents that have been shown to inhibit HCMV attachment and entry in vitro.
The adsorption of HCMV virions to cell surface heparan sulfate proteoglycans (HSPGs) is mediated by positively charged regions of the viral gM/gN complex and is essential for stabilizing virions at the cell surface prior to the engagement of entry receptors [4]. Several experimental inhibitors of HCMV attachment have been characterized, including sulfated polysaccharides, lactoferrin, and peptide-derivatized dendrimers. Negatively charged polyanions, such as sulfated polysaccharides from bacteria, algae, and animals and semisynthetic compounds, such as dextran sulfate and pentosan polysulfate, disrupt the electrostatic interactions between the positively charged region of HCMV envelope glycoproteins and the negatively charged sulfate/carboxyl groups of heparan sulfate (HS) chains in HSPGs; these compounds show potent anti-HCMV activity against laboratory strains and clinical isolates [155]. HSPGs can also be bound by the N-terminal region of lactoferrin, an iron-binding glycoprotein that exists in most mucosal secretions and body fluids, suggesting that it acts by preventing virions from tethering to the cell surface [156].
Dendrimers are synthetic hyperbranched molecules that may have potential applications as antivirals, based on their small size (nanomolar), ease of preparation, and ability to display multiple copies of surface groups (multivalency) that are required for recognition, including the initial interactions that occur between an infecting virus and the target cell [157]. Recently, two peptide-derivatized dendrimers, SB105 and SB105_A10, were shown to inhibit HCMV replication directly by preventing viral adsorption to HSPGs onto cells [158, 159].
The use of compounds that target viral attachment could be curbed by the cell-to-cell spread of clinical HCMV isolates. In a normal host, however, the release of cell-free virus depends on the site of infection; whereas cell-free viral transmission during hematogenous dissemination is believed to be unlikely (because HCMV replication is highly cell associated), cell-free virus is commonly found in body fluids, such as urine, saliva, and breast milk, often at high titers [160]. Thus, molecules that block viral adsorption may be used to prevent HCMV transmission via such excretions.
HCMV-exploits its coding capacity for glycoproteins to form different envelope complexes [3]. The gH/gL heterodimer can participate in two distinct glycoprotein complexes; it can associate with gO to form a heterotrimeric complex that regulates pH-independent fusion at the cell surface in fibroblasts or it associates with pUL128, pUL130, and pUL131 to form a pentameric complex, required for entry by endocytosis, followed by low pH-dependent fusion in endothelial and epithelial cells, DCs, and monocytes [67, 161163]. gB is also required for viral entry and cell-to-cell spread [164]. Thus, compounds that bind to virion components that mediate entry or interfere with the protein-protein interactions required to induce membrane fusion can be termed HCMV entry inhibitors.
Experimental agents that have been shown to interfere with HCMV entry include CFI02, β-peptides, and CpG ODNs. gB is the target of a small-molecule thiourea derivative, CFI02, which suppresses HCMV replication. Mechanism-of-action studies indicate that CFI02 acts at an early stage in HCMV replication by inhibiting gB-mediated fusion of the virion envelope to the cell membrane [165]. Further, heptad repeat motifs, characteristic of α-helical coiled-coil interactions, have been identified within gB and gH. Peptides that correspond to these regions have been shown to inhibit the entry of clinical and laboratory HCMV strains, thus providing the proof of concept that blocking the coiled-coil interactions required for viral entry is a feasible strategy of preventing HCMV infection [166]. These potential new targets for therapeutic intervention have been exploited, based on the development of oligomers of β-aminoacids (β-peptides) that mimic the heptad repeat domain of gB and block viral infection during virus-cell membrane fusion [167]. β-peptides showed to be more potent than gB-derived α-peptides and blocked the activation of the type I IFN pathway in HCMV-infected fibroblasts [21], suggesting that β-peptides can impede both HCMV replication and viral-induced immunopathogenesis.
Short synthetic oligodeoxynucleotides that contain deoxycytidyl-deoxyguanosine motifs (CpG ODNs) can mimic bacterial and viral DNA to stimulate TLR9 and activate innate responses [168, 169]. Their antiviral activity has been proposed to be secondary to CpG-induced IFN responses that are triggered through TLR9 activation. Luganini et al. [170] recently reported, however, that in vitro replication of HCMV was suppressed by several CpG ODNs in a TLR9-independent mechanism. The B-class prototype CpG ODN 2006 was shown to prevent the nuclear localization of pp65 and input viral DNA, thus suggesting that it inhibits HCMV entry [170]. Notably, when added after the onset of HCMV replication, CpG ODN 2006 stimulates viral replication [31], as discussed, indicating that once the virus establishes its transcriptional programs, it takes advantage of the TLR9 stimulation pathway to propagate. These findings also suggest that CpG ODNs should be considered for antiviral intervention solely to prevent HCMV infection.
Yet, the window of opportunity for the mentioned experimental compounds that target the attachment and entry phases of HCMV infection is narrow. Their development as candidate drugs for future intervention should be considered in combination with conventional anti-HCMV therapeutics, such as ganciclovir and foscarnet that inhibit viral replication.
Conversely, intravenous immunoglobulins that are enriched for antibodies against HCMV (HCMV-IVIG) have been approved for use in preventing HCMV diseases in transplant recipients. The rationale for their clinical application lies in their ability to neutralize the virus and prevent entry into several cell types. Therefore, HCMV-IVIG represents the first example of a drug capable of blocking a pre-IE event that has been extensively used in patients at risk of HCMV disease. Further, the immunomodulatory activity of IVIG [171] might help reduce HCMV-induced immunopathology. However, in spite of their widespread clinical application, the role of HCMV-IVIG in the prevention of HCMV infection and disease remain to be fully elucidated. In fact, prophylactic administration of HCMV-IVIG has been associated with improved total survival, reduced HCMV disease, and lower HCMV-associated deaths in solid organ transplant recipients [172], whereas in patients who are undergoing hematopoietic stem cell transplantation, routine prophylaxis with HCMV-IVIG remains controversial [173]. Moreover, observational clinical studies indicate that administration of HCMV-IVIG to pregnant woman with primary HCMV infection may be effective in treating and preventing fetal infection [174].
The low neutralization potency of these preparations, however, may limit their clinical use. Thus, human monoclonal antibodies (mAbs) that neutralize HCMV infection have recently garnered interest as more effective and safer passive immunotherapeutic agents. Panels of human mAbs against gB and gH [175] or those that recognize conformational epitopes that require two or more proteins of the gH/gL/pUL128-131 pentameric complex [176] were developed from immortalized memory B cells of HCMV-immune donors. Notably, the human mAbs against the UL128-131 locus gene products [161] showed a neutralizing activity 2-3 logs more potent than neutralizing mAbs directed to gB or gH [176]. Although their protective activity in vivo remains to be investigated, these new human mAbs are promising next-generation immunotherapeutic compounds for the therapy/prophylaxis of HCMV infection and disease.
6. Concluding Remarks
The complex interaction between HCMV and the host begins immediately on viral contact with many cell types, including innate immune cells. Virion recognition and binding and entry-related events induce inflammation and IFN responses, the latter upregulating restriction factors that, in turn, contribute to the creation of an intracellular antiviral state. However, the induction of the IFN response is modulated by many counteracting viral mechanisms, as well as the inactivation of IFN restriction factors and modulation of innate cell functions that facilitate evasion of host intrinsic and innate immunity.
The identification of the mechanisms of host-HCMV interactions during attachment and entry has provided the rationale for the design of novel experimental compounds that target these events. Blocking the early phases of infection may provide a window of opportunity that allows such interventions to inhibit HCMV gene expression and replication and modulate inflammatory and IFN host responses, thus hindering viral-induced immunopathogenesis.
HCMV uses several immunoevasion strategies to evade host NK cells and APCs, most of which involve protein products of L viral genes that are used to complete the viral cycle. Novel therapeutics that block the viral cycle before the late stages of replication might also prevent HCMV from exploiting such strategies, thus increasing the immunocompetence of the host.
Acknowledgments
This work was supported by an RFO from the University of Bologna (to M. P. Landini and S. Varani); the Piedmont Region Ricerca Sanitaria Finalizzata (to G. Gribaudo); the Italian Ministry of Education, University, and Research MIUR (PRIN 2008 and FIRB-Futuro in Ricerca), Fondazione Banca Popolare di Novara BPN (to S. Landolfo); the Italian Association for Cancer Research (AIRC), the Italian Ministry of Education, University, and Research (MIUR), and Sapienza University of Rome (to C. Cerboni and A. Santoni). The authors thank Vittorio Sambri for critical review of this paper.
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Miners exposed to high levels of diesel exhaust have three to five times the risks of lung cancer as workers in occupations where diesel exhaust exposures are less intense, according to a study by
The former chief of security for the Upper Big Branch mine, where 29 miners died in an explosion in 2010, was sentenced Wednesday to three years in prison.
A gas explosion in a southwest China coal mine Friday killed 11 workers and injured six others, state press said in the latest accident to hit the nation’s dangerous mining industry.
The Ship Breaking and Ship Recycling Rules-2011 herein after referred to as `Rule' has been formulated in pursurance of the Hon'ble High Court Division of Supreme Court, in writ petition No. 7260 of 2008 dated May 24, 2011 taking into consideration the directions contained in the order.
NEW DELHI, 19 OCT: Managing environmental impacts has emerged as a big challenge in India’s fast and high growth rate development, the labour and employment minister, Mr Mallikarjun Kharge, said to
Read the draft Bio-Medical Waste (Management and Handling) Rules, 2011 notified by the MoEF under Environment (Protection) Act, 1986 to replace earlier rules (1998) and the amendments thereof.
South Asian countries must not allow their beaches to be used as dumping grounds for the chemically contaminated and extremely hazardous ships from the western countries.
Shanghai's environmental watchdog ordered two factories in its suburbs to halt production pending an investigation into the source of lead poisoning among children in a nearby village.
This recent CAG report on Corporate Social Responsibility of Coal India Ltd reveals that that 239 mines which have been in operation since before 1994, do not have prior environment clearance.
A report of the Comptroller and Auditor General of India (CAG) on the performance audit of the activities of Corporate Social Responsibility by Coal India Ltd (CIL) during April 2004 to March 2010 in areas like environmental protection, safety requirement, occupational health of the workers and community development shows low allocation of funds
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Bibliography: Skybreaker
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Title: Skybreaker
Author: Kenneth Oppel
Year: 2005
Type: NOVEL
Storylen: juvenile
Series: Matt Cruse
Series Number: 2
Language: English
Wikipedia Entry: http://en.wikipedia.org/wiki/Skybreaker
ISFDB Record Number: 172008
Note: Winner of the 2006 Ruth and Sylvia Schwartz Children's Book Award.
User Rating: This title has fewer than 5 votes. VOTE
Current Tags: young-adult (1), Young Adult (1), alternate history (1) Add Tags
Awards:
Publications:
Reviews:
Copyright (c) 1995-2011 Al von Ruff.
ISFDB Engine - Version 4.00 (04/24/06)
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Bitter cup of Coffee Party: Digging up the AstroTurf on Annabel Park and the Obama Administration
Posted by Conservative Generation for Left Coast Rebel
The NY Times is out today promoting the Coffee Party as a grassroots movement with ideologies similar to the Tea Party, but with a more palatable message. NY Times is so enamored with this group that they offered the Coffee Party free advertising space for their events. However, the media template echoing through the MSMs and portraying Coffee Party leader Annabel Park as an inexperienced and reluctant grassroots organizer is egregiously misleading. In truth, she has a large number of leftist connections and one in particular that leads to the Obama Administration.
NY Times quoted Annabel’s assessment of her role in the Coffee Party movement:
“I’m in shock, just the level of energy here,” said the founder, Annabel Park, a documentary filmmaker who lives outside Washington. “In the beginning, I was actively saying, ‘Get in touch with us, start a chapter.’ Now I can’t keep up. We have 300 requests to start a chapter that I have not been able to respond to.”
The Washington Post said the following of Annabel Park:
She's exhausted, overcommitted, passing whole days on Facebook, not collecting a paycheck, hopping between conference calls, sending e-mails at 4 a.m., smoothing out conflicts over strategy. She has been swept up in this project, and so have others.
In truth, Annabel is a longtime political wonk with a large number of leftist political connections.
Jacobson broke Annabel’s association with United for Obama, but there are far more. According to the Washington Post in 2008, she started by campaigning for Democrat Senator James Webb in 2006. Since then, she has been associated with a large number of leftist groups including, Asian Pacific Americans for Progress, Asian Americans for Obama, and she started the 121 Coalition. If you include her friend Eric Byler, co-director with Annabel of “9500 Liberty” a Michael Moore-esk liberal documentary, you can add in APA Five and Asian-Nation.org. Eric Byler also did the filming for the Coffee Party’s homepage video. Both Annabel and Eric recently did a workshop for the APA Five on community activism. Perhaps, there was something already in the works?
As has been pointed out by many today, Annabel also has a large number of MSM contacts. She previously worked as a Strategy Analyst for the NY Times and has been interviewed a number of times prior to the Coffee Party’s break out Washington Post article. Here is Annabel being interviewed in the Washington Post in 2008. Here she is with Anderson Cooper of CNN.
The Washington Post reported that Annabel started the Coffee Party movement with a Facebook post expressing her frustration with the Tea Party Movement. The Washington Post goes on:
Friends replied, and more friends replied. So last month, in her Silver Spring apartment, Park started a fan page called "Join the Coffee Party Movement."
The real question Annabel needs to answer is, "exactly who on Facebook answered your post?" Or perhaps, that question is better left to Kalpen Modi (stagename Kal Penn), who is a friend listed on her Facebook page. See screenshot below; click to enlarge
When last we saw Kalpen, he was sailing off into the rising Obama sun and leaving his acting job as Kutner on Fox’s TV show House to take a position in the Obama administration. Kalpen Modi is now the associate director for the Office of Public Engagement. The purpose of Kalpen’s job is to coordinate with organizations associated with Asian Americans and Pacific Islanders. An itinerary for a conference call sent by Kalpen to Asian Pacific Americans for Progress gives you an idea of how Obama is coordinating with these organizations. Kalpen's job in the Obama administration put him directly in contact with the same groups, organizations, and personal contacts as Annabel.
We broke the story last week that Annabel Park and Eric Byler had interviewed Kal Penn Modi for Obama propaganda during the 2008 campaign. However, Annabel’s friend Eric Byler may have known Kalpen previously (Perhaps while they were both speaking at an APA Five conference together).
This Annabel-Kalpen connection creates a series of conundrums for the salivating MSMs, like the Washington Post and NY Times, who want to spin this AstroTurf group, the Coffee Party, as grassroots. How could the MSMs have known? Annabel said herself that the Coffee Party was grassroots, why do any investigative journalism?
Even without Annabel’s links to the Obama Administration, her associations don’t pass the sniff test. If it smells bitter, tastes bitter (h/t to Jacobson again), it must be Coffee Party.
Via Memeorandum
Other Bloggers on the trail of the Coffee Party
The Other McCain
The Lonely Conservative
Legal Insurrection
Another Black Conservative
American Power
Riehl World View
Update:
Jacobson was called by the writer of the NY Times fluff piece on the Coffee Party, Kate Zernike. When Jacobson asked why the author had not further disclosed Annabel’s ties with the Obama administration, the author replied that she was limited to 700 words.
Kate, just a suggestion. Less publicity (via her article):
The party (coffeepartyusa.org) is planning nationwide coffee houses for March 13, where people can gather to decide which issues they want to take on and even which candidates they want to support.
This summer, Ms. Park said, the party will hold a convention in the Midwest, with a slogan along the lines of “Meet Me in the Middle.”
More journalism.
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The free office suite
Download LibreOffice
LibreOffice Mac OS X (PPC), version 4.0.2, Slovak. Not the version you wanted? Change System, Version or Language
You need to download and install these files in order:
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LibreOffice is an open source project and you can therefore download the source code to build your own installer.
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Entropy 2012, 14(12), 2478-2491; doi:10.3390/e14122478
Article
Maximum Entropy Gibbs Density Modeling for Pattern Classification
1 Laboratoire de recherche en imagerie et orthopédie, Centre de recherche du CHUM, École de technologie supérieure, Pavillon J.A. de Sève, 1560, rue Sherbrooke E., Y-1615, Montreal (QC),H2L 4M1, Canada 2 Institut national de la recherche scientifique, INRS-EMT, Place Bonaventure, 800, de La Gauchetière O., Montreal (QC), H5A 1K6, Canada 3 École de technologie supérieure, 1100, Rue Notre-Dame O., Montreal (QC), H3C 1K3, Canada
* Author to whom correspondence should be addressed.
Received: 25 September 2012; in revised form: 19 October 2012 / Accepted: 30 November 2012 / Published: 4 December 2012
(This article belongs to the Special Issue Maximum Entropy Production)
Download PDF Full-Text [250 KB, uploaded 4 December 2012 09:36 CET]
Abstract: Recent studies have shown that the Gibbs density function is a good model for visual patterns and that its parameters can be learned from pattern category training data by a gradient algorithm optimizing a constrained entropy criterion. These studies represented each pattern category by a single density. However, the patterns in a category can be so complex as to require a representation spread over several densities to more accurately account for the shape of their distribution in the feature space. The purpose of the present study is to investigate a representation of visual pattern category by several Gibbs densities using a Kohonen neural structure. In this Gibbs density based Kohonen network, which we call a Gibbsian Kohonen network, each node stores the parameters of a Gibbs density. Collectively, these Gibbs densities represent the pattern category. The parameters are learned by a gradient update rule so that the corresponding Gibbs densities maximize entropy subject to reproducing observed feature statistics of the training patterns. We verified the validity of the method and the efficiency of the ensuing Gibbs density pattern representation on a handwritten character recognition application.
Keywords: maximum entropy; Kohonen neural network; Gibbs density; parameter estimation; pattern classification; handwritten characters
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Cite This Article
MDPI and ACS Style
Mezghani, N.; Mitiche, A.; Cheriet, M. Maximum Entropy Gibbs Density Modeling for Pattern Classification. Entropy 2012, 14, 2478-2491.
AMA Style
Mezghani N, Mitiche A, Cheriet M. Maximum Entropy Gibbs Density Modeling for Pattern Classification. Entropy. 2012; 14(12):2478-2491.
Chicago/Turabian Style
Mezghani, Neila; Mitiche, Amar; Cheriet, Mohamed. 2012. "Maximum Entropy Gibbs Density Modeling for Pattern Classification." Entropy 14, no. 12: 2478-2491.
Entropy EISSN 1099-4300 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
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