In silico mapping provides an expedient way to associate the natural diversity of phenotypic traits with ancestrally inherited polymorphisms for the purpose of dissecting genetic traits. In mouse, the current single nucleotide polymorphism (SNP) data have lacked the density across the genome and coverage of enough strains to properly achieve this goal. To remedy this, 470,407 allele calls were produced for 10,990 evenly spaced SNP loci across 48 inbred mouse strains. Use of the SNP set with statistical models that considered unique patterns within blocks of three SNPs as an inferred haplotype could successfully map known single gene traits and a cloned quantitative trait gene. Application of this method to high-density lipoprotein and gallstone phenotypes reproduced previously characterized quantitative trait loci (QTL). The inferred haplotype data also facilitates the refinement of QTL regions such that candidate genes can be more easily identified and characterized as shown for  A large resource of genetic markers, typed in 48 strains of mice, combined with statistical techniques, allow "in silico" mapping of genetic regions involved in interesting traits in mice The combined efforts of the public and private mouse genome sequencing consortiums have yielded important advances in understanding the structure and content of the genome . IdentifThe utility of this sequence-variation data is 2-fold. First, the single nucleotide polymorphisms (SNPs) identified by these sequencing projects provide denser coverage marker sets that are well suited for high-throughput genotyping systems. Currently, these benefits have only been available for crosses between the relatively few strains for which substantial polymorphism discovery has been undertaken.Second, the distribution of SNPs between any two strains, or more precisely, the lack of SNPs between two mouse strains, indicates regions of their genomes that were inherited from a common ancestor. Phenotypic differences that are traditionally mapped in QTL studies are almost exclusively due to loci inherited from different ancestral progenitors rather than new mutations . Thus, ahttp://www.jax.org/phenome) have collated multistrain phenotype data. We demonstrate that when using these datasets, in combination with new analysis methods, statistically significant associations between discrete genomic regions and biologically important phenotypes can be identified. Confirmation of these associations was obtained by comparison to data from traditional QTL mapping methods.To overcome the barriers to in silico mapping, 10,990 SNP assays have been typed against 48 mouse strains in this study. These assays provide an extensive polymorphic marker set enabling expansion of traditional mapping efforts to other strains. Wide-ranging phenotyping projects that have been coordinated by The Jackson Laboratory ((Mus spretus), represent a different species of mouse from the other lines tested. Not surprisingly, fewer genotypes were obtained from these two because of the divergent genomes  track with the phenotypic outcome with the idea that these haplotypes contain causative mutations. For in silico mapping to be successful, a requirement is that the SNP data accurately represent this ancestral relationship of the mice at the genomic level. At a gross level, this was examined by comparing branches of the phylogenic tree generated from this SNP dataset with the known breeding histories of the strains used in this study . An inspMus musculus Chromosome 9 (MMU9) (At a more detailed level the specific genomic contributions of mouse strains derived as hybrids of other common laboratory strains can be estimated. For example, DBA/2J is considered to have contributed approximately 16% of the genomic content to the C57BLKS/J mouse . Compari9 (MMU9) B. Based A lack of sufficient underlying SNP data to this point have prevented the thorough development and testing of an algorithm to carry out in silico mapping . Previouhttp://jaxmice.jax.org/jaxmice-cgi/jaxmicedb.cgi). Albino mice were excluded from the mapping of nonagouti because the nature of the phenotype prevents the ascertainment of agouti or nonagouti coat colors. In each case, the appropriate locus for the gene responsible for the particular trait was identified from this SNP collection with the most significant p-value  statistical model. Briefly, at each three-SNP window, a modified F-statistic based on the true genotype–phenotype pairings is calculated  and gallstone development. The average HDL values from 10-wk-old mice fed on a normal chow diet were taken from the Mouse Phenome Database . Becausep-value, a recently described method by p-value = 0.000506; −log[p] = 3.2958) was used , and seven of these regions fell within the range of traditionally identified QTL for gallstone formation or mucin accumulation, which is considered a precursor to gallstone formation , located at 89.55 Mb, is expressed in the liver and adipose tissue (http://symatlas.gnf.org) and functions by producing cyclic adenosine monophosphate . This excludes the MMUX in silico QTL since they cannot be verified from current traditional QTL data. The false positive cutoff employed here is very restrictive and could be relaxed to find additional real associations, but the chances of including false positives would then increase. For the gallstones phenotype the concordance is not demonstrated to the same level; however, the top-ranked loci still show overlap with previously defined QTL. What about the loci that do not show overlap; are they still real? From a statistical analysis it is unlikely they are false positives. In these results, 25 strains are simultaneously combined, unlike standard QTL mapping using two-strain comparisons, and some phenotype–genotype associations may occur that have not been observed by classical methods. Contributions from diverse strains that have not normally been used in F2 crosses or available in RI lines can now be incorporated.The SNP data here provide new resources for traditional mapping projects and enable development of inbred strain haplotype methods for QTL detection. The analyses presented here indicate that the inferred haplotype structures derived from this dataset provide sufficient estimation of genetic diversity/similarity to map Mendelian traits to within 1-Mb intervals. QTL can also be defined as inferred haplotype loci of several megabases in size. The analysis for QTL provides a rank order of significant phenotype/genotype associations, and using the gFWER method of controlling for multiple-testing error, the loci reported as statistically significant are very likely to be biologically relevant. This point is borne out by the high concordance between the in silico QTL and the traditionally determined QTL . The traEven showing that this method does find significant associations, the question arises about its general utility and applicability. The methods of in silico mapping as described here should be viewed as a complement to, and not a substitute for, traditional methods for mapping QTL. Although we have demonstrated a robust approach to in silico mapping, it would certainly not be expected to find all QTL for a given phenotype. Major contributors to phenotypic variation will show up, but weaker contributors would be expected to be lost because of the limited power of 25 strains. It would also be expected to miss QTL resulting from recent strain-specific mutations or low-frequency haplotypes. Traditional QTL methods will still be required to identify the more subtle interactions, including those involving epistasis and modifying genes.However, these methods would provide a useful starting point for a new phenotype that is being investigated, where often the first step for any QTL analysis is a strain survey to quantify the range of the phenotype. Additionally, if these analyses are overlaid with the results from a traditional two-strain QTL mapping, one of the major advantages to be gained from this approach is that associative loci are defined in terms of a few megabases instead of tens of centiMorgans.M. m. domesticus. To be successful, phenotypes must have a low intrastrain variation but sufficient variance within the strain set selected. This however, is not a requirement restricted to in silico mapping.The number and selection of strains and appropriate phenotype are also important considerations. Here we have limited our analysis of the complex traits, HDL and gallstones, to 25 strains—those that are best interrogated by this SNP set. While it is true that more strains have the potential to add greater statistical power to resolve QTL, this potential is limited by our ability to accurately represent the ancestral relationship of those additional strains. If we add more strains, but cannot accurately infer haplotype structures in those strains, we only add more noise to the analysis. The ability to detect all possible haplotypes in the utilized strains from the SNP data suffers from the availability of sequence data, currently from only four strains of mice. Because the source SNPs come from the sequencing of only four closely related strains, this current set is biased toward interrogating ancestry of The overall power of this method will only improve as the biases and limitations of the SNP panel are addressed and additional strains are genotyped and phenotyped. Unique strains would become more useful if all possible SNPs are known and the mapping is then done directly with the causative polymorphism or at least with a large unbiased set of SNPs. As resequencing of other mouse genomes progresses, the ability to correctly infer the complete number and structure of haplotypes will improve, and the number of QTL regions reaching statistically significant levels will increase.Recently, two similar studies of haplotype structures across 5-Mb regions were published, although they produced differing conclusions on how their findings might affect in silico mapping efforts . Yalcin This is not to suggest that fragmented small haplotypes are not common in the genome of the inbred mouse. This clearly does mean that there will be regions of the genome that will not be interrogated well by an in silico method. This approach is still limited by the density of this SNP map and can only be expected to visualize inferred haplotype patterns of approximately 1 Mb in size, and therefore smaller haplotype structures are hidden and potential phenotype–genotype associations will be missed. Here future, larger SNP sets that will allow more SNPs to infer haplotype will become important. However, this is the best resolved whole genome view of the diversity of the commonly used inbred strains to date.The algorithms employed here provide a starting point for further development of in silico mapping. We have shown that they can be used to identify Mendelian traits and replicate classical QTL associations. Clearly, the next goals are to validate some of the previously unreported associations, and this work is ongoing.Tas1r3 locus were gathered from sequence from multiple strains available in GenBank (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=nucleotide&cmd=search&term=tas1r3). All physical positions presented in the paper are from the Celera Mouse Genome Assembly R13.SNPs for use in genotyping were selected on a weighted basis from the Celera Mouse SNP Database containing data from the DBA/2J, A/J, C57BL/6J, 129S1/SvImJ, and 129X1/SvJ strains. Sufficient SNPs were selected for coverage of at least one SNP per 300 kb on average. The 129S1/SvImJ and 129X1/SvJ strains were considered as the same strain when their alleles agreed; preference was given first to SNPs where each allele of the SNP was represented by two strains. This was done to favor selection of SNPs representing ancestral inheritance, not recent strain-specific mutations, and to favor real SNPs as opposed to errors in sequence annotation. Additional selective value was incorporated based on whether the SNP was in a gene, how many sequencing runs supported the presence of the SNP, and the proximity of the SNP to previously selected SNPs. Additional SNPs used to characterize the Primers for PCR and single-base extension were designed by using the SpectroDESIGNER software package . Assay designs are available as Supporting Information. All SNP assays were named for their position in the genome in the following format: the chromosomal location, the Mb position on the chromosome, and the kb position with a period separating each number.Taq , 0.625 μl of 10X HotStar PCR buffer containing 15 mM MgCl2, 0.5μl PCR primers mixed together at a concentration of 1.25 μM for multiplexed reactions, and 0.325 μl of 25 mM MgCl2. Reactions were heated at 95 °C for 15 min followed by 45 cycles at 95 °C for 20 s, 56 °C for 30 s, and 72 °C for 1 min and a final incubation at 72 °C for 3 min. After PCR amplification, remaining dNTPs were dephosphorylated by adding 1.5 μl of water, 0.17 μl of homogeneous mass extend reaction buffer (Sequenom), 0.3 units of shrimp alkaline phosphatase (Sequenom), and 0.03 μl of 10 units/μl exonuclease (USB Corporation, Cleveland, Ohio, United States). The reaction was placed at 37 °C for 20 min, and the enzyme was deactivated by incubating at 85 °C for 15 min. After shrimp alkaline phosphatase treatment, the genotyping reaction was combined with 0.76 μl of water, 0.2 μl of 10X Termination mix (Sequenom), 0.04 μl of 0.063 units/μl Thermosequenase (Sequenom), and 1μl of 10 mM extension primer. The MassEXTEND reaction was carried out at 94 °C for 2 min and then 99 cycles of 94 °C for 5 s, 52 °C for 5 s, and 72 °C for 5 s The reaction mix was desalted by adding 3 mg of a cationic resin, SpectroCLEAN (Sequenom), and resuspended in 30 μl of water. Completed genotyping reactions were spotted in nanoliter volumes onto a matrix arrayed into 384 elements on a silicon chip (Sequenom SpectroCHIP), and the allele-specific mass of the extension product was determined by matrix-assisted laser desorption ionization time-of-flight MS. Analysis of data was by automated allele calling from the SpectroTYPER software. All SNP data are available at NCBI dbSNP (http://www.ncbi.nih.gov/entrez/query.fcgi?db=snp) and The Jackson Laboratory Mouse Phenome Database (http://www.jax.org/phenome). Placement of the SNP data across the genome and major and minor allele distributions can be visualized using SNPview (http://snp.gnf.org).For SNP genotyping, genomic DNA from pedigreed mice (Mouse DNA Resources, The Jackson Laboratory, Bar Harbor, Maine, United States) was diluted to 10 ng/μl, and 1 μl of DNA was combined with 2.45 μl of water, 0.1μl of 25 mM dNTPs , 0.03μl of 5 units/μl HotStar The use of a single marker is restrictive in the sense that it only allows a representation of the genome as diallelic. The use of windows of multiple markers enables the visualization of more complex genomic relationships between multiple strains. This more accurately models actual haplotype patterns than does a binary approach. In determining the size of the SNP window to use as a definition of inferred haplotype for purposes of the algorithm, sizes of two, three, four, and five SNPs were examined. A window of only two SNPs was still found to be too limiting. Windows of three, four, and five SNPs produced similar results, but as window size is increased biologically meaningful patterns become fragmented, creating more single-strain inferred haplotypes, resulting in an increase in noise. Singly represented haplotypes can never be informative in this analysis because the commonality of haplotypes is required to achieve significant association with a phenotype. Three SNP windows were also analyzed across the whole genome to identify mirror loci. This would be a locus that has exactly the same strain distribution pattern across all 25 strains used in an in silico run. There were no mirror loci, or 1-off, or 2-off mirror loci  that occurred outside of a 5-Mb interval.Defining the genetic measure as a categorical unit necessitated the use of an ANOVA-based model. The type of ANOVA to use was determined by the characteristics of the phenotypic values.The phenotypes studied here fell into two categories: binary or continuous. The coat color phenotype was considered as binary, where phenotypic values were set to 1 and 0. The HDL phenotype is an example of a continuous phenotype since the phenotypic values are measured on a continuous scale. Two different statistical methods were employed based on this distinction.glm with the parameter family set to binomial was used. This was followed by an application of anova.glm with the parameter test set to Chisq.When phenotypic values are binary, the appropriate statistical approach involves first fitting a binomial generalized linear model to the data. An analysis of deviance table is then computed for the fitted model. The R language function For continuous phenotypic values, a log transformation was applied to reduce the effects of outliers in the phenotypic data. Next, an F-statistic weighted for the genotypic diversity of the strains within an inferred haplotype group was used. The weighted F-statistic had the following form:whereandgn is the number of strains in a given inferred haplotype, gμ is the mean of phenotypic values in a given inferred haplotype , Tμ is the mean of all phenotypic values, k is the number of inferred haplotypes, N is the total number of data values, and gw is the weight representing the genetic diversity of the inferred haplotype. The genetic diversity ratio (gw) between two strains is the number of SNPs genome-wide in which both strains have genetic information and they disagree, divided by the total number of SNPs in which they both have genetic information. The genetic diversity coefficient for an inferred haplotype in the weighted F-statistic is the average gw between all strain pairs contained in the inferred haplotype.where p-value was computed by dividing the number of random F-statistics larger than the true F-statistic by the total number of random F-statistics (million). In this way 200 p-values were collected. The vertical heights reported in the bar graphs (see p) transform of the median of these 200 p-values. A 95% confidence interval (CI) for the p-value at this window was also calculated from this bootstrap distribution.The weighted F-statistic calculated at each SNP window determines if at least one of the inferred haplotypes has an average phenotypic value significantly different from the other inferred haplotypes. To assess the significance of the computed value, the null distribution of the weighted F-statistic was simulated at each SNP window by taking a million bootstrap samples of the phenotypic values. As in the algorithm used for binary phenotypes, inferred haplotype patterns present in only one strain were not included in the calculation because they are not informative in elucidating shared ancestral blocks. From this distribution of a million random F-statistics, 200 bootstrap samples of size 1 million were computed. For each bootstrap sample, a aphs see  are the To estimate the overall false positive rate for this type of calculation, calculating a significance threshold based on the family-wise error rate (FWER) has been proposed . Others p-values, the gFWER method requires an estimate of the “supremum” (least upper bound) of expected values reported at each locus, so the most significant value is reported from the 200 bootstrap p-values  corresponding to the (1 − α) percentile was added to the null distribution  is further complicated. Nearly 11,000 hypothesis tests (one at each three-SNP window) are conducted in a single run of the algorithm. Therefore, equations that currently exist for the estimation of power for QTL mapping by traditional methods cannot be applied here because they assume that the test statistic has some previously defined parametric form. Code for the above described algorithms is available upon request.For calculation of the significance of the number of in silico QTL that overlapped with previously identified QTL for the HDL phenotype, a binomial distribution was usedp = probability of success of 0.42 (overlap with HDL QTL in previous literature).given q = probability of failure; the 0.0025 result is the probability of at least nine successes in ten trials. Only ten loci could be assessed for this result as no information is available for traditional HDL QTL present on the X chromosome.Therefore For the mapping of the retinal degeneration traits, 37 strains were used. This represented all of the strains for which information existed in The Jackson Laboratory database minus the most divergent wild-derived strains for which inference of haplotype would be expected to be most inaccurate. These strains were A/J, AKR/J, BALB/cByJ, BUB/BnJ, C3H/HeJ, C57BL/10J, C57BL/6J, C57BLKS/J, C57BR/cdJ, C57 l/J, C58/J, CBA/J, CE/J, DBA/1J, DBA/2J, FVB/NJ, I/LnJ, KK/HlJ, LG/J, LP/J, MA/MyJ, NOD/LtJ, NON/LtJ, NZB/BlNJ, NZW/LacJ, PERA/EiJ, PL/J, RIIIS/J, SEA/GnJ, SJL/J, SM/J, ST/bJ, SWR/J, WSB/EiJ, ZALENDE/EiJ, 129S1/SvImJ, and 129X1/SvJ. Because of the added complexity of the coat color traits, mapping was restricted to the 25 most related strains for which coat color phenotype could clearly be determined. For the albino analysis, 129S1/SvImJ, A/J, AKR/J, BALB/cByJ, C3H/HeJ, C57BL/10J, C57BL/6J, C57BLKS/J, C57BR/cdJ, C57 l/J, C58/J, CBA/J, DBA/1J, DBA/2J, I/LnJ, LP/J, MA/MyJ, NZB/BlNJ, NZW/LacJ, PERA/EiJ, PL/J, SEA/GnJ, SM/J, WSB/EiJ, and ZALENDE/EiJ strains were used. For the nonagouti mapping, the same strain set as the albino mapping was used except for the mice presenting the albino phenotype. The strains were 129S1/SvImJ, C3H/HeJ, C57BL/10J, C57BL/6J, C57BLKS/J, C57BR/cdJ, C57 l/J, C58/J, CBA/J, DBA/1J, DBA/2J, I/LnJ, LP/J, NZB/BlNJ, PERA/EiJ, SEA/GnJ, SM/J, WSB/EiJ, and ZALENDE/EiJ. Any mouse showing an agouti coat color was considered to be agouti for this analysis regardless of genotype at the agouti locus. Only limited phenotype data were available for saccharin preference, so again all strains with available data except the most divergent wild-derived strains for which inference of haplotype would be expected to be most inaccurate were used. These strains were A/J, AKR/J, BALB/cByJ, BUB/BnJ, C3H/HeJ, C57BL/6J, C57 L/J, CBA/J, CE/J, DBA/2J, FVB/NJ, I/LnJ, KK/HlJ, LP/J, NOD/LtJ, NZB/BlNJ, PL/J, RIIIS/J, SEA/GnJ, SJL/J, SM/J, ST/bJ, and SWR/J. For the mapping of the other complex traits, only the 25 strains with the closest ancestral relationship were used. These strains were 129S1/SvImJ, A/J, AKR/J, BALB/cByJ, BTBR T+ tf/J, C3H/HeJ, C57BL/10J, C57BL/6J, C57BLKS/J, C57BR/cdJ, C57 l/J, C58/J, CBA/J, DBA/1J, DBA/2J, I/LnJ, LP/J, MA/MyJ, NZB/BlNJ, NZW/LacJ, PERA/EiJ, PL/J, SEA/GnJ, SM/J, and WSB/EiJ.Dataset S1(16.1 MB XLS).Click here for additional data file.Figure S1M. m. domesticus strains show a much closer relationship than the other wild-derived strains to the common laboratory strains.Ancestral relationships between strains can be seen within clusters of the tree such as the fact that BALB/cByJ is a progenitor strain to SEA/GnJ. The bias of the SNP set can also be viewed by the exaggerated distance between the C57 and 129 clusters and the DBA and A/J cluster. The wild-derived strains make up the outermost cluster, but the three (2.2 MB EPS).Click here for additional data file.Figure S2p-value at each three-SNP window was calculated and plotted along the x-axis. Because any log p-value below 3 will not reach significance, calculations are halted at any locus once obtaining a log p-value of 3 becomes impossible in order to increase computational throughput. As such all log p-values below 3 are reported at 3. The false positive cutoff established by the gFWER calculation is indicated by a horizontal red line. Every quantitative trait was run twice through the algorithm to ensure consistency of results.The log (5.8 MB EPS).Click here for additional data file.Figure S3p-value is reported from each run. The significance cutoff is indicated by the vertical red line.To estimate an appropriate false positive cutoff, 10,000 genome scans are conducted on randomized datasets and the 99.5 percentile log (3.1 MB EPS).Click here for additional data file.Table S1(44 KB XLS).Click here for additional data file.Table S2(34 KB XLS).Click here for additional data file.http://www.jax.org/phenome) accession numbers for the phenomes discussed in this paper are MPD:29 and MPD:99.The Mouse Phenome Database (


The Healthcare Commission started work on 1 April 2004, taking over all the functions of the former Commission for Health Improvement , which had released the corresponding PCT ratings for 2002/2003 in July 2003.On 21 July 2004, the Healthcare Commission , with handy functions like map search (by PCT name or part of it). The maps feature a colour-blind friendly quadri-colour scheme to represent PCT star ratings. Clicking a PCT on any of the maps will display the detailed performance report of that PCT for the corresponding year.We produced two Web-based interactive maps of PCT star ratings, one for 2003 and the other for 2004 Using our Web-based interactive maps, users can visually appreciate at a glance the distribution of PCT performance across England. They can visually compare the performance of different PCTs in the same year and also between 2003 and 2004 (by switching between the synchronised 'PCT Ratings 2003' and 'PCT Ratings 2004' themes). The performance of many PCTs has improved in 2004, whereas some PCTs achieved lower ratings in 2004 compared to 2003. Web-based interactive geographical interfaces offer an intuitive way of indexing, accessing, mining, and understanding large healthcare information sets describing geographically differentiated phenomena. By acting as an enhanced alternative or supplement to purely textual online interfaces, interactive Web maps can further empower organisations and decision makers. The Healthcare Commission started work on 1 April 2004, taking over all the functions of the former Commission for Health Improvement , which had released the corresponding PCT ratings for 2002/2003 in July 2003 [On Wednesday 21 July 2004, the Healthcare Commission A star rating scheme is adopted. PCTs with the highest levels of performance in the measured areas are awarded a rating of three stars. PCTs with mostly high levels of performance, but which are not consistent across all measured areas, are awarded a rating of two stars. PCTs where there is some cause for concern about particular areas of measured performance are awarded a rating of one star. PCTs that have shown the poorest levels of measured performance or little progress in implementing clinical governance receive a rating of zero stars . and . This "geographic search" does not allow any visual appreciation of PCT performance levels, or any visual comparisons to be made between PCTs or between 2003 and 2004 result sets.The performance ratings Web pages of the Healthcare Commission and the former Commission for Health Improvement offer a very limited "geographic search" restricted to browsing results by Strategic Health Authority (SHA) . The maps use a yellow-green-blue quadri-colour scheme to represent PCT star ratings. Users can switch between the two map sets or themes, 'PCT Ratings 2003' and 'PCT Ratings 2004', within the same pane  can be organised and navigated based on their geographical attributes [Using our Web-based interactive maps, users can quickly and intuitively locate any PCT and retrieve detailed performance information about it. They can also visually appreciate at a glance the distribution of PCT performance across England; for example, one can instantly note that there were no three star (dark blue) PCTs in the London region in 2002/2003 and that this has remained unchanged in 2003/2004. Users can visually compare the performance of different PCTs in the same year and also between 2003 and 2004 (by switching between 'PCT Ratings 2003' and 'PCT Ratings 2004' themes). The performance of many PCTs has improved in 2004, whereas some PCTs, e.g., Northumberland Care Trust – Figure Web-based interactive geographical interfaces offer an intuitive way of indexing, accessing, mining, and understanding large healthcare information sets describing geographically differentiated phenomena, and can act as an enhanced alternative or supplement to purely textual online interfaces. Geographical interfaces enable instant visual comparisons to be made between different geographical areas and over time (when information sets and maps for successive periods of time are available), thus empowering organisations and decision makers. and the Healthcare Commission  respectively. The Internet addresses (URLs) of the corresponding detailed reports of PCT performance were also harvested from the same sources.Star ratings of English PCTs for the years 2002/2003 and 2003/2004 were obtained from the Web sites of the former Commission for Health Improvement . We used the 2001 Census PCT (post April 2002 change) boundary dataset, which is the copyright of the Crown/Ordnance Survey , and is freely available to the UK academic community from EDINA UKBORDERS service with the support of the ESRC and JISC . The names/boundaries and labels (codes) of few PCTs changed between 2003 and 2004, but this was properly cared for in our exercise.The maps were created in ESRI ArcView GIS Version 3.1 We inserted four new fields in the original PCT boundary dataset table to store the 2003 and 2004 star ratings and corresponding detailed report URLs for all English PCTs. The PCTs in the output maps are coloured according to the values in their star rating fields (0, 1, 2, or 3 corresponding to the number of stars awarded), with light colours for low ratings to dark colours for high ratings. and [We used ColorBrewer  and ) to sele and Figure ).The online interactive maps were then produced using the Demo version of alta4 HTML ImageMapper 3.5 extension for ESRI ArcView GIS 3.x .It is noteworthy that HTML ImageMapper does not require any server side software installation, and as such is much simpler to use than some other Internet GIS solutions like the client/server version of ALOV Map/TimeMap  and JShape  Java applets, which don't require any server side setup, are limited by the fact that they need to download the whole map shapefile from the Web server before they can start on the client side, and so are not suitable for large datasets (our PCT boundary dataset in ESRI shapefile format is about 50 MB in size). Other options for generating interactive Web maps from a desktop GIS are discussed in [The standalone versions of ALOV Map/TimeMap


A primary goal of acute treatment for depression is clinical remission of symptoms. Most meta-analyses of remission rates involve randomized controlled trials (RCTs) using patients from psychiatric settings, but most depressed patients are treated in primary care. The goal of this study was to determine remission rates obtained in RCTs of treatment interventions for Major Depressive Disorder (MDD) conducted in primary care settings.Potentially relevant studies were identified using computerized and manual search strategies up to May 2003. Criteria for inclusion included published RCTs with a clear definition of remission using established outcome measures.A total of 13 studies (N = 3202 patients) meeting inclusion criteria were identified. Overall remission rates for active interventions ranged between 50% and 67%, compared to 32% for pill placebo conditions and 35% for usual care conditions.Remission rates in primary care studies of depression are at least as high as for those in psychiatric settings. It is a realistic goal for family physicians to target remission of symptoms as an optimal outcome for treatment of depression. Major depressive disorder (MDD) is one of the most common and disabling of medical conditions . The CanThere are many effective treatments for MDD, including psychotherapy and antidepressants. Traditionally, efficacy in randomized controlled trials (RCTs) for depression has been determined on the basis of score changes in rating scales such as the Hamilton Depression Rating Scale (HDRS)  or the MSuch findings suggest that the goals of acute treatment (approximately the first 8–12 weeks or so of treatment) for MDD should be clinical remission, a clinical state distinguished by minimal residual symptoms, rather than just response -13. ClinIncreasing numbers of treatment studies are now explicitly reporting both clinical response and remission rates in assessment of outcome. A meta-analysis of 8 antidepressant studies of venlafaxine versus selective serotonin reuptake inhibitors [SSRIs] and placebo reported mean remission rates of 45%, 35%, and 25%, respectively . A subseAll the studies in these systematic reviews involved patients in psychiatric or mixed settings. However, most people suffering from MDD will be managed in the primary care setting . ApproxiPotentially relevant studies were identified using computerized and manual search strategies. The computerized search conducted in June, 2003 included the databases: Medline, Psych Info, Embase, Biosis, Cochrane Database of Systematic Reviews, and Cochrane Controlled Trials Register and Current Controlled Trials (1981–May 2003). The search terms used were 'depressive disorder' or 'depression' combined with 'primary care' and 'remission' and/or variants. The bibliographies of relevant articles were also manually searched. Two reviewers (MYD and RWL) collected and independently assessed abstracts for inclusion criteria. Disagreements were resolved with consensus.Studies were included if they were RCTs with original data comparing one or more interventions  and published in English. Only studies of predominantly adult populations, as opposed to exclusively child or elderly patient populations, were included. Although the focus was principally upon patients with MDD , the criteria for a diagnosis of MDD was intentionally broad in order to capture the heterogeneity of the sample and allow the results to be as generalizable as possible. Included studies also had to use a standardized outcome measure (e.g., HDRS, MADRS, Beck Depression Inventory [BDI] ) and proTwo independent reviewers (MYD and EEM) extracted data from studies using a checklist developed for this study, with disagreements resolved by a third reviewer (RWL). A conservative measure of remission rate was calculated from each study using an intent-to-treat analysis , even ifThe type of intervention was classified as placebo, "usual care" by clinician (standard treatment by a patient's own physician), psychotherapy treatment only, antidepressant treatment only, psychotherapy plus antidepressant treatment, or program intervention .Each set of rates was pooled based on a Bayesian approach to meta-analysis using the Fastpro software program (version 1.7) by Eddy and Hasselblad. Readers interested in a more detailed discussion of this approach should refer to Eddy et al . The pooThe initial electronic and bibliographic search found 63 articles of which 47 warranted more detailed review based on the published abstract. Of these, 34 articles were excluded due to methodology (not RCTs, 4 studies), lack of remission criteria 18 studies), diagnostic criteria (not MDD, 11 studies) and age criteria (geriatric, 4 studies)  ranged between 50% and 67%. These rates are equivalent to, or indeed greater than, those reported in meta-analyses of studies examining pharmacological or psychological interventions for depression in psychiatric populations, in which the overall remission rates ranged between 35% and 46% -24. On tsymptoms .While it was not within the scope of the current study to compare the effectiveness of different treatment interventions in improving remission rates, we can report on the trends we observed in the data. Antidepressant and psychotherapy interventions delivered in isolation showed similar remission rates (54% for both). Combination antidepressant plus psychotherapy interventions showed somewhat higher rates (67%), although this category included only 1 arm with only 35 patients. Program interventions had a mean remission rate of 50%, and all treatment interventions fared better than either placebo (32%) or usual care (35%).The studies identified in our review were quite heterogeneous in nature, ranging from those that looked solely at the effects of a particular pharmacological agent, through to complex program initiatives that incorporated a variety of interventions at different levels of care. This heterogeneity limits our ability to make broad comments about remission rates in primary care, but was not unexpected, as we wanted to capture the diversity of treatment interventions for depression currently being tested in this setting. Other potential limitations of the study include that fact that we only assessed published studies written in English and that we used a conservative measure of remission rate. Finally, we also used the definition of remission as specified by each individual study. While these definitions were similar to those widely used in RCTs conducted in psychiatric settings, and thus are useful for comparison, there is current controversy about depression scales and which cutoff scores indicate true remission of symptoms .This meta-analysis serves to answer an important clinical question about the feasibility of obtaining remission of symptoms of MDD in primary care patients. Our results indicate that this is a realistic goal in this population, although further research is still required to determine whether certain treatment modalities (or combinations of treatment interventions) are superior to others in achieving higher remission rates. Future research should also focus upon developing pragmatic strategies for general practitioners to implement evidence-based guidelines concerning the treatment of depression to clinical remission.MYD and EEM conducted the data extraction, wrote the initial draft of the manuscript, interpreted results, and revised the manuscript. PW provided statistical consultation and analysis, and revised the manuscript. JEA interpreted the results and revised the manuscript. RWL conceived the initial idea, developed the method, interpreted results, revised the manuscript, and provided financial resources for the study. All authors read and approved the final manuscript.RWL is on advisory/speaker boards or has received research funds from: AstraZeneca, Biovail, Canadian Network for Mood and Anxiety Treatments, Eli Lilly, GlaxoSmithKline, Janssen-Ortho, Litebook, Inc., Lundbeck, Merck, Organon, Roche, Shire, Servier, and Wyeth.The pre-publication history for this paper can be accessed here:Studies excluded from the reviewClick here for file


Annotation of sequences that share little similarity to sequences of known function remains a major obstacle in genome annotation. Some of the best methods of detecting remote relationships between protein sequences are based on matching sequence profiles. We analyse the superfamily specific performance of sequence profile-profile matching. Our benchmark consists of a set of 16 protein superfamilies that are highly diverse at the sequence level. We relate the performance to the number of sequences in the profiles, the profile diversity and the extent of structural conservation in the superfamily.ROC10, varying from 0.95 down to 0.01. These large differences persist even when the profiles are trimmed to approximately the same level of diversity.The performance varies greatly between superfamilies with the truncated receiver operating characteristic, Although the number of sequences in the profile (profile width) and degree of sequence variation within positions in the profile (profile diversity) contribute to accurate detection there are other superfamily specific factors. Currently some of the best methods for detecting relationships between protein sequences below the so-called twilight zone of sequence similarity are offered by iterative search algorithms such as PSI-BLAST  which, iHere, we examine how the performance of remote homolog detection by profile-profile methods varies between particular superfamilies. Since superfamilies are believed to constitute sets of remote homologs, detection of same-superfamily relationships is an important task for bioinformatics, and with the increasing number of structures becoming available, improvement in this area will help build a complete structural map of sequence space. In this paper, we use a set of superfamilies that are very sequence diverse to benchmark profile-profile methods. By sequence diverse, we mean that the superfamily has many domains that show no detectable sequence similarity to each other; this lack of detectable sequence similarity means this set is a difficult benchmark for remote homolog detection methods.profile width, we mean the number of sequences in the profile, defined in contrast to profile length and by diversity we mean the degree of sequence variation within positions in the profile. In particular, Panchenko suggested that there may be an optimum level of profile diversity [Previous work has shown that the performance of profile-profile methods is chiefly determined by the width and diversity of the profiles. By iversity , whilst iversity .We examine the performance of profile-profile matching with regard to specific superfamilies with both the full profiles generated from a PSI-BLAST search, and with profiles that are trimmed to similar width and diversity. Significant differences in recognition performance exist between superfamilies for both the full and trimmed profiles. This suggests that performance of profile-profile matching is not simply a function of profile width and diversity. We examine how the performance relates to the structural diversity of superfamilies and find that structurally conserved superfamilies are recognised more successfully than structurally diverse superfamilies.Table ROC10 (see Methods for definition) for each superfamily. The figure shows that there is a large variation in performance with respect to superfamily for both the full profiles and the trimmed profiles.Figure ROC10 values ≥ 0.7, the fibronectin, thioredoxin-like, (trans)glycosidases, immunoglobulin and FAD/NAD(P)-binding have ROC10 > 0.2 and the remaining 8 superfamilies all perform poorly, having a performance less than 0.1.For the full profiles, the alpha/beta-Hydrolases, Cytochrome c and S-adenosyl superfamilies perform well, all having with ROC10 values greater than 0.2, while the rest are still less than 0.1.After trimming, although performance is reduced, the overall pattern of performance still remains. All the well recognised superfamilies (with the exception of the (trans)glycosidases and thioredoxin-like) still show ROC10 of 0.7 when using trimmed profiles despite having, on average, less than 20 sequences in the profile.The fact that the performance varies greatly between superfamilies despite the trimming of the profiles indicates that the profile generation is not the only limiting step in the performance of profile-profile methods. One might have thought that, for instance, the bad recognition of 4-helical cytokines is due to the small number of homologs drawn from the profile-building stage. Whilst this still may be true, it is not necessarily true: the Cytochrome c superfamily still shows a Figure ROC10 for each superfamily. The figure shows a correlation between the mean RMSD of each super-family and its ROC10 value. The figure shows that superfamilies with a mean RMSD of less than 2 Å tend to be well recognised by profile-profile methods, whilst the structurally diverse superfamilies are not.Figure It may be the case that despite the absence of any discernible global sequence similarity within our dataset some local patterns of conservation do exist. These patterns may be present more strongly in some superfamilies than in others. In order to examine this possibility we constructed multiple structure based sequence alignments for each of the 16 superfamilies and then looked down the columns of the multiple sequence alignments to examine the extent of conservation at each position (see Methods section).ROC10) versus conservation. Apart from the cytochrome c superfamily (an outlier with a high ROC10 of 0.7 despite a conservation score of 0.2 because the superfamily has a conserved CxxCH motif that facilitates detection), the well performing superfamilies -binding and fibronectin with ROC10 values for the trimmed profiles of at least 0.25) have conservation measures of greater than 0.25. This suggests that some superfamilies although highly sequence diverse, may retain some patterns of conservation that facilitate recognition. Further investigation of the functional implications of this variation would be a next step.Figure ROC10). The P-loop and Viral coat superfamilies have low conservation scores and and large structural diversity reflected by high RMSD values. In contrast, the fibronectin and immunoglobulin superfamilies have higher conservation values (both around 0.28) and lower RMSDs (around 1.5Å). However the figure does not show any clear correlation between conservation and RMSD.Figure Our results suggest that profile profile methods can detect remotely related sequences for some superfamilies significantly better than for others. In our dataset the sequence identity between domains in all the superfamilies is low (not greater than 10% as defined by the ASTRAL). Although the mean width and diversity of the profiles varies across the superfamilies this does not appear to be the only factor contributing to the differences in detection.ROC10 dropping from 0.70 to 0.22. However trimming had no effect on performance for the FAD/NAD(P)-binding superfamily, and only resulted in a small reduction in performance for the immunoglobulins and the cytochrome c superfamilies. Importantly the membership of the top ranking superfamilies in terms of performance did not change after trimming.The effect of the trimming varied depending on superfamily. For the best performing profile  the trimming reduced the performance by about 50% (from 0.95 to 0.43) but the effect on the rank was small dropping from first place to second. Similarly the trimming impacted significantly on the performance of the S-adenosyl methyl transferases with Although the overall level of sequence similarity within our dataset is low (not more than 10% identity) the different superfamilies exhibit different levels of conservation at positions within the multiple structure based alignments. These conserved positions may facilitate recognition. The extent to which they constrain the structures leading to less diverse alignments is unclear. We recognise also that our measure of conservation and also the use of RMSD as a measure of structural diversity both have their shortcomings. It would be interesting to identify and extract a conserved core and represent structural profiles as combination of core profiles separated by regions of variable length.There exist large superfamily specific differences in the performance of profile profile matching for the detection of remote sequence relationships. Some superfamilies can be detected far more successfully than others. The width and diversity of the profiles are important factors in successful recognition. However these are not the only factors that contribute to these superfamily specific differences.We took release 1.63 of ASTRAL  which prFor each domain of each of the 16 superfamilies we executed a five round PSI-BLAST [To produce trimmed profiles, we take the full profile and remove the bottom sequence (corresponding to the most remote homolog) until a stopping criterion is reached. The stopping criterion is based on Neff, a statistic previously used for this task ,6,7. NefWe use the program COMPASS  to perfoi of superfamily j, , the sequence profile corresponding to  was not included in the sequence profile library. This procedure was carried out twice: firstly with the full profiles, and the again with the trimmed profiles.To assess the performance of profile-profile matching, each domain of each of the 16 superfamilies was used as a query and its sequence profile was matched against a library of sequence profiles representing the dataset. A profile database was then created using the 543 profiles. When matching the profile of domain ROC10 as a statistic that describes the performance of the profiles for a particular super-family. ROCn is defined as , where T is the total number of true hits possible and ti is the number of true positives with a score better than the ith false hit. Variance in the ROC10 statistic was calculated using the method given in [We use To evaluate the structural diversity within each superfamily, each member of a superfamily was structurally compared to every other member. For all the domains in a superfamily we perform pairwise structural alignments using the program SAP  to all oi and j at positions xi, xj in the superposition, is defined to be ((1 + RMSD)(1 + |xi - xj|))-1. A detailed explanation and analysis of this method is given in [To create a structure based multiple alignment of a superfamily, we first made all pairwise structural comparisons between all pairs within a superfamily using SAP ,12. We tgiven in .We used the Taylor Venn diagram  to assigJAC carried out the benchmarking and wrote the necessary code and helped to prepare the manuscript. MASS conceived of the study, provided input into the design and refined the manuscript. All authors read and approved the final manuscript.


Whiskers don't fossilize, so it's hard to say when they first evolved. But it's quite likely they emerged along with mammals, over 200 million years ago. To elude the eye (and feet) of ungainly dinosaurs, it's thought these shrew-like prototypes foraged at night and sought refuge underground, where the sensory advantages of whiskers would come in handy. Nocturnal animals use whiskers much like the blind use walking sticks: to navigate their surroundings, explore close objects, and avoid running into things.Whiskers, or vibrissae, connect to nerves, blood vessels, and muscles. These special connections allow rats, for example, to actively “whisk” the surface of objects and discern fine differences in texture, just as we move our fingertips along a surface to pick up details. In the wild, whisking helps rats navigate unfamiliar terrain to find food. But how does the brain know what the animal is touching?Rat whiskers scan surfaces in a rhythmic motion that excites sensory receptor cells embedded in their whisker pad. Receptors in each whisker shaft are innervated by several hundred “first-order neurons” that relay sensory signals to second-order neurons in the brain stem, then on to third-order neurons in the thalamus, and finally on to the cortex, where sensory stimuli are integrated in cell clusters called barrels.Ehsan Arabzadeh, Erik Zorzin, and Mathew Diamond work with rats to investigate how sensory receptors extract fundamental features from complex and diverse stimuli to encode texture. Not much is known about how receptor and cortical neurons respond to active whisking along irregular surfaces, though responses to simple stimuli  suggest that neurons might represent texture by encoding kinetic features of whisker vibrations, in particular, velocity. In a new study, Diamond and colleagues investigate the connection between textures, whisker vibrations, and neural codes: do distinct textures produce distinct vibrations? If so, how are these vibrations encoded and reported?The authors first collected kinetic data of whiskers moving across different textured surfaces. Stimulating cranial nerve VII of anesthetized rats (the motor nerve) generated whisking movements akin to those seen in conscious rats; the kinetics of these movements and the vibrations of the whisker shafts were measured under different conditions, including no contact with objects (“free whisk”), contact with smooth objects, and contact with various grades of sandpaper. These vibrations were then “played back” to other rats, while measuring the neuronal activity at two critical stages in the sensory pathway: the first-order neurons that innervate the whiskers and the barrel cortex neurons that integrate the incoming signal.Altogether, the authors collected a neural dataset consisting of first-order recordings, barrel cortical cluster recordings, and simultaneous paired recordings from both sites, all in response to playback of the library of texture-related vibrations. This approach afforded the opportunity to directly compare encoding of information at both levels in the sensory pathway. These recordings show, the authors argue, that temporally distinct firing patterns in the trigeminal ganglion (the cell bodies of the first-order neurons) and cortex captured the kinetic features of the texture-induced vibrations. Each texture's “kinetic signature” is encoded by a characteristic, temporally precise firing pattern associated with whisker movement. Compared to free whisking, coarse sandpaper produced irregular bursts of high and low velocity, and both first-order and cortical neurons fired far more impulses for coarse sandpaper than for free whisks. The authors then used stimuli consisting of random velocities to uncover the “tuning curves” of neurons, and simulations showed that these neuronal tuning curves could perfectly predict the real neural responses to textures.Noting the close match between the simulated and natural responses, Diamond and colleagues conclude that the texture-induced firing patterns observed in the first-order and cortical neurons suggest that neurons selectively encode elemental kinetic features—namely, high velocity—to tell rats what they're whisking. This selectivity allows even a single whisker to transmit significant bits of texture-specific information to the brain. Interesting as rat whisking may be, these findings have relevance beyond the world of whiskered beings, shedding light on the underlying neural processes that translate touch into recognition.


RNA interference (RNAi) can potently reduce target gene expression in mammalian cells and is in wide use for loss-of-function studies. Several recent reports have demonstrated that short double-stranded RNAs (dsRNAs), used to mediate RNAi, can also induce an interferon-based response resulting in changes in the expression of many interferon-responsive genes. Off-target gene silencing has also been described, bringing into question the validity of certain RNAi-based approaches for studying gene function. We have targeted the plasminogen activator inhibitor-2 (PAI-2 or SERPINB2) mRNA using lentiviral vectors for delivery of U6 promoter-driven PAI-2-targeted short hairpin RNA (shRNA) expression. PAI-2 is reported to have anti-apoptotic activity, thus reduction of endogenous expression may be expected to make cells more sensitive to programmed cell death.As expected, we encountered a cytotoxic phenotype when targeting the PAI-2 mRNA with vector-derived shRNA. However, this predicted phenotype was a potent non-specific effect of shRNA expression, as functional overexpression of the target protein failed to rescue the phenotype. By decreasing the shRNA length or modifying its sequence we maintained PAI-2 silencing and reduced, but did not eliminate, cytotoxicity. ShRNA of 21 complementary nucleotides (21 mers) or more increased expression of the oligoadenylate synthase-1 (OAS1) interferon-responsive gene. 19 mer shRNA had no effect on OAS1 expression but long-term selective pressure on cell growth was observed. By lowering lentiviral vector titre we were able to reduce both expression of shRNA and induction of OAS1, without a major impact on the efficacy of gene silencing.Our data demonstrate a rapid cytotoxic effect of shRNAs expressed in human tumor cell lines. There appears to be a cut-off of 21 complementary nucleotides below which there is no interferon response while target gene silencing is maintained. Cytotoxicity or OAS1 induction could be reduced by changing shRNA sequence or vector titre, but stable gene silencing could not be maintained in extended cell culture despite persistent marker gene expression from the RNAi-inducing transgene cassette. These results underscore the necessity of careful controls for immediate and long-term RNAi use in mammalian cell systems. Gene silencing is a powerful tool with which to study protein function. Gene inactivations in mice have revolutionised the way we study both basic biology and a plethora of disease types ,2. Gene Maintaining RNAi-inducing dsRNA below 30 nucleotides in length was thought to avoid activation of the interferon-induced anti-viral response. PKR is a key anti-viral regulator and its expression can be induced by the interferon response . PKR is In this work we used a simple approach for gene silencing in human tumor cell lines, using lentiviral vectors for stable delivery of shRNAs. We aimed to study the effects of targeting the plasminogen activator inhibitor-2 (PAI-2 or SERPINB2) mRNA on cell survival in the presence of pro-apoptotic stimuli. In addition to its inhibitory activity on the urokinase plasminogen activator, PAI-2 is thought to have anti-apoptotic properties ,25. Its Using lentiviral vectors for delivery of RNAi-inducing expression cassettes we achieved potent PAI-2 gene silencing, accompanied by a rapid cytotoxic effect. The degree of cytotoxicity was proportional to shRNA length and induction of an interferon response gene could be detected when shRNA of 21 complementary base pairs or more was expressed. The phenotype was not target gene specific, as PAI-2 overexpression failed to rescue cytotoxicity and control hairpins were also cytotoxic. Using lower vector titre, reduced shRNA expression and interferon response induction was measured without compromising gene silencing. Using a 19 complementary base pair shRNA expression vector, which reduced PAI-2 expression and induced no initial cytotoxicity or interferon response, transduced cell marker gene expression was maintained but gene silencing lost in long-term cell culture. Our results highlight the need for careful controls to monitor specificity and maintenance of gene silencing when using RNAi for stable loss-of-function studies in mammalian cells.Lentiviral vectors were generated which deliver an expression cassette for human U6 promoter-driven expression of short hairpin RNA (shRNA), with exact homology to the human PAI-2 mRNA. The vector expression cassette also bears the enhanced green fluorescent protein (GFP) gene under the control of the EF-1α promoter, and an internal ribosome entry site (IRES) sequence  also reduced PAI-2 mRNA and protein levels. Equal sample loading for immunoblots was confirmed by Ponceau S staining of nitrocellulose membranes (data not shown) and the presence of equal amounts of a PAI-2 monoclonal antibody-reactive non-specific band in each blot . In parallel, we transduced IS-1 cells with the sh119 vector or a GFP control vector. We achieved high percentage transduction rates which were monitored for over two weeks Figure . GFP posHere we report the use of lentiviral vectors for the delivery of expression cassettes designed for RNAi-induced stable knock-down of gene expression. We undertook this approach because of the promise of RNAi to easily create cell lines that are specifically deficient in one protein component. By using lentiviral vectors for shRNA expression, high transduction efficiencies can be achieved, avoiding effects due to clonal selection of phenotypically different cells.We successfully targeted the PAI-2 mRNA with the aim of studying the effects of reducing PAI-2 activity on cell sensitivity to apoptosis-inducing stimuli. PAI-2 activity has previously been implicated in protection from apoptosis . The cytWe were able to detect increased expression of an interferon response gene, OAS1, in cells transduced with all hairpins of 21 or more base-paired nucleotides. A 21 mer hairpin induced the most potent OAS1 induction. These results, and those of others , suggestOverexpression of functional PAI-2 did not rescue the cytotoxic effects or OAS1 induction observed in cells transduced with a series of vectors for expression of different length shRNAs. This result, the cytotoxicity associated with scrambled sequence hairpin-encoding constructs, and the same selective pressure seen on the growth of transduced HeLa cells, which do not express detectable PAI-2 mRNA or protein (Table IS-1 cell transduction, using one PAI-2 targeting vector (sh119), initially appeared stable, compared to control-transduced cells. PAI-2 protein levels were clearly reduced 10 days after transduction and selective pressure on cell growth appeared to be minimal, as the percentage of sh119 GFP positive cells was apparently stable at about 80 %, 17 days after transduction. However, after cell sorting of GFP positive cells and prolonged cell culture of over one month after transduction, the PAI-2 antigen measured in sh119s (s for selected) cells was restored to control-transduced cell levels. Despite maintenance of transduction marker expression, gene silencing was absent. The GFP expression levels in selected sh119s cells was reduced after one month of growth, compared to cells monitored three days after transduction. It is possible that we selected cells with a greatly reduced, non-cytotoxic shRNA expression level, as we have detected a reduced number of integrated vector copies. The negative selective effect on cells transduced with the sh119 vector was not due to the suppression of PAI-2 expression, as PAI-2 overexpressing cells showed the same negative selection.In all experiments in which a selective pressure on growth was apparent on shRNA-expressing cells, reduced percentage GFP positive cells and reduced GFP expression in transduced cells was measured over time. We hypothesised that very high expression levels of the various shRNAs is cytotoxic. This could occur via high numbers of transcriptionally active vector integration events or integration at transcriptionally active chromosomal regions. Both might be controlled using a tightly regulatable expression system, which has been described , but mayOur data demonstrate the importance of appropriate controls for using RNAi, as proposed in a recent editorial . These iIn the light of our own data and other recent reports -22, soluOur study demonstrates vector-derived RNAi in tumor cell lines and points towards the necessity of careful, but clearly feasible, controls when using RNAi for stable gene suppression in short- and long-term experiments.Isreco-1 (IS-1) cells were a gift from Dr. B. Sordat (ISREC, Lausanne). 293T cells were a gift from Dr. D. Trono . HeLa cells were purchased from the European collection of cell cultures, ECACC number: 93021013. All cells were maintained in DMEM supplemented with 10 % Fetal Bovine Serum and 10 mM HEPES pH 7.4 (IS-1 medium) (purchased from Invitrogen).PEF-1α-iresGFP region of the plasmid. A schematic of the gene transfer cassette is given in Figure Gene transfer plasmids, for RNA interference using lentiviral vectors, were constructed using the backbone of ploxEWiresGFP, a gift from Dr. P. Salmon, Geneva University Medical Centre. The human U6 gene was amplified by PCR using HeLa cell genomic DNA as template and the oligonucleotides U6-ClaI-F 5' GATC ATCGATAAGGTCGGGCAGGAAGAGGGCCTATTTCCC 3' and U6-ClaI-R 5' GATCATCGATTGGTAAACCGTGCACCGGCGATAAACG 3'. The 483 base pair PCR product was digested with ClaI, inserted into the ClaI site of pTRE2 hyg (Clontech), and its sequence verified by DNA sequencing. The U6 promoter and gene sequence corresponded to nucleotides 65 to 527 of Genbank accession number M14486. This plasmid was used as template for PCR reactions to amplify U6 promoter-driven expression cassettes. Each PCR product included the U6 promoter with shRNA-encoding sequences beginning at the U6 +1 site, and a run of 6 or 7 thymidine bases for an RNA polymerase III transcription termination signal. Each PCR introduced flanking ClaI restriction sites. PCR products were directly cloned into the pGEM Teasy (Promega) plasmid and sequenced. ClaI fragments of positive clones were excised and ligated into the unique ClaI site of ploxEWiresGFP. The U6 promoter cassettes in the resulting lentiviral vector plasmids were verified by DNA sequencing. Each construct used for vector production had the same orientation of the U6 expression cassette with respect to the 4 or 1 × 105 cells/well. Upon addition of vectors, plates were centrifuged for one hour at 1000 g in the presence of 8 μg/ml polybrene (hexadimethrine bromide, Sigma). After 24 hours, target cells were washed twice with PBS and cultured in IS-1 medium until analysis. Where indicated, viral titre was determined by transducing cells with 10 μl of lentiviral vector conditioned medium from 293T cell producer cells and measuring the % of GFP positive cells by flow cytometry. We routinely achieve 106 effective transducing units per ml of producer cell conditioned medium, which results in a typical multiplicity of transduction of 10. Comparisons between cell populations transduced with different vectors is made by flow cytometry analysis of GFP positive cells.Lentiviral vectors were produced by three plasmid co-transfection of 293T cells, essentially as described previously . VectorsAnti-human PAI-2 monoclonal antibody 3750 was purchased from American Diagnostica. PE-labelled goat anti-mouse antibodies were purchased from Pharmingen. HRP-conjugated goat anti-mouse antibodies were purchased from Bio-Rad.Flow cytometry was performed using Becton Dickinson FACScan, FACStrack or FACScalibur instruments at the Geneva University Medical Centre flow cytometry facility. GFP was detected in cells detached and resuspended in FACS buffer comprising 1 % BSA in PBS supplemented with 0.05 % sodium azide. For the detection of intracellular PAI-2, a method adopted from Dr. M. Ranson  was used. Cells were detached and fixed in 0.25 % paraformaldehyde (PAF)/PBS for one hour on ice. Cells were permeabilised in 0.1 % saponin/PBS for 30 minutes at room temperature. Fixed, permeabilised cells were incubated for 30 minutes in PBS/0.5 % BSA/0.1 % saponin containing 2 μg/ml 3750 anti-PAI-2 monoclonal antibody, washed twice in PBS/0.1 % saponin and incubated for 30 minutes in 0.1 % saponin/0.5 % BSA/PBS/goat anti-mouse-PE antibodies. Finally, cells were washed twice in 0.1% saponin/PBS, twice in PBS and resuspended in 2.5 % PAF for flow cytometric analysis.Sorting of live GFP positive cells was performed using a FACStar+ instrument (Becton Dickinson).2, 5 mM DTT and 1 mM PMSF for one hour on ice. Lysates were centrifuged at 16000 g for 5–10 minutes to remove nuclei and precipitates. Supernatant protein concentrations were measured using the Bio-Rad protein assay with BSA in lysis buffer as a standard. Cell lysates were separated by reducing SDS-PAGE and transferred to nitrocellulose membranes. Equal total protein lysate was used for each blot, between 2.5 and 10 μg depending on the assay. Membranes were blocked for 1 hour at room temperature in TBS-0.1 % Tween 20/5 % non-fat milk, and probed using antibodies in TBS-0.1 % Tween 20/5 % non-fat milk. The 3750 anti-PAI-2 antibody was used at a concentration of 1 μg/ml.Cells in suspension were lysed in 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 0.5 % NP-40, 3 mM MgClPhase contrast microscopy was performed using a Zeiss Axiovert 100 M instrument on live cells. Images were collected using a Hamamatsu CCD camera (ORCA-100).To measure PAI-2 functional activity, IS-1 and IS-1 PAI-2 cells were lysed in 1 % NP-40, 150 mM NaCl and 50 mM Tris pH 8.0. Thereafter, 6 μg of cleared total cell lysate was incubated with and without 10 U of low molecular weight urokinase (u-PA). 3 μg total cell lysate samples were then subjected to reducing SDS-PAGE and immunoblotting as for other PAI-2 immunoblots.® reagent (Invitrogen). cDNA was generated using ImpromII® reverse transcriptase (Promega) and random hexamers, according to the manufacturers instructions, typically using 1 μg of total RNA per reaction. Quantitative PCR was performed using an Applied Biosystems Prism 7000 instrument using Applied Biosystems SYBR® green master mix reagent and oligonucleotide pairs to detect hypoxanthine phosphoribosyl transferase (HPRT), PAI-2 and oligoadenylate synthase-1 (OAS1) cDNA. 5' to 3' primer sequences were as follows: HPRT forward TATTGTAATGACCAGTCAACAG, HPRT reverse GGTCCTTTTCACCA GCAAG, PAI-2 forward GGGTCAAGACTCAAACCAAAG, PAI-2 reverse CCTTTGAAGTAGACAGCATTC, OAS1 forward AGGTGGTAAAGGGT GGCTCC and OAS1 reverse ACAACCAGGTCAGCGTCAGAT. Data were analysed using Applied Biosystems Prism software and the ΔCT method. Briefly, target gene expression was normalised to the HPRT endogenous reference gene for each sample. The difference between mean threshold PCR cycle values for target and control genes gave the ΔCT value. This was then calibrated to the control sample in each experiment to give the ΔΔCT value, where the control had a ΔΔCT value of 0. The fold target gene expression, compared to the calibrator value, is given by the formula 2-ΔΔCT. Error bars represent the standard deviation of each target gene value, after evaluating the expression 2-ΔΔCT+s and 2-ΔΔCT-s, where s is the standard deviation of the ΔΔCT value. All reactions were performed in duplicates or triplicates.Total RNA was isolated from cells using RNA preparation kits from Qiagen or TRIZOLmirVana™ Probe Construction and miRNA Detection kits from Ambion (Austin), according to the manufacturers instructions. These kits employ in vitro transcription for radiolabelled probe generation and an RNase protection protocol for detection of small RNA expression, respectively. Briefly, radiolabelled RNA probes incorporating α-32P-UTP (Amersham) were constructed using T7 polymerase-driven transcription templates. Templates were designed to generate RNAs which hybridize to the endogenous Mir-16 miRNA, as a control, or the first 19 nucleotides of the sh325 shRNA (see table Expression of shRNAs was detected using the RF carried out all of the experiments in this study and contributed to its conception, design and description. EKOK conceived the study and participated in its design and description. Both authors approved the final manuscript.


Conservative treatment remains the standard of care for treating mild to moderate carpal tunnel syndrome despite a small number of well-controlled studies and limited objective evidence to support current treatment options. There is an increasing interest in the usefulness of wrist magnetic resonance imaging could play in predicting who will benefit for various treatments.Two hundred patients with mild to moderate symptoms will be recruited over 3 1/2 years from neurological surgery, primary care, electrodiagnostic clinics. We will exclude patients with clinical or electrodiagnostic evidence of denervation or thenar muscle atrophy.We will randomly assign patients to either a well-defined conservative care protocol or surgery. The conservative care treatment will include visits with a hand therapist, exercises, a self-care booklet, work modification/ activity restriction, B6 therapy, ultrasound and possible steroid injections. The surgical care would be left up to the surgeon (endoscopic vs. open) with usual and customary follow-up. All patients will receive a wrist MRI at baseline.Patients will be contacted at 3, 6, 9 and 12 months after randomization to complete the Carpal Tunnel Syndrome Assessment Questionnaire (CTSAQ). In addition, we will compare disability (activity and work days lost) and general well being as measured by the SF-36 version II. We will control for demographics and use psychological measures  as well as EDS and MRI predictors of outcomes.We have designed a randomized controlled trial which will assess the effectiveness of surgery for patients with mild to moderate carpal tunnel syndrome. An important secondary goal is to study the ability of MRI to predict patient outcomes. Carpal tunnel syndrome (CTS) is the most common peripheral nerve entrapment syndrome, with an annual incidence of 50–150 cases/100,000. It is an important cause of workplace morbidity ). TypicaA Cochrane literature review  for randAlthough there is generally a lack of rigorous scientific support for non-surgical treatments for CTS , there iAlthough not commonly used, Ebenbichler  found thDiagnostic criteria for grading CTS severity as "mild" and "moderate" are not well established. Electrodiagnostic studies (EDS) do not correlate well with clinical severity and have not been shown to accurately predict outcomes for patients with mild to moderate CTS -42.We designed a randomized clinical trial to compare surgical release to non-surgical treatment for patients with mild to moderate CTS. We will examine the association between outcome, as measured by symptoms and functional status, and baseline variables such as symptoms, function, occupational risk factors, EDS measures, demographics, signs and symptoms, and prior treatments. Our primary endpoint is at 12 months.In addition, our study will evaluate the ability of high resolution magnetic resonance imaging (MRI) of the wrist to predict who will benefit from surgical release. MRI has the potential to offer new insight into the diagnosis and management of patients with hand and wrist neurological symptoms. Unlike electrodiagnostic studies, MRI directly visualizes the median nerve and can detect abnormalities of both configuration (nerve compression) as well as signal  -46. EithFinally, we will gather utilization data for each arm of the study to test the hypothesis that the incremental cost-effectiveness ratio of surgery falls below conventional cost-effectiveness thresholds.We have designed a multi-center, randomized trial comparing surgical release to a multi-component, non-surgical therapy. The study protocol was approved by the University of Washington Human Subjects Division and all participants provide written informed consent.The two major goals of this study are to determine: 1) if surgery compared with conservative therapy benefits patients with mild or moderate carpal tunnel syndrome, and, 2) if high resolution magnetic resonance imaging (MRI) of the median nerve can identify patients for whom early surgery might be more efficacious than conservative therapy.Patients with mild to moderate CTS are recruited from six participating sites in the Puget Sound region of Washington State: the University of Washington (UW) affiliated practice sites , Virginia Mason Medical Center (Seattle), the Seattle Hand Surgery , Proliance Surgeons , and Management Services Organization of Washington in Tacoma. We recruit patients within the primary care clinics as well as the specialty referral clinics that treat patients with carpal tunnel syndrome. These clinics include neurological surgery, neurology, orthopedic surgery, and physical medicine and rehabilitation. Additionally, we identify potential subjects in the electrophysiology laboratories at each of our participating sites.Prior to study participation, patients are required to have a physician confirmation of suspected CTS and to obtain an EDS with or without an electromyeogram (EMG). We define mild to moderate carpal tunnel syndrome based on electrodiagnostic studies (EDS) and clinical findings. Specifically, patients are eligible for the study if EDS demonstrates any one of the following: (1) wrist median motor nerve conduction latency greater than or equal to > = 4.4 milliseconds (ms), (2) a 10 cm (thumb to wrist) median to radial sensory nerve ratio difference of > 0.5 ms, (3) an 8 cm mid-palm median to ulnar sensory nerve difference > 0.3 ms, (4) a 14 cm (digit four to wrist) median to ulnar difference of > 0.4 ms, or (5) a combined sensory index ,48 ≥ 1.0Other study inclusion criteria are: age 18 years or older, no previous hand or wrist surgery on the study hand, no previous carpal tunnel release on the contralateral hand in the previous 6 months, symptoms in at least two digits in a median motor nerve pattern, able and willing to answer research questionnaires in English, and classification using the hand diagrams as at least "possible CTS",50. ExclPatients with evidence of severe CTS on EDS, EMG, or clinical findings are excluded from the study. Severe CTS is defined as a median motor amplitude of ≤ 3.8 mV, EMG evidence of denervation, or thenar atrophy.Study participants are required to have not improved after a minimum of two weeks of standard non-surgical treatment  and be willing to schedule surgery within one week if randomized to the surgical arm of the trial. Lack of improvement with conservative care is defined by any of the following: (1) < 0.75 point improvement  on the CFor patients with bilateral CTS, we designate a "study hand" based on the following priorities: 1) most severe according to patient reporting, 2) most severe based on electrodiagnostic reports, and 3) the dominant hand.We use a 50/50 computer-generated block randomization, stratified by enrollment site. The block size is randomly varied between 4 and 12 to reduce the potential for clinicians or research staff to predict treatment allocation. The treatment assignment is centrally administered and concealed in consecutively numbered opaque envelopes.Patients are randomized to receive either a surgical release of the median nerve or a package of multiple, non-surgical treatments tailored for individual patients. For those randomized to surgery, we attempt to schedule the surgery within two weeks of allocation or as soon as possible. Surgery is performed by a board-certified neurological surgeon or orthopedic surgeon. Either open or endoscopic surgery can be performed, depending on the surgeon's preference. Surgical patients receive clinical follow-up just as they would if not in the study. Typically, this includes a follow-up visit within one week for wound and suture management and several post-operative follow-up visits with a hand therapist to perform median nerve and tendon gliding exercises.We created expert focus groups and reviewed the relevant body of literature to develop the non-surgical treatment arm of study. Our focus group included experts in orthopedics, neurosurgery, physical medicine and rehabilitation, hand therapy, biostatistics, health services, behavioral science, and health economics.The non-surgical treatment is directed by a hand, occupational, or physical therapist and includes a minimum of three visits, separated by 6 weeks each. At the first visit, each patient receives an educational booklet, a prescription for NSAIDs (if they have not previously tried them), and specific exercises for their hand. The educational booklet details the hand exercises, describes the causes of carpal tunnel syndrome, and lists resources for obtaining additional information. Work and activity modifications are prescribed at the discretion of the hand therapist and additional hand therapy visits are prescribed as needed. Patients allocated to the non-surgical arm who have already undergone extensive physical therapy can opt to get ultrasound treatment immediately (see below).2, in pulsed mode 1:4, and 15-minutes each. Three months after randomization, patients return for the final non-surgical evaluation. If improved, they are instructed in a self-care maintenance program and advised to return to their provider if their symptoms worsen. If symptoms are not improved, patients are referred to a study surgeon for evaluation for crossover to receive surgery or corticosteroid injection.Patients in the non-surgical arm return six weeks after the randomization for a study visit. If a patient reports improvement, no changes to their therapy are made. For patients who do not improve, we offer oral vitamin B-6 supplements (100 mg per day) and ultrasound in addition to the existing therapy regimen. The ultrasound regimen used in this study consists of up to 12 sessions per week (for 6 weeks) of focused ultrasound at 1 Mhz, 1.0 W/sqr cmOutcomes measures are collected for patients in person at baseline and via telephone interviews at 3, 6, 9, and 12 months after randomization. Although it is impossible to blind providers and patients to the treatment the assignment, the telephone interviewer is blinded to study participants' treatment assignment.Carpal Tunnel Syndrome Assessment Questionnaire (CTSAQ) is the primary outcome measure. The CTSAQ is a self-report CTS functional status and symptom severity questionnaire with established validity, reliability and responsiveness [The functional status scale of the siveness ,53. The SF-36 Health Survey version 2  has been used to assess general health status in samples of patients with a variety of diseases, including CTS [The ding CTS . It consSymptom Check List SCL-90 12-item Somatization and 13-item Depression scales [Study participants also complete the n scales -57. PartPain Catastrophizing Scale is used as both a predictor and a secondary outcome. A substantial volume of research had consistently found substantial associations between pain-related catastrophizing and pain-related disability [The 13-item sability -62. We aIn addition to the outcome measures, we obtain information on several other variables at baseline. This includes information on demographics, occupational risk factors, medical co-morbidity, hand pain history, work status, and litigation/compensation issues. The first three items of the Alcohol Use Disorders Identification Test (AUDIT) are administered to assess the frequency of alcohol use, amount of alcohol consumption, and binge drinking -65.resource use questionnaire (RUQ) at 3, 6, 9, and 12 months post-enrollment.We are also collecting information on the costs of care following enrollment. We aim to estimate the cost to health purchasers and society based on billing and medical records, and a detailed We attempted to either measure or abstract from the medical records information that is generally included in the hand physical examination at the time of enrollment. The hand physical included measurements of patient height, weight, dominant hand, 2-point discrimination (measured at digits #2 and #5), wrist width and thickness (for MRI correlation), 2-point pinch strength (average of three efforts), Semmes-Weinstein monofilament test (from 1.65 to 6.65), Tinel sign, Phalen sign (held greater than 1 minute), and flick test. Not every site routinely completed all sensibility and/or strength tests.Mackinnon-Dellon disk-criminators™ are used to test the static two-point discrimination at the second and fifth digits as a measure of sensibility to correlate to the MRI findings. We tested each digit a minimum of three times and until the tester was confident that a clear endpoint was reached. The test was performed on both hands even in patients with unilateral disease. The disk prongs are held perpendicular to the long axis of the finger. The prongs are placed upon the skin only with sufficient pressure for a patient to determine that he is being stimulated. The three-point pinch strength is tested bilaterally, with the non-study hand being first. We recorded the mean of three serial efforts of the key pinch measurement  using a B&L Engineered (B&L Engineering 3002 Dow Ave, Suite 416, Tuscin CA 92780) 30 lbs. pinch gauge calibrated to +/- 1% accuracy.Tinel sign is positive if tapping over the median nerve at the distal wrist crease for approximately 10 seconds reproduces the pain, numbness and or tingling in the patients hand or wrist. Phalen sign is positive if while holding both wrists flexed at 90 degrees with the dorsum of the hands in opposition to each other for a minimum of 1 minute produces dysesthesias and pain. A flick test is positive if in their response to being asked "What do you actually do with your hand(s) when your symptoms are at their worst?" a patients gestures by flicking the wrist(s) -74.All subjects who enter the study undergo wrist MRI except for subjects who have MRI contraindications , are claustrophobic, who exceed the weight limit of the MRI table, who have scheduling difficulties, or refuse the MRI. We use phased-array surface coils to obtain high resolution images of the carpal tunnel and median nerve. The imaging protocol consists of a fast T1-weighted gradient echo coronal localizer, an axial T1-weighted series and a short-tau inversion recovery (STIR) T2-weighted series. This protocol has established reliability and, because of the short imaging times, patients usually are in the MR scanner for only 15–20 minutes. Imaging parameters for the axial series are listed in table The MRI is interpreted by a neuroradiologist without knowledge of the demographic data, clinical findings, or electrodiagnostic study findings. The key imaging variables are the degree and length of signal abnormality within the median nerve, flattening or swelling of the median nerve, although other qualitative and quantitative measurements will be made as well table .Patients receive a follow-up interview via telephone at 3, 6, 9 and 12 months. Hand specific symptom and functional disability scores, along with general health and psychological instruments are collected. For each interview, we attempt to contact participants a minimum of three times, varying the day and time of the call. In instances where we are unable to reach a person by phone, the survey instruments are mailed along with a postage paid return envelope. Some patients also consent to allow us to contact them for follow-up using e-mail.All data are collected onto hardcopies and entered into an MS Access database using a web-based data entry system that requires double entry of data to reduce errors in transcribing. Error reporting to identify out of range answers, inconsistent replies and compliance monitoring is routinely performed.We developed stopping rules using CTSAQ symptom scores, disability reporting, adverse event rates and rates of "red flag" answers to a question asking about thoughts of suicide on the SCL-90. A safety officer monitors these variables for group differences, and also monitors response variables, missing data, and protocol compliance.A formal evaluation of efficacy will be conducted after 100 patients have been randomized to test for group differences in the CTSAQ symptom severity score. We adopted O'Brien-Fleming boundaries for discontinuation, and therefore maintain an overall type I error rate of 5%. Additional stopping criteria include a suicidal ideation rate difference of 10%, a difference in the change from baseline rate of functional disability  of 20%, or a difference in the number of days lost from work of 20%. The nature and severity of the adverse events will also be considered individually by the safety officer. Finally, individual item response rates on all answered questionnaires with less than 20% being incomplete, failure to collect greater than 75% at three-month follow-up, or enrollment rates below the expected rate by more than 25% provided grounds for the safety officer to recommend changes or stop the study.In the primary analysis the CTSAQ functional score at 12 months will be compared between the surgical and non-surgical treatment arms, using conventional t-tests and ANCOVA techniques to adjust for baseline values. The analysis will be conducted on an intention-to-treat basis.Secondary analyses will include a comparison of secondary outcome measures  for the two treatment arms. Adjusted analyses of CTSAQ functional status and secondary outcomes at 1 year will be conducted using linear regression methods. To characterize the time evolution in the primary and secondary outcomes we will use linear mixed models  to analyze the repeated measures obtained at all follow-up visits. Finally, we will use exploratory regression methods to determine if baseline disability, psychological factors, MRI variables, and electrodiagnostic measures correlate with clinical outcomes.We will use aggregate results of patient outcomes on the CTSAQ and SF-36 scores to perform a descriptive analysis, uncontrolled for other baseline factors. We will explore factors that predict improvement in CTSAQ  and general health scores as measured by the SF-36. Specific analysis and tests used will depend on the distribution of the tested values. We will also test the independent associations of the various mental health domains, the work-related risk factors, and physical findings to changes in the CTSAQ. Finally, we will use linear regression to test relevant associations while controlling for baseline demographics, co-morbidity and other important variables to identify factors that may predict improved outcomes at 1 year.As important sub-analyses we will also determine the reliability of quantitative MR median nerve measurements, determine the correlation of symptoms and function in patients with these quantitative MR measurements as well as with electrodiagnostic studies (EDS), and construct a cost-effectiveness model to test the hypothesis that MRI is an efficient method for selecting patients with mild or moderate CTS who are likely to benefit from surgery.The prospective cohort study by Katz et al. provided data on outcome differences between surgical and non-surgical patients . They alIf we consider the difference in means observed for the pilot data (m1-m0 = 0.554) then 48+48 = 96 subjects are adequate for 80% power and a total of 64+64 = 128 are required for 90% power (refer to following table). However, since we are recruiting patients with less severe disease, we would expect smaller improvements in the scores from baseline to follow-up and hence, smaller effect sizes. Thus, it is reasonable to power the study to detect a difference in means of 0.4 rather than 0.5. Since our primary analysis will use the more efficient ANCOVA, these sample size estimates based on observed change scores (post-pre) are slightly conservative. This should allow for a small percentage of subjects lost to follow-up. Similar power calculations were conducted to assess the power to detect an impact on CTSAQ symptom scores. The pilot data suggest a 0.67 point greater reduction in symptoms for the surgical patients, and variance estimates lead to sample sizes of 30+30 = 60 total patients, and 40+40 = 80 total patients to detect 0.67 point difference (table An RCT offers the best chance of answering, in an unbiased fashion, the relative efficacies of surgery compared with conservative therapy for patients with mild to moderate CTS. Our study is designed to test two main hypotheses: 1) that patients with mild or moderate CTS benefit from surgery more than conservative therapy, and; 2) that high-resolution wrist MRI accurately identifies which patients with mild to moderate symptoms are more likely to benefit from surgery. We expect our findings to be more generalizable to the primary care clinicians and include milder cases of CTS than Gerritsens' study. No previous study has sufficiently considered conservative treatment options of vitamin B6, ultrasound, anti-inflammatory drugs, and hand exercises in combination versus surgical treatment. Furthermore, our study offers the unique use of wrist MRI data to establish the diagnostic and predictive understanding of carpal tunnel syndrome.The pre-publication history for this paper can be accessed here: