Species phylogenies are not estimated directly, but rather through phylogenetic analyses of different gene datasets. However, true gene trees can differ from the true species tree (and hence from one another) due to biological processes such as horizontal gene transfer, incomplete lineage sorting, and gene duplication and loss, so that no single gene tree is a reliable estimate of the species tree. Several methods have been developed to estimate species trees from estimated gene trees, differing according to the specific algorithmic technique used and the biological model used to explain differences between species and gene trees. Relatively little is known about the relative performance of these methods.We report on a study evaluating several different methods for estimating species trees from sequence datasets, simulating sequence evolution under a complex model including indels (insertions and deletions), substitutions, and incomplete lineage sorting. The most important finding of our study is that some fast and simple methods are nearly as accurate as the most accurate methods, which employ sophisticated statistical methods and are computationally quite intensive. We also observe that methods that explicitly consider errors in the estimated gene trees produce more accurate trees than methods that assume the estimated gene trees are correct.Our study shows that highly accurate estimations of species trees are achievable, even when gene trees differ from each other and from the species tree, and that these estimations can be obtained using fairly simple and computationally tractable methods. The most frequently used approaches for estimating species phylogenies compute alignments on each gene, concatenate these alignments into one super-alignment, and then estimate a tree from the super-alignment. However, these “combined analysis” methods do not have good statistical properties because different regions of the genome can have different evolutionary histories. More generally, it is increasingly clear that gene trees can be different from species trees due to a number of biological processes. One of the dominant causes for this incongruence between gene and species trees is ng (ILS) , a populng (ILS)  deeply ing (ILS) , but ILSng (ILS) .Several methods have been developed that estimate species trees from estimated gene trees under the ILS model. One main approach is to find the species tree that implies the fewest number of deep coalescent events, a computational problem called “MDC” , a probAlternative techniques that are based upon statistical models for ILS include GLASS , STEM 1, *BEAST Other new methods have been developed and studied for estimating species trees from incongruent gene trees, among them the method by Yu et al.  . The twIn this paper we report on a simulation study using ROSE  in whichOur simulation protocol is substantially more complex  than most prior studies. Except for the 17-taxon datasets  tree. For the RAxML analysis, we use RAxML-75%, the 75%-bootstrap tree . Finally, FastTree outputs support values for every branch in the trees it produces; thus, we can also use FT-75%, the tree produced by contracting all branches with support less than 75% in the FastTree output. Note that the MP-maj, RAxML-75%, and FT-75% trees are not likely to be fully resolved; therefore, these point estimations of gene trees can only be used with species tree estimation methods that permit unresolved gene tree inputs.Although MrBayes and RAxML with rapid bootstrapping produce a sample of different trees, we also use them to produce point estimates of the gene tree for those species tree estimation methods that require single trees for each gene. In the case of the MrBayes (MB) analysis, we output the majority consensus tree (MB-maj) , and the maximum a We explore seven methods for estimating species trees from estimated gene trees or gene tree distributions. Four of these are based upon ILS, and include Glass (from Phylonet v. 2.3), Phylonet-MDC v. 2.3 , iGTP-MDC v. 1.1, and BUCKy v. 1.4.0. The other three methods are Greedy, iGTP-Dup v. 1.1 and iGTP-Duploss v. 1.1. All of these methods, except BUCKy, take as input a single tree for each gene. In contrast, BUCKy operates in two steps: first it uses a technique (the default is MrBayes) to produce a distribution of estimated gene trees for each gene, and then it uses these distributions to infer the species tree. In fact, though, BUCKy produces two different trees–the “concordance” tree (which we refer to as “BUCKy-con”) and the population tree (which we refer to as “BUCKy-pop”) . The keyWe describe methods for species tree estimation by indicating the technique used to estimate the gene trees (or their distributions) and the technique used to compute the species tree from the gene trees. For example, Greedy(FT-75%) refers to the method that computes the FT-75% tree on every gene sequence alignment, and then combines these  trees using Greedy. Similarly, BUCKy-pop(MB-spa) refers to BUCKy-pop run on a sparse sample of the MrBayes distributions for each gene, BUCKy-con(MB-full) refers to BUCKy-con run on the full sample of the MrBayes distributions, and Phylonet(MB-map) refers to Phylonet run on the MAP trees from the MrBayes distributions. Not all species trees we compute are fully resolved; hence, the Robinson-Foulds distance , is inappropriate for evaluating accuracy, as it is biased in favor of unresolved trees . TherefoWe began by exploring the computational performance of the methods we studied on 12 100-taxon datasets, half with 25 genes and half with 50 genes. These studies showed that these different pipelines  vary substantially in terms of computational effort.The most computationally intensive methods we explored use MrBayes or RAxML to estimate distributions on gene trees, but MrBayes analyses are particularly expensive. Our analyses (data not shown) indicated that enforcing a requirement that all MrBayes analyses reach convergence was not achieved on any dataset we examined, even after at least a week for each alignment . Therefore, each 17-taxon 32-gene dataset we examined could easily require a year  of analysis in order for each MrBayes analysis to reach convergence.Restricting the MrBayes runs so that each gene is analyzed in at most a day greatly reduces the running time, and allows the first step (computing gene tree distributions for each gene) to complete in as many days as there are genes . These analyses will still be computationally intensive, but not as intensive as waiting for all MrBayes analyses to converge. However, early terminations of MrBayes analyses are likely to make the analyses “invalid”, and potentially therefore reduce the accuracy of the resultant estimated species phylogeny. RAxML with bootstrapping can also be computationally intensive, so that analyses of single 100-taxon datasets with 1000 bootstrap replicates took on average almost 10 hours, making an analysis of 50 genes require 20 days just to obtain the initial set of tree distributions. Limiting RAxML to only 100 bootstrap replicates greatly reduces the running time to 1.5 hours on average per sequence alignment with 100 taxa, thus making it possible to obtain RAxML distributions on 50 genes in about 3 days.The only computationally intensive method for estimating species trees from gene trees is BUCKy . BUCKy’s computational challenges are very much impacted by the total number of taxa and total number of trees in its input. We compared BUCKy analyses based upon sparse samplings (at most 2000 trees per gene) from the MrBayes distributions as input to BUCKy, in comparison to larger numbers of trees  on six 100-taxon datasets with 25 and 50 genes. This modification resulted a tremendous reduction in the computational effort, especially for the 50 gene datasets. The BUCKy analyses of these 50 gene datasets with this sparse sampling of MrBayes trees completed on average in about one day and had peak memory usage averaging 33 GB, while BUCKy analyses using the “full” MrBayes runs averaged one week per analysis and had average peak memory usage of 150 GB. Several of the BUCKy analyses of the full MrBayes sample used more than 200 GB of main memory, but no analysis of the sparse sample used more than 36 GB. The memory requirement for these full MrBayes analyses is truly prohibitive, since most investigators will not have computational resources of this scale available, but the sparse MrBayes analyses are computationally much less troublesome.Except where indicated, the analyses in this study are based upon “fast” versions of MrBayes and RAxML with bootstrapping, as follows. MrBayes analyses were performed with only 1M iterations and we used only 100 bootstrap replicates for RAxML. Under these settings, single 100-taxon sequence alignments could be analyzed using MrBayes in under a day and using RAxML in about 1.5 hours. Therefore, analyses of 50 gene sequence alignments could be performed in 1-2 months using MrBayes and in about 3 days using RAxML. These settings made it possible to perform analyses of multiple datasets, each with tens of genes, in reasonable timeframes.The fast methods we studied are all based upon FastTree, a method that is deterministic and very fast on even very large datasets. In our studies, even on the 500-taxon datasets with 50 genes, all the fast methods finished in under two hours and used less than 9 MB of peak memory.Figure We begin the study with examining two specific algorithm design choices that were made in order to improve topological accuracy and/or running time.Recall that Phylonet was designed to solve the MDC problem, and that when used with incompletely resolved gene trees, it is designed to solve the Constraint-MDC problem. The motivation for the Constraint-MDC problem is that low support edges are often not reflective of true history . The hypWe also wished to evaluate the consequences of using only a sparse subset of the MrBayes distribution as input for BUCKy, instead of the full distribution. The advantage of using the sparse distribution is the decrease in running time and memory usage ; however, the concern is that using a sparse distribution might reduce accuracy of BUCKy analyses. We therefore wish to determine whether there is a statistical difference between BUCKy(MB-full) and BUCKy(MB-sparse).In each experiment below, we began by comparing Phylonet(FT-75%) to Phylonet(FT) to see if there was a statistically significant difference. For those datasets in which we ran BUCKy(MB) analyses, we also compared BUCKy-con(MB-full) to BUCKy-con(MB-spa) and BUCKy-pop(MB-full) to BUCKy-pop(MB-spa). These pairwise comparisons were performed using Wilcoxon signed-ranks tests. Other comparisons between methods were performed using Wilcoxon signed-ranks tests, and then corrected for multiple tests using Bonferroni’s correction.p < 0.01 for each condition, Wilcoxon signed-rank test). We observed a statistically significant improvement of Phylonet(FT-75%) over Phylonet(FT) on all the datasets without ILS datasets for both true and MAFFT alignments . For the ILS datasets, we observed a statistically significant improvement for all 17-taxon ILS datasets . On the ten 100-taxon ILS datasets, we also saw a statistically significant improvement of Phylonet(FT-75%) over Phylonet(FT) .We compared Phylonet(FT) to Phylonet(FT-75%) on every dataset we generated, and there was a statistically significant improvement obtained in every model condition (Since Phylonet(FT-75%) shows a statistically significant improvement over Phylonet(FT) in every model condition, it seems likely that contracting low support edges in FastTree trees does improve the species tree accuracy produced by Phylonet.p = 0.002, Wilcoxon signed-rank test), and no condition that resulted in a statistically significant change for BUCKy-pop.We now evaluate the statistical significance of changes in tree error that result from using BUCKy to analyze a sparse subset of the MrBayes distribution instead of the full distribution. These comparisons were done on only a few datasets . Of these three model conditions, there was only one condition (17 taxa with 32 genes) in which there was any statistically significant difference for BUCKy-con , and so there was no need to estimate the alignment on any dataset.We begin with a discussion of Glass’s performance. First, Glass can be run on distance matrices estimated directly from an alignment or from a tree computed on an alignment. We evaluated the accuracy for trees returned by Glass on 25 datasets, each with 32 genes per dataset, using three different ways of calculating distances on 25 datasets: the logdet  distanceFigure Among the “fast” methods , Phylonet(FT-75%), Greedy(FT), and Greedy(FT-75%) are the best three for both numbers of genes, and iGTP-dup the least accurate for both numbers of genes. Finally, for both numbers of genes, the most accurate of the iGTP methods was iGTP-duploss.We now compare six representative methods, BUCKy-con(MB-spa), BUCKy-con(MB-full), BUCKy-con(RAxML), Phylonet(FT-75%), Greedy(FT-75%), and iGTP-duploss(FT). The difference in error rate between the best fast method and the best slow method is about 5%, which in relative terms is large – but for datasets of this size, this is about 0.7 of an edge on average .After using Bonferroni’s correction for multiple tests, we observed that only one pair of representative methods (iGTP-duploss(FT) and BUCKy-con(MB-spa)) had a statistically significant difference on 8 genes. For 32 genes, we saw some additionally statistically significant pairwise comparisons, all involving BUCKy-con(MB-spa) or BUCKy-con(MB-full). However, all the other methods could not be distinguished from each other. Furthermore, three fast methods–greedy(FT-75%), Phylonet(FT-75%), and iGTP-duploss(FT)–were statistically indistinguishable from BUCKy-con(MB-spa), BUCKy-pop(MB-spa), and BUCKy-pop(MB-full).For the 100-taxon datasets, in order to run the computationally intensive methods, we explored performance on only 10 datasets, each with 25 genes. Because these datasets are large, we only evaluated BUCKy(MB-spa), terminating MrBayes at 1M iterations, and giving a sparse sample of 2000 trees for each gene to BUCKy. These datasets evolved under a model with substitutions as well as indels, and so we discuss results for both the true and MAFFT alignment on each dataset.Glass, run on the logdet distance matrix computed directly from the sequence alignments, had very high error rates for both true and MAFFT alignments , much higher than any other method (none of which had error rate above 9%). Error rates did not vary much between the MAFFT and true alignments, so that with the exception of Glass, error rates did not change by more than half a percent. Therefore, we focus our discuss on the results for the true alignment, and omit results for Glass. On the true alignments  and BUCKy-pop(RAxML)).Since a systematist will not always know the cause for incongruence between estimated gene trees, species tree estimation methods that are designed to handle ILS need to be able to reconstruct species trees when incongruence is due to other causes. Here we examine the case where all incongruence between estimated gene trees is due to estimation error.The true gene trees for these datasets have identical topologies but different branch lengths, thus allowing us to investigate the ability of methods to reconstruct the true species tree from gene tree estimates when no ILS occurs. These datasets evolved with indels as well as substitutions, thus requiring the estimation of alignments for the gene sequence datasets. When alignments are not highly accurate, however, gene trees will also have error, leading to the estimated gene trees exhibiting increased incongruence. We explored this question on datasets with 100 and 500 taxa. Trends for 500 taxa were very similar to those we observed for 100 taxa, and so we focus our discussion on the 100 taxon datasets. As before, Glass(logdet) had very high error, and we omit Glass from further discussion. The most striking observation is that the error rates of fast methods on the MAFFT alignment are much higher than error rates on the true alignment Fig.  suggestiAll fast methods (besides iGTP-dup(FT)) have excellent accuracy on the true alignment, with error rates that vary from 3.4-4.9%; thus, the difference between the most accurate method (greedy(FT)) and the other fast methods (except for iGTP-dup(FT)) is very small. However, we note that the three fast methods with best accuracy are the usual ones – greedy(FT) with 3.4% error followed by greedy(FT-75%) with 3.6% error and Phylonet(FT-75%) with 4.0% error. After correcting for multiple tests, all but two comparisons are statistically significant; the only ones that aren’t are Greedy(FT) vs. Greedy(FT-75%) and iGTP-duploss(FT) vs. Phylonet(FT-75%).The situation changes for the MAFFT alignments, where error rates for the fast methods are much higher  which has 29% error), and the differences between methods somewhat greater. On these alignments, the most accurate method is Greedy(FT) at 18.0%, followed by iGTP-duploss at 18.4%, and then by Greedy(FT-75%) at 18.5%. Phylonet(FT-75%) is in fourth place with 20.0% error. Thus, the results for the MAFFT alignment are similar, but show greater differences between methods, and also show that iGTP-duploss(FT) can give very good results as well. After correcting for multiple tests, we find that the top three methods, Greedy(FT), Greedy(FT-75%), and iGTP-duploss(FT), cannot be distinguished statistically, nor can iGTP-mdc(FT) be distinguished from Phylonet(FT-75%) or Phylonet(FT).did improve the error, but not dramatically. The best accuracy for methods that use MrBayes was obtained by Greedy(MB-maj) (2.2% FN), followed closely by BUCKy-con(MB) at 2.5%. The best accuracy for methods based upon RAxML was BUCKy-pop(RAxML) at 2.7% FN, followed closely by BUCKy-con(RAxML) at 2.8%. The most accurate methods that use FastTree were Phylonet(FT-75%), Greedy(FT-75%), and Greedy(FT), with error rates of 3.5%, 3.5%, and 3.3%, respectively. iGTP-duploss(FT) had the lowest error of the three iGTP methods (3.7% FN rate), and iGTP-dup(FT) had the worst accuracy of all methods (7.0% FN rate). Not surprisingly, after correcting for multiple tests, none of the differences were statistically significant.In order to evaluate the computationally intensive methods, we used a subset of 12 datasets . Average error rates on the six datasets with the true alignment ranged from 2.2-3.0% when based upon MrBayes distributions (full or sparse), from 2.7-4.5% when based upon RAxML bootstrapping analyses, and from 3.3-7.0% when based upon FastTree . The best results for MrBayes analyses were obtained by BUCKy-con(MB-full) (14.0%), the best results for RAxML-based analyses were obtained by BUCKy-con(RAxML) , and the best results for FastTree-based analyses were obtained by Greedy(FT-75%) (at 14.8% FN rate). Here, too, iGTP-dup(FT) had the worst accuracy (29.5% FN rate) of all methods, and iGTP-duploss(FT) had the least error of all three iGTP methods (15.3% FN rate). Again, not surprisingly, after correcting for multiple tests, none of these differences were statistically significant.The experiments we reported have datasets that range in terms of the causes for incongruence between estimated gene trees , rates of evolution (the 17-taxon and 100-taxon non-ILS datasets have higher rates than the 100-taxon ILS datasets), presence of indels, number of taxa, and type of alignment (true or MAFFT). Thus, the relative performance between methods varies with the models. However, certain trends hold throughout.The first observation is that BUCKy analyses of MrBayes or RAxML distributions produces highly accurate trees, either tied with the best or close to the best, even for the suboptimal way in which we ran these methods .The second observation is that several fast methods , Phylonet(FT-75%), and Greedy(FT)) provide very good results, coming close to BUCKy analyses of MrBayes and RAxML distributions , and with greatly reduced computational requirements. Furthermore, these fast trees have very low computational requirements, completing in at most two hours and with peak memory usage of at most 9 MB, even on our largest datasets containing 50 genes and 500 taxa. Therefore, there are feasible alternatives to methods like BUCKy, which offer statistically-based estimation and high accuracy but at a computational cost that may be prohibitive.The fast methods we studied are all based upon FastTree, a heuristic for ML that uses a deterministic hill-climbing heuristic to find locally optimal solutions to ML, and thus is likely to end up “stuck” in local optima. It is therefore possible that even better accuracy might be obtained, albeit at a computational cost. The remaining observations have to do with algorithm design. First, our study shows that methods, like Phylonet and BUCKy, that by design explicitly accommodate error in the input gene trees can produce more accurate trees than methods that assume all the input gene trees are correct. We have also shown that modifications to MrBayes (such as sparsification of its output distribution) can be made without compromising the accuracy of BUCKy’s estimated trees. These, and other approaches, could easily result in additional improvements for species tree estimation methods.Our study evaluating the computationally intensive methods was necessarily limited in number and in scope. Nevertheless, the experiments we performed show that some very fast methods for estimating species trees from gene sequence alignments can come close to the accuracy of the best methods, while taking dramatically less time , and with much smaller peak memory requirements . Furthermore, while we observed differences in accuracy between methods, some of which were substantial, the data suggests that in many cases, the differences between the best fast methods and the best computationally intensive methods we studied are not statistically significant. Additional experiments are clearly needed in order to evaluate whether this is true, or whether this is a consequence of the limited number of datasets we evaluated. It is also important to determine if these trends hold generally, or if there are conditions where the computationally intensive methods offer substantially improved accuracy.Our study has many ramifications for simulation studies. Because alignment estimation error has an impact on both the absolute and relative performance of methods, future simulation studies should include indels in their models and use both estimated and true alignments. Gene tree estimation error has a very large impact on species tree estimation, and so the best methods for estimating gene trees should be used. This study shows that relative and absolute performance is also impacted by the number of taxa, and so datasets with larger numbers of taxa ought to be included. Finally, since simple methods (like the greedy consensus of estimated gene trees) are often quite accurate, these methods should be included in the methods that are compared.Although we initiated the study in order to evaluate methods for estimating species trees in the presence of ILS, the observations in this study are also relevant to estimating species trees in the presence of gene duplication and loss. In particular, the poor performance of iGTP-dup for non-ILS datasets, in comparison to simple methods like Greedy, suggests that there is need for substantial improvement in methods for estimating species trees in the presence of gene duplication and loss. Perhaps the improvements for species tree estimation we observed by developing methods that explicitly handle error  can be obtained for gene duplication and loss scenarios as well.http://www.cs.utexas.edu/users/phylo/datasets/ILS.All datasets  used in this study are available at The 17-taxon datasets with 8 and 32 genes are from , and werWe simulated ten 100-taxon 25-gene datasets that evolve with ILS as follows. A single 100-taxon model tree from  was usedms 100 25 -T -I 100 1 1 ... 1 -ej <t> <from> <to> ...This produces 25 gene trees, each with branch lengths. We simulated sequence evolution down each gene tree under a GTR+Gamma+Gap model (with parameters derived from the model tree in ) using RWe used model species trees from  (see disThe authors declare that they have no competing interests.TW conceived and designed the study; JY implemented the analysis tools, produced the data, and created the figures and tables; TW and JY analyzed the data; TW wrote the paper.


Critically ill patients, mostly on positive pressure ventilation, are at higher risk of pneumothorax as well as their need for a central venous line (CVL) to optimize fluid status, CVP measurement, and so forth, and where the CVL is not being placed in the best circumstances with the patients being critically ill, unstable and with higher chances of error predisposed by pre-existing lung disease, obesity or whatever the admitting diagnosis. Before CVL placement was a blind technique relying on the anatomical positions identifying the position of major blood vessels and thus post-insertion X-ray was needed to confirm correct placement and to assess for pneumothorax. But with ultrasound (US) being more widely available, and most CVLs placed as US guided, the ultimate question develops: is post-insertion chest X-ray still needed?A retrospective study of 856 lines placed in 602 patients being evaluated over a period of 11 months. All cases were performed in a controlled ICU environment. Chest X-rays were performed 30 minutes post-insertion in the D0 adult ICU unit in a tertiary medical center in Abu Dhabi, UAE. The D0 ICU has a capacity of 24 beds with an average admission rate of 55 to 60 patients per month. Records were assessed and evaluated, and data collected and statistically studied.A total of 856 lines performed in 602 patients were evaluated. In 607 US-guided cannulating internal jugular veins with only four cases of malposition, there were no cases of pneumothorax recorded. A total of 161 subclavian veins were cannulated with no US, of which six cases of pneumothorax were reported; two cases needed intercostal tube insertion. Eighty-eight femoral vein cannulations with no US were performed and no complications were recorded.Chest X-ray is not necessary after US-guided CVL placement. Cutting out the chest X-ray procedure post insertion proved to be cost-effective.


Prospective evaluation of the effectiveness of the WHO-recommended standardized retreatment regimen for tuberculosis by Edward Jones-López and colleagues reveals an unacceptable proportion of unsuccessful outcomes. Each year, 10%–20% of patients with tuberculosis (TB) in low- and middle-income countries present with previously treated TB and are empirically started on a World Health Organization (WHO)-recommended standardized retreatment regimen. The effectiveness of this retreatment regimen has not been systematically evaluated.p<0.0001). Multidrug-resistant TB at enrolment was the only common risk factor for death during follow-up for both HIV-infected  and HIV-uninfected  individuals. Other risk factors for death during follow-up among HIV-infected patients were CD4<50 cells/ml and no antiretroviral treatment  and Karnofsky score <70 ; and among HIV-uninfected patients were poor adherence   and duration of TB symptoms .From July 2003 to January 2007, we enrolled smear-positive, pulmonary TB patients into a prospective cohort to study treatment outcomes and mortality during and after treatment with the standardized retreatment regimen. Median time of follow-up was 21 months (interquartile range 12–33 months). A total of 29/148 (20%) HIV-uninfected and 37/140 (26%) HIV-infected patients had an unsuccessful treatment outcome. In a multiple logistic regression analysis to adjust for confounding, factors associated with an unsuccessful treatment outcome were poor adherence  1.10–5.22), HIV infection , age , and duration of TB symptoms . All patients with multidrug-resistant TB had an unsuccessful treatment outcome. HIV-infected individuals were more likely to die than HIV-uninfected individuals , and 5%–10% of HIV-uninfected individuals will go on to develop disease and become infectious. The risk of progression from infection to disease in HIV infected is much higher. If left untreated, each person with active TB may infect 10 to 15 people every year, reinforcing the public health priority of controlling TB through adequate treatment. Patients with a previous history of TB treatment are a major concern for TB programs throughout the world because these patients are at a much higher risk of harboring a form of TB that is resistant to the drugs most frequently used, resulting in poorer treatment outcomes and significantly complicating current management strategies. More then 1 million people in over 90 countries need to be “re-treated” after failing, interrupting, or relapsing from previous TB treatment.One-third of the world's population is currently infected with Every year, 10%–20% of people with TB in low- and middle-income countries are started on a standardized five-drug retreatment regimen as recommended by the World Health Organization (WHO). Yet, unlike treatment regimens for newly diagnosed TB patients, the recommended retreatment regimen  has never been properly evaluated in randomized clinical trials or prospective cohort studies. Rather, this regimen was recommended by experts before the current situation of widespread drug-resistant TB and HIV infection.WHO surveillance data suggest that the retreatment regimen is successful in about 70% of patients, but retrospective studies that have evaluated the regimen's efficacy showed variable treatment responses with success rates ranging from 26% to 92%. However, these studies have generally only assessed outcomes at the completion of the retreatment regimen, and few have examined the risk of TB recurrence, especially in people who are also infected with HIV and so are more likely to experience TB recurrence—an issue of particular concern in sub-Saharan Africa. Therefore, in this study based in Kampala, Uganda, the researchers conducted a prospective cohort study to assess treatment and survival outcomes in patients previously treated for TB and to identify factors associated with poor outcomes. Given the overwhelming contribution of HIV infection to death, the researchers categorized their survival analysis by HIV status.The researchers recruited consecutive smear-positive TB patients who were admitted to Mulago Hospital, Kampala, Uganda, for the retreatment of TB with the standard retreatment regimen between July 2003 and January 2007. Eligible patients received daily directly observed therapy and after hospital discharge, were seen every month during their 8-month TB-retreatment course. Home health visitors assessed treatment adherence through treatment card review, monthly pill counts, and patient self-report. After the completion of the retreatment regimen, patients were evaluated for TB recurrence every 3 months for a median of 21 months. The researchers then used a statistical model to identify treatment outcomes and mortality HIV-uninfected and HIV-infected patients.p<0.0001), and having multidrug resistant TB at enrollment was the only common risk factor for death during follow-up for both HIV-infected and HIV uninfected patients. Other risk factors for death among HIV-infected patients were CD4<50 cells/ml and no antiretroviral therapy treatment and among HIV-uninfected patients were poor adherence and duration of TB symptoms.The researchers found that 29/148 (20%) of HIV-uninfected and 37/140 (26%) of HIV-infected patients had an unsuccessful treatment outcome. Factors associated with an unsuccessful treatment outcome were poor adherence, HIV infection, increasing age, and duration of TB symptoms. All patients with multidrug resistant TB, a form of TB that is resistant to the two most important drugs used to treat TB, had an unsuccessful treatment outcome. In addition, HIV-infected subjects were more likely to die than HIV-uninfected subjects , the standard retreatment regimen had low treatment response rates and was associated with poor long-term outcomes in certain subgroups of patients, particularly those with multidrug resistant TB and HIV.These findings indicate that the standard retreatment approach to TB as implemented in low- and middle-income settings is inadequate and stress the importance of a new, more effective, strategies. Improved access to rapid diagnostics for TB drug-resistance, second-line TB treatment, and antiretroviral therapy is urgently needed, along with a strong evidence base to guide clinicians and policy makers on how best to use these tools.http://dx.doi.org/10.1371/journal.pmed.1000427.Please access these Web sites via the online version of this summary at TB, TB retreatment, and multidrug-resistant TBThe World Health Organization has information on TB/HIV coinfectionWHO also provides information on the global plan to stop TBThe Stop TB Partnership provides information on  Each year, 10%–20% of patients with tuberculosis (TB) in low- and middle-income countries present with previously treated TB and are started on therapy empirically with a standardized five-drug retreatment regimen as recommended by the World Health Organization (WHO). It is estimated that annually more than 1 million people in over 90 countries are treated with the 8-mo regimen  after failing, interrupting, or relapsing from prior treatment Unlike treatment regimens for new TB patients, the currently recommended retreatment regimen  has never been evaluated for efficacy in randomized clinical trials or prospective cohort studies On the basis of WHO surveillance data, retreatment regimens successfully treat approximately 70% of cases As part of a larger study to assess a new management strategy for DR-TB in resource-limited settings, we conducted a prospective cohort study in Kampala, Uganda to assess outcomes in previously treated TB patients, with particular reference to the impact of drug resistance on treatment outcomes and survival. We sought to identify factors associated with poor outcomes and separated our survival analysis by HIV status, given the overwhelming contribution of HIV infection to early death, and in order to evaluate the impact of antiretroviral treatment (ART) and CD4 count on mortality among TB/HIV coinfected patients. When we began our study in 2003, Uganda lacked the information, training, and resources required to address the problem of DR-TB. As part of this study, we helped the Uganda National Tuberculosis and Leprosy Programme (NTLP) develop the pillars of a rational and sustainable program to control DR-TB in Uganda, which included the provision of treatment with second-line TB drugs when possible.The AIDS Research Sub-Committee of the Uganda National Council of Science and Technology and the Institutional Review Boards at the University of Medicine and Dentistry of New Jersey and the London School of Hygiene & Tropical Medicine approved the study.This study was conducted at the 85-bed inpatient TB ward of the NTLP Chemotherapy Centre at Mulago Hospital in Kampala, Uganda. The NTLP Centre serves both as a local treatment clinic  and as the national referral centre . Most (95%) of the retreatment cases seen at the NTLP Centre were hospitalized to facilitate administration of streptomycin when clinically possible; however, patients with suspected or known MDR-TB were managed as outpatients. Uganda is on the WHO list of high-burden countries with a 2007 estimated annual TB  incidence rate of 330 cases per 100,000; 39% with HIV infection and a prevalence of MDR-TB in new and retreatment TB cases of 0.5% and 4.4%, respectively Mycobacterium tuberculosis in culture. We used a standardized questionnaire to collect demographic and clinical information. At presentation, participants were classified according to the WHO definitions of prior treatment outcomes  All consecutive TB patients hospitalized for retreatment between July 2003 and January 2007 were eligible for inclusion in the study, provided they were 18 years or older and gave written informed consent. Eligible individuals were included in the study if they provided sputum specimens that were positive for acid-fast bacilli (AFB) on smear microscopy and TB was confirmed subsequently by growth of Second-line drugs for the treatment of MDR-TB were not available under routine care by the NTLP when the study was initiated. After discussions with collaborators, officials from the Ugandan Ministry of Health, NTLP, WHO, and other stake holders, we agreed to use our study as an opportunity to build the necessary foundations for a sustainable MDR-TB program in Uganda. These included measuring the prevalence of DR-TB, developing the laboratory capacity for standard and rapid DST, obtaining preferentially priced second-line TB drugs, training medical and nursing personnel in MDR-TB management, developing a community-based DOT program, and establishing hospital infection control measures. When second-line drugs became available in 2005, treatment for MDR-TB was begun as a pilot treatment program and phased in as drugs became available and conditions allowed. Divided into two groups, a total of 12 MDR patients received individualized treatment with second-line drugs (seven of them were included in this study and five were not included); the first group of patients began treatment in October 2005 and the second in August 2006. All MDR patients in this study were initially treated with the standard retreatment regimen at the time they first presented to the clinic (before their MDR status was known); they all had an unsuccessful treatment outcome and were included in the survival analysis. The results from individualized treatment with second-line drugs are not included in this analysis.Patients received daily DOT while admitted to the hospital. After hospital discharge, participants were seen monthly during their 8-mo TB-treatment course, at month 9, and quarterly thereafter for the duration of the study. Patients who did not keep their scheduled visits were traced to their homes. During follow-up visits, home health visitors assessed treatment adherence through treatment card review, monthly pill counts, and patient self-report. Adherence was categorized into a five-level ordinal scale . The worst assessment at any visit was taken as the summary measure of adherence for each participant. Because of small numbers of patients in some categories, patients were classified as either “mostly adherent” (consisting of the first two categories) or “missed half or more” (consisting of the remaining three categories). For individuals who died or were lost to follow-up before the month-3 visit, we were unable to assess adherence.During follow-up, two sputum samples were collected for AFB smear microscopy and culture at each of five predefined time points ; additional samples were collected at later visits if patients had a productive cough. Following WHO treatment outcome definitions M. tuberculosis complex was determined by either the BACTEC NAP test (Becton Dickinson Diagnostic Instrument Systems) or PCR for IS6110 as previously described M. tuberculosis isolates obtained from each patient at the time of recruitment were subjected to DST for S, R, H, E, and Z using BACTEC 460 or MGIT 960. For quality assurance of DST, 245 specimens were also tested at the CDC with over 95% concordance. The radiometric BACTEC 460 was used through July 2006 when it was replaced by the fluorometric MGIT 960. Most (87%) baseline specimens were cultured using the BACTEC 460 system and the majority of the initial isolates tested for drug susceptibility with the 460. The isolation rate and DTP liquid culture for baseline specimens are comparable with the BACTEC 460 and MGIT 960 systems. Likewise the DST results are highly correlative between the two systems. Thus, the change in methodologies during the study had no impact on the interpretation of results.Sputum specimens were processed with the standard digestion and decontamination method using NALC/Na citrate/NaOH. Centrifuged pellets were resuspended in a phosphate buffer solution and used to prepare smears and cultures on 7H10 agar and liquid media. Sputum smear microscopy was performed using auramine O fluorescent stain and reported according to the US CDC microscopy grading scheme (negative or 1+ to 4+) M. tuberculosis at any time after completing treatment. For each patient with TB recurrence, we compared the patient's initial and recurrent isolates using IS6110 restriction fragment length polymorphism (RFLP) analysis to differentiate participants with relapse  from those with reinfection (different isolates).We stored the baseline isolate for each participant and all isolates from patients who had a culture positive for HIV testing was offered to all participants; HIV infection status was determined using a serial rapid test algorithm in concordance with international guidelines The objectives of the statistical analysis were to describe the treatment outcomes and mortality of HIV-uninfected and HIV-infected patients and to find factors associated with treatment outcome and mortality. Logistic regression was used to identify factors associated with unsuccessful treatment outcome. Factors were considered for inclusion as risk variables or confounders in models on substantive grounds, i.e., on the basis of prior knowledge or biological plausibility Survival analysis methods were used to investigate factors associated with mortality. Kaplan Meier survival curves were derived to compare mortality between HIV-infected and HIV-uninfected patients, and to evaluate the impact of baseline CD4 count and ART treatment on mortality among those HIV-infected. Cox proportional hazards models were fitted to find factors associated with mortality. Separate models were fitted for HIV-uninfected and HIV-infected individuals to allow for inclusion of CD4+ cell count and ART-status in the model for HIV-infected individuals. A composite five-level variable for CD4+ cell count and ART was constructed , because of the strong correlation between CD4+ cell count and ART. The composite variable of CD4+ cell count and ART use was analyzed as a time-varying covariate as individuals initiated ART at different times during follow-up. Baseline CD4+ cell count was used for all analyses. The proportional hazards assumption was checked using graphical methods as well as the test of Grambsch and Therneu A total of 288 eligible patients, 148 (51%) HIV-uninfected and 140 (49%) HIV-infected, were enrolled in the study between July 2003 and January 2007. Their baseline characteristics are summarized by HIV status in M. tuberculosis isolate that was resistant to at least one first-line antituberculosis drug: 18 (6%) had MDR, 34 (12%) had mono-resistance to isoniazid, and two (1%) individuals had mono-resistance to rifampicin. The remaining 12 (4%) individuals had mono-resistance to streptomycin (eight), ethambutol (three), or pyrazinamide (one). The proportion of individuals with MDR-TB at baseline was similar for HIV-uninfected and HIV-infected patients, 11 (7%) and seven (5%), respectively (p = 0.40).66 (23%) participants had an Follow-up results during the 8-mo TB-treatment phase are summarized by HIV status in p = 0.64).Patients were admitted for the 2-mo course of injectable streptomycin treatment for a median duration of 57 d (IQR 53–61 d), during which time all patients had daily DOT. After discharge, 217 (75%) individuals were fully adherent or missed a few doses  and 59 (21%) missed half or more of their doses. Adherence could not be measured in 11 (4%) patients as they either died or were lost to follow-up before the month-3 visit. Adherence to TB treatment after hospital discharge did not differ between HIV-uninfected (77%) and HIV-infected patients (74%)  individuals were studied for TB recurrence ; of these, 13 (6%) had recurrent TB , a TB recurrence rate of 4.2 per 100 person-years of observation (PYO). Among the 12 patients with recurrent TB and available RFLP results, seven (58%) had reinfection (two HIV-uninfected and five HIV-infected) and five (42%) had relapse .p<0.0001) than HIV-uninfected (5.9 per 100 PYO)  died during 511 PYO; 38 (13%) individuals died during the 8-mo TB-treatment course  and 29 (100 PYO) . Of the In unadjusted analyses , a histoIn this prospective cohort study of previously treated pulmonary TB patients managed under programmatic conditions in Uganda, we found that 70%–80% of patients with and without HIV coinfection had a successful treatment outcome at the conclusion of antituberculous therapy . Although this overall response compares well with many other studies, we found that the retreatment regimen has unacceptably low treatment response rates in certain subgroups of patients and is associated with poor long-term outcomes, particularly in MDR-TB and in HIV-infected patients. Initiation of treatment with ART in eligible patients mitigated much of the excess mortality associated with HIV infection alone, in agreement with a recent clinical trial showing that use of ART concurrently with antituberculous therapy in TB patients is safe and improves survival In accordance with the recent review by Menzies and colleagues p<0.0001). In this study, all patients with MDR, especially those with HIV, not surprisingly did poorly given the composition of the retreatment TB regimen. However, comparatively few patients had baseline MDR and thus, the overall attributable risk of MDR to poor TB-treatment outcomes or death was only 27%. Furthermore, other forms of DR-TB such as mono-resistance to isoniazid or rifampicin, present in approximately 20% of the cohort, did not predict treatment outcome or survival.This wide variability is mainly explained by differences in the relative frequency of three prognostic factors across study populations, which include the levels of treatment adherence, MDR-TB, and HIV infection. Among these factors, MDR-TB seems to have a prominent influence on the efficacy of the retreatment regimen as shown in the ecological study by Mak and colleagues M. tuberculosis, resulting in acceptable clinical outcomes in most retreatment cases at the conclusion of TB therapy. However, despite these relatively short-term optimistic findings, this cohort of retreatment TB patients experienced poor long-term outcomes. In this sub-Saharan African programmatic setting, of the patients studied for TB recurrence after satisfactory completion of TB treatment, 6% had TB recurrence while a further 9% died, and 18% were lost to follow-up. In addition seven patients who had an unsatisfactory outcome at month 8 (either treatment failure or unknown outcome) later died. This finding, which has been previously reported Taken together, the available data show that the retreatment regimen is effective in treatment of adherent patients with pan-susceptible or mono-resistant The poor prognosis of retreatment TB patients is usually ascribed to undiagnosed DR-TB and this is leading to a long overdue improvement in laboratory and treatment capacity in resource-limited settings. However, there is a need to further understand if the rapid death (within 1–3 wk after presentation) observed in some patients with HIV/AIDS and DR-TB is a consequence of advanced HIV disease, suboptimally treated TB, or both At the annual collaborator's meetings in Kampala, our group had long and impassioned discussions regarding one very difficult ethical dilemma we faced: in a setting such as Uganda where second-line TB drugs are not routinely available, can research in MDR-TB be ethically sound? Although a profound debate on this issue would be beyond the scope of this manuscript, we believe it is important to provide the reader with a summary of our thinking and our actions. Our discussions focused on possible alternatives to our study design and the provision of treatment with second-line drugs considering the time, cost, and complexities involved in building an MDR-TB–treatment program. We considered three alternate designs. Excluding known or suspected MDR patients (and other resistance patterns that are likely to fail treatment or relapse with the retreatment regimen) would have significantly reduced the generalizability of our results and excluded the very population that “exposes” the limitations of the regimen we intended to evaluate. We also considered withholding DST results performed at enrolment, but we reasoned this would have only artificially lessened the ethical concerns we had. Finally, we considered discontinuing the study of MDR-TB patients at the time their DST results became available. During our discussions, it became evident that none of these options would have improved the care of study participants and rather, would prevent them from the benefits of research participation, including the eventual access of some of them to second-line drugs. Even before second-line drugs became available through our program, we are confident patients benefited from study participation both directly (improved treatment and reduced toxicity) and indirectly (patient education and reduced transmission) when compared to existing standard of care in Uganda. With no access to second-line drugs and a prevalence three times higher then expected Our results should be interpreted in the context of the study design and its limitations. Our design may have biased findings towards improved outcomes when compared to true programmatic conditions for several reasons. Firstly, because the clinic attempted to manage patients with suspected MDR-TB as outpatients in an effort to reduce nosocomial transmission, this study did not include all MDR-TB patients diagnosed at this site during the study period. Inclusion of the entire cohort of MDR-TB patients we have reported elsewhere The results of this prospective study provide evidence that the standard retreatment approach to TB, as implemented in low- and middle-income settings, is inadequate. Improved access to rapid diagnostics for TB drug resistance, second-line TB treatment, and antiretroviral therapy is urgently needed, but the evidence base guiding the utilization of these tools by clinicians and planners remains sparse. As has been advocated Table S1Clinical outcomes according to HIV and potential prognostic variables after completing the standard 8-mo retreatment regimen in 288 patients in Kampala, Uganda, 2003–2007.(0.09 MB PDF)Click here for additional data file.Table S2Mortality by HIV status and potential explanatory factors.(0.09 MB PDF)Click here for additional data file.


Deleted in Azoospermia gene family encodes three germ cell-specific RNA-binding proteins  that are essential for gametogenesis in diverse species. Targeted disruption of Boll in mice causes male-specific spermiogenic defects, but females are apparently fertile. Overexpression of human BOLL promotes the derivation of germ cell-like cells from genetically female (XX), but not male (XY) human ES cells however, suggesting a functional role for BOLL in regulating female gametogenesis in humans. Whether BOLL is expressed during oogenesis in mammals also remains unclear. We have therefore investigated the expression of BOLL during fetal oogenesis in humans and mice. We demonstrate that BOLL protein is expressed in the germ cells of the human fetal ovary, at a later developmental stage than, and almost mutually-exclusive to, the expression of DAZL. Strikingly, BOLL is downregulated, and DAZL re-expressed, as primordial follicles form, revealing BOLL expression to be restricted to a narrow window during fetal oogenesis. By quantifying the extent of co-expression of DAZL and BOLL with markers of meiosis, we show that this window likely corresponds to the later stages of meiotic prophase I. Finally, we demonstrate that Boll is also transiently expressed during oogenesis in the fetal mouse ovary, but is simultaneously co-expressed within the same germ cells as Dazl. These data reveal significant similarities and differences between the expression of BOLL homologues during oogenesis in humans and mice, and raise questions as to the validity of the -/-Boll mouse as a model for understanding BOLL function during human oogenesis.The  Deleted in Azoospermia (DAZ) gene family encodes three conserved RNA-binding proteins , the expression of which is largely restricted to germ cells. DAZ-family proteins are essential for germ cell development in diverse organisms, and inactivating mutations in members of the DAZ family result in a failure of germ cell development in either or both sexes [The th sexes -4. DAZ-fth sexes , mRNA trth sexes  and mRNAth sexes . The steth sexes ,8-11.DAZ gene is present in multiple copies on the Y chromosome of humans and Old World monkeys [AZFc region of the Y chromosome, an area frequently deleted in men with severe oligozoospermia or azoospermia [DAZ arose from a duplication of the autosomal homologue Dazl (Deleted in azoospermia-like) [BOLL , is the ancestral member of the family, with orthologues throughout the metazoa [Boule-deficient flies display defective spermatogenesis [Boll in mice results in male-specific infertility [Boll-deficient female mice are apparently fertile however, leading to the hypothesis that Boll is dispensable for mammalian oogenesis [The prototypic  monkeys , and wasospermia . DAZ aroia-like) , the expia-like) ,11,15-20 metazoa ,22. Boulogenesis , arisingogenesis . Targeteertility . Boll-deogenesis ,22. Introgenesis , suggestBoll transcripts have been reported in the mammalian fetal ovary [Although al ovary ,24,25 thal ovary , and havDAZL and BOLL during human fetal ovary development, using qRT-PCR  and late (17-20wga), and compare this to the expression of BOLL. In the first trimester (8-9wga), DAZL expression is low and BOLL undetectable. In the early second trimester (14-16wga) both DAZL expression significantly increases, and BOLL mRNA first becomes detectable (p<0.001). At both 14-16 and 17-20wga weeks BOLL mRNA levels were lower than that of DAZL, relative to the internal housekeeping control RPL32. Consistent with previous immunohistochemical studies [BOLL mRNA in the human fetal testis across this developmental window (data not shown).We first investigated the expression of  qRT-PCR . Previouelopment , however studies  we were The expression and distribution of DAZL protein in the human fetal ovary has been reported previously . HoweverWe confirmed the specificity of the DAZL and BOLL antibodies used in this study . To establish if the timing of the switch from DAZL to BOLL during human fetal oogenesis occurs at a specific stage of the first meiotic prophase, we performed triple immunofluorescence analysis for DAZL, BOLL and the meiosis marker Synaptonemal Complex Protein 3 . We detsed DAZL . In contsed DAZL , consistQuantification of this confirmed the extensive overlap of BOLL and SYCP3 expression ; 69% SYCGiven the striking switch between DAZL and BOLL expression during human fetal ovarian germ cell development, the lack of extensive co-expression of both proteins within the same germ cells, and the apparent lack of an oogenesis defect in Boll-deficient mice, we sought to establish whether the expression patterns of DAZL and BOLL are conserved during fetal oogenesis in humans and mice. We therefore performed single-antigen immunofluorescence to detect either Dazl or Boll on sections of fetal mouse ovaries at embryonic day (e) 13.5, e15.5, e18.5 and postnatal day 0 P0; , which cConsistent with previous studies, germ cells in the fetal mouse ovary expressed Dazl at all stages examined . At e13.The distribution of Dazl and Boll in the germ cells of the fetal mouse ovary at e15.5  suggesteDAZL and BOLL gene expression overlaps using gestational age as the discriminator, DAZL and BOLL proteins are in fact expressed by distinct populations of germ cells at different stages of maturation. We find a progressive pattern of expression as germ cells mature, with DAZL expressed in germ cells prior to, and in the early stages of meiotic prophase I, after which it is down-regulated and BOLL expressed. BOLL in turn is also down-regulated and DAZL re-expressed at the time that germ cells in the human fetal ovary form primordial follicles , but by 49dpc DAZL expression is greater than that of BOLL, with both genes maximally expressed at 56dpc, corresponding to the early stages of meiotic prophase I. Interestingly, BOLL transcript levels, unlike those of DAZL, appear to decline slightly between 14-16wga and 17-20wga . This likely reflects a progressive reduction of the number of BOLL-positive germ cells in the fetal ovary at later gestations, as oocytes downregulate BOLL and re-express DAZL as they form primordial follicles, although this may result from a dilution of BOLL mRNA due to the emergence of additional BOLL-negative germ cells at this stage.Previous work from our laboratory reported developmental changes in rimester . Human orimester . The datal ovary -35. The ep ovary . In thatDAZL and BOLL gene expression overlap temporally, our data clearly demonstrate that the spatial distribution of DAZL and BOLL proteins within the human fetal ovary is quite distinct, with DAZL-expressing germ cells towards the periphery, and BOLL-expressing germ cells towards the centre of the ovary. This radial distribution of germ cells reflects a gradient of differentiation, with smaller, less mature germs in the peripheral cortex, and larger, more mature germ less localised in the central medullary region [Although the patterns of y region ,36,37. Oy region . The tray region , revealiy region . The dat+-only population of germ cells, coupled with assessment of co-expression of SYCP3 with proteins known to be expressed around the time of follicle formation, may provide insight into the developmental stage of these cells.An interesting observation of this study is the identification of a subpopulation of germ cells that express SYCP3, but do not express DAZL or BOLL. It is tempting to speculate that the population of BOLL-negative, DAZL-negative, SYCP3-positive germ cells identified here represents germ cells that are at a stage of development immediately preceding follicle formation, in which BOLL has been downregulated in anticipation of germ cell nest disassembly and follicle formation, but which have not yet proceeded far enough in the follicle assembly process to have reactivated DAZL. However, these germ cells may instead be destined for loss during the extensive waves of apoptosis that occur prior to, and during, follicle formation ,39,40. DDAZL and BOLL gene expression in the human fetal ovary used in their study display similarly overlapping expression to our qPCR data, the immunohistochemical data we present here disputes their model’s prediction, revealing that BOLL is expressed in a different population of germ cells to those expressing DAZL, and the onset of BOLL protein expression occurs only well after meiosis has initiated, precluding extensive physical interaction between the two proteins. Importantly, this underlines the importance of relating transcript data derived from whole organs comprised of multiple different cell types and stages of development to the spatial and temporal distributions of the transcripts and the proteins they encode within the organ.Zheng et al. (2011) recently reported the construction of a mathematical model of the human fetal ovary, that integrates microarray data on gene expression during human fetal ovarian development with gene ontology and information on protein function, and from this proposed that BOLL may function co-operatively with DAZL to prepare germ cells in the human fetal ovary for entry into meiosis . Whilst in vivo [Boll-deficient males [Dazl-deficient mice on various genetic backgrounds [boule-deficient Drosophila by human BOLL [DAZL and BOLL are RNA-binding proteins, which act to promote the translation of mRNA into protein by binding specific motifs in the 3’ UTRs of target transcripts . Numerouin vivo ,10. In cin vivo , and whein vivo . The seqnt males ,22 compakgrounds -18,20,49man BOLL ) indicatBoll-knockout mouse as a mouse as a model for understanding BOLL function during human female oogenesis. It is tempting to speculate that the simultaneous co-expression of Dazl and Boll in the germ cells of the fetal mouse ovary may present a mechanism by which Dazl could compensate functionally for the absence of Boll, permitting oogenesis to proceed normally in mice. Indeed, such functional redundancy has been proposed as one explanation for the relatively late spermiogenic phenotype seen in Boll-/- male mice, which only becomes manifest at a developmental stage after which Dazl is no longer expressed [BOLL mutations may contribute to fertility defects in human females.The data presented here raise important questions about the validity of the xpressed . The seqEthical approval for this study was obtained from Lothian Research Ethics Committee (study code LREC 08/S1101/1). All participants gave informed written consent in accordance with national guidelines. Experiments involving mice were approved by the University of Edinburgh Animal Research Ethics Committee and performed according to the UK Animal (Scientific Procedures) Act 1986.SRY gene [Human fetuses ) were obtained after elective termination of pregnancy. Terminations were for social reasons, and all fetuses used in this study were morphologically normal. Gestational age was determined by ultrasound scan, and confirmed (for second trimester fetuses) by direct measurement of foot length. The sex of the first trimester fetal gonads was determined by PCR for the SRY gene . Extra-oad libitum according to UK Home Office and local University of Edinburgh ethical standards. The day of vaginal plug detection was designated as embryonic day (e) 0.5. Gonads were isolated from fetuses at e13.5, e15.5 and e18.5, and from neonatal mice on the day of birth (P0), and fixed in 4% NBF for 3 hours before processing into paraffin blocks.C57BL/6 mice were housed on a 12 hour light/dark cycle and fed 2O were prepared as negative controls.RNA was extracted from 8-12wga human fetal ovaries using the RNeasy Micro Kit  and from 13-20wga ovaries using the RNeasy Mini Kit (QIAGEN) according the manufacturer’s instructions. cDNA was synthesized using the SuperScript VILO cDNA Synthesis Kit  following manufacturer’s instructions. Identical reactions in which the Reverse Transcriptase (RT) enzyme mix was replaced by nuclease-free dHDAZL, BOLL and RPL32 were generated by mixing 1µl serially diluted cDNAs  with 0.2µl Forward/Reverse primers (25mM stocks), 5µl SYBRGreen Master mix (Life Technologies) and 3.6µl nuclease-free dH2O, and run on ABI7900HT thermal cycler (Life Technologies). Ct values were plotted against log concentration, and the resulting slope for each was used to calculate expression within each sample. To permit comparison between individual samples, gene expression was calculated relative to that of the housekeeping gene RPL32 . The PCR programme used was 50°C for 2 minutes (min), 95°C for 10min, then 40 cycles of 95°C for 15 seconds (sec), 60°C for 1min, and ended with a dissociation stage  to check the specificity of PCR products. Quantitation of gene expression in experimental samples was performed using the same conditions as used for standard curves, but diluting the cDNAs 1/10. Results were analysed using ABI SDS2.4 software, Microsoft Excel 2003 and GraphPad Prism 5 software. The sequences of the oligonucleotide primers used in this study can be found in For measurement of gene expression by qRT-PCR, standard curves for Human or mouse fetal ovaries were dissected, fixed in Bouins or 4% NBF and processed into wax blocks by standard methods . 5µm secFor double-immunofluorescence, the primary antibody to the first antigen was removed by microwaving the slides in 400ml 0.05M citrate buffer for 2.5min, cooled for 1 hour and then blocked in serum block for 30min. The second primary antibody incubation was performed exactly as for the first, as above. On day three, tissues were stained with either peroxidase-conjugated secondary antibody and TSA (as on day 2), or with a fluorophore-conjugated secondary antibody at room temperature for 1h.For triple-antigen immunofluorescence, the tissues were microwaved and serum blocked again after detection of the second primary antibody, and the third primary antibody incubation performed as for the first and second ones. The signal was detected as on day 3, and finally the tissues were counterstained with DAPI. Images were captured using a 710 Confocal Microscope  and Zen 2009 software.t tests, or ANOVA with Neumann-Keuls post hoc testing. P values less than 0.05 were considered statistically significant.Tiled images were taken for stained sections using 710 Confocal Microscope (Carl Zeiss) and Zen 2009 software, and different areas include cortex and center of each image were selected randomly for further analysis. For germ cell nuclear diameter measurement, two perpendicular diameters of the germ cell nucleus were measured using Image-Pro Plus , then averaged and related back to a scale bar to convert pixels to μm. For cell counting, the plugin cell counter of ImageJ  was used and cell numbers were counted manually. The data were analysed using Microsoft Excel 2003 and GraphPad Prism 5 software. Data were analysed using Figure S1Validation of anti-DAZL and anti-BOLL antibodies for immunofluorescence.HEK293 cells were transfected with either pCMV6-DAZL or -BOLL vectors. Anti-DAZL antibodies detected epitopes (green) in pCMV6-DAZL transfected cells, but not in pCMV6-BOLL transfected or untransfected cells (left and centre columns). Mouse anti-BOLL antibody detected epitopes (green) in cells ectopically-expressing BOLL only (right column). Blue: DAPI. Scale bars: 20µm in all panels.(TIF)Click here for additional data file.Figure S2Specificity of DAZL and BOLL antibodies.Each blot shows cell lysates of HEK293 cells either mock transfected (transfection agent only) or transfected with vectors expressing hDAZL (pCMV6-hDAZL) or hBOLL (pCMV6-hBOLL). α -tubulin  was used as loading control. A) and B) Both anti-DAZL antibodies (mouse and rabbit as indicated) detected a band at ~40-45kDa in only hDAZL-transfected cells (consistent with the predicted molecular weight of DAZL plus the Myc- and FLAG- tags of around 40kDa). C) anti-BOLL antibody detected a band of ~39kDa only in hBOLL-transfected cells. No comparable bands were found in mock transfected cells.(TIF)Click here for additional data file.Figure S3Co-localisation of phospho-ATM with DAZL or BOLL.A) immunofluorescent co-localisation of phospho-ATM with DAZL or BOLL in the human fetal ovary (14 weeks gestation). DAZL shows limited co-expression with phospho-ATM, whereas almost all the phospho-ATM+ cells are also BOLL+. Arrows indicate germ cells co-expressing of phospho-ATM and DAZL or BOLL, and arrowheads indicate germ cells expressing phospho-ATM only. Scale bars = 20µm. B) Quantification of phospho-ATM co-localisation with DAZL and BOLL. BOLL is expressed in ~80% of phospho-ATM+ cells, which is significantly higher than the proportion of DAZL/phospho-ATM double positive cells .(TIF)Click here for additional data file.Methods S1This file contains details of the methods used to generate the results in (DOCX)Click here for additional data file.Table S1Oligonucleotide primers used for qRT-PCR.(DOCX)Click here for additional data file.Table S2Antibodies used for Immunofluorescence.(DOCX)Click here for additional data file.Table S3Antibodies used for Immunoblotting.(DOCX)Click here for additional data file.


CVC care started from quarter 7 and a total of 1274 nurses were trained by their supervisors using a web-based, modular, e-learning programme. The study included 3952 patients with 6353 CVCs accumulating 61,366 catheter-days. Hospital-wide, 106 patients had 114 CLABSIs with a cumulative incidence of 1.79 infections per 100 catheters. We observed a significant quarterly reduction of the incidence density  after adjusting for multiple confounders. The incidence densities (n/1000 catheter-days) in the first and last study year were 2.3/1000 and 0.7/1000 hospital-wide, 1.7/1000 and 0.4/1000 in the intensive care units, and 2.7/1000 and 0.9/1000 in non-intensive care settings, respectively. Median time-to-infection was 15 days . Our findings suggest that clinically relevant reduction of hospital-wide CLABSI was reached with a comprehensive, multidisciplinary and multimodal quality improvement programme including aspects of behavioural change and key principles of good implementation practice. This is one of the first multimodal, multidisciplinary, hospital-wide training strategies successfully reducing CLABSI.Central line-associated bloodstream infection (CLABSI) is the major complication of central venous catheters (CVC). The aim of the study was to test the effectiveness of a hospital-wide strategy on CLABSI reduction. Between 2008 and 2011, all CVCs were observed individually and hospital-wide at a large university-affiliated, tertiary care hospital. CVC insertion training started from the 3 Central vascular lines are indispensable in hospital care, but the major potential complication of their use is central line-associated bloodstream infection (CLABSI) This prospective study was conducted between 2008 and 2011 at the University of Geneva Hospitals, Geneva, Switzerland, a 1908-bed primary and tertiary care centre. In 2011, there were 48,112 admissions accounting for 671,709 hospital-days. All adult inpatients with a CVC were eligible for study inclusion. The policies regarding management of suspected CLABSI and the method for obtaining blood cultures did not change during the study period. Outcomes were stratified by department .The study was approved by the institutional review board of the University of Geneva Hospitals, Geneva, Switzerland (protocol number 07023). The institutional review board waived the need for written informed consent from the participants.www.carepractice.net). All modules featured detailed procedure sequences and were animated by short movies, icons, and keywords.In 2007, existing protocols related to CVC insertion and care were reviewed and updated by an interdisciplinary study group, which included members from anaesthesiology, infection control, and the nursing department. A detailed insertion checklist was defined by the study group based on evidence in the literature and by repeated practice testing in daily routine. The complete insertion procedure from patient preparation until dressing application was filmed for training purposes. For catheter care, a modular e-learning programme was developed, including assistance with CVC insertion, infusate preparation, CVC manipulation, dressing change, CVC removal, and clinical surveillance and documentation . The workshop was divided into 3 sequences: 1) lecturing on CVC insertion and CLABSI prevention; 2) filming of participants inserting a CVC; and 3) giving feedback based on best practice recommendations. Between October 2009 and March 2010, all nurses in the medical and surgical wards were trained by their supervisors using the “carepractice” e-learning programme. Supervisors were familiarized with the e-learning programme in focus groups where they also had to prove their capacity to organize appropriate training sessions. All new medical and nursing staff were trained using the tools as described above.After a baseline of 6 months, physicians working in the operating theatre and intensive care were trained during 4-hour workshops at the hospital simulator training centre  in the presence of a CVC with no other apparent source was the primary outcome. For coagulase-negative staphylococci (CoNS), two positive blood cultures or a complete course of antibiotic therapy adjusted for susceptibility testing were required On the basis of the baseline incidence of 4.2 CLABSI episodes per 1000 catheter-days (15 CLABSI episodes per 426 CVCs) observed during the pilot study Categorical variables were compared using the chi-square test; continuous variables were summarized as medians and compared using the Wilcoxon rank sum test. CLABSI incidence rates were studied across time by using a mixed-effects Poisson regression analysis and reported as incidence rate ratios (IRR). As patients could be hospitalized several times and receive multiple CVCs, the model took into consideration a random effect at the individual patient level. All risk factors were assessed first by univariable analysis and variables of clinical interest were included in the multivariable analysis. Potential confounders for adjusted time trend analysis were gender, patient age, Charlson co-morbidity index P-values of less than 0.05 were considered to indicate statistical significance. All statistical analyses were conducted using Stata software, version 12.0 (StataCorp).All-cause mortality was assessed up to day 28 after removal of the last CVC using logistic regression analysis adjusted for gender, patient age, Charlson co-morbidity index, emergency admission, ICU stay, CLABSI, and number of CVCs during hospital stay. Results are reported as odds ratio (OR) and their 95% confidence interval (95% CI). Two-sided Between June 2008 and November 2011, 146 physicians were trained in 36 simulator-based workshops . BetweenA total of 189,643 patients were admitted to our institution during the study period. Among these, 3952 (2.1%) received at least one CVC. A total of 6352 CVCs were placed in 3952 patients during 4452 hospitalizations. CVC characteristics with unadjusted yearly time trends are shown in nd and 4th quartiles  for which a trend was calculated  as the very low incidence reported in the literature by Timsit and colleagues in the intervention arm of a large randomized, controlled study demonstrating the benefit of chlorhexidine-impregnated dressings Our intervention programme addressed catheter care by taking into consideration aspects of behaviour change and key principles of good implementation practice P<.001) and 71%  has been described in the ICU setting, but not in other wards www.carepractice.net) comprehensively addressed every detail of catheter care. The design and structuring prevented the tool from looking overloaded and simplified navigation through the modules. Informal feedback about the tool from healthcare professionals was highly favourable.Simulation-based training of catheter insertion allowed the adoption of evidence-based techniques in a stress-free environment, similar to other reports where it effectively improved knowledge and insertion technique nd quartile of catheter dwell-time (7 to 12 days). CVCs with longer dwell-times accumulated more catheter-days and although more CLABSIs were identified, the incidence density was lower. CVCs with lower dwell-times accumulated less catheter-days, but also had less CLABSIs, which resulted in a lower incidence density as well. Our data suggest that the highest risk is for catheters in place for 7 to 12 days and support the idea of leaving CVCs in place until they are no longer required.Similar to a recent study among haemodialysis patients Crude mortality in our study (17.7%) was higher than in the hospital-wide Thai study (11–12%) Our prevention programme can and should be applied by other hospitals with quality improvement interest; in particular, because the core element of our programme “carepractice.net” is freely accessible. We believe that the concept of combining practical training for a smaller specialized group with e-learning for a larger group can be adapted to other areas in infection control, such as the prevention of catheter-associated urinary tract infection or surgical site infection.Our study has limitations. First, the study was non-controlled and observational, and thus, a regression-to-the-mean-effect may have occurred. However, the baseline incidence density was not unusually high (2.3/1000 catheter-days) for a tertiary-care hospital in a high-income country and the final analysis was adjusted for a large number of potential confounders, such as gender, age, comorbidities, catheter type and site placement, professionals placing the catheter, and variations of dwell-time. Although confounders could be better controlled in a randomized controlled trial, a study addressing behaviour change of entire groups of professionals cannot be conducted in a single centre using such a design. Second, generalisability is limited based on data coming from a single centre. However, the University of Geneva Hospitals is a large institution providing both primary and tertiary care with a broad patient population case- mix. Third, unfortunately, there were no consistent process indicators; this was mainly due to the high workload for catheter surveillance. Although process measures would have added strength to the findings of the study, the results of the multivariable analysis adjusting for a number of case-mix variables and the fact that no technology  was introduced during the four-year study period makes the intervention very likely to be responsible for a large proportion of the positive effect.Our findings suggest that clinically relevant reduction of hospital-wide CLABSI was reached with a comprehensive, multidisciplinary and multimodal quality improvement programme including aspects of behavioural change in a practical manner and key principles of good implementation practice. The content of our training programmes was comprehensive and addressed all single steps in the procedure of CVC insertion and catheter care. In this sense, our strategy is more in-depth than some promoted bundles


MicroRNAs (miRNAs) are small, non-coding, single-stranded RNAs between 18-22 nucleotides long that regulate gene expression. Expression of miRNAs is altered in tumor compared to normal tissue; there is some evidence that these changes may be reflected in the serum of cancer cases compared to healthy individuals. This has yet to be examined in a prospective study where samples are collected before diagnosis.We used Affymetrix arrays to examine serum miRNA expression profiles in 410 participants in the Sister Study, a prospective cohort study of 50,884 women. All women in the cohort had never been diagnosed with breast cancer at the time of enrollment. We compared global miRNA expression patterns in 205 women who subsequently developed breast cancer and 205 women who remained breast cancer-free. In addition within the case group we examined the association of miRNA expression in serum with different tumor characteristics, including hormone status  and lymph node status.Overall, 414 of 1,105 of the human miRNAs on the chip were expressed above background levels in 50 or more women. When the average expression among controls was compared to cases using conditional logistic regression, 21 miRNAs were found to be differentially expressed (P≤.05). Using qRT-PCR on a small, independent sample of 5 cases and 5 controls we verified overexpression of the 3 highest expressing miRNAs among cases, miR-18a, miR-181a, and miR-222; the differences were not statistically significant in this small set. The 21 differentially expressed miRNAs are known to target at least 82 genes; using the gene list for pathway analysis we found enrichment of genes involved in cancer-related processes. In a separate case-case analyses restricted to the 21 miRNAs, we found 7 miRNAs with differential expression for women whose breast tumors differed by HER-2 expression, and 10 miRNAs with differential expression by nodal status.miRNA levels in serum show a number of small differences between women who later develop cancer versus those who remain cancer-free. MicroRNAs (miRNAs) are small, non-coding, single-stranded RNAs ranging in size between 18 and 22 nucleotides; they are typically excised from longer, 60- to 110-nucleotide stem-loop precursors . A singlAberrant expression of miRNAs in cancer was initially identified in B-cell chronic lymphocytic leukemia , and miRmiRNAs have recently been found in human serum and plasma, where they appear to be resistant to RNAase degradation and thus relatively stable, even in stored samples . This stHere, we report on a study that prospectively collected serum samples from 205 women who subsequently developed breast cancer and 205 women who remained cancer-free and that used microarrays to comprehensively assess known miRNAs.The Sister Study  is a pron = 242). We excluded 29 cases who lacked a serum sample or whose sample had integrity issues during collection and shipping and eight cases whose sample had limited volume, leaving 205 cases that are the focus of our study. For each case, a matched control was selected from the 50,884 participants on the basis of the following criteria: no history of cancer (other than non-melanoma skin cancer), having completed enrollment by August 2008, an available blood sample, same race , similar age at enrollment (within 5 years), and similar date of blood draw (within 2 months). Three replicate serum samples from three women  who were not participants in the study but who provided blood samples that were collected and processed in the same manner as Sister Study participants were used to provide technical replicates.We designed a matched-pair nested case control study. We selected patients who had confirmed invasive breast cancer, who completed enrollment by August 2008, and whose diagnosis occurred within 18 months following blood draw  in a manner designed to ensure a balance of these same characteristics. The nine replicates (described above) were assigned to the same batch and chip lot. Laboratory personnel were blind to case control status and other phenotype information.Total RNA was extracted in batches by using a Total RNA purification kit . In accordance with the manufacturer's recommendation not to exceed 200 µL per column, 400 µL of total serum from each individual was split into two equal 200-µL aliquots and then processed separately following the manufacturer's recommended protocol for total RNA purification from serum. An on-column DNase digestion was added before sample elution by using an RNase-Free DNase I Kit , and the two aliquots were subsequently pooled. Fixed volumes rather than fixed amounts of RNA were used in accordance with other studies .Total RNA (8 µL) was directly labeled by using Flash Tag Biotin HSR Labeling kits  in accordance with the instructions of the manufacturer. RNA was heated to 80°C for 10 minutes before labeling to inactivate any residual DNase activity. RNA was hybridized for 42 hours to the GeneChip miRNA 2.0 array . The GeC. elegans miScript miRNA Mimic . Synthetic cel-39 was spiked-in at a final concentration of 0.25 fmol/µL prior to extraction and used as a PCR normalization control. The RNA concentration, reverse transcription, and pre-PCR steps were carried out in accordance with a previously published protocol [An independent set of 10 women were used to validate selected miRNAs via quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Five women who provided consent and blood samples but who developed breast cancer prior to completing enrollment were selected as cases, along with five controls who also provided consent and blood samples and who were cancer-free but did not complete enrollment. Total RNA was extracted from serum samples of these women as described above with the addition of Synthetic protocol . ExoSAP-protocol . Primersgene−Ctnorm).To determine the best candidate miRNA for PCR normalization in our data set, we ran the array expression data from the 47 miRNAs expressed in almost all individuals through the NormFinder software . NormFinmiRNA expression intensity values were background-corrected and normalized across arrays by using the robust multichip average method . The intP value of less than 0.06 was used to identify miRNAs above background. Subsequent analysis was restricted to 414 miRNAs that exceeded background levels in at least 50 women. Conditional logistic regression was used to identify differentially expressed miRNA probes between cases and controls for those 414 probes. Because analysis of circulating miRNAs in prospectively collected samples is still exploratory, we - like some other investigators of circulating miRNAs [P values that are unadjusted for multiple comparisons.For each array, the miRNA probe set signals were compared with the distribution of signals for anti-genomic probes that had matching GC content , and in accordance with the recommendation of the manufacturer, Wilcoxon rank-sum test of g miRNAs ,31 - regThe association between miRNAs and the tumor characteristics of hormone receptor status  and lymph node status was tested in a case-only logistic analysis, in which race was adjusted for. Chip lot and batch were specified as random effect variables. All statistical analyses were performed by using R 2.15.miRNAs found to be significantly associated with case control status were further analyzed with ingenuity pathway analysis (IPA) . Using In = 205) and controls (n = 205) were analyzed in this study; baseline characteristics of the cases and controls are summarized in Table In total, 410 serum samples from breast cancer cases n = 05 and coP ≤0.05) (Table P <0.05). Differential miRNA expression was not stronger in women close to their time of diagnosis, but sample size was small and all cases were diagnosed within 18 months of blood draw (data not shown). Using qRT-PCR on a small independent replication set of five cases and five controls, we further examined the three miRNAs  with the highest expression in cases. As predicted, all three miRNAs showed higher levels in cases, although none was statistically significant in this small set of women  Table . The difTo explore potential biological associations, we ran IPA on the 82 experimentally verified mRNA targets of the 21 differentially expressed miRNAs. Sixteen IPA canonical pathways, including molecular mechanisms of cancer, were enriched as were other cancer-related pathways, including p53 signaling, cyclins and cell cycle regulation, and Myc-mediated apoptosis signaling ; one miRNA was overexpressed and six miRNAs were underexpressed in the HER-2-positive tumors ; five were overexpressed and five were underexpressed in node-positive tumors  [miR-18a, miR-181a, and miR-222 showed the highest percentage difference between cases and controls in our study; qRT-PCR of these miRNAs in a small independent replication set of cases and controls, though not statistically significant, replicated the direction of change for all three. These three miRNAs have been suggested to act as oncogenes through regulation of their potential target mRNAs. miR-18a is part of the oncogenic miR 17-92 cluster, which is often overexpressed in solid tumors, including breast . OverexpPDCD4) , which iPDCD4) . In breaPDCD4) . miR-222PDCD4) , and expPDCD4) .Interestingly, two recent case control studies have provided evidence that both miR-222 and miR-181a are overexpressed in the serum of patients with breast cancer. One used sequencing by oligonucleotide ligation and detection (SOLiD) of serum samples obtained prior to surgery from 13 breast cancer cases compared with samples from 10 healthy controls and found 26 miRNAs that were overexpressed in cases, including miR-222 and miR-181a; overexpression of miR-222 was validated in an independent group of 50 cases and 50 controls by using qRT-PCR . A seconAmong cases, we compared the serum miRNA profiles of women with different tumor characteristics, including hormone status  and nodal status. Although there were no differences in ER or PR status, there were differences in HER-2 and lymph node status. Of the seven miRNAs differentially expressed in the serum of women who developed HER-2-overexpressing breast tumors, miR-93, miR-183, and miR-29a have been reported to be associated with breast cancer in previous studies ,43,44. IAlthough miRNAs that are differentially expressed between tumor and normal tissue are more frequently downregulated in tumor tissue , our stuWe find some evidence of differences in miRNA serum levels between women who subsequently developed cancer compared with women who remained clinically cancer-free. The magnitude of these differences is small, and this may limit their clinical application as circulating early-detection markers for breast cancer. This is the first study to use prospectively collected samples; limitations include a relatively short follow-up period and a sample size that is not large enough to fully explore etiologic versus diagnostic relevance. Our study was carried out within a cohort of women who each had a sister with breast cancer, putting the former at about twofold increased risk, and so the differences that we observed may not be generalizable to women without a similar family history.ER: estrogen receptor; HER-2: human epidermal growth factor receptor 2; IBC: inflammatory breast cancer; IPA: ingenuity pathway analysis; miRNA: microRNA; ncRNA: non-coding RNA; PCR: polymerase chain reaction; PR: progesterone receptor; qRT-PCR: quantitative reverse transcription-polymerase chain reaction.The authors declare that they have no competing interests.ACG carried out sample extraction and sample preparation, did some statistical analysis, and helped to design the experiments, interpret the results, and write the manuscript. ZX performed the majority of the statistical analysis. CRW and PAW participated in study design. LDR participated in study design and provided patient data and study variables. DPS participated in study design and collected samples and data. RCG provided study reagents and processing as well as critical advice on study design, QA, and data interpretation. JAT helped to design the experiments, interpret the results, and write the manuscript. All authors read and approved the final manuscript.Spearman correlation coefficient values for technical replicates of arrays. Three replicate serum samples from three women  were processed and hybridized to arrays as described for samples in the main study. Spearman correlation coefficients were calculated for the three pairings of replicate samples for each woman and averaged. One array from Individual 1 appeared to be an outlier but was included in the results shown above. Exclusion of this array resulted in correlation coefficients of greater than 0.97 in all three categories of probes for Individual 1.Click here for fileEfficiency of the four polymerase chain reaction (PCR) assays. The efficiency of PCR amplifications for the normalization control and three target microRNAs (miRNAs) was calculated by using DART-PCR version 1.0. The average efficiency of each of three independent PCRs was similar across all four miRNAs.Click here for fileQuantitative reverse transcription-polymerase chain reaction (qRT-PCR) validation in 10 cases and controls. Serum levels of five cases and five controls were examined by using qRT-PCR for miR181a, miR18a, and miR-222. Box plots show normalized relative expression. Normalization was carried out by using the mean of miR-1825 and spiked in cel-39. The horizontal line represents the mean for each sample.Click here for fileExperimentally observed targets enriched for cancer and signaling pathways. Identified ingenuity pathway analysis (IPA) canonical pathways enriched by the experimentally observed targets of the 21 miRNAs differentially expressed between the cases and non-cases. The negative log (10) false discovery rate-corrected P values are shown. Note that this test has not corrected for possible dependencies across the mRNAs considered and that statistical significance may be overstated. HER-2, human epidermal growth factor receptor 2; ILK, integrin-linked kinase; PTEN, phosphatase and tensin homolog.Click here for fileList of differentially expressed serum microRNAs (miRNAs) based on HER-2/NEU expression.Click here for fileList of differentially expressed serum microRNAs (miRNAs) based on nodal status.Click here for file