Copy number variants (CNV) can be called from SNP-arrays; however, few studies have attempted to combine both CNV and SNP calls to test for association with complex diseases. Even when SNPs are located within CNVs, two separate association analyses are necessary, to compare the distribution of bi-allelic genotypes in cases and controls (referred to as SNP-only strategy) and the number of copies of a region (referred to as CNV-only strategy). However, when disease susceptibility is actually associated with allele specific copy-number states, the two strategies may not yield comparable results, raising a series of questions about the optimal analytical approach. We performed simulations of the performance of association testing under different scenarios that varied genotype frequencies and inheritance models. We show that the SNP-only strategy lacks power under most scenarios when the SNP is located within a CNV; frequently it is excluded from analysis as it does not pass quality control metrics either because of an increased rate of missing calls or a departure from fitness for Hardy-Weinberg proportion. The CNV-only strategy also lacks power because the association testing depends on the allele which copy number varies. The combined strategy performs well in most of the scenarios. Hence, we advocate the use of this combined strategy when testing for association with SNPs located within CNVs. Different types of variations occur in the human genome, ranging from single nucleotide base changes to copy changes of entire chromosomes . Single nucleotide polymorphisms (SNPs) are single base changes of the DNA sequence that cover less than 1% of the human genome. Structural variations are larger polymorphisms that involve contiguous sequences of nucleotides . CNVs arMany SNPs have been shown to contribute to a range of complex diseases involving both genetic and environmental factors through complex mechanisms. Although the role of common CNVs in human diseases has been less investigated, their contribution to complex diseases is probably substantial. Indeed, CNVs often include important functional elements of the DNA such as genes ,4. In adThe recent developments of SNP genotyping technologies have resulted in the large scale analysis of genotypes in genome-wide associations (GWAS), which have identified over 1200 new loci associated with human diseases or traits ,9. Data Given that CNVs are distributed across the human genome, a significant proportion of the SNPs are located in CNVs. While it is biologically plausible that both number of copies and the actual alleles could play a role in disease susceptibility ,15, veryThe objective of this work is to assess the performances of classically used statistical models to estimate the effect of the allele or the number of copies for a disease susceptibility SNP, when it is located within a CNV. The performances of various strategies to simultaneously test for both effects are investigated by simulations of allele-specific copy number states  in different scenarios of genotype frequencies and inheritance models and compared on real data from HapMap.allele and RRCN be respectively the relative risks for the disease associated to one extra B allele and one extra copy number. Using this model, we simulated allele-specific copy number states in 1,000 cases and 1,000 controls, as well as in 5,000 cases and 5,000 controls to see how results were affected by the sample size. These allele-specific copy number states were either analyzed directly or after condensing to bi-allelic genotypes  (Allele (multi) or Allele (bi) strategies), a trend effect of the number of copies (the CN strategy), or both allelic and copy number effects (the Joint and Codominant strategies) (Allele (multi), CN, Joint and Codominant strategies), or the bi-allelic genotype information inferred from it (the Allele (bi) strategy) . All the strategies used a likelihood-ratio test (LRT) and their null hypotheses are shown in We modeled disease susceptibility across a genomic region in which deletions and duplications occur independently with respective probabilities f(del) and f(dup) to give rise to 5 possible copy-number states . In each genomic region, we assumed there was a SNP with two alleles A and B with allele B conferring an increased risk for the disease under study. We assumed that the effects of the B allele and the copy-numbers were multiplicative and we let RRmissing) . Five asategies) . The strFirst, we focused on the situation in which the full information on allele-specific copy number states was available for both cases and controls. This implied that these states were reconstructed from the observed Log R ratio and B allele frequencies using a CNV calling algorithm. For the current model, we assumed here that these reconstructions were unambiguous but we recognize that this is not generally the case on real data as discussed below.allele=1 and RRCN=1), under most of the scenarios investigated, type-one error rates were equal to their nominal values (Codominant strategy) with a low frequency of duplications. This could be due to the high number of degrees of freedom and the low number of observations for some allele-specific copy number state categories, but the inflation was small (the maximum type-1-error was 7.4% instead of 5%).When case-control data were simulated under the null hypothesis of no effect of the allele or of the number of copies . In these situations the most powerful strategies to detect the association were, respectively, the Allele (multi) and CN strategies that were also the ones best fitting the simulated model of association. In the dataset of 1,000 cases and 1,000 controls, the power was maximum (100%) for the relative risks of 1.5 and above. This was true when both deletions and duplications were present (Joint and the Codominant strategies) performed well and the loss in power compared to the best strategy was relatively low. This was especially true for the Joint strategy relative power in comparison to the most powerful strategy  strategy when RRallele>1 and RRCN=1) was always greater than 0.76. Its relative power even reached 1 when the simulated relative risks, RRCN and RRallele respectively, were greater than 1.5 (Allele (multi) strategy when copy-number effects were simulated and the CN strategy when B allele effects were simulated) were the less powerful but interestingly the power was not null, thus detecting part of the other effect. This was due to the fact that when a trend on the copy-numbers was simulated, individuals with 4 copies of the B allele were at higher risk than individuals with 3, 2, 1 or 0 copies, and inversely, when a trend on the number of B alleles was simulated, individuals with 4 copies were at higher risk than individuals with 3, 2, 1 or 0 copies. Thus the estimations provided by the CN and Allele (multi) strategies are biased , the most powerful strategy was always the Joint strategy testing for both effects, regardless of the strengths of the effects, the frequencies of both the B allele and the copy numbers, and the sample size . This pattern was observed when both deletions and duplication were simulated . Thus, we concluded that the best strategy showing the higher performances over all the investigated scenarios was the Joint strategy.When a combined effect of the number of copies and of the number of B alleles was simulated  strategies are estimates of the marginal risks of the number of copies and of the allele respectively and are thus biased estimates of the effect sizes  was available as it is the case when investigators are interested in testing for association only with SNPs. To reconstruct the bi-allelic genotype information from the allele-specific copy-number states, we needed to evaluate the probabilities, given the allele-specific copy-number states, of the different bi-allelic genotypes. To estimate these probabilities, we used empirical data from the Spanish Bladder Cancer (SBC) / EPICURO study  showing that the SNP callings were almost never ambiguous when the number of copies was 1 or 2 and without almost missing data whereas the callings were really messy when the number of copies was 3, 4 or 0 with very high missing rates . InteresAllele (bi) strategy, showed lower performances in comparison to the Allele (multi) strategy that tested for the same effect but using the allele-specific copy number states. This could be seen when both deletions and duplications were present , and regardless of the sample size, the power of the Allele (bi) strategy was null, meaning that the strategy could not detect the effect of the number of copies while the Allele (multi) strategy could (see the first columns of the Allele (bi) strategy had a tendency to decrease when the B allele frequency increased. This was due to the fact that a significant protective effect of the B allele was detected when the B allele frequency was low (data not showed). Regarding the effect estimation, we could observe that the effect of the B allele was under-estimated in most of the investigated scenarios, especially when deletions were simulated. Inversely, in the scenarios where duplications were simulated and the frequency of B was higher than 20%, the effect of the B allele tended to be overestimated (data not shown).Because the bi-allelic dataset contained partial information on the allele and a high rate of missing genotypes, the association test of the effect of the B allele based on this information, evaluated through the  present , but als present , and dupJoint strategy. This power loss was higher when the sensibility was lower and when the CNV types were deletions only. Interestingly, in the presence of both deletions and duplications, even with a sensitivity for CNV detection as low as 0.2, the power was still higher than the power of the Allele (bi) strategy using the bi-allelic genotypes in a SNP only analysis. When there were deletions only, depending on the sensitivity for CNV detection, the SNP only analysis using the Allele (bi) strategy could however perform better than the Joint strategy , the Allele (bi), and the CN strategies respectively , and the Allele (bi) strategies except for 10 and 13 SNPs with a significant ORallele in the Joint strategy that were not detected with the Allele (multi) and with the Allele (bi) strategies respectively. Both the Allele (multi) and the Allele (bi) strategies detected 2 SNPs that did not reach the Bonferroni-corrected significance level for the Joint strategy but were close to significance. The overlap in the results obtained using the Joint and the CN strategies was poor as shown in CN were very different. A striking example is SNP_A-8568797 with an estimated ORCN of 0.42 with the CN strategy and 6.73 with the Joint strategy (CN strategy). However, it is the opposite when looking within strata with a fixed number of A or B alleles and with allelic variation, i.e. within {A, AB, ABB}, {B, AB} and {BB, ABB} strata. In these strata, cases have more copies than controls (this explains the result obtained for the Joint strategy).Finally, we applied the different methods on real CNV data from the HapMap project on chromosome 22 in 113 CEU individuals and 127 Yoruba . Based on the frequency of the allele and of the abnormal number of copies, we retained a total of 136 SNPs for the analysis . These SNPs appeared to be located in 3 copy-number regions in which the individual CNV breakpoints could be slightly different. Among these SNPs, 98, 90, 87 and 16 gave a significant result  with the ectively . If we cstrategy . This imIn this work, we investigated various strategies for association testing when a SNP is located within a CNV region. We simulated allele-specific copy number states for two different sample sizes of cases and controls , as well as the corresponding bi-allelic genotypes obtained in a classical SNP analysis in which all individuals are assumed to have two alleles. We considered a wide range of scenarios for both copy numbers and allele frequencies in relation to the tested strength of the association signal. We compared association testing strategies, either analyzing the effect of the allele, the effect of the number of copies, or both effects, either using the complete information of the alleles and the number of copies contained in the allele-specific copy number states or using the partial information on the allele only contained in the bi-allelic genotypes.We found that, using the complete information of allele-specific copy number states, the model that performed best over all the considered scenarios was the one testing simultaneously for both the number of copies and the allele effects through two terms, the sum and the difference of the two allele counts, a model previously proposed by Korn et al., 2008 . We showWe also observed that testing the effect of the allele using partial information on the allele state contained in the bi-allelic genotypes led to a small loss of power in comparison to testing the effect of the allele using the complete information of allele and copy number contained in the allele-specific copy number states. This was true under most of the investigated scenarios. The B allele effect was not correctly estimated but was either under or over-estimated depending on the frequencies of deletions and duplications, respectively. Additionally, and on the basis of real data, we observed that, for a SNP located in a CNV region, the missing rate for bi-allelic genotypes was often quite high, and there were significant departures from Hardy-Weinberg equilibrium. Thus, and as previously mentioned , SNPs loIn this paper, we investigated various possibilities for common CNV frequency, from high (66%) to relatively low (20%), including various types of CNV, either deletions only, duplications only or both deletions and duplications. We also investigated a large range of possibilities for the SNP allele frequency, from rare (5%) to common (50%). Regarding the pattern of association, we were interested in both the effect of the copy number and the effect of the allele, thus, we simulated these two effects combined in an multiplicative manner, considering for each effect, the null effect of no association and various strengths of association, from small (relative risk of 1.2) to high (relative risk of 2). We believe these scenarios cover a wide range of possible situations. However there are also some limitations in the models we considered. In particular, we made the assumption that for a given allele-specific copy number state, for example BBB, the risk was the same for all possible combination of state on the two chromosomes . Further work will be needed to investigate more complex scenarios and taking into account the repartition of the alleles across the two chromosomes, but this was beyond the scope of this paper.Joint strategy was found to give more significant results than the Allele (multi) and Allele (bi) strategies, and few significant results were obtained with the CN strategy. It is true however that the Joint strategy can only be performed on a small proportion of the SNPs as it required that the studied SNPs fall within a common CNV. Of the 11,307 SNPs located in chromosome 22 in the HapMap data, only 136 were retained. It is thus difficult to compare the power of the classical test of association that will be performed on all the SNPs and the one of the Joint test that will only be performed on such a limited number of SNPs and will thus require a less stringent multiple testing correction. For these SNPs however that fall in common CNV and could be detected by using the Joint strategy, there is a risk that their effect on the disease will not be evidenced by the classical test and there is thus a gain of using the proposed strategy. Moreover, in the presence of both an effect of the number of copies and of the allele, effects will not be well estimated by the tests that only consider one of the effects. This was clear from the simulated and was also illustrated on the HapMap data where some SNPs showed very different ORCN estimates with the Joint and the CN strategies, supporting the importance of considering jointly the effect of the allele and of the number of copies.By applying the studied strategies on HapMap chromosome 22 data, we were able to identify SNPs with numbers of copies and/or of the allele frequencies that varied between the CEU and Yoruba populations. Consistent with the simulations results, the Joint strategy using allele-specific copy number states is more powerful than performing a SNP-only analysis that does not take into account the total number of copies. With the availability of sequence data and the development of efficient algorithms to call allele and copy number with increasing accuracy, investigators are likely to become more and more interested in testing both effects and this work could then provide some insights into the statistical methodology to use.The calling of allele-specific copy number states needs a previous identification of the individual CNV regions to identify the total number of copies at each SNP. This CNV calling can be unreliable as shown by the high discordance rates observed in duplicates . NeverthWe considered a SNP with two alleles A and B, B being the susceptibility allele, located within a CNV region that can be present in a variable number of copies, from 0 to 4. We simulated the allele-specific copy number states for this SNP for a set of 1,000 cases and 1,000 controls, as well as for a set of 5,000 cases and 5,000 controls, with various scenarios of genotype frequencies and inheritance models. Each scenario was replicated 10,000 times.The frequency of each allele-specific copy number state in the control population was computed from the frequencies of each possible number of copies, from 0 to 4, and the frequencies of each possible allele, A and B . On the CN for the risk associated to an increase of 1 copy (reference is CN=2), and RRallele for the risk associated to an increase of 1 B allele . Assuming that the allele and copy number effects were multiplicative, we derived the RR for each allele-specific copy number state as detailed in The frequencies of the allele-specific copy number states in cases were calculated given the allele-specific copy number state frequencies in controls, and the relative risks (RRs) of the allele-specific copy number states versus the reference genotype see . We chosIn order to investigate the impact of performing a classical SNP analysis in which only information on bi-allelic genotypes or missing genotypes were available, we reconstructed from the simulated allele-specific copy number states, the bi-allelic genotypes. To estimate the probability of the different bi-allelic genotypes  given an allele-specific copy number state, we used real data from the Spanish Bladder Cancer (SBC) / EPICURO Study with more than 2000 individuals genotyped with the Illumina 1M array , and forJoint strategy. In this analysis, we investigated 4 scenarios, the CNV type being either deletion only or both deletion and duplication, the frequency of normal chromosome carrying one copy of the CNV being either 0.33 or 0.8, the frequency of the B allele being 0.2 and the relative risks being 1.2.Because the CNV detection sensitivity of SNP arrays is low, we also simulated allele-specific copy number states in which CNV calling errors were introduced. Based on previous works, we specified a sensitivity of 0.2 or 0.5 and a specificity of 0.99 for CNV detection ,14. For Allele (multi) strategy) or the bi-allelic genotypes (Allele (bi) strategy). Second we tested for the effect of the number of copies only assuming a trend on the number of copies. We used a logistic regression model with the number of copies as a continuous variable (CN strategy). Finally, we used two logistic regression models that took into account the full information of allele and number of copies contained in the allele-specific copy number states. The first one was proposed by Korn et al. 2008 [Joint strategy). The two terms of the model are the sum and the difference of the number of copies of each allele. The second model is a general co-dominant model assessing the effect of each allele-specific copy number state (Codominant strategy), and testing for the association using a LRT whose the number of degrees-of-freedom depends on the number of observed allele-specific copy number states. In the Joint strategy, we then focused on both coefficients to detect an eventual bias in the estimations of the allele and copy number effects. For each scenario and each association strategy, the power was calculated as the percentage of replicates for which the p-value was lower than 0.05.Allele and copy number effects were tested by different association strategies . All theal. 2008  and testhttp://www.r-project.org).Simulations and statistical analysis were performed with R version 2.13.0 (Joint strategy and estimate odds ratios (Script S1). We also provide R functions to perform simulations and compare strategies (Script S2).We provide some R functions to analyze case-control data with the http://www.hapmap.org) and annotated as “included”, meaning the data were of good quality. We included 118 CEU individuals and 120 YOR individuals. The SNP calling was performed using Birdseed [http://www.openbioinformatics.org/penncnv/) [Joint strategy implemented in Script S1, as well as the CN, the Allele (multi), and the Allele (bi) strategies were run using the population as phenotype in the association analysis . The Joint, the CN and the Allele (multi) strategies were run on each of the 136 SNPs considered for this analysis, whereas the Allele (bi) strategy was run on a subset of 122 SNPs for which less than 5% of the genotypes were missing in the sample HapMap data. Bonferroni correction for multiple testing was applied for each strategy according to the respective number of tests. The Joint and the Allele (multi) strategies were corrected for 136 tests. The Allele (bi) strategy was corrected for 122 tests. Because the numbers of copies are highly correlated between SNPs, we applied a method similar to the method proposed by Li and Ji, 2005 [CN strategy. The original method was based on the eigenvalues of the matrix of genotype correlation at the different markers. Here, we consider the matrix of correlation of the number of copies for the 136 SNPs instead. The effective number of independent test was estimated to be 16 and a Bonferroni correction for 16 tests was thus applied for the CN strategy.We used publicly available genetic data from unrelated HapMap individuals from two populations, the CEU-CEPH population (CEU) and the Yoruba population (YOR). The genetic data were generated with the Affymetrix 6.0 SNP-array. We considered the individuals whose CEL files were available for download on the HapMap website  strategies.Coefficients estimated in the (PDF)Click here for additional data file.Text S2Joint strategy.Coefficient estimated in the (PDF)Click here for additional data file.Text S3Calculation of the allele-specific copy number state frequencies in cases.(PDF)Click here for additional data file.Figure S1Type-1-errors.Type-1-errors were estimated under the null hypothesis of no association, for the 36 investigated scenarios of frequencies, when simulating 1,000 cases and 1,000 controls (first row), and when simulating 5,000 cases and 5,000 controls (second row).(PDF)Click here for additional data file.Figure S2Summary of the results of the association analysis on HapMap data.This flowchart summarizes the results obtained by applying the Joint, the CN, the Allele (multi) and the Allele (bi) strategies to the chromosome 22 HapMap data.(PDF)Click here for additional data file.Figure S3Manhattan and CNV plots of the association analysis on HapMap data.The -log10 of the p-values of the Joint (panel A), the CN (panel B), the Allele (multi) (panel C) and the Allele (bi) (panel D) strategies are reported for the probes located in the 3 copy-number variant regions identified on chromosome 22 in the HapMap data. Panel E displays the detected CNVs in these 3 regions in the HapMap individuals used in the analysis - CEU as cases and YOR as controls - deletions are displayed in red and duplications in green.(PDF)Click here for additional data file.Table S1Joint strategy.Relative power of the For each of the 36 frequency scenarios investigated, this table displays the relative power of the Joint strategy, first relatively to the CN strategy when there is only and effect of the number of copies, and second relatively to the Allele (multi) strategy when there is only and effect of the allele.(PDF)Click here for additional data file.Table S2Joint model.Estimations of the coefficients and the odds ratios using the This table displays the means and the mean squared errors (MSE) of the Joint model coefficient estimates and of the odds ratios for the allele and copy number effect after transforming the Joint model coefficients, computed for each of the 16 scenarios of association strengths, over 360,000 replicates  considering a sample of 1,000 cases and 1,000 controls.(PDF)Click here for additional data file.Table S3Bi-allelic genotype probabilities.(PDF)Click here for additional data file.Table S4Results of the association analysis on HapMap data.This table displays the results of the Joint, the CN, the Allele (multi) and the Allele (bi) strategies applied to HapMap data, restricting the analysis to the chromosome 22. The SNPs excluded from the Allele (bi) strategies because of a missing rate greater than 5% have no results for this strategy (displayed with “-“).(XLSX)Click here for additional data file.Table S5Details of the computation of the allele-specific copy number states frequencies and relative risks.The notations f(del), f(norm) and f(dup) refer to the frequencies of chromosomes carrying respectively 0, 1 and 2 copies of the CNV, f(B) is the frequency of the allele B. RRallele|CN and RRCN|allele are the relative risks associated respectively to an increase of one allele B, and an increase of one copy.(PDF)Click here for additional data file.Table S6Expected allele-specific copy number states frequencies.The frequencies are displayed for each of the 36 frequency scenarios investigated.(PDF)Click here for additional data file.Script S1Joint strategy.R scripts implementing the computation of the allele-specific copy number states and the association analysis using the This compressed file contains 1- an R script (AssociationSNPinCNV.R), 2- a manual describing the arguments and values of the R functions implemented in AssociationSNPinCNV.R , 3- a tutorial  and input files  as an example to run the functions implemented in the R script AssociationSNPinCNV.R.(ZIP)Click here for additional data file.Script S2R scripts to simulate and analyze allele-specific copy number states and bi-allelic genotypes for datasets of cases and controls.This compressed file contains 1- an R script (ASCNsimulationsAndAssociationStrategies.R), 2- a manual describing the arguments and values of the R functions implemented in ASCNsimulationsAndAssociationStrategies.R , 3- a tutorial as an example to run the functions implemented in the R script ASCNsimulationsAndAssociationStrategies.R .(ZIP)Click here for additional data file.


Indeed, it is one of the most exceptional examples of convergent evolution in the history of life. The repeated and rapid evolution of Kranz-type C4 suggests that it may be a derivative of a conserved developmental pathway that is present in all angiosperms. Here, I argue that the Kranz-type C4 photosynthetic system is an extension of the endodermis/starch sheath, that is normally only found in the roots and stems, into photosynthetic structures such as leaves. Support for this hypothesis was recently provided by a study that showed that the same genetic pathway that gives rise to the endodermis in roots, the SCARECROW/SHORT-ROOT radial patterning system, also regulates the development of Kranz anatomy and C4 physiology in leaves. This new hypothesis for the evolution of Kranz-type C4 photosynthesis has opened new opportunities to explore the underlying genetic networks that regulate the development and physiology of C4 and provides new potential avenues for the engineering of the mechanism into C3 crops.Kranz-type C A new revolution in agriculture is needed to keep pace with the demands of humanity in the next century . More huCrop breeding and biotechnology in the last century have altered plants in many drastic ways. Breeders were able to increase the harvest index, growth rate, biomass accumulation, disease and pest resistance, biotic and abiotic stress tolerance, and nutrient use efficiency, while also extending the climatic range of crops into previously unproductive regions . However3 crops, by introducing the C4 photosynthetic mechanism, could be an effective way to increase yields by boosting productivity per unit area of land as well as reducing the amount of water and nitrogen used in achieving those yields , Nicotinamide adenine dinucleotide-malic enzyme (NAD-ME), or phosphoenolpyruvate carboxykinase (PEPCK) types  types . HoweverK) types . Within K) types . However3 and C4 plants, vein patterning is regulated by auxin gradients generated by both synthesis and transport  and Short-root (SHR), are expressed in roots, stems, and leaves  mutant of maize also supports the endodermal development model for C4 BS in leaves. The lsn mutant phenotype mimics the abnormalities observed when auxin transport inhibitors are applied to developing leaves . What is interesting in the lsn mutant is the formation of normal BS and M, both structurally and physiologically, around the distorted vascular core in the leaves , and both BS and M plastids appear normal in transmission electron microscopy (TEM) analysis . This finding conflicts with the cell lineage models that have previously been proposed for the development of the C4 BS which suggested that BS and M cells arose from organized cell division patterns , the founding member of the gene family, results in altered growth and flowering time  domains , which h4 in monocots appears to be ancient, arising with the grasses and sedges as they began to diverge from the other monocots ? This morphological shift reduces many of the dicot leaf blade anatomical features, the most profound being the elimination of reticulate vein patterning. The formation of parallel veins in monocots is presumed to be derived from the merger of two sides of a previously radial-organized veins in the stems and petioles  are usually separated by one or many layers of non-photosynthetic parenchyma cells . The surrounding photosynthetic layers, one on either side of the leaf, start to invade the region of the central vascular strands . This anatomical adjustment would also bring the outer photosynthetic layer of cells in direct contact with the endodermis/starch sheath, allowing the two programs to interact. Thus, the morphological shifts that lead to the emergence of the grasses and sedges could also have been the events that pre-conditioned Kranz-type C4 within these clades.However, most of the monocots utilize the C strands , most li3 instead of C4? Under the phyllode theory of monocot evolution, the ancestors of rice may have been pre-conditioned for C4 metabolism in the same manner as other C4 grasses and sedges. However, it is important to remember that when compared to C3 photosynthesis, the C4 mechanism is energetically more expensive. It takes 18 ATP to fix one CO2 molecule in the C3 mechanism and 30 ATP in the C4 system  transgene were grown, prepared, and visualized as described in The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In the treatment of clavicle fractures, the choice of procedure depends on the possibility of restoring the anatomical functional integrity of the shoulder.We examined 71 patients  who were affected by clavicle fracture sequelae. Demographic and clinical data and the site of the lesion were recorded for each partecipant. The dissatisfaction of the patient was determined by the presence of 1 or more affirmative answers on the Simple Shoulder Test. The Constant Shoulder Score was also included in the functional and clinical exams. We measured the length of the healthy clavicle and the previously fractured clavicle, and we expressed the difference in length in mm and in percentage shortening. We then examined the correlations between the shortening of the bone and the clinical and functional outcomes of the patients.Sixty patients had a lesion of the diaphysis, 8 patients had a lesion of the lateral third of the clavicle, and 3 patients had a lesion of the medial third of the clavicle. The mean Constant Shoulder Score was 77.9, and 51 of the 71 patients were satisfied with their treatment. Radiography showed a mean clavicle shortening of 10 mm (mean percentage 6.5%). In the 20 dissatisfied patients, the mean clavicle shortening was 15.2 mm (9.7%). In these patients, we found a highly significant association between dissatisfaction with treatment and the amount of bone shortening, (p < 0.0001), as well as with a diaphyseal location (p < 0.05) and with the female sex (p = 0.004). No other variable related to the patient, the type of treatment or the fracture characteristics correlated with the treatment outcome.In the literature, measurements of the shortening of the bone segment following a fracture range between 15 and 23 mm, and marked shortening is correlated with the failure of conservative treatment. However, these data need to be reinterpreted in light of the physiological variability of the clavicle length, which ranges from 140 to 158 mm in the healthy population. Shortening of the bone by more than 9.7% should be the cut-off for predicting failure of conservative treatment. Optimal shoulder function is achieved by the interaction of four joints  working in biomechanical harmony. It is impossible to intervene at one joint without affecting the efficiency of the other three . The firFractures of the clavicle are common, comprising up to 5% of all skeletal lesions in adults . ClaviclMean post-traumatic shortening the fractured clavicle is approximately 1.2 cm, but a reduction of up to 3 cm has been described . In the The aim of our work was to evaluate shoulder function and patient satisfaction in relation to the shortening of the fractured segment in patients suffering a clavicle fracture that was treated conservatively with the figure-of-eight bandage.This clinical retrospective study included patients who were undergoing conservative treatment with a figure-of-eight bandage for a clavicle fracture during the period from 2004 to 2010 at our Operative Unit of Orthopaedics and Traumatology. All of the patients received information about the aims of the study and signed an informed consent form prior to undergoing the clinical and diagnostic tests that were included in the study protocol. The study was authorised by the Ethics Committee of the General Hospital of Bari and was performed in accordance with the ethical standards of the 1964 Declaration of Helsinki.Inclusion criteria were as follows:- a blunt, uncomplicated clavicle fracture with a single focus, which was treated within three days of diagnosis- conservative treatment with a figure-of-eight bandage (FEB)Exclusion criteria were as follows:- bilateral clavicle fractures- a previous clavicle fracture or pseudoarthrosis- an exposed clavicle fracture or associated fracture of the coracoid, an acromioclavicular or sterno-clavicular luxation, floating shoulders, lesions of the plexus and concomitant fractures in other sites- the presence of disease impairing the function of either shoulderNinety-three partecipants met the inclusion criteria. Twenty-two patients decided not to participate because of travel or the time that was required to attend the clinical exam; therefore, a total of 71 participant participated in this study.All of the patients were clinically reviewed, and the following were considered:- demographic  and clinical data  and history - the site of the lesion  according to the 1988 Edinburgh classification  in which- a self-administered questionnaire defining one or more affirmative answers on the Simple Shoulder Test as unsatisfactory, which verifies the presence of resting pain, aesthetic deformity, pain when leaning on the affected shoulder, differences in the range of movement and force as compared to the contralateral side and the loss of functional autonomy of the upper limb in daily activities ,10-13- the Constant Shoulder Score, which assesses pain, degree of function, ROM and muscular force - an AP chest X-ray with a specific clavicle projection that is obtained by cephalic angling of the tube at 45° to avoid superimposition of the clavicle and ribs at a 1:1 scale Successful consolidation of the fracture was determined by the formation of a bone callus and the presence of a trabecular bridge over the fracture gap at the periosteal and endosteal level within 6 months of the trauma . After cQuantitative variables were expressed as means and standard deviations (SD); categorical variables were expressed as frequency distributions. A t-test was used to compare the age difference between male and female patients, the Simple Shoulder Test results and the difference in follow-up time between satisfied and dissatisfied patients. The Wilcoxon test was used to compare the age and the Constant Scores of satisfied and dissatisfied patients. To assess the differences in frequency distributions, the Chi-square test was employed. To reveal correlations between shortening of the limb , a logistic regression model was applied to the Constant Scores and R2 was calculated. A multivariate logistic regression was used to analyse factors that were associated with satisfied patients.Significance was set at p < 0.05; Fisher Exact test was used when appropriate. Data processing was performed with Epi-Info 6.00 software .A total of 71 eligible patients who were affected by clavicle fractures and were treated within 72 hours of the trauma were recruited. All of the patients had been treated with FEB for a mean of 28 days  and had done hanging-type exercises and active and passive joint ROM for two weeks after the removal of the bandage. No patient presented with pseudoarthrosis complications, lesions of the brachial plexus or thoracic outlet syndrome.The study sample included 51 males (71.8%) and 20 females (28.2%). The mean age was 38.9 years (SD = 13.3), 37.2 years (SD = 10.9) for females and 39.6 years (SD = 14.1) for males . The fracture occurred on the clavicle of the dominant arm in 47.9% of the patients (n = 34), 45.1% of the males (n = 23/51) and 55% (n = 11/20) of the females .In 59 patients, the lesion was at the level of the diaphysis 83.1%). Three (4.2%) patients had a lesion of the medial site, and 9 (12.7%) patients had a lesion of the lateral segment. There was not a significant difference in the distribution of the fracture site between genders  declared that they did heavy physical work or a considerable amount of sport, 22 (30.9%) led a sedentary life, while no type of physical activity was recorded for 18 patients (25.3%).The mean follow up time was 32.7 months  with no significant difference between males and females . The mean Constant Score was 77.9  and was lower in males  than in females . The mean clavicle shortening was 10 mm , 10.7 mm (SD = 5.3) in males and 8.2 mm (SD = 3.7) in females ; the mean percentage was 6.5% , with no significant difference between males  and females . Twenty-two patients (30.9%) had a shortening of the clavicle that was less than 5%, 39 patients (54.9%) had shortening between 5 and 10% and 10 patients (14.1%) had shortening greater than 10%.According to the criteria described above for the Simple Shoulder Test, 51 partecipants (71.8%) were satisfied with the conservative treatment, and 21 (29.6%) patients were dissatisfied. There were no significant differences in follow up time  or in the mean age between dissatisfied and satisfied patients   reported dissatisfaction as compared to men 19/51; 37.2%). For this comparison, Chi-square = 7.4 and Fisher exact p = 0.0004  table .The percentage of dissatisfied patients was 25.6% among the patients with a percentage of clavicle shortening of 5-10% and 100% among the patients with a percentage of clavicle shortening that was greater than 10%; none of the patients with a clavicle shortening of less than 5% were dissatisfied .In satisfied patients, the mean bone segment shortening was 7.9 mm (5.3%) versus 15.2 mm (9.7%) in patients who complained of functional dissatisfaction Figures .We also performed a multivariate logistic regression analysis using "patient satisfied" as the dependent variable table .The Constant Score was correlated with the shortening value expressed both in mm  or a triangular bandage to restore the retro-positioning of the shoulder, resolving the superimposition of the stumps and limiting clavicular shortening. Surgery is usually reserved for the treatment of exposed lesions or highly displaced fractures to stabilise the bone malalignment with the use of screws, plates, endomedullary wires or bands .Previous studies have analysed the risk of dysfunction after conservative treatment, which can be due to severe shortening of the bone segment, residual bone deformity, loss of force and persistent pain ,9. For tSome studies have observed fewer cases of consolidation defects after surgery (2.2%) as compared to conservative therapy (15.1%) , whereasAccording to the literature, the incidence of failure of conservative treatment of clavicle fractures ranges from 4.4% to 31% in terms of pain, loss of force, rapid fatigue, paresthesia, pain when lying on the affected shoulder and aesthetic defects ,25. In oNeer suggests a statistical association between the degree of shortening of the bone segment and poor clinical results, with an increased risk of evolution to pseudoarthrosis ; in our There is still no consensus in literature as to whether conservative treatment of clavicle fractures is the optimal treatment in most cases  or if suThe results of the present study demonstrate poorer outcomes when the fracture occurs at the midshaft and when the shortening of the bone segment is more than 9.7% as compared to the original length. We also observed a greater degree of dissatisfaction in male patients.However, our study has limitations. The study sample is relatively small, and there is no surgical control group. The Constant Score application at heterogeneous follow up points diminished its utility. Because this was a retrospective study, no functional data about the pre-treatment Constant Score were available. This data could have served to calculate the post-treatment improvement and better quantified each patient's recovery. Another limitation is that the data received a post hoc analysis.Moreover, our results have been interpreted in the context of those reported by other authors who used different criteria for assessing the degree of patient satisfaction. Finally, AP chest radiography is unable to study shifts of the bone stumps in the sagittal plane; imaging of this type of shift requires a CT scan. It would be useful to design a prospective study with a specified minimal follow up time for administration of the survey to improve the results of our research.While conservative treatment remains the gold standard for minimally displaced clavicle fractures, in cases with severe dislocation of the focus, surgery may be indicated, depending on the clinical-instrumental characteristics of the case. The present study assessed the reliability of using the percentage shortening of the bone segment as a means of predicting the failure of conservative treatment of a clavicle fracture. Although our results cannot be generalised, the validity of basing the therapeutic decision on the percentage shortening value as compared to the initial length of the segment could be validated in multi-centric studies using larger population samples.FEB: figure of eight bandage; SD: standard deviation; CT: computed tomography.The authors declare that they have no competing interests.DGS and NA gave substantial contributes in the drafting the manuscript and in the revising it for the intellectual content. SG and ML participated in the acquisition of data of case reports. TS participated in the design of the study and performed the statistical analysis. MB participated in the analysis and interpretation of data, and reviewed the manuscript. All authors read and approved the final manuscript.


The Approximate Bayesian Computation (ABC) approach has been used to infer demographic parameters for numerous species, including humans. However, most applications of ABC still use limited amounts of data, from a small number of loci, compared to the large amount of genome-wide population-genetic data which have become available in the last few years.We evaluated the performance of the ABC approach for three 'population divergence' models - similar to the 'isolation with migration' model - when the data consists of several hundred thousand SNPs typed for multiple individuals by simulating data from known demographic models. The ABC approach was used to infer demographic parameters of interest and we compared the inferred values to the true parameter values that was used to generate hypothetical "observed" data. For all three case models, the ABC approach inferred most demographic parameters quite well with narrow credible intervals, for example, population divergence times and past population sizes, but some parameters were more difficult to infer, such as population sizes at present and migration rates. We compared the ability of different summary statistics to infer demographic parameters, including haplotype and LD based statistics, and found that the accuracy of the parameter estimates can be improved by combining summary statistics that capture different parts of information in the data. Furthermore, our results suggest that poor choices of prior distributions can in some circumstances be detected using ABC. Finally, increasing the amount of data beyond some hundred loci will substantially improve the accuracy of many parameter estimates using ABC.We conclude that the ABC approach can accommodate realistic genome-wide population genetic data, which may be difficult to analyze with full likelihood approaches, and that the ABC can provide accurate and precise inference of demographic parameters from these data, suggesting that the ABC approach will be a useful tool for analyzing large genome-wide datasets. In evolutionary biology and population genetics, several approaches for inferring demographic or genetic parameters are based on Bayesian statistical inference -3. BayesP(θ|D) is the conditional distribution of some parameter of interest (θ) given the data (D), P(θ) is the prior distribution of the parameter, and P(D|θ) is the probability of the data given the parameter (the likelihood function: L(θ) = P(D|θ). According to the expression above, the conditional distribution P(θ|D) of the parameter given the data, which is called the posterior distribution, is proportional to the prior distribution and the likelihood. For most practical cases in evolutionary biology and population genetics, the likelihood function is very difficult to compute because of the large amount of data and the potentially complex models, and exact approaches requiring evaluation of the full likelihood are often restricted to simple evolutionary models . The estimate of the migration rate m12 turned out to be very close to the true value of m12 , but its 95% credible interval was about as wide as for m21.For the simple isolation with migration model 1, the parameter estimation turned out to be quite accurate. Table N1' and N2' were much more narrow compared to 95% credible intervals of N1 and N2, indicating that present population sizes are more difficult to estimate. Table N1' and N2'), and the present population sizes (N1 and N2). Figure For model 2, the means of the posterior samples of past population sizes were slightly closer to the true values than the means for the present population sizes, but the 95% credible intervals of Model 3 - a combination of model 1 and model 2 - is more complex, but also more flexible than models 1 and 2. For model 3, we estimated seven parameters compared to the three and the four parameters in models 1 and 2, respectively. A summary of the posterior samples for the parameters in model 3 is given in Table T, N1', N2', were still quite close to the true values even for the complex model 3, but the migration rates and present population sizes were more difficult to estimate as illustrated by the wide credible intervals . However, the means of the posterior samples of s Figure .N1' ranged from 200 to 9,800, the true current population size N1 ranged from 11,000 to 198,000, the true migration rate m21 ranged from 0.1 to 4.9, and the true divergence time T ranged from 0.01 to 0.49 . For these cases, the set of summary statistics for accepted parameter-values included one or more extreme outlier, which in turn caused the local linear regression to produce a wide range of adjusted parameter-values since the normal least-square estimation for the regression model is non-robust to outliers . The ABC procedure uses fixed values for the mutation and the recombination rates (θ = 5 and ρ = 40). In the case that the mutation rate or the recombination rate of the ABC was similar to the mean of the distribution of the mutation rate or the recombination rate, the ABC estimates were not affected much  for model 3. All other parameter settings of the model and the priors were the same as in Table T for this case where the prior distribution does not cover the true parameter values. The posterior sample was limited to the range of the prior distribution and the mean of the posterior sample for each case hit the upper bound of the prior distribution  for the case of the true T = 0.7, and repeating the ABC analysis , the mean of the posterior sample (0.7274) was quite close to the true value, and the 95% interval  was fairly narrow around the true value.In order to evaluate how a poor choice of prior affects the inference based on the ABC approach with local linear regression adjustment, we set a number of true values of the divergence time on Table . For comon Table . These oT. We investigated the complex model 3 by simulating 49 "observed" datasets from a set of known parameter values using the same approach as described above. The ABC with the local linear regression adjustment was used to infer the population divergence time T. The mean difference between the true and the estimated T, and the mean width of the 95% credibility interval of the posterior sample of T is shown in Figure -18). Moreover, pairs of summary statistics generally improved the accuracy of the parameter estimation compared to single summary statistics; the mean difference between true and estimated T was greater than 0.070 for all single summary statistics , whereas the mean difference was less than 0.070 for 68% of the pairs of summary statistics . However, there were pairs of summary statistics that performed poorly - at the level of single summary statistics - for example, pairs that include ST Fgenerally performed poorly, as well as pairs that included similar types of data, such as the number of distinct haplotype-alleles (NOA) and the number of private haplotype-alleles , but several pairs of summary statistics performed at the same level. Although this comparison of summary statistics was by no means exhaustive, we noted that i) combining summary statistics generally provided more accurate inference, and ii) there was a large variation in performance across pairs of summary statistics. These two observations suggested that combining several summary statistics that capture different population-genetic phenomena may be a powerful approach for making accurate inferences at the same time as keeping the number of summary statistics low, both important features for any ABC investigation [We investigated the performance of each summary statistic, and combinations of summary statistics, for estimating the divergence time T = 0.01, 0.02, ..., 0.49; m12 = 0.1, 0.2, ..., 4.9; and m21 = 0.1, 0.2, ..., 4.9, and the other true parameters were set as in Table T and migration rates. For increasing numbers of loci, Figure T and the migration rate m21 (the results for m12 were very similar to m21). The mean values of the posterior samples rapidly approach the true parameter value when the number of loci increases from 100 to 1,000 and the width of the 95% credible intervals rapidly decrease until about 2,000 loci, and continue to decrease at a low rate for increasing numbers of loci.We used a simulation approach to investigate the impact of the number of loci on accuracy of the ABC estimation, in particular the accuracy of the divergence time estimate and the migration rate estimates. We simulated 147 "observed" dataset from model 1 . For example, for model 3, five of the seven parameter estimates were substantially improved if 50,000 replicates were used instead of 10,000 replicates, and the remaining two parameter estimates (the two current population sizes) were very similar regardless of the number of replicates. This result confirms that we can overcome some of the difficulties in parameter estimation for complex models by increasing the number of simulation steps in the ABC procedure, but it will mainly decrease the Monte Carlo error and allow a reduction of the tolerance level. However, the number of replicate simulations can be a limiting factor for ABC analyses of large scale genome-wide data because of computing time. In our case, approximately 8-9 minutes of CPU time was needed to generate one simulated dataset  for model 3 using ms , which cm12 = 2.0, and m21 = 1.0). However, the precision of the parameter estimates were clearly more accurate based on means compared to using variances . If we use both the mean and the variance, the precision of the parameter estimates were quite close to the result based only on means, but with larger 95% credible intervals . Note also that the number of summary statistics for the combined case is twice the number of summary statistics for the case based on means. The lower quality of the estimates may be related to increasing the dimensionality of the problem as the number of summary statistics increases when considering both means and variances [Among the seven parameters in our models, the migration rates and present population sizes were the most difficult to estimate. A divergence model without migration and an island migration model may result in quite similar gene genealogies for sampled individuals and there will be little information contained in most types of summary statistics to distinguish the differences . MoreoveIn models 2 and 3, it is assumed that the population sizes grow exponentially and the populations spend a very short amount of time with a size close to the 'present population size', hence the populations are far from being at an equilibrium. The summary statistics that we used capture events over a long period of time, and during most of this time, the populations have much smaller sizes compared to the present population sizes. Therefore, little information about present population sizes was contained in the summary statistics, which could explain the difficulty in estimating present population sizes compared to past population sizes.We investigated cases where the true parameter value was outside the range of the prior distribution Table  in orderet al. 2011 [Huang al. 2011  demonstrBoth the mutation and the recombination rates are likely to vary across the genome. However, we assumed that the mutation and recombination rates did not vary along the genome, and they were fixed to a known value for the simulations used in the ABC. We further demonstrate that this assumption works well even if the mutation and the recombination rates vary around some mean values as long as these mean values are similar to the fixed values used in the ABC. To make the simulations in the ABC approach even more realistic, we could draw mutation and recombination rates for each genome-region from some distribution and potentially estimate the empirical mutation and recombination rates. Alternatively, the mutation and recombination rates could be treated as nuisance parameters that are only included to make the simulated data better resemble the empirical data.To conclude, we find that increasing the amount of data from a few loci, or a few hundred loci, to thousands of loci can substantially improve the accuracy of parameter estimation using ABC. In contrast to many full-likelihood inference approaches, the ABC approach is well suited for analyzing large amounts of population genomic data, using for example haplotype-based summary statistics.SL conducted the simulations and the analysis, MJ conceived and supervised the project, and SL and MJ wrote the manuscript. Both authors read and approved the final manuscript.Table S1. Outline of the investigations of the performance of ABC using simulated datasets  to mimic empirically observed data. Table S2. The mean difference between the true and estimated divergence time T (across 49 choices of true T) and the mean width of the 95% credible interval of the posterior sample given by single summary statistics, pairs of summary statistics, and the combination of all eight summary statistics. The results are based on model 3. See also Figure Click here for file


Malaria intermittent preventive treatment of malaria in infant with sulphadoxine-pyrimethamine (IPTi-SP) reduced the incidence of malaria and anaemia by 30% and 20% respectively. The strategy is now a recommended policy for malaria control. However, there was no published study on the impact of the strategy on mortality. The present study assessed the impact of the implementation of IPTi-SP in health services in Mali on all-cause mortality.The 22 health sub-districts of the district of Kolokani were randomized at a 1:1 ratio to either receive IPTi-SP or to serve as a control. The IPTi-SP was implemented for two years starting December 2006. Information on births and deaths through 31 March, 2009 was collected on all children who reached four months of age on 1 December, 2006, likely to be exposed to the intervention in 75 localities randomly selected in each zone.A total of 5,882 children  who reached four months of age between 1 December, 2006 and 1 December, 2008 were surveyed between the age of four months to the age of 18 months from 1 December, 2006 to 31 March, 2009. In the cohort of four to 18 months of age, the mortality rate per 1,000 children was 2.53 in the intervention zone compared to 3.46 in the nonintervention zone, gender and season adjusted mortality rate ratio (MRR) = 0.73 . In the cohort of the four to 12 months of age, mortality rates per 1,000 children were 2.22 in the intervention zone and 3.13 in the non-intervention zone, MRR = 0.71  adjusted for gender and season.The implementation of the IPTi-SP resulted in a substantial reduction in all-cause mortality in children. The results of this study support the adoption and the implementation of IPTi-SP as malaria control strategy.ClinicalTrials.govNCT00766662 It is estimated that about one million of people in sub-Saharan Africa died directly from malaria in 2008, of which 800,000 were children under five years . The num2, divided into 22 health sub-districts. Each health sub-district covers several villages . The total population was 208,317 inhabitants with children under one year of age representing about 4% of the total population. Malaria is hyperendemic in the region with parasite prevalence in children under five years of 45% during the dry season and above 70% during the rainy season [The study was conducted in the district of Kolokani, Mali. The district covers 14,380 kmy season . Each suThe study was a cluster randomized trial. The 22 health sub-districts were randomized in a 1:1 ratio with the intervention in 11 health areas and the other 11 serving as controls for the assessment of the impact of IPTi implementation on the mortality. The intervention consisted of the administration of a half tablet of SP along with EPI vaccines: second and third dose of the DTP at the age of three and four months respectively and measles and yellow fever vaccines at nine months of age. The implementation started in December 2006 after the training of the health personnel and provision of SP to the health centres. This implementation was co-supervised by the Malaria Research and Training Center (MRTC) of the University of Bamako and the district health services through December 2007. The implementation continued in the intervention zones through December 2008 under the supervision of the health services. Sulphadoxine-pyrimethamine used during the second year was provided by UNICEF to the district health services. A cross-sectional survey conducted after one year of the implementation in December 2007 found that 77% of children who received the EPI vaccines also received the IPTi-SP in the intervention zone while this proportion was less than 3% in the non-intervention zone [Using an approximation of arc-sinus transform formula, assuming an expected mortality rate of 106 for 1,000 person-months in children aged four to 18 months, to detect a 30% difference in mortality between the two zones with an alpha error of 5% and a power of 80%, about 1,263 subjects in each zone were needed if a simple random sampling method was used. Assuming a cluster effect of 1.5, taking into account the cluster sampling and a proportion of missing data of 5%, a total of 1,995 subjects per zone were needed. With a total population estimated at 214,509 inhabitants and a proportion of children aged four to 18 months of 4%  about half the total population  was to be surveyed.A total of 150 localities were randomly selected and each household was visited. A simple questionnaire was designed and used collect information in all the children, who reached age of four months between 1 December, 2006 and 1 December, 2008  likely to be exposed to the intervention between 1 December, 2006 and 31 December, 2008. Data were collected by interview and using the child's birth certificate, vaccination card or mother antenatal consultation records. Information collected include the locality, the sub-district, the compound, the household number and subject number in the household, date of birth, vital status (died or not) and date of death if applicable.Data were collected on forms, were transferred in Bamako and double entered using Microsoft Access and reconciled. Statistical analysis was performed using Stata, version 10 . The primary endpoint was the all-cause mortality between 1 December, 2006 and 31 March, 2009 in children aged four to18 months (cohort 1). The secondary endpoint was the all-cause death rate between 1 December, 2006 and 31 March, 2009 in children aged four to 12 months (cohort 2). Cohort 2 was included in cohort 1. The death rates in the two zones were compared using Fischer exact test. Cox regression model adjusted for gender and stratified by trimester was used to adjust for the potential effect of the transmission season and gender. The starting point was the date of age of four months (to start the exposition to the intervention) and the ending point was either: i) the date of age of 18 months or 12 months for cohort 1 and cohort 2 respectively, or ii) date of death, or iii) 31 March, 2009. The duration of follow-up (period at risk) for each subject was the number of days between the starting and the ending points.Initial and successive amendments of the protocol were examined and approved by the Ethical Committee of the Faculty of Medicine, Pharmacy and Dentistry of the University of Bamako. Informed consents were obtained from parents prior to interview.A total of 5,882 children that reached four months of age between 1 December, 2006 and 1 December, 2008 in the study area were surveyed, including 2,869 from 75 localities in the intervention zone and 3,013 from 75 villages in the non-intervention zone. The gender distribution was similar between the two zones . The exact date of death was unknown for two subjects  who were excluded from the analysis.Among the 5,880 children, 198 died at the age of four to 18 months and 119 died at the age of four to 12 months. Of the children who died at age of four to 18 months, 86 (2.86%) lived in the intervention and 112 (3.91%) lived in the non-intervention zone . Of those who died at the age of four to 12 months, 51 (1.69%) lived in the intervention zone and 68 (2.37%) lived in the non-intervention zone . The mortality rate, mortality rates ratios and reduction in mortality are presented in Table This study estimated the effect of two years of IPTi implementation on mortality in children and has found a reduction in all-cause mortality by 27% and 29% in children four to 18 months of age and those of four to 12 months of age, respectively. Use of insecticide-impregnated bet nets (ITN) for malaria control in endemic areas was associated with a reduction of 17% in all-cause mortality [Demonstrating the effect of a malaria control strategy on malaria specific mortality has always been difficult due to the requirement of large sample size and factors that can confound the relationship between the intervention and the malaria-specific mortality. For example, the impact of the use of the ITN on malaria-specific mortality could not be demonstrated initially in individual, large, randomized control trials ,10, but In the present study, the reduction in all-cause mortality may be due to a combination of both the direct and indirect effect of the IPTi on malaria and the indirect effects on other causes of death. Previous evaluations have shown that the implementation of IPTi has resulted in a significant increase in coverage of EPI vaccines, an increase that was more marked in the intervention zone . The higIPTi has now been adopted by the WHO as a policy for malaria control despite the lack of data on the impact of the strategy on mortality ,21,22.This study has two major strengths. First, it is a randomized controlled study, which ensures the comparability between the two arms. Second, the IPTi was implemented within the health care system, better reflecting the environment in which the strategy will be implemented. Although the data were collected by individuals not involved in the implementation and who were not aware if a locality was in the intervention or non-intervention zone, it is difficult to rule out the possibility of observer bias, since it would be easy to find out if a locality was in the intervention or non intervention zone. The mortality rate observed in the present study was lower than expected  resulting in relatively lower power and wider confidence intervals. Reassuringly, the mortality rates in this study are consistent with those reported elsewhere in the West African region ,23,24. AThe implementation of the IPTi has resulted in a 27% reduction of the all-cause mortality in children aged four to 18 months. The results of this study support the adoption of IPTi as malaria control strategy and its implementation as a tool for malaria control.The authors declare that they have no competing interests.AD, OD, JT, RS, CR conceived and designed the study. AD, OD, MK, DT planned, organized, supervised the collected data. Data were analysed by AD and CR. All authors read and approved the final manuscript


The implementation of an in-hospital rapid response system (RRS) could improve the outcome of a deteriorating patient but could increase the medical emergency team (MET) and ICU staff workload ,2.A retrospective analysis of the years pre, during and post implementation of a RRS in a 480-bed hospital with a mean of 17,500 admissions/year.The number of MET calls initially increased from 34 to 56 and then decreased to 39 calls/1,000 admissions/year. Most of the calls were from the emergency department and less from medical and surgical wards. The number of ICU admissions did not increase Figure . FinallyThe implementation of RRS could result in a temporary increase of MET calls but not of ICU admissions; moreover, it could lead to a reduction of mortality and hospital LOS, but not of ICU LOS.


Strigolactones (SLs) are a new group of plant hormones, which have been intensively investigated during the last few years. The wide spectrum of SLs actions, including the regulation of shoot/root architecture, and the stimulation of the interactions between roots and fungi or bacteria, as well as the stimulation of germination of parasitic plants, indicates that this group of hormones may play an important role in the mechanisms that control soil exploration, and the root-mediated uptake of nutrients. Current studies have shown that SLs might be factors that have an influence on the plant response to a deficiency of macronutrients. Experimental data from the last four years have confirmed that the biosynthesis and exudation of SLs are increased under phosphorus and nitrogen deficiency. All these data suggest that SLs may regulate the complex response to nutrient stress, which include not only the modification of the plant developmental process, but also the cooperation with other organisms in order to minimize the effects of threats. In this paper the results of studies that indicate that SLs play an important role in the response to nutrient stress are reviewed and the consequences of the higher biosynthesis and exudation of SLs in response to phosphorus and nitrogen deficiency are discussed. Orobranche and Striga) was postulated [Arabiopsis thaliana, indicated some additional functions of these hormones in plants [Strigolactones (SLs) are a new group of plant hormones that have been intensively investigated during the last few years. In the first report describing SLs from 1966, their role as stimulators of the germination of parasitic plants : CCD7 attacks the 9′ and 10′ bond of β-carotene and then CCD8 cleaves the obtained compounds into carlactone, which is the precursor of SLs [d27, whose phenotype can be reversed by treatment with GR24, which is the synthetic analogue of SLs [MAX1gene, identified in Arabidopsis [SLs are secondary metabolites, which are synthetized from carotenoid precursors mainly in the roots . In riceplastids ,20. Thise of SLs . The Arae of SLs  and the e of SLs . The lasbidopsis   is one of the most important macronutrients that is required for plant growth and development. It plays a crucial role in many metabolic processes and regulatory pathways, such as phosphorylation reactions, and it is also a structural component of fundamentally important macromolecules such as nucleic acids, ATP and membrane lipids . NitrogeTrifolium pretense L., which is known to be a host for the parasitic plants and AMF. In this case, the level of orobanchol was analyzed under low P, N, K, magnesium (Mg) and calcium (Ca) conditions. A higher production of orobanchol (20 times higher) was observed only under P deficiency, whereas the other analyzed nutrients did not affect the biosynthesis of SLs [Sorghum bicolor L. under P and N deficiency revealed that the production of 5-deoxystrigol, one of the major SLs identified in this species, was almost 30 times higher during the response to nutrient stress conditions than in the control plants [Striga hermonthica [S. hermonthica seeds. Results obtained for sorghum and red clover indicated that the response of the exudation of SLs to nutrient availability varies across plant species. One of the hypotheses assumed that the observed differences were due to different strategies of macronutrient uptake in legumes (red clover) and non-legumes (sorghum) [Studies on different plant species have revealed that the level of macronutrients in the soil is a powerful regulator of the biosynthesis and exudation of SLs. The first reports about the correlation between nutrient deficiency and a higher secretion of SLs were published in 2007 ,40. The s of SLs  (Table 2sorghum) ,40.Astragalus sinicus L. (Chinese milk vetch) and Medicago sativa L.  from Fabaceae, Lactuca sativa L. (lettuce) and Calendula officinalis L. (marigold) from Asteraceae, Solanum lycopersicum L. from Solanaceae and Triticum aestivum L. from Poaceae. Two of these species, alfalfa and Chinese milk vetch, belong to legumes, whereas four others, lettuce, marigold, tomato and wheat, are non-legumes. As was expected, P deficiency induced the production and exudation of SLs in all of the analyzed species, whereas the effect of N starvation on the biosynthesis and exudation of SLs was not observed in alfalfa and, surprisingly, also not in tomato [Pisum sativum L. [To confirm this hypothesis, Yoneyama and coworkers  tested tn tomato   under nutrient stress conditions was measured [D10, D17 and D27 increased under P deficiency, whereas the expression of these genes decreased after P supplementation [The increased production of SLs in response to P and/or N deficiency has been described in many species, but the question of whether it is the result of a higher expression of the gene encoding proteins involved in the biosynthesis of SLs or the higher activity of proteins already present in a plant still remains. To answer this question, the expression of three rice genes involved in the production of SLs  and biosynthesis (d10) did not show this inhibition under a low level of P and the outgrowth of tiller buds was higher than in a wild-type grown in control conditions [max1, max2 and max4, were analyzed [Shoot branching is correlated with many different external factors such as sunlight, water or plant density. However, the modification of shoot architecture has also been reported in the case of a P deficiency . A low lnditions . Similaranalyzed . Under canalyzed . These rBut why does the plant strategy to survive under a P deficiency include decreasing shoot branching? The answer could be that plants under a low P level have to limit the production of new tillers/branches in order to protect the required minimum amount of this macronutrient for the shoots that have already developed. The SLs produced during stress response play a key role in the inhibition of branching under P starvation. The lack of the correlation between the P concentration and the number of tillers/axillary branches in the mutants with affected SL biosynthesis and signaling clearly showed that both the production and perception of SLs are required for the suppression of tiller bud outgrowth under a P deficiency ,47. AddiAs was already mentioned, P is immobile in the soil and for the uptake of higher amounts of this macronutrient, plant roots have to explore new areas in the soil. This is why the P concentration influences the root system architecture; in the case of a low level of this macronutrient a higher number of longer lateral roots (LRs) is present, while the development of primary roots (PRs) is repressed . Moreovemax3-11) and signaling (max2-1) revealed more a branched phenotype of root systems that was caused by a higher number of LRs than in the wild-type (Col-0). The treatment of plants with GR24 reduced the density of LRs in Col-0 and mutants that were deficient in SL synthesis, but did not affect the lateral root formation in the max2-1 mutant [max2-1) were insensitive to the applied hormone. Nevertheless in the case of absence of GR24 no differences in RH phenotype between mutants and wild-type plats were observed [A new role of SLs in plant root growth and development has been proposed ,50. An aobserved . All of max2-1  and max4-1 (biosynthesis) mutants, the density of RHs remained unchanged after 48-hours post germination (hpg) regardless of the P concentration and was similar to that of the wild-type plants in control P conditions [max4 under a low P level increased to the value observed in Col-0, while in max2 the number of RHs remained unchanged. In the case of SL-deficiency mutants, the exogenous hormone restores the wild-type phenotype, whereas in the case of insensitive mutants, no effect was observed. On the other hand, wild-type plants treated with the AbamineSG, that may inhibit the SLs production via abscisic acid [There are at least three recently published papers describing the potential role of SLs in the response to P starvation in the below-ground part of plants ,52,53. Asic acid , showed max mutants under control conditions, a higher number of LR primordia was observed, which indicates that endogenous SLs may prevent the outgrowth of LRs [The second aspect of the adaptation of the root architecture to P starvation is an increase in the number of LRs that can be observed in different species . In Arabh of LRs . Howeverh of LRs .max mutants of Arabidopsis revealed a reduction in the efficiency of a response to nutrient stress conditions in the case of SL mutants [max2-1 mutant in comparison to Col-0. These results were obtained for ACP5 (encoding APase), IPS1 (transcription factor) and three P transporters , all of which are highly involved in the mobilization of P in plants in nutrient stress [ACP5 and PHT1;4 genes was observed in the max4-1 mutant, which indicates differences in the response to P starvation between signaling- and biosynthesis-related SL mutants; however, in both of the cases, this response is reduced in comparison to the wild-type [PHT1;4 and PHT1;5 genes in the max4-1 mutant, while the inhibitor of SLs biosynthesis caused a lower induction of ACP5, PHT1;4, PHT1;5 in the wild-type under P stress [The plant response to a low P concentration includes the modification of the shoot and root architecture as well as different molecular mechanisms that are involved in the acquisition and mobilization of P in plants during the adaptation of a plant to stress . An anal mutants . In the t stress . During ild-type . The rolP stress .Lotus japonicus root exudate was described as a stimulating factor for the hyphal branching of the fungal partner [Petunia hybrida mutants pdr1 carrying lesions in the gene encoding SLs transporter, revealed the additional role of SLs in stimulation of intracellular hyphal growth within the roots [The history of studies on SLs began over 60 years ago with a search for the factor that was exudated by plant roots and that induced the germination of the seeds of parasites . Further partner . The pro partner –65. Analhe roots . A detaihe roots ,5,34,66.he roots . All therms1 and rms4 were analyzed in order to test their ability to establish interactions with AMF [rms1), but also in the case of rms4, the SLs insensitive pea mutant. This observation cannot be explained by a lower level of SLs in the tissue and/or root exudate because the production of this hormone in rms4 is not different than in wild-type plants. A hypothesis about the endogenous role of SLs during mycorrhizae has been proposed. It is suggested that the perception of SLs in plant tissues is involved in the promotion of symbioses with AMF [d3-1 carrying lesions in the gene encoding F-box protein, the orthologue of pea (RMS4) [d14-1, showed an enhanced colonization of AMF, which was correlated with a higher production of SLs [In order to explore the role of SLs in the regulation of mycorrhizae, the pea mutants with AMF . Surpriswith AMF . Similara (RMS4) . Anothern of SLs . These rn of SLs .Gigaspora rosea, the fungi partner of pea, in comparison to the exudates of control roots [DAD1) was repressed under high P conditions. The obtained data indicate that a low P level may induce the transcription of genes encoding enzymes that produce SLs, which are responsible for the promotion of mycorrhizae. However, treatment of plants grown at a high level of P with GR24 did not increase AMF colonization, which indicates that SLs are not the only factor regulating mycorrhizae [Experiments carried out on pea plants that grew under low, medium and high P concentrations confirmed that the exudates obtained from roots after P starvation increased the branching of ol roots . The numol roots . Howeverol roots . This meol roots ,70. Addiol roots . Additioorrhizae . All of orrhizae ,65,69,71Plant roots may interact not only with the fungi in the rhizosphere, but also with other microorganisms. Legumes are a group of plants that developed the strategy of N uptake via symbioses with N-fixing rhizobial bacteria, which are housed in special root nodules . This inMedicago sativa plants showed that GR24 increased the number of nodules, but that this promotion of nodulation was not caused by any influence on bacteria growth or metabolism [rms1 mutant, in which SL biosynthesis is inactive, clearly pointed to the important role of SLs in nodulation. In control conditions, the number of nodules in the rms1-1 mutant was almost 40% lower than in wild-type plants and the application of GR24 increased the nodulation in both [rms1 was due to an SL-regulated process that occurs in roots, but was not due to root morphology [ccd8 was analyzed in terms the stimulation or inhibition of nodulation under different N conditions. The results of the assay indicated that SLs are not required for nitrogen to regulate nodule number, although they do promote nodulation [An analysis of tabolism . A genet in both . A graftrphology . The finrphology ,41. To adulation .All of the presented data confirm that under P and N starvation SLs are exuded into the soil and act as signal molecules that increase the symbioses with AMF and N-fixing rhizobial bacteria in order to obtain an additional source of the macronutrients . TherefoThe role of SLs in the response to salt stress was proposed. As symbioses with AMF may also increase plant resistance to soil salinity, lettuce plants grown under a high concentration of NaCl were analyzed in terms of the exudation of SLs . In the max2, that is more tolerant to the oxidative stress in comparison to the wild-type [MAX3 and MAX4 genes, which encode the proteins that are involved in the transformation of carotenoids and the production of SLs, remained unchanged [The defense and adaptation strategies of plants to abiotic and biotic stresses are based mainly on the redox signaling network . Plant hild-type ,80, makeild-type . Moreoveild-type ,83. Sincnchanged .SLs are a group of plant hormones that have been intensively investigated during the last years. Each year new roles of SLs are discovered. In this manuscript, the potential functions of SLs in response to nutrient stress were discussed. Present knowledge indicates that SLs comprehensively influence plant development allowing the plant to adapt to the nutrient deficiency.Many different aspects of SLs’ functions in plants are being intensively investigated. One of the powerful environmental factors, that affects plant growth and shoot branching, is light. First reports describing correlation between light and SLs have already been published. In 2010 analysis of microarray data indicated that SLs have a positive effect on expression of light harvesting genes in tomato . SLs havEvery year of research on SLs reveals new enzymes involved in their production and players of SL perception. Research in the next few years should shed new light on the SLs biosynthesis, signaling pathways, and the functions of these hormones in plants, as well as the cross-talk with other plant hormones.


Infections Related to Health Care (IRHC) today are considered as a public health problem worldwide, therefore it is essential that institutions focus their efforts on the continuous improvement of quality of care using the IRHC indices as indicators of quality, validated and recognized for this purpose, but in Brazil the comparison of IRHC (benchmarking) with the purpose of evaluation is arduous task, since there is no national database for external comparisons, is even scarcer such information in pediatrics and neonatology.To compare rates of IRHC among intensive care units (ICU) neonatal and pediatric, to improve the quality of care and support for the proposed construction of new national public policies.Prospective cohort study, longitudinal, September 2012 and partial evaluation in December 2012, and the data were grouped into two distinct groups , with distinct purpose of comparisons.The distribution of hospitals according to geographic region of the country: 50% North, Northeast 42%, 8% Midwest, according to the type of ICU: 67% Neonatal, Pediatric 33%. The average density Incidence of Primary Bloodstream Infection / 1000 CVC days in the group of Pediatrics ranged from 3.82 to 34.13 and Neonatology group ranged from 2.58 (in the weight range E) to 164 6 (in the weight range D) and the average density of Pneumonia Incidence / 1000 days of MV in the group of Pediatrics ranged from 5.32 to 24 and Group of Neonatology ranged from 3.42 (in the weight range C) at 194 (the weight range B), whereas the density of Urinary Tract Infection / 1000 SVD days ranged from 4.40 to 23.65 (Pediatrics) and 0 in all weight ranges in the Group of Neonatology 11.78 weight in the range of E; density Enterocolitis in neonatology group ranged from 0.17 (weight range D) to 3.24 (the weight range D).This study has pointed to the possibility of knowing the reality of care in the Pediatric ICU and Neonatal in Brazil, through indicators that monitor important adverse events such as infection, even in the face of different realities within a country of continental dimensions and not always homogeneous.None declared


A commentary onRe-examining the role of hydrogen peroxide in bacteriostatic and bactericidal activities of honeyby Brudzynski, K., Abubaker, K., St-Martin, L., and Castle, A. (2011). Front. Microbiol. 2:213. doi:10.3389/fmicb.2011.00213In the trend of the use of antimicrobial compounds from natural and renewable resources, natural antimicrobial compounds, particularly found in food and with potential biomedical applications, are of highest interest. In this context, the contribution by Brudzynski et al. to our special issue on “antimicrobial compounds from natural sources” deserves a special attention not only for giving insights into the natural antimicrobial components present in the honey, but also for exploiting their potential mechanism of action.The antibacterial properties of honey have been well documented. The hydrogen peroxide has been described as the main compound responsible by the antibacterial activity of honeys. The hydrogen peroxide is a potent antimicrobial agent, produced mainly during glucose oxidation catalyzed by the action of the bee enzyme, glucose oxidase, which is introduced into honey during nectar harvesting by bees. The hydrogen peroxide concentration in honey is determined by the rate of its production by glucose oxidase and its destruction by catalases. Thus, the hydrogen peroxide levels in different honeys may differ considerably from honey to honey. In this study the authors re-examined the role of the hydrogen peroxide as component responsible for the antibacterial activity in honey.The correlation between the endogenous hydrogen peroxide concentration and the inhibitory activity of bacterial growth by honey is well established. Indeed, honeys with a high concentration of hydrogen peroxide have higher antibacterial activity. However, honey is a complex chemical milieu composed of over 100 different compounds , which can interact with the hydrogen peroxide, affecting the oxidizing activity of the honey. Consequently, this interaction may result in increase or decrease of the antimicrobial activity of honey.2O2 are used (0.8–8 M) and H2O2 antimicrobial activity has been verified against several medical important bacteria species, including Staphylococcus spp., Streptococcus spp., Pseudomonas spp. and Bacillus spores. The bactericide activity of hydrogen peroxide is related to the accumulation of irreversible oxidative damages to the membrane, proteins, enzymes, and DNA.Hydrogen peroxide alone is commonly used as disinfectant compound in medical equipment in hospitals. For disinfection of these materials high concentrations of HIn vitro studies showed that the cell death of cultured mammalian, yeast and bacterial cells is observed with higher than 50 mM of H2O2 and was associated with chromosomal DNA degradation. However, this concentration is still about 50-fold higher than the concentration of hydrogen peroxide found in honey.The hydrogen peroxide content in honey is about 900-fold lower than the concentration commonly used for disinfecting medical equipments. The authors hypothesized that the oxidizing action of honey's hydrogen peroxide on bacterial cells may be modulated by the presence of other bioactive molecules in honey and therefore, may differ from the action of hydrogen peroxide alone. Thus, the aim of the study was to critically analyze the effects of hydrogen peroxide on growth and survival of bacterial cells in order to analyze its putative role as a main component responsible for the antibacterial activity of honey.Bacillus subtilis and Escherichia coli as bacterial reference strains, to determine the correlation between the hydrogen peroxide concentration and the bacterial growth inhibition ability. Moreover, the treatment of honey samples with catalase allowed verifying the effects of the inhibition of oxidizing activity of the endogenous hydrogen peroxide on antibacterial activity and on DNA degradation of the bacterial cells.Several natural and artificial honey samples were evaluated by the authors, using 2O2 showed a concentration-dependent inhibition of E. coli and B. subtilis growth of about 90% at 1.25 mM and 2.5 mM, respectively. However, among the different honeys samples analyzed in this study, the concentration of hydrogen peroxide ranged from 0.248 ± 0.02 to 2.68 ± 0.04 mM. Endogenous H2O2 found in Canadian honeys inhibited the growth of E. coli in a concentration-dependent manner, and its MIC90 was two-fold higher compared to that determined for exogenous H2O2. In contrast, honey H2O2 was ineffective to inhibit B. subtilis growth, suggesting that other honey compounds or physical features, such as high osmolarity, were responsible for the antimicrobial properties of honeys. Paradoxical growth stimulation of B. subtilis was also observed at high honey dilutions (16-fold and over) and in the presence of high levels of H2O2. According to the authors, these differences could be related to difference in the bacterial susceptibility profile to the oxidative action of hydrogen peroxide and/or interference from other honey compounds.The antibacterial susceptibility tests with exogenous H2O2 triggered cell death of cultured mammalian, yeast, and bacterial cells was associated with chromosomal DNA degrades, the authors also examined the action of honey H2O2 in bacterial DNA degradation, which is a lethal event that ultimately kills the cell. In general, despite the presence of antioxidants in honey such as catalases, polyphenols, Maillard reaction products, and ascorbic acid that would lower the oxidative stress to cells, DNA degradation of E. coli cells exposed to exogenous or endogenous H2O2. In fact, the lower concentrations of honeys H2O2 required to effectively degrade chromosomal DNA led the authors to suggest that the oxidizing effect of H2O2 was augmented by other honey components such as transition metals  also commonly present in honeys.Little is known about the role of the honey's hydrogen peroxide to bacterial cell death. Considering that the HIn conclusion, this study represents an important step in understanding the antibacterial properties of honey. The findings described in this study not only revise the old views but also provide novel information on the role of hydrogen peroxide in the regulation of bacteriostatic and bactericidal activities of honey. The identification of the main components responsible for the antibacterial activity of honey and the mechanism of action underlying this effect bring new knowledge to the field that will greatly contribute to understand the mechanisms by which honey compounds lead to bacterial growth inhibition and bacterial death.