Although IS 6110 has been used extensively as an epidemiological marker, the identification of the precise chromosomal insertion sites has been limited by technical challenges. Here, we present IS-seq, a novel method that combines high-throughput sequencing using Illumina technology with efficient combinatorial sample multiplexing to simultaneously probe 519 clinical isolates, identifying almost all the flanking regions of the element in a single experiment.The insertion element IS6110 flanking regions on the different isolates. When validated using reference strains, the method had 100% specificity and 98% positive predictive value. The insertions mapped to both coding and non-coding regions, and in some cases interrupted genes thought to be essential for virulence or in vitro growth. Strains were classified into families using insertion sites, and high agreement with previous studies was observed.We identified a total of 6,976 ISM. tuberculosis strains.This high-throughput IS-seq method, which can also be used to map insertions in other organisms, extends previous surveys of in vivo interrupted loci and provides a baseline for probing the consequences of disruptions in  Mycobacterium tuberculosis, is an intracellular pathogen that has infected humans since ancient times [In spite of effective chemotherapy against tuberculosis, this disease is still a global health problem and a leading cause of death worldwide . The caunt times ,3. Tubernt times ,5. Activnt times . In mostnt times .M. tuberculosis which is thus considered to have a clonal population structure, with strains having almost identical nucleotide sequences [6110 is an important source of genomic variability for M. tuberculosis and is distributed in different positions across the bacterial chromosome [6110 has been widely used as an epidemiological marker in tuberculosis by DNA fingerprinting using Restriction Fragment Length Polymorphism (RFLP) [M. tuberculosis genome raises the possibility that it might also drive phenotypic variability or affect strain fitness. It has been demonstrated that IS 6110 transposition can be stimulated by specific stress conditions and thus contribute to genetic diversity in circulating M. tuberculosis clinical isolates [6110 insertions can affect gene expression by interrupting protein-coding genes, by mediating recombination events that result in deletions and inversions, and by up-regulating the expression of nearby genes due to a promoter located within the transposable element [6110 is primarily thought of as a valuable epidemiological tool, its prevalence and effect on genome function prompted us to take a deeper look at the distribution and patterns of IS 6110 insertions by conducting a broad survey of circulating strains.There is no evidence of recent genetic exchange in equences . Howeverequences -11. The romosome . The copm (RFLP) ,14. Thesisolates -17. IS61 element ,16,18,196110 insertion sites in 161 M. tuberculosis isolates, which was accomplished by PCR amplification of the target sequences followed by cloning and sequencing, identified 294 unique insertions sites in only 100 genes, suggesting that a large gene repertoire is required for human infection [6110 insertions during infection and transmission, we used Illumina® sequencing technology to identify IS 6110 flanking regions in more than 500 M. tuberculosis isolates from representative collections of clinical isolates from Europe and South America. This high-throughput approach obviates the need for cloning and allows analysis of many strains in a cost-effective manner. We were able to identify approximately 7,000 IS 6110 insertion sites that interrupt almost 300 genes, which together represent <10% of the M. tuberculosis genome. Thus, we substantially increased the survey of naturally occurring in vivo IS 6110 transpositions, providing new insights regarding their distribution, diversity and role in strain classificationA previous examination of ISnfection . HoweverM. tuberculosis strains for analysis of the IS 6110 insertion sites (see Methods for strain selection criteria). This set included the reference strain M. tuberculosis H37Rv (Colombian isolate) and six other strains  that were used to validate the method by verification of insertion sites using PCR (see Table IS6110 RFLP and spligotyping.We developed a method for the simultaneous identification of insertion sites (IS-seq) in up to 600 different strains by barcoding samples and using Illumina® sequencing technology to sequence the genomic DNA flanking both ends of an insertion sequence Figure . We sele6110 were then amplified in a nested PCR using primers that incorporate additional barcodes. Finally, the amplified products were pooled, purified and sequenced. The resulting fragments contain either the 3’ or 5’ terminal sequence of IS 6110, the flanking genomic sequence, and two different barcodes at each terminus. Since most of the enzymatic steps are “add only” reactions, there is no material loss due to purification steps and the protocol can be easily automated and performed in 96-well plates. Furthermore, reactions are pooled after both barcodes have been added, so that the gel purification of hundreds of libraries can be performed using a single gel lane.To generate a sequencing library we randomly sheared genomic DNA and ligated barcoded adapters to the fragment ends. Fragments that include the terminal end of the ISM. tuberculosis genome. All assembled contigs were mapped to at least one of the available genomes. The sequences that did not assemble in contigs were further mapped as single reads to the different genomes and less than 5% of these reads could not be mapped. Based on an estimate of the average sequence coverage obtained per IS 6110 element (i.e. number of reads per RFLP band), a cutoff point was defined (see Methods) to remove strains with low coverage, which are likely to produce unreliable results  and obtained only few and scattered reads without significant mapping. These results demonstrate the efficiency and specificity of the protocol and primer design.Using this protocol, we obtained more than 14 million “paired-end” sequences using an Illumina® GA II machine. The sequence reads contained an even distribution of barcodes  had 16 IS 6110 elements, with an extra copy being part of a doublet in positions 3,552,584 and 3,552,713 [The accuracy of IS-seq was assessed using seven CR Table . These t,552,713 , highlig6110 element insertion is the generation of a 3-4 bp duplication at the insertion site [6110 element on both termini (73.1%). In 91.85% of these cases we identified a 3 bp (75.56%) or 4 bp (16.29%) duplication. Taken together, these results demonstrate that the IS-seq method accurately and reproducibly identifies and maps IS 6110 insertion sites.A hallmark of the ISion site . We expeM. tuberculosis genome. An analysis of the distribution and frequency of independent insertion sites throughout the genome also showed a significant location bias. A Poisson test for the distribution of insertion sites showed that there were forty-four 2 Kb-windows (~2.5% of the genome) with significantly more insertions than expected .The IS-seq method identified 6,976 insertion sites in the 519 strains analyzed , which is thought to be involved in fatty acid transport, or plcD, which encodes a phospholipase C, and intergenic regions like dnaA-dnaN or the region between genes Rv2813-2816  (http://www.ncbi.nlm.nih.gov/sutils/coxik.cgi?gi=135) and the Tuberculist database (http://tuberculist.epfl.ch/index.html). Disrupted genes were broadly distributed across functional categories, with a significant over-representation of insertions in genes belonging to three categories: 1) PE/PPE, 2) genes in COG from the category “unknown” and 3) Cell wall/membrane/envelope biogenesis genes  to identify the co-occurrence or absence of insertion sites in order to generate a dendrogram using 504 of our strains, which belonged to the M. tuberculosis Euro-American lineage  and the Beijing family , which was also used as an outgroup . The us dnaAdnaN and the R region . Despitelineages , we propp Figure . We also6110. Interestingly, we were able to identify the insertions in the DR region that are responsible for the spoligotype patterns of the different members of the Haarlem family  . No association was found between insertion sites present in different phylogenetic families and a specific biological trait, suggesting that it is unlikely that exact, yet independent insertions could occur in different families that confer a fitness advantage  the use of two molecular barcodes per sample to multiplex samples on an Illumina® sequencer, allowing the analysis of a combinatorial number of samples (600) with just 25 different barcodes; ii) a high-throughput library construction protocol that uses minimal amounts of starting material, and iii) a novel computational strategy that maximizes the efficiency of sequence identification by assembling reads prior to mapping against the M. tuberculosis sequenced genomes. This study, combined with the advances on sequencing technologies, provides a highly cost-efficient method for M. tuberculosis strain identification and typing that could be extended to other target amplifications and sequencing. While the use of 4 bp barcodes allows multiplexing of up to 600 samples per sequencing reaction, 5 bp barcodes may allow pooling up to 9,216 samples. Additionally, the requirement of just 100 reads per insertion site for accurately mapping, combined with new technologies such as MiSeq that achieve up to 4 million reads in just a matter of hours, can revolutionize the field of tuberculosis strain characterization.The IS-seq method was effective at detecting, in a single reaction, multiple IS6110 element present in the same locus could be due to convergent evolution, which in time could be reflected as insertion hot spots (i.e. independent insertions within few nucleotides in a given locus)[IS-seq effectively detected loci with multiple independent insertions or insertions in the same location in many strains, and identified new and previously characterized hotspots and insertions that could be considered ancestral. Although an ISen locus),38 and een locus)-41. HoweM. tuberculosis isolates significantly extends previous surveys by identifying more that 900 new sites and provides, for the first time, a comprehensive overview of the gene repertoire that can be interrupted in vivo by IS 6110. Rarefaction curves indicated that additional genes could still be identified, especially in strains that were not significantly represented in our dataset. However, the number of loci predicted to sustain in vivo insertions was close to the maximum of about 300 genes previously predicted based on an analysis of 161 strains [6110 elements in the PE/PPE gene family reinforces their previously proposed role in immune evasion and is consistent with previous reports [6110 insertions in genes reported to be essential is not implausible, since insertions as well as deletions of some of these genes have already been described [6110 insertions identified here can also result in transcription up-regulation of downstream genes, as has been reported for an insertion in the promoter region of the two-component system gene phoP[phoP promoter was observed here in a fast evolving clade of Haarlem 1 in Latin America  of M. tuberculosis clinical isolates. This is an innovative approach that combines an efficient barcoding strategy with a 96-well plate automated library preparation and massive parallel sequencing in a single Illumina sequencing reaction. Our results showed about 7,000 different insertions in the whole panel of analyzed isolates, which is more than has been identified up to date. Among the interrupted genes we identified some that were proposed to be essential for virulence and in vitro growth. These results will be useful for researchers in the field of tuberculosis including the development of new drugs and vaccines.This article describes the simultaneous identification of IS6110 RFLP, as described [http://www.pasteurguadeloupe.fr:8081/SITVITDemo/index.jsp) [The strains for this study were selected from three different collections in Colombia, Argentina and Spain and were isolated from patients between 1995 and 2008. Isolates were selected to represent the main circulating lineages as well as strains with particular characteristics, such as strains involved in TB epidemics, or strains that acquired multidrug resistance. All isolates were typed by spoligotyping and ISescribed  and weredex.jsp) .The study and protocols for collection of bacterial strains from patients were approved by the ethics committees at each institution: the Ethics Committee at the Universidad de Antioquia and local health authorities in Medellín, Colombia , the Ethical Committee of the Aragon Government for strains collected at the Hospital of Getafe, Hospital Universitario Miguel Servet and Hospital Universitario Lozano Blesa in Spain, and the Ethical Committee of the Instituto Malbrán in Argentina. Written informed consent was provided by the subjects in Colombia and Spain. In Argentina this was waived because the investigators performing the study received secondary data about human subjects and microbiological strains, which had been originally isolated elsewhere from human biological samples for diagnostic purposes, but no possible personal identifiers were transferred to the researchers. The source of the data was disclosed to the Committee in the application.Extraction of genomic DNA was performed by the chemical lysis method . In brieThe DNA from each sample was quantified by spectroscopy . Four hundred nanograms of each DNA were diluted in 10 μl of sterile water and sonicated at maximum power for 1 min  to yield fragments with an average size between 200–400 bp. End repair of the DNA was done in a final volume of 20 μl using 10 μl of sonicated DNA, 0.05 mM dNTPs, 1x T4 DNA ligase buffer (NEB), 5 U of Klenow DNA Polymerase (NEB) and 10 U of T4 PNK (NEB). The reaction was incubated for 30 min at 25°C followed by 20 min at 75°C. To remove unused dNTPs, 11U of SAP (Promega) were added to each sample and incubated 30 min at 37°C followed by 30 min at 80°C to inactivate the phosphatase. Addition of a 3’ terminus adenine to the fragments was done in 30 μl using 21 μl of the previous reaction, 0.1 mM dATP, 10 U of Klenow exo minus (Epicentre) and 3 μl of 1x T4 DNA Ligase buffer; the reaction was incubated for 30 min at 37°C and then 20 min at 75°C.Adapters were annealed by combining 25 picomoles of the forward and reverse adapters , and Nested_Primer1 that anneals on the adapter (underlined) and contains the reverse Illumina PE PCR primer 1.0  (5’ AATGATACGGCGACCACCGAGATCTACATCTTTCCCTACACGAC 3’). IS-specific primers were designed to specifically anneal the terminal direct repeats of the IS 6110 leaving a CA dinucleotide at the 3’ end for quality control. The PCR was performed using 5 U of Platinum Taq (Invitrogen), 1x PCR Buffer, 0.5 mM dNTPs, 0.5 μM of Adapter Primer1and 0.5 μM of an equimolar mix of the IS specific primers, 2 mM MgCl2, 0.5 M Betaine and 120 ng  of the purified DNA in a final volume of 25 μl. The thermal cycling program consisted of 33 min at 93°C for initial denaturation and 35 cycles of 2-step PCR amplification using 93°C for 45 sec and 6 min annealing/amplification. The first 5 cycles were done at 55°C, the remaining were done at 68°C.For the amplification of regions flanking the ISCAAGCAGAAGACGGCATACGAGAT CGGTCTCGGCATTCCTGCTGAACCGCT3’) that anneals on the adapter (underlined) and incorporates the Illumina PE PCR Primer 2.0 . This was done using 2 μl of the initial product; all the reagents had the same concentration as the initial PCR. The amplification was done as before for 20 cycles with an annealing temperature of 62°C. From this PCR, 2.4 μg of DNA from each reaction (100 ng/sample) were pooled together in a single tube. Purification of the fragments between 200 – 500 bp was done by gel extraction  and the purified DNA was sequenced in an Illumina® Genome Analyzer (GA) II sequencer. Two lanes in independent flow cells were performed. The first one generating 2 x 35 bp PE sequences and the second one generating 2 x 60 bp PE sequences. Generated sequences were deposited in NCBI Short Read Archive accession number SRA030755.Nested_Primer1 was then used in a nested PCR reaction with Nested_Primer2 (5’ M. tuberculosis H37Rv (Colombian Isolate) with known insertion sites were used to validate the method. Primers were designed to PCR-amplify genes and regions flanking the insertion sequence , the other file has sequences containing the primer barcode, the IS-Primer, a CA dinucleotide characteristic of the insertion site and the target sequence .http://laurikari.net/tre/) for fuzzy string matching with up to 2 errors in the IS primer and posterior masking. A Smith-Waterman fast alignment was then used to search for and trim parts of adapters or primers. Any sequence with terminal consecutive N’s or more than 1 consecutive internal N was removed. After trimming, only sequences with exact matches to the barcodes and with a length, after barcode trimming, longer than 12 nucleotides were kept and stored in an independent file per sample.The reads were first classified according to the barcodes, filtered for low quality data and the IS primer was masked. Trimming of possible sequencing primers was done using agrep . All hits were retrieved and organized by genome according to the detail level of annotation. If the contig and the consensus hit the same genome in opposite strands and with a maximum distance between the hits of 600 bp (since the fragments sequenced ranged between 200–500 bp), the hit was reported in a table as a “specific” hit where the coverage, genome, position and orientation of the hit were reported. Otherwise, the contig was dissolved and the long reads sent to the leftover file. Leftover reads were mapped individually to all the sequenced reference genomes. All hits within 10% of the score of the best hit were kept for each read. Each region in the reference genomes longer than 25 bp with a minimum coverage of 9 reads was reported. In this case, since there is no evidence for the exact insertion site location, the fragments are labeled as “approximate”. Both reports were combined and a table for each strain was generated with the coverage, position, strand, genome and the annotation for the position.For the remaining contigs, the consensus of the alignment and the contig sequence were used as queries against the 6 complete and annotated Since both ends of each insertion element were ideally amplified and sequenced, we further parsed the data in order to search for segments located in close proximity but in opposite directions. This parsing generated 5 categories of results based on the confidence of the mapping. C0 corresponded to IS located in the CRISPR element of the genome known as the direct repeat (DR) region, which due to tandem 36 bp repeats needed special parsing for accurate mapping. C1, which has the highest confidence, consisting of a pair of “specific” fragments each corresponding to an end of the IS element. C2 consisted of a pair of fragments, one” specific” and one “approximate” in close proximity and opposite directions. C3 was formed by two” approximate” fragments in close proximity and opposite directions. C4 indicated that only one “specific” fragment could be mapped. C5 include those hits where only an “approximate” fragment was mapped. These last 2 categories had the highest probability of being false positives but they were kept since they could also represent regions where one of the flanking sides of the IS element is absent from all 5 reference genomes due to strains specific insertions or deletions.6110 element. This also implies that there will be no increase of false positives with higher depth of sequencing.To see how well our sequence reads were able to detect IS insertion sites we looked at the correlation between expected (based on RFLP data) and observed sites (our IS-seq protocol). The relationship between the coverage obtained using IS-seq (reads per RFLP band) and the difference between observed and expected sites showed that our method was prone to identify fewer insertion sites than number of bands identified by RFLP when the coverage was below 100 reads per RFLP band, hence decreasing the sensitivity . Unique sites with more than 20 sequences were checked manually for consistency and to verify that it was not composed of more than one unique site within the parameter of 30 bp used above.Since complete genome sequences of all the strains under study are unavailable, and thus estimation of true negatives is unfeasible, we calculated the sensitivity and positive predictive values (PPV) for validation analysis using the following formulas:We assumed that each insertion observed appeared either independently or that all the insertions in a given position of the genome appeared only once in a common ancestor. Although both assumptions are unlikely, they nevertheless give the lowest and highest maximum number of insertion events that can be measured. To rarefy this number of insertion events respective to the number of genes affected, random sub-sampling of the distribution of insertions was performed continually from a total of 10 insertions to the total number of insertions available. A step of 20 insertions was chosen and 10 replicates per step were performed. The data was plotted in MATLAB and a non-linear fit to an exponential decay function was performed using the formula:M. tuberculosis genome, the number of independent insertions observed in non-overlapping windows of 2 and 50 Kb were determined. Given the total number of independent insertions and the length of the genome, an average number of insertions in a 2 or 50 Kb window can be determined by,To examine the bias in genomic distribution of IS sequences in the We tested the probability that the observed number of insertions in a given window followed an expected Poisson distribution. Bonferroni correction of the threshold was performed to assess the significance of the hit.6110 elements. The Fisher exact test was performed on each category to estimate the probability of obtaining by chance that number or more insertions in a particular category, given the length of the genes. Bonferroni correction for the threshold was performed. The genes involved in virulence, detoxification and adaptation were retrieved from http://tuberculist.epfl.ch/index.html .COG categories and gene lengths were retrieved from the NCBI entry for each reference genome . Genes with independent insertions were assigned to the different categories, filtering out those belonging to the PE/PPE family or those corresponding to IS6110 insertion patterns, we applied mutual information measurements. In order to generate a metric that will allow us to build hierarchical clustering trees, we used the derived metric known as Variation of Information where the distance between any two samples d is given by: Equation 1:To determine the relatedness among the different isolates given by ISwhere I, or the mutual information between X and Y, is defined as Equation 2:and H(X) is defined as Equation 3:6110 insertion sites were highly associated with strain classifications we used Mutual Information (Equation 2) to determine the association between a given insertion in a loci and a specific family. Mutual Information values can be used to determine approximate p-values for the association as follows:Once we determined that the ISwhere N(X) is the number of elements of X.We used Conditional Mutual Information, which allows us to compensate for the correlation between insertion sites and phylogeny, to test for correlation between biological traits (X) and the presence or absence of a particular insertion site (Y) given that the strain belongs to a phylogenetic family (Z), using the following formula:M. tuberculosis genomes present in the PATRIC database [M. tuberculosis '98-R604 INH-RIF-EM', M. tuberculosis 02_1987, M. tuberculosis 210, M. tuberculosis 94_M4241A, M. tuberculosis C, M. tuberculosis CDC1551, M. tuberculosis CPHL_A, M. tuberculosis EAS054, M. tuberculosis F11, M. tuberculosis GM 1503, M. tuberculosis H37Ra, M. tuberculosis H37Rv, M. tuberculosis K85, M. tuberculosis KZN 1435, M. tuberculosis KZN 4207, M. tuberculosis KZN 605, M. tuberculosis KZN R506, M. tuberculosis KZN V2475, M. tuberculosis T17, M. tuberculosis T46, M. tuberculosis T85, M. tuberculosis T92, M. tuberculosis str. Haarlem. IS 6110 elements were identified in each genome using blast with an e-value threshold < 1e-30; 200 bp of the up-stream and down-stream flanking sequences were extracted for each insertion identified. The positions of these insertions were identified running blast of the flanking sequences against the H37Rv and CDC1551 genomes (NCBI). The list of insertions and positions was compared against the family-specific markers identified and were classified accordingly. A subset of these strains has been used recently and classified using SNPs  declare that they have no competing interests.Conceived and designed the experiments: AR, AS, KEV, MMZ, RDM, PDP. Performed the experiments: AR, AS, AC, IH. Analyzed the data: AR, AS, SS, CM, MJG, VR, LL, JR, MMZ, RDM, PDP. Wrote the paper: AR, AS, AC, IH,SS, CM, MJG, VR, JR, MMZ, RDM, PDP. All authors read and approved the final manuscript.Table S1. RFLP, barcode and sequencing information for strains . Table S2 Loci with identified IS 6110 insertions. Table S3 2kb and 50Kb window analysis for IS distribution . Table S4 Loci with insertion sites in multiple strains . Table S5 Characteristic insertion sites associated with specific M. tuberculosis lineages. Table S6 Classification of strains based on spoligotyping, RFLP and IS-seq . Table S7 Classification of sequenced M. tuberculosis genomes using IS-seq data . Table S8 Association between characteristic IS 6110 and biological traits by mutual information. Table S9 Barcodes used in primers and adapters. Table S10 Primers used for insertion site validation.Click here for fileFigure S1. Distribution of Barcodes in Sequenced Samples. Out of approximately 14 million reads (2 x 35/60 bp), 13,243,263 contained a barcode from the adapter (A) and 8,313,986 contained the barcode and the IS 6110 specific primer (B). Different colors represent different barcodes used. The barcode in the IS 6110 specific primer was more evenly distributed  than the barcode in the adapter . In the latter case the outlier barcodes corresponded to CCGG and CACGA that can potentially generate a hairpin with the adapter sequence, thus hampering the ligation reaction.Click here for fileFigure S2. - Detection of Insertion Sites Using IS-seq. The difference between observed (Number of insertions determined by IS-seq) and expected (Number of RFLP bands) sites is plotted against the coverage (reads per strain) obtained with IS-seq. Error bars indicate standard deviation. Red line shows the limit at which lower coverage resulted in reduced specificity of detection.Click here for fileFigure S3. Rarefaction Analysis of Insertion Sites. Random subsampling of the number of insertion sites were performed at different depths and plotted against the number of genes interrupted. Plots for all the insertions observed (green) or for unique insertions (blue) were performed. Non-linear fits for an exponential decay function were estimated (orange and black lines). Coefficient of determination (R2) for each regression is shown as well as the estimator for the K parameter, where K represents the maximum theoretical limit for each regression model, which corresponds to the lower and higher limits of the maximum number of genes, predicted to be susceptible to IS6110 in vivo.Click here for file


Two previous genome-wide association studies (GWAS) of high myopia in a Japanese population found several single nucleotide polymorphisms (SNPs) associated with the disease. The present study examined whether these markers are associated with myopia in a Chinese population.2 test.Individuals with or without complex myopia were recruited from Chinese university students, and probands with early onset high myopia were identified in the Pediatric and Genetic Eye Clinic of the Zhongshan Ophthalmic Center. DNA was prepared from venous leukocytes. Three SNPs, rs577948 and rs11218544 at chromosome position 11q24.1 and rs2839471 at chromosome position 21q22.3, were genotyped. The allele and genotype frequencies of these SNPs were compared between the myopia cases and controls using a χA total of 2,870 subjects were examined in this study, including 1,255 individuals with complex myopia (−10.00 diopter (D)<spherical refraction≤-4.00 D), 563 with early onset high myopia , and 1,052 healthy controls . There were no statistically significant differences found for the genotype or allele frequencies of the three SNPs between the myopia cases and controls in the Chinese population under study.We did not find evidence for the association of myopia with rs577948, rs11218544, or rs2839471 in the Chinese population studied. Myopia is the most common cause of vision impairment worldwide, with a prevalence of approximately 25.4%–80% –3. The prs577948 and rs11218544 at chromosome 11q24.1 and rs2839471 at chromosome 21q22.3  are listed in Restriction fragment length polymorphism (RFLP) analysis was used to detect the SNP rs577948 and rs11218544 were genotyped by nested polymerase chain reaction (nest-PCR) combined with fluorescence-labeled allele-specific PCR (AS-PCR). Briefly, two pairs of primers were used. The first pair of primers was designed to amplify fragments encompassing the SNPs. The amplicons were then used as templates for a second round of PCR, in which three internal primers and a labeled common primer were used: two SNP allele-specific primers with a length difference of 4 bp, a reverse specific primer with an M13 tail, and a 5′-FAM-labeled M13 probe  direct sequencing of the first round of PCR products from four samples in each plate (96 samples); and 2) any sample that was read differently by the two researchers was confirmed by sequencing.2 test. The average age was compared between the myopia cases and healthy controls with a t-test. The distribution of all three SNPs was evaluated with respect to Hardy–Weinberg equilibrium (HWE). The frequencies of alleles and genotypes in the early onset high myopia and complex myopia groups, as well as in the classified spherical refraction groups were compared with those in the healthy control group using a χ2 test. A p<0.05 was used as the level of statistical significance (α), and a Bonferroni correction was applied for multiple testing, correcting the threshold for significance from 0.05 to 0.017 (α/3=0.05/3).The distribution of sexes was compared between the myopia cases and healthy controls with a χrs2839471, rs577948, and rs11218544 was successfully performed in the 2,870 unrelated subjects , the difference was not significant after Bonferroni correction with a corrected significance level of p=0.017.The basic clinical information for all 2870 subjects is listed in subjects . The gensubjects . Althougrs2839471 is located in the region of the uromodulin-like 1 (UMODL1) gene, which may be associated with extracellular matrix and involved in the formation of sclera [rs577948 and rs11218544 are located in a region that includes two genes, BH3-like motif containing protein (BLID) and LOC39959. BLID is expressed in mitochondria, and may function in apoptosis in pathological myopia [LOC399959 is a hypothetical noncoding RNA [rs577948) also failed to confirm the reported association [In this study, we genotyped three SNPs that were previously reported to be significantly associated with myopia. The SNP f sclera ,38. The l myopia . LOC3999ding RNA . Furtherding RNA ,30. In tding RNA ,43. The ociation . Althougociation ,30, the The distribution of sexes was not completely matched in the myopia and healthy control groups. However, this does not affect our conclusions because the results were unaffected when male subjects were randomly excluded to reach a balanced proportion of sexes. In addition, on average our study subjects were younger than those in the Japanese study for both cases and controls. This should not influence our conclusion because myopia is an early onset disease, and the degree of refractive error in myopia usually increases with age –48. In oMyopia is a genetically heterogeneous disease, and both early onset and complex traits have been suggested to be associated with myopia. Traditionally, myopia has generally been classified based on degrees of spherical refraction or their closely related axial length ,30. In t


Visceral leishmaniasis (VL) remains as one of the most neglected tropical diseases with over 60% of the world’s total VL cases occurring in the Indian subcontinent. Due to the invasive risky procedure and technical expertise required in the classical parasitological diagnosis, the goal of the VL experts has been to develop noninvasive procedure(s) applicable in the field settings. Several serological and molecular biological approaches have been developed over the last decades, but only a few are applicable in field settings that can be performed with relative ease. Recently, loop-mediated isothermal amplification (LAMP) has emerged as a novel nucleic acid amplification method for diagnosis of VL. In this study, we have evaluated the LAMP assay using buffy coat DNA samples from VL patients in Bangladesh and compared its performance with leishmania nested PCR (Ln-PCR), an established molecular method with very high diagnostic indices.L. donovani kinetoplast minicircle DNA under isothermal (62 °C) conditions in a heat block. For Ln-PCR, we used primers targeting the parasite’s small-subunit rRNA region.Seventy five (75) parasitologically confirmed VL patients by spleen smear microcopy and 101 controls  were enrolled in this study. LAMP assay was carried out using a set of four primers targeting LAMP assay was found to be positive in 68 of 75 confirmed VL cases, and revealed its diagnostic sensitivity of 90.7% , whereas all controls were negative by LAMP assay, indicating a specificity of 100% . The Ln-PCR yielded a sensitivity of 96%  and a specificity of 100% .High diagnostic sensitivity and excellent specificity were observed in this first report of LAMP diagnostic evaluation from Bangladesh. Considering its many fold advantages over conventional PCR and potential to be used as a simple and rapid test in the VL endemic areas of the Indian subcontinent, our findings are encouraging, but further evaluation of LAMP is needed. Leishmania donovani and transmitted exclusively by the bite of the sandfly, Phlebotomous argentipes. The annual incidence worldwide is approximately 0.2 to 0.4 million cases with mortality rates of 1.5% (93 deaths/6224 VL cases from 2004–2008) in Bangladesh [Visceral leishmaniasis (VL) is considered as one of the most neglected tropical diseases. In the Indian subcontinent VL is mainly caused by the intracellular protozoan parasite Diagnosis of VL is still a great challenge and is cumbersome, especially in the resource limited settings of the Indian subcontinent. Definitive diagnosis of VL is of paramount importance to provide a specific treatment regimen as well as in understanding the disease epidemiology. Parasitological confirmation of VL through microscopy still relies on invasive procedure like spleen, bone-marrow or lymph-node aspiration. Involvement of substantial risk and skilled personnel are among the major limitations associated with these procedures, so access to such diagnostic facilities is far beyond the reach of most of the poor people affected with VL living in the remote rural areas.Leishmania DNA from peripheral blood and buffy coat which is ideal for minimal invasive procedure [Leishmania donovani DNA from blood samples of VL patients and the results were comparable with that of conventional PCR [Efforts are evolving continuously to develop diagnostic methods for VL but existing ones still have some limitations. Serological methods like direct agglutination test (DAT) and rK39 (recombinant k39 antigen-based immunochromatographic strip test) dipstick are now well standardized , but therocedure -12. Howerocedure . LAMP asonal PCR .L. donovani DNA from buffy coat of confirmed VL patients and to examine its efficacy as a diagnostic alternative to PCR.This prospective study was designed to evaluate the diagnostic accuracy of LAMP for rapid and sensitive detection of Ethical clearance was obtained from the International Centre for Diarrhoeal Disease Research, Bangladesh , ethical review committee. Informed written consent was obtained from each patient or from their legal guardian before splenic aspiration and venipuncture. Written consent was also obtained from all controls before including their samples in the study.Leishmania amastigotes in the splenic aspiration smear.A total of 75 confirmed VL patients were enrolled in the study. All of the subjects were admitted to Rajshahi Medical College Hospital (RMCH), Bangladesh from January 2010 to October 2011. The definitive diagnosis of VL was based on the microscopic demonstration of A total of 101 subjects were enrolled in this study as control. The controls were divided into three categories. Twenty five (25) endemic healthy controls were collected from Godagari sub district, a highly endemic area of VL in the Rajshahi division, Bangladesh. Twenty six (26) apparently healthy controls without any signs and symptoms of present VL or past history of VL were also enrolled from VL non endemic areas. Fifty (50) disease controls, including 25 tuberculosis patients confirmed as sputum positive for acid fast bacilli (AFB) by microscopy from the National Institute of Diseases of Chest and Hospital (NIDCH), Mohakhali, Dhaka and another 25 patients with other febrile diseases  having fever for more than 2 weeks and admitted into different wards of RMCH were also included in the study.L. donovani amastigote.Confirmed VL patients were selected based on splenic smear positive for et al.[L. donovani amastigotes by an experienced microscopist at the department of Microbiology of Rajshahi Medical College (RMC). Presence of LD bodies was graded on a scale from 1+ to 6+. If the number of amastigotes counted per field was >100, 10–100 or 1–10, it was graded as 6+, 5+ and 4+ respectively. Similarly, 1–10 amastigotes in 10, 100 or 1000 fields were graded as 3+, 2+ and 1+ respectively [Splenic aspiration was performed after excluding contraindications for the procedure in each case by an experienced doctor following standard technique as described by Bryceson et al.. In everectively Maintaining good clinical practice, 3.0 mL of venous blood was collected in vacuette (K3 EDTA tube) from all subjects. Immediately after collection, blood was centrifuged by density gradient centrifugation with an equal volume of Histopaque 1119 . After centrifugation, plasma and buffy coat samples were transferred into two different sterile 1.5 mL micro-centrifuge tubes and stored at −20°C at RMC until shipment to ICDDR, B. The samples were transferred periodically to ICDDR, B maintaining cold chain.For all the subjects, rK39 was performed from plasma samples according to manufacturer’s instruction provided as product inserts .DNA was extracted from buffy coats using the commercially available QIAamp DNA Blood Mini Kit  according to the manufacturer’s instructions. After extraction, DNA was eluted in a final volume of 0.2 mL of AE buffer . Purity of DNA was measured in a Nano-drop (~5μL of DNA) and was found to be satisfactory as an OD ratio at A260/A280 was within the range of 1.7-1.9 in all DNA samples.Leishmania specific nested PCR (Ln-PCR) was performed with primers targeting the parasites' small-subunit rRNA (SSU-rRNA) region with some minor modifications as described in a previous study [2, 2.5units of pure Taq DNA polymerase, BSA and stabilizers for each reaction. Prior to the second amplification or nested PCR, the amplified products from the first run were diluted at 1:20 with molecular grade water. An additional 2.0 mM MgCl2 was added to the final reaction mixture prior to the second round of amplification. The final amplicons were visualized as 358 bp on 1.5% agarose gel.For detection of parasite DNA, us study . For botFor the second round of amplification, 35 cycles were used consisting of denaturation at 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s followed by a final extension at 72°C for 5 min, in a BioRad’s MyCycler.et al.[4)2SO4, 10 mM KCl, 2 mMMgSO4, 0.1% Triton X-100, pH 8.8, 25°C at final 1X and an additional 6 mM MgSO4 and 8 units of Bst polymerase large Fragment , and 2 μL of buffy coat DNA. Each tube containing the reaction mixture was sealed with paraffin (as LAMP causes massive amplification which might result in contamination of DNA) and incubated in a heat block at 62°C for 1 hour and 30 min. During the course of the reaction, the binding of a reaction by-product (pyrophosphate ions) to magnesium ion results in a white precipitate, entailing the reaction fluid turbid. At the end of incubation, turbidity was measured visually and the results were interpreted as positive or negative based on turbidity  maintained in Novy-MacNeal-Nicolle medium (NNN medium) by subpassaging was utilized for extraction of DNA. After extraction, the concentration of DNA was measured by Nano-drop. A serial dilution of cultured promastigote DNA starting from 100 pg to 0.1 fg were prepared in TE buffer . The dilutions were amplified by both LAMP and Ln-PCR and products were visualized by agarose gel electrophoresis .Sensitivity and specificity were calculated using the online Clinical Calculator 1. Data were entered and clinical parameters were analyzed using the SPSS software (version 11.5) for Windows. Sensitivities of LAMP and Ln-PCR were compared by McNemar’s test using a online calculator , while all controls were found negative, yielding a specificity of 100% . Table .Among 75 confirmed VL cases, 72 were found to be positive by Ln-PCR and all the controls were negative, resulting in a sensitivity of 96%  and specificity of 100% . Whereas, 68 of 75 VL cases were found to be positive by LAMP and all the controls gave negative results, which yielded a sensitivity of 90.7%  and a specificity of 100%  . As all the controls were negative by both the methods, the McNemar test was not performed for specificity.et al.[Loop-mediated isothermal amplification originally developed by Notomi et al. is a leset al.-18, as wet al.-23. Despet al.[et al.[In this study, we have validated the LAMP assay developed by Takagi et al., with 75l.[et al., diagnosl.[et al.. To compl.[et al.-27.Although in this study we observed the performance of LAMP to be slightly lower than Ln-PCR (sensitivity 96% and parasite detection rate is 0.1 parasite against 1 parasite by LAMP), yet it achieved better results than shown in two previous studies. The reason for this could be the use of buffy coat instead of whole blood. The specificity of the LAMP assay has been shown to be very high as the assay utilizes four sets of primers targeting six distinct target DNA sequences ,28,29. IThe very exciting features of LAMP assay include requirement of just a heat block or even a simple water bath instead of a costly thermal cycler, UV gel documentation and transilluminator, which all are essential components of conventional PCR ,24,30,31In addition, results of LAMP assay are interpreted by observing turbidity ,31,32 thBst polymerase enzyme used in LAMP assay is active at relatively high temperatures, which minimizes the possibility of non-specific priming [Furthermore, the  priming  and is m priming . Alongsi priming .Several reports have indicated the usefulness of using heat-treated samples as a template DNA source without compromising the sensitivity, and thus eliminating the need for DNA extraction which reduces both time and cost ,19,28,31To achieve the goal of a VL elimination program, development and introduction of rapid and less expensive diagnosis through non-invasive or minimally invasive clinical samples has no alternative and LAMP method has the potential to full-fill this requirement. So we recommend LAMP as a future novel VL diagnosis tool in the field condition, but before being introduced in the field further evaluation is necessary with larger case and control groups using heat-treated blood samples.VL: Visceral leishmaniasis; LAMP: loop-mediated isothermal amplification; Ln-PCR: Leishmania nested PCR.We declare that we have no competing interests. The funding body had no role in study design, data collection and analysis, preparation of the manuscript or decision to publish.Study was designed by MGMK and DM. Sample collection spleen smear microscopy and clinical examinations were done by MAS. Laboratory experiments were done by KRHB, MGMK and TA. All authors equally contributed in drafting and revising the manuscript. All authors read and approved the final version of the manuscript.STARD checklist for reporting of studies of diagnostic accuracy.Click here for file


Staphylococcus aureus (MRSA) is increasingly being described as a cause of acute sinusitis. We present a patient with acute MRSA sinusitis complicated by rapid intracranial extension, marginal vancomycin susceptibility (MIC = 2 mg/L), delayed drainage of intracranial abscess, and subsequent development of rifampin resistance. Given the relatively high risk of intracranial extension of severe acute bacterial sinusitis and high mortality associated with invasive MRSA infections, we suggest early surgical drainage of intracranial abscesses in these circumstances. We believe this is important given the limited intracranial penetration of currently available treatment options for MRSA, especially those with a vancomycin minimal inhibitory concentration (MIC) of ≥2 mg/L.Methicillin resistant  Our patient, a previously healthy 12-year-old male, presented with acute MRSA sinusitis and rapid intracranial extension. The clinical case was complicated by a marginal vancomycin susceptibility (MIC = 2 mg/L), delayed drainage of intracranial abscess, and development of rifampin resistance. This case is illustrative of community-associated MRSA as a potential cause of acute sinusitis leading to intracranial extension, the challenges of antibiotic management of intracranial MRSA abscess, and the hazards of delayed drainage of intracranial MRSA abscess.A previously healthy 12-year-old male with a history of intermittent migraines was admitted with acute onset of altered mental status and facial swelling. The patient had symptoms of headache, “upset stomach,” increasing fatigue, and tactile fever for two days prior to admission. On the day of admission he was found to be minimally responsive with significant swelling to the left aspect of the face, yellowish discharge from the left eye, and a protuberance from the forehead. A noncontrast head CT scan done at an outside health care facility demonstrated bilateral orbital cellulitis, pansinusitis, and possible venous sinus thrombosis prompting transfer to a pediatric hospital.μL with a manual differential of 8% bands, 3% metamyelocytes, 74% neutrophils and 9% lymphocytes, hemoglobin (gm/dL)/hematocrit (%) of 8.2/24.3, and platelet count of 101 × 103/μL. Additionally, a coagulation panel revealed a prolonged prothrombin time (PT) and activated partial thromboplastin time (aPTT) of 19 seconds and 42.3 seconds, respectively, as well elevated D-dimer levels of 11.11 mg/L suggestive of disseminated intravascular coagulation in this patient. A brain MRI showed a superior midline epidural fluid collection, measuring 8.6 cm anteroposterior × 3.1 cm transverse × 1.0 cm craniocaudal, following the dorsal aspect of the superior sagittal sinus  revealed a white blood cell count of 8400 cells/ Figures . The fluNeurosurgical consultation was requested for surgical drainage of the intracranial abscess, but operative intervention was refused citing that the collection was epidural and not contributing to a mass effect. Neurosurgical consultants questioned whether the collection was an abscess, despite the patient's history, symptomatology, and imaging strongly suggesting that this was a case of acute bacterial sinusitis with rapid extension intracranially, orbitally, and subcutaneously. μg/mL. Initial peripheral blood culture was positive at 14 hours of incubation and identified as MRSA. Significant concern was raised about the MRSA having an MIC of 2 mg/L, which is the upper limit of susceptible, given the challenges of vancomycin penetration into an intracranial abscess. Because the epidural MRSA abscess was not drained initially, rifampin was added. It took 4 days to achieve a target vancomycin trough level of 17 The patient remained febrile, clinically unstable, and intubated during the first three days of hospitalization. A repeat MRI on day three of hospitalization was interpreted by the radiologist as a mild increase in size of the epidural abscess in the superior midline, but surgical intervention was again deferred by neurosurgical consultants. The patient's clinical status did not improve on antibiotics, and a head MRI on the sixth day of hospitalization showed an increased size of the epidural hematoma with a new abscess along the outer table of the left frontal bone and a small subdural fluid collection along the anterior left frontal lobe. A craniotomy procedure with abscess drainage was then performed recovering purulent fluid, all cultures of which grew MRSA, demonstrating the persistence of the organism in the intracranial abscess. On hospital day ten otolaryngology surgeons performed bilateral maxillary antrostomies, bilateral ethmoidectomies, bilateral frontal sinus drainage, and left orbital subperiosteal abscess drainage. MRSA recovered from the frontal sinus demonstrated new resistance to rifampin, which was then discontinued. On hospital day thirteen a right-sided thoracotomy was performed to drain an empyema, which grew MRSA. After four weeks of hospitalization the patient was discharged home on antibiotics and has subsequently made a complete recovery. Although a formal evaluation of the patient's immune system was not performed, he had a normal globulin fraction of 3 gm/dL suggesting that a major deficiency of antibody production was unlikely.Staphylococcus aureus has long been appreciated as a causative agent of chronic bacterial sinusitis [Community-associated MRSA has been the predominant cause of skin and soft tissue infections in North America for the past decade , 2. Stapinusitis , but MRSinusitis , 5. To oinusitis , 7. Recoinusitis  or invasinusitis –11. GiveThis case additionally illustrates the importance of timely drainage of an intracranial MRSA abscess. MRSA subperiosteal abscesses in orbital infections have been noted to be increasing in incidence and are associate with a more aggressive disease course than for other organisms, leading to recommendations for empiric antibiotic coverage with a very low threshold for surgical intervention –15. It iDelayed surgical intervention has also been associated with increasing MIC values for vancomycin leading to the development of resistance (VISA) and heteroresistance (hVISA) . Althoug


In Brazil, the main cause of death is the coronary heart disease and the surgical treatment used is the Myocardial Revascularization (MR). Patients undergoing to MR through bypass saphenous vein have been developed dehiscence in 10%. Dehiscence of surgical incision through Bioestimulation treatment with Low Level Laser in patients that underwent to MR seems to be an unprecedented new treatment and less invasive technique in the medical armamentarium.It was analyzed 10 diabetic patients, mean age 65,3 years that after surgery of MR evaluated for dehiscence surgical saphenectomy in lower member as well as edema and pain. Diode Laser C.W. (685 nm wavelength) with Power= 25 mW, Time= 30 s, Fluency= 4J/cm2 was applied, punctually surrounding surgical wound’s sore every 2 cm.It revealed early reduction of the pain, as well as improved the health appearance of granulated tissue, decrease inflammatory process, wound’s size and reduction fibrin since the very first application. In superficial wounds only 3 sessions was required, while in the extensive wounds 8-10 applications was necessary.LLLT were effective in tissue repair, closing the saphenectomy dehiscence with a substantial improvement of patient’s quality of life, that benefits patients and Institutions, with cost-effectiveness, beyond denoted to be a less invasive treatment.


Vigna. unguiculata ssp. sesquipedialis) is a distinctive subspecies of cowpea [Vigna. unguiculata (L.) Walp.] that apparently originated in East Asia and is characterized by extremely long and thin pods and an aggressive climbing growth habit. The crop is widely cultivated throughout Asia for the production of immature pods known as ‘long beans’ or ‘asparagus beans’. While the genome of cowpea ssp. unguiculata has been characterized recently by high-density genetic mapping and partial sequencing, little is known about the genome of asparagus bean. We report here the first genetic map of asparagus bean based on SNP and SSR markers. The current map consists of 375 loci mapped onto 11 linkage groups (LGs), with 191 loci detected by SNP markers and 184 loci by SSR markers. The overall map length is 745 cM, with an average marker distance of 1.98 cM. There are four high marker-density blocks distributed on three LGs and three regions of segregation distortion (SDRs) identified on two other LGs, two of which co-locate in chromosomal regions syntenic to SDRs in soybean. Synteny between asparagus bean and the model legume Lotus. japonica was also established. This work provides the basis for mapping and functional analysis of genes/QTLs of particular interest in asparagus bean, as well as for comparative genomics study of cowpea at the subspecies level.Asparagus bean ( Vigna unguiculata L. Walp.) (2n = 2x = 22), an important annual grain legume, fodder and vegetable crop in many tropical/subtropical regions of the world, is a diploid species belonging to the Phaseoleae with an estimated genome size of ∼630 Mb Vigna angularis Willd. Ohwi & Ohashi) and mungbean (Vigna. radiata L. Wilczek) of the genus Vigna, as well as species of the genus Phaseolus and Glycine, including the model crop legume soybean (Glycine max L. Merr). There are two main cultivated divisions of cowpea worldwide, i.e. the dominant subspecies unguiculata, which is widely cultivated in Africa, India, Middle East and the Americas for dry grain and which is a major source of dietary protein for millions of people in developing countries sesquipedialis, which is also known as asparagus bean or ‘yard long’ bean in eastern and southern Asia, for production of immature green pods unguiculata, and is considered one of the top ten Asian vegetables. Diverse landrace forms of asparagus bean are found throughout Asia Cowpea  and for different uses (primarily dry grain and fodder in Africa compared with tender pods at the immature stage in Asia) unguiculata by narrow kidney-shaped seeds. In contrast to the aim of gaining higher yield of seeds with improved nutrition for ssp. unguiculata, the main objective in asparagus bean breeding is to obtain more pods per plant and to slow the rate of seed development to prolong the production of attractive immature pods favourable for vegetable use.The extent of genome conservation/diversification between asparagus bean and ssp. unguiculata was constructed based on 928 EST-derived SNP markers, providing a solid basis for gene/QTL mapping and genome characterization unguiculata gene pool Recently, a consensus genetic map of cowpea ssp. unguiculata DNA sequences, which overall showed more than 98% transferability in asparagus bean Thus far, there has been no genetic map for asparagus bean. In previous work, we developed over 1000 SSR markers from ssp. P<0.05). Twelve markers had a minor allele frequency (MAF) value lower than 0.2, demonstrating significant segregation distortion. There were four multi-locus SSR markers , meaning they detected more than one polymorphic locus each.Amplification of DNA of the mapping parents using 1372 SSR markers gave an overall technical success rate of 92.3% and detected 210 polymorphic loci, providing an average polymorphic rate of 16.5%. Without considering SSRs from Li et al (2001) unguiculata RIL populations used in Muchero et al. (2009) unguiculata, in agreement with previous genetic diversity studies using AFLP and SSR markers The GoldenGate assay harboring 1378 technically acceptable EST-derived SNPs had 206 polymorphic loci, showing a polymorphism rate of 14%. This value is higher than that of EST-derived SSRs (9.1%), but much lower than those observed in the six ssp. Three hundred seventy-one markers targeting 375 discrete loci were mapped onto 11 linkage groups (LGs) corresponding to the chromosome pair number of asparagus bean. For consistency, the nomenclature of LG1 to LG11 was assigned according to SNP markers shared with the cowpea consensus map of Muchero et al. (2009) Four high marker density regions were found on three LGs, one occurring in interstitial regions in LG2, one in interstitial region in LG6 and the remaining two near telomeres of LG3 in opposite directions . There aThree regions of segregation distortion (SDR) were mapped onto two LGs . The LG2unguiculata mapping populations used in Muchero et al. (2009) unguiculata SNP maps unguiculata consensus map linkage groups, with the exception of LG2 and LG5, which are quite similar between the two maps. The tagging of more chromosomal regions, in particular non-coding regions by SSR markers on the ZZ map are considered the reason for these length variations. We note that some of the SSR markers, together with 21 new SNP markers that were not included in the published consensus map, extend the map coverage in some LGs. For example, as shown in unguiculata is indicative of a low rate of recombination. In maize, rates of recombination for specific chromosome segments also are known to vary greatly in different mapping populations The ZZ map, which is 745 cM in length, is only a little longer than the 680 cM consensus map. This map, which is single-population based and thus is not dependent on integration of markers from different populations, may be more accurate in marker order at some positions. The number of 191 mapped SNP markers is much less than that in any of the individual ssp. unguiculata and its close relative Vigna radiataA closer look at marker order in the two maps revealed high conservation but also possible differences in genome structure between the two subspecies. Among the shared SNP loci, 90% are in the same order. Of the remaining 10% loci showing inconsistency, 30 (90%) were due to additional crossovers observed in ZZ population thus is not the reflection of genome reorganization. Three markers, i.e., 1_0308 (LG1), 1_1059 (LG2) and 1_1298 (LG6) show slight change in orders between the two maps; however, since these markers are randomly distributed, such changes maybe due to the presence of additional flanking SSR loci or different mapping LOD threshold used. In fact, minor variations of marker order and position are common when comparing genetic maps constructed from different mapping populations Medicago truncatula has been reported L. japonica has not yet been established. Here, we show that the genomes of asparagus bean and L. japonica are highly syntenic, with macrosynteny punctuated by rearrangements frequently involving translocation and inversion of chromosome arms (L. japonicusL. japonicus chromosome 1 (hereafter referred to as LjChr) exhibits the largest number of matched asparagus bean marker loci and is syntenic to as many as five asparagus bean chromosomes . LjChr4 and 5 each corresponds to only one asparagus bean chromosome, which may reflect their relative shorter physical lengths L. japonicus provides a framework foundation for further marker and gene identification in asparagus bean through the use of sequence data from L. japonica as a genomic model.The macrocolinerity between cowpea and the warm-season model legumes soybean and ome arms . The ‘chPhaseoleae group. Similarly, SDRs in syntenic regions were also observed in related species of PoaceaeThe SDRs in ZZ map were compared with those mapped in soybean genetic map. Yamanaka et al. (2001) reported two SDRs with three or more loci in soybean LG D2 and LG J unguiculata to asparagus bean made the mapping project very efficient and ‘parallel’ to the international ssp. unguiculata mapping work, allowing comparison of genetic maps between the subspecies. The development and application of SSR markers, some of which are from EST sequences and others from genomic sequences, complemented the relatively low polymorphism rate of EST-derived SNPs between the mapping parents, and more importantly, enabled us to detect more loci including non-coding regions which are absent in the consensus map. Although SNP assays are becoming more cost-efficient, in some situations SSR markers may be more practical for marker-assisted breeding unguiculata consensus map with the newly available SSR markers and to recalculate the consensus map, by which more informative results could be obtained from comparing the updated maps of the two subspecies.We present here the first genetic map of asparagus bean that establishes a foundation for genome characterization and molecular breeding of this specialized cowpea subspecies. This map, comprised of two types of DNA markers has some advantages. The successful transfer of high-throughput SNP genotyping platform developed from ssp. MedicagoL. japonica (this report) would contribute to the detailed analysis of genome evolution of model legumes. With more sequences and mapping data from other important crop legumes such as mungbean The ZZ map also provides a significant resource for functional analysis of traits of interest as well as comparative genomics studies. A functional classification of the mapped loci shows a broad range of functional types among these loci. This information is particularly important in gene/QTL mapping of traits such as pod length and climbing habit to help discover candidate genes . EST-der7:8 recombinant inbred lines (RILs) developed by single-seed descent from a cross between ‘ZN016’, a rustic landrace asparagus bean  variety originating from southern China and ‘Zhijiang282’, a commercial asparagus bean cultivar in China, as well as the mapping parents were used in this study.The mapping population, which consists of 114 FGenomic DNA for SSR analysis was extracted from leaves of two-week-old seedlings using either a modified CTAB method Procedures of the high-throughput SNP genotyping assay and raw data processing employing a 1536-SNP Illumina GoldenGate platform was the same to that as described in Muchero et al (2009) One thousand three hundred and seventy-two microsatellite markers developed in the authors' lab, including 494 EST-SSRs, 632 GSS-SSRs and 200 SSRs derived from BAC end sequences, in addition to 46 SSR markers developed by Li et al (2001) 2, 0.2 U rTaq DNA polymerase  and 1× PCR buffer supplied together with the enzyme. The PCR cycles were 94°C 3 min, 36 cycles of 94°C 20 s, 48–52°C 30 s depending on primers used, 72°C 40 s, and a 5 min 72°C extension. The PCR products were separated in 8% non-denaturing polyacrylamide gels (Acr:Bis  = 19∶1 or 29∶1) at room temperature in vertical gel apparatus (170×150×1.0 mm) with 1× TBE buffer and visualized by silver staining Polymerase chain reactions (PCR) were carried out in 12.5 µl reactions containing 10 ng of each of the template DNA, 2.5 pmol of each of the primers, 2.5 nmol of each of the dNTPs, 18.6 nmol MgClP<0.05) was defined as a SDR Linkage maps were constructed using JoinMap 3.0  incorporating the available SNP and SSR markers data. A LOD score of 5.0 with a maximum recombination of 45% was used to assign markers to linkage groups with the exception for LG6 and LG11, where a LOD score of 8.0 and 9.0, respectively, was applied to break spurious linkage according to Muchero et al. (2009). LOD threshold for map calculation was set as 3.0 except for LG2, LG8 and LG4, in which the LOD scores of 2.0, 2.0 and as low as 0.1 were used respectively, in order to allow the inclusion of more distant loci. Other important mapping parameters included: Kosambi's mapping function L. japonica was determined by blast N searches against the genome sequence of L. japonica  using source sequences of mapped asparagus bean SNP and SSR markers as queries. The cut-off value for significance was set as e-20 in at least a 120bp overlap.Synteny between asparagus bean and the model legume Table S1Sequences of SSR primers developed in the authors' lab and used in the current study.(XLS)Click here for additional data file.Table S2Position, type and functional annotation of mapped loci.(XLS)Click here for additional data file.Table S3Synteny of mapped loci to the L. japonica pseudochromosomes.(XLS)Click here for additional data file.


Fetal movement counting is a method used by the mother to quantify her baby's movements, and may prevent adverse pregnancy outcome by a timely evaluation of fetal health when the woman reports decreased fetal movements. We aimed to assess effects of fetal movement counting on identification of fetal pathology and pregnancy outcome.In a multicentre, randomized, controlled trial, 1076 pregnant women with singleton pregnancies from an unselected population were assigned to either perform fetal movement counting from gestational week 28, or to receive standard antenatal care not including fetal movement counting (controls). Women were recruited from nine Norwegian hospitals during September 2007 through November 2009. Main outcome was a compound measure of fetal pathology and adverse pregnancy outcomes. Analysis was performed by intention-to-treat.The frequency of the main outcome was equal in the groups; 63 of 433 (11.6%) in the intervention group, versus 53 of 532 (10.7%) in the control group [RR: 1.1 95% CI 0.7–1.5)]. The growth-restricted fetuses were more often identified prior to birth in the intervention group than in the control group; 20 of 23 fetuses (87.0%) versus 12 of 20 fetuses (60.0%), respectively, [RR: 1.5 (95% CI 1.0–2.1)]. In the intervention group two babies (0.4%) had Apgar scores <4 at 1 minute, versus 12 (2.3%) in the control group [RR: 0.2 (95% CI 0.04–0.7)]. The frequency of consultations for decreased fetal movement was 71 (13.1%) and 57 (10.7%) in the intervention and control groups, respectively [RR: 1.2 (95% CI 0.9–1.7)]. The frequency of interventions was similar in the groups.Maternal ability to detect clinically important changes in fetal activity seemed to be improved by fetal movement counting; there was an increased identification of fetal growth restriction and improved perinatal outcome, without inducing more consultations or obstetric interventions.ClinicalTrials.govNCT00513942 Maternal perception of a gradual diminishment of fetal activity is a significant marker of a vulnerable fetus and can indicate chronic fetal compromise per se may serve as an organized daily effort to ensure vigilance to fetal movements, and thus, improve the ongoing maternal self-screening Fetal movement counting is a means of screening fetal status. It is developed as a simple, inexpensive and easily accessible tool to support the mother in monitoring her baby's well-being to identify alarming behavior in time to intervene There is a lack of evidence of the sensitivity and specificity for the variety of quantitative definitions of decreased fetal movement that has been proposed over the years and there is no conclusive evidence that any of them reduce perinatal morbidity and mortality The protocol for this trial and supporting CONSORT checklist are available as supporting information; see www.clinicaltrials.gov protocol registration system (number NCT00513942).Ethical approval was obtained from The Regional Committee for Medical Research Ethics (reference S-07188a) 7 May 2007, and by The Norwegian Data Inspectorate and Directorate for Health (reference 07/2504) 19 July 2007. The study was registered in In a randomized, controlled trial pregnant women were allocated to one of two groups, the intervention group who were instructed to perform fetal movement counting from gestational week 28, and the control group who received standard antenatal care according to the Norwegian guidelines. In Norway, antenatal care is a public health care service free of charge to which almost all pregnant women adhere. Participants were approached during their regular ultrasound screening in pregnancy weeks 17–19 : emergency Caesarean section on fetal indication; (iii) oligohydramnios (as defined by the clinicians); (iv) pathological blood flow in arteria umbilicalis; (v) maternal perception of absent fetal movements for more than 24 hours before admission to hospital, or (vi) perinatal death. Secondary outcomes were (i) Apgar scores <4 at 1 and 5 minutes; (ii) fetal growth restriction <2.5th centile unidentified prior to birth; (iii) the total number of consultations for decreased fetal activity; (iv) use of health resources in evaluation of these pregnancies, and (v) interventions prior to or during delivery. Fetal growth restriction was defined as a birth weight <2.5th centile adjusted for maternal height and weight in early pregnancy, and baby's sex th percentile birth weight (i.e. <21.5% negative deviation), or a negative trend on serial antenatal ultrasounds.Primary outcome was a compound measure of the following: (i) fetal growth restriction <2.5general information: age, educational level, marital status, Body Mass Index, nicotine and alcohol consumption, and country of origin; (ii) obstetric risk factors: previous pregnancy with fetal growth restriction, stillbirth >21 weeks of gestation, preterm delivery, serious preeclampsia or malformations; (iii) pre pregnancy risk factors: hypertension, chronic renal or coronary disease, known diabetes type I or II, inflammatory and rheumatoid diseases, coagulopathy, epilepsy or hypothyreosis, and (iv) maternal complications identified during pregnancy: hypertension, preeclampsia, preterm contractions, prolonged preterm rupture of membranes, haemorrhage >27 weeks of pregnancy, diabetes (any type), urinary tract infection or other relevant infections.Maternal characteristics included demographic information and risk factors according to Norwegian Guidelines As an indicator of the user-friendliness of the counting chart, compliance was measured. A total of 427 (78.5%) women returned the chart. Of these, 331 women (77.5%) completed the counting chart more than 50 per cent of the days during the period and at least two days each week. Compliance did not vary between any subgroups.Sample size calculation was made using the computer program PS Power and Sample Sizes The data were analyzed using SPSS 17.0  and Episheet The significance level was set at p = 0.05. All analyses were performed according to the intention-to-treat principle. Analyses were performed by the researchers without blinding for group assignment. This manuscript is in compliance with the CONSORT statement for reporting trials We obtained written informed consent from 1155 women. As demonstrated in the study profile , 1076 woThe frequency of the primary outcome was equal between the groups; 63 of 433 (11.6%) in the intervention group, versus 53 of 532 (10.7%) in the control group [RR: 1.1 95% CI 0.7–1.5) (p = 0.652)]. The proportion growth-restricted fetuses was similar between the groups , but theThe frequency of consultations because of maternal concern for decreased fetal movements did not differ between the groups, 71 of 542 pregnancies (13.1%) in the intervention group, versus 57 of 532 pregnancies (10.7%) in the control group [RR: 1.2 (95% CI 0.9–1.7) (p = 0.228)]. The mean gestational age at the time of maternal report was similar between the groups: mean gestational day (SD) was 254 (range 196–295) (SD 27.6) and 258 (range 198–296) (SD 25.9) in the groups, respectively (p = 0.402). Among the consultations for decreased fetal movements, more often a fetus with a <−10% weight estimate was identified in the intervention versus the control group .There were no statistically significant differences between the groups regarding the frequency of interventions prior to or during delivery, neither in the total sample  nor amonThis study has suggested that an intervention involving fetal movement counting, compared to no intervention, was associated with improved identification of fetal growth restriction and a reduction in fetuses with severely low Apgar scores – both known to be associated with further adverse neonatal and childhood outcomes et al. et al. did not identify any significant reduction in the in rates of unexplained stillbirth for women using a counting chart. Several methodological issues have been identified that have raised questions about the validity of the results and conclusions In 1989, Grant undetected antenatally have a higher mortality than those that are detected prior to delivery This study was underpowered to detect any difference in perinatal mortality rates. However, we identified improved identification of fetal growth restriction, which is the most frequently reported association to decreased fetal activity and adverse outcomes The universal self-screening performed as pregnant women perceive decreased fetal movements, may be the first identification of fetal compromise The frequency of consultations because of maternal report of decreased fetal movements in the current study is approximating previous studies One of the factors associated with decreased fetal activity is a low Apgar score By using a clinically oriented definition of fetal growth restriction, including significant deviation from normal growth by serial ultrasound, an intervention that led to large proportions of pregnant women being examined by serial ultrasound would provide skewed results inflating detection rates. This was not the case in this study, as the use of ultrasound was identical in both arms of the trial. Although the consultation rates were similar between the groups, we found an improved identification of fetal growth restriction and perinatal outcome in the intervention group. Uniform information and systematic monitoring of fetal activity in this group may have improved the mothers' ability to distinguish normal variations in fetal activity from changes representing risk. Thus, the women contacting the delivery clinics with their concern for decreased fetal activity in the intervention group may have been better selected.The strength of this study lies in its experimental design. Furthermore, the sample size was sufficiently large to permit cautious generalization of the findings, although the primary outcomes are composed of few cases, and size of the effect must be interpreted with caution. The sample was representative for the total population of pregnant women in Norway with respect to demographic characteristics, although the lower proportions of smoking women in the study sample may indicate a bias towards healthier pregnancies. Subgroup analyses were not pre-specified in the protocol, as the actual groups are too small to have enough power to proclaim any secure effects. Therefore, subgroup analyses have not been performed, according to the CONSORT statement The population of pregnant Norwegian women is relatively homogeneous; the women who participated were predominantly employed, cohabiting, white, and well-educated; a typical Scandinavian population. Therefore, generalizations should be limited to similar populations.It is impossible to restrict the information about fetal movement completely to only one part of a population. Pregnant women are frequent users of social networks, and share their personal experiences and views Although, all women must have heard about fetal movement counting in the recruitment process, only 143 (30.2%) reported that they knew about fetal movement counting when asked directly in a questionnaire. Of the women who knew about fetal movement counting, 62 (43.4%) had heard or read about it in the information brochure, whereas 81 (56.6%) had obtained information from the Internet, friends, or their midwife or physician. However, only one woman in the control group (0.2%) had used a fetal movement chart, indicating a clear separation between the groups.Knowledge about the assignment group could potentially affect the observation, examination or the intervention of the participants. However, there was no difference between the intervention and control groups with regard to the frequency of use of any kind of observation or examination or follow-up, whether in the total sample or among the women reporting decreased fetal activity and such bias thus seems unlikely.The overall goal for fetal movement counting is reduced fetal mortality. As the perinatal mortality rates are as low as 4.4/1000 in our population Regardless of the debate on the effect of fetal movement counting, in current antenatal care, monitoring fetal activity is ongoing, largely as an unstructured self-screening procedure, administered and interpreted by the pregnant women individually Maternal ability to detect clinically important changes in fetal activity seemed to be improved by fetal movement counting; there was an increased identification of fetal growth restriction and improved perinatal outcomes, without inducing either more frequent consultations at hospitals or increased frequency of interventions before or during delivery. Further research is needed to assess the effects of fetal movement counting on hard outcomes such as stillbirth rates.Checklist S1CONSORT Checklist.(DOC)Click here for additional data file.Protocol S1Trial Protocol.(DOC)Click here for additional data file.


To identify the value of unilateral pedal lymphangiography (LAG) with non-contrast CT in the location and treatment decision of idiopathic chylothorax after failure of thoracic duct ligation.Twenty four patients aged 9–84 year old (median 44 yr) who had idiopathic chylothorax were involved, and unilateral pedal LAG with non-contrast CT was performed in every patient. All patients failed to previous right supra-diaphragmatic thoracic duct ligation.P = 0.02). Meanwhile, non-operative therapy had significantly higher successful rate .The amount of iodized oil used was 6–14 ml with no related complications. LAG demonstrated 8 patients with thoracic duct leaks and 10 patients with leaks elsewhere, but no visible chylous leak in 6 patients. Ligation of thoracic duct was performed as the primary treatment in all 8 cases as having thoracic duct leakage and cured 7(87.5%) patients. For 8 patients not having thoracic duct lesion under LAG, the successful rate of thoracic duct ligation was 25% (2 out of 8 patients), which was significantly lower than patients due to thoracic duct lesions (Unilateral pedal LAG with non-contrast CT could identify the causes and locate the leaks of idiopathic chylothorax in 75% of patients after failure of thoracic duct ligation. Two thirds of patients were found not to have thoracic duct leakage and would be better managed by non-operative treatment. Chylous pleural effusion, or chylothorax, is defined as the accumulation of chyle-containing lymphatic fluid within the pleural space . It has Despite of its rarity, extensive chyle leakage from idiopathic chylothorax can also have deleterious physiologic consequences in the form of hypovolemia and hypoproteinemia resulting from severe fluid and protein loss . HistoriIf conservative treatment of chylothorax is unsuccessful, or in the event of high flow chylothorax surgical ligature of the thoracic duct must be undertaken . The purFrom year 2000 to 2011, twenty-four patients aged 9–84 year old (median 44 yr) who had idiopathic chylothorax were involved in this series, including 10 males and 14 females. All patients failed to their previous right supra-diaphragmatic thoracic duct ligations. Main clinical manifestations were dyspnea, shortness of breath or unavailable of prostration. The presence of chylothorax was defined by 1 or more of the following inclusion criteria: (1) a pleural fluid triglyceride level of 110 mg/dL or greater; (2) the presence of chylomicrons in the pleural effusion. Other causes of chylothrax, such as malignancies, trauma, tuberculosis or filariasis, were excluded. After their previous thoracic duct ligations, 20 patients had unilateral (9 in left and 11 in right side) and 4 patients had bilateral chylothorax with duration of 3 days to one year. Conventional unenhanced MRI was done for each patient but only located suspected leak sites of thoracic duct in 4 patients. Thus, patients were screened for contraindications to LAG, including cardiovascular and pulmonary disease  [Unilateral pedal LAG was performed according to the following steps. First, 2 ml of methylene blue, a dye that stains the lymphatics, was injected into the web space between the first and second toes of one foot. Thirty minutes later, linear cut-down was performed on the dorsum of the foot below the ankle, and the lymphatic vessel was isolated. After cannulation of a lymphatic vessel on the dorsum of foot using a 30 gauge needle, iodized oil , which is a contrast agent for LAG, was injected at a rate of 0.1 ml/min, not exceeding a total volume of 14 ml.Completion radiographs were obtained to assess for immediately apparent chyle leaks and to document the filling phase of the LAG. Each patient had follow-up nodal phase radiographs 24 h after the initial procedure. In these nodal phase radiographs, the pleural space, peritoneal space, and urinary collecting system were examined for signs of contrast extravasation. Any readily apparent leaks were reassessed and any newly apparent leaks were documented. In all cases, a non-contrast CT scan was also performed at that time to confirm the result of LAG or determine the exact level of the lymphatic leakage.All 24 patients underwent unilateral pedal LAG with the amount of iodized oil from 6 to 14 ml. LAG was successful in all patients with no related complications. Of these patients, LAG showed chylous leakage from thoracic duct in 8 (including those 4 patients whose chylothorax was detected by MRI) and from intra- or retroperitoneal lymphatics in 7 and 2 patients respectively, whereas 6 patients showed no chylous leak by LAG with CT. The remaining patient, a 9-year-old boy who had bilateral chylothorax was detected to have extensive pleural lymphatic leak Figures . The durP = 0.02). The remaining 8 patients not due to thoracic duct leaks received non-operative therapy with thoracentesis. Its cure or improve rate was 87.5% (7 out of 8 patients), which was significantly higher than thoracic duct ligation  (Table LAG with non-contrast CT properly located chyle leak in 18 (75%) of all 24 cases, whereas 6 patients showed no leakage. . We suggest that unilateral pedal LAG with non-contrast CT is valuable to determine underlying lymphatic vessel leak and localize the leakage site for surgical therapy. It could also indicate those patients suitable for non-operaitve treatment to avoid unnecessary surgical interventions.Infection and pain are the most common complications seen with LAG, but serious complications have also been reported, including intra-alveolar hemorrhage, allergic reactions to Lipiodol or methylene blue, extravasation of contrast into the deep tissues of the foot, and oil emboli to the lungs, brain, kidney, and liver ,8,18-20.The limitation of this study is the limit number of patients with no randomized control comparison, predominantly due to the rarity of idiopathic chylothorax. A multicenter, randomized control study with large number of patients is necessary to investigate the exact value of unilateral pedal LAG with CT in the location and management of idiopathic chylothorax.Unilateral pedal LAG with non-contrast CT could identify the causes and leakage sites in 75% of patients with idiopathic chylothorax after failure of thoracic duct ligation. Two thirds of patients were found not to have thoracic duct leakage and would be better managed by non-operative treatment.The authors declare that they have no competing interests.DL participated in the design of the study, LAG performing and helped to draft the manuscript. YS participated in the design of the study, the statistical analysis and draft the manuscript. JS performed thoracentesis and thoracic surgeries. All authors read and approved the final manuscript.