Development of cost-effective SNP assays for chickpea genome analysis and breeding

A total of 1499 ESTs generated from 26 different Cicer species, available in the public domain at the time of analysis, were used for in silico identification of SNPs using the bioinformatic tools developed at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) (http://hpc.icrisat.org/PBSWeb). Cluster analysis provided a total of 118 clusters, of which 11 clusters contained sequences from more than one Cicer species. Further, these clusters were assembled into 19 contigs and 184 putative SNPs were identified in 15 contigs. However, only 73 SNPs involved restriction enzyme sites for development of the CAPS assays as identified through the SNP2CAPs program. Primer pairs were designed for only 8 contigs (CL3a, CL3c, CL3d, CL3e, CL4a, CL10, CL20 and CL99) which had SNPs, resulting in putative recognition sites to commonly used restriction enzymes. Results of the demonstrates the utility of Cicer EST resources and the availability of bioinformatics analysis pipelines for the large-scale identification of SNPs on the HPC (High Performance Computer) at ICRISAT and the development of costeffective CAPS assay for SNP genotyping. It is anticipated that the availability of large number of ESTs from more than one genotype of cultivated chickpea (C. arietinum) in the near future will make it possible to develop larger number of SNPs in cultivated chickpea germplasm for genome analysis and breeding application

DESIGN, SYNTHESIS AND IN VITRO ANTI-CANCER ACTIVITY OF NOVEL 1,2,4-TRIAZOLE DERIVATIVES

Objective: DNA topoisomerase is one of the important targets for anticancer agents. Many triazole derivatives have been shown to possess cytotoxic activity. In this paper, we present the design and in silico docking of a virtual library of molecules with DNA topoisomerase II along with their synthesis and In vitro cytotoxicity profile. Methods: Sybyl X 2.1 programmesss were used to perform the docking experiments on DNA topoisomerase II using etoposide as ligand. In vitro anticancer activity was carried out by trypan blue exclusion assay against EAC cells. DNA fragmentation studies were performed by Gel electrophoresis to identify the cause of cell death induced by these compounds. Results: Among the compounds studied for docking, 12c generated the highest docking score (13.66) and showed hydrogen bonding interactions with glycine 778 at a distance of 1.879 A˚. the compounds 12c & 12g showed the highest level of cytotoxicity with IC50 value of 0.55 μM and 0.62 μM respectively. Compounds 12c and12g were subjected to DNA fragmentation studies to identify the cause of cell death induced by these compounds. Gel electrophoresis of these compounds showed a typical feature of apoptosis ladders in agarose gel. Compound 12c was able to induce apoptosis at a concentration of about 3 μM. Conclusion: A series of bis-triazoles were synthesized targeted to DNA topoisomerase II and evaluated their In vitro cytotoxicity. The compound 12c was found to be most active and also exhibited apoptosis inducing potential

An Integrated Pipeline of Open Source Software Adapted for Multi-CPU Architectures: Use in the Large-Scale Identification of Single Nucleotide Polymorphisms

The large amounts of EST sequence data available from a single species of an organism as well as for several species within a genus provide an easy source of identification of intra- and interspecies single nucleotide polymorphisms (SNPs). In the case of model organisms, the data available are numerous, given the degree of redundancy in the deposited EST data. There are several available bioinformatics tools that can be used to mine this data; however, using them requires a certain level of expertise: the tools have to be used sequentially with accompanying format conversion and steps like clustering and assembly of sequences become time-intensive jobs even for moderately sized datasets. We report here a pipeline of open source software extended to run on multiple CPU architectures that can be used to mine large EST datasets for SNPs and identify restriction sites for assaying the SNPs so that cost-effective CAPS assays can be developed for SNP genotyping in genetics and breeding applications. At the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), the pipeline has been implemented to run on a Paracel high-performance system consisting of four dual AMD Opteron processors running Linux with MPICH. The pipeline can be accessed through user-friendly web interfaces at http://hpc.icrisat.cgiar.org/PBSWeb and is available on request for academic use. We have validated the developed pipeline by mining chickpea ESTs for interspecies SNPs, development of CAPS assays for SNP genotyping, and confirmation of restriction digestion pattern at the sequence level

Uniaxial Tensile Stress-Strain Relationships of RC Elements Strengthened with FRP Sheets

The shear behavior of fiber-reinforced-polymer–strengthened reinforced concrete (FRP-strengthened RC) members is not fully developed and accurately predicted because of the lack of accurate constitutive laws for the components of the composite members. This paper presents experimental and analytical investigations of tensile stress-strain relationships of concrete and steel in FRP-strengthened RC members. These stress-strain relationships are required in formulations of softened truss models to predict the shear behavior of the FRP-strengthened RC element. Thirteen full-scale FRP-strengthened RC prismatic specimens with different FRP reinforcement ratios, steel reinforcement ratios, and FRP wrapping schemes were tested under uniaxial tension loading. The results show that the tensile behavior of the concrete and steel is altered because of the externally bonded FRP sheets. Modified constitutive laws are proposed and incorporated in the softened membrane model (SMM) to demonstrate through two tests the behavior of FRP-strengthened RC element subjected to pure shear. Moreover, crack spacing and crack width were studied and compared with existing code provisions

Comparative Genomic Characterization of the Highly Persistent and Potentially Virulent Cronobacter sakazakii ST83, CC65 Strain H322 and Other ST83 Strains

Cronobacter (C.) sakazakii is an opportunistic pathogen and has been associated with serious infections with high mortality rates predominantly in pre-term, low-birth weight and/or immune compromised neonates and infants. Infections have been epidemiologically linked to consumption of intrinsically and extrinsically contaminated lots of reconstituted powdered infant formula (PIF), thus contamination of such products is a challenging task for the PIF producing industry. We present the draft genome of C. sakazakii H322, a highly persistent sequence type (ST) 83, clonal complex (CC) 65, serotype O:7 strain obtained from a batch of non-released contaminated PIF product. The presence of this strain in the production environment was traced back more than 4 years. Whole genome sequencing (WGS) of this strain together with four more ST83 strains (PIF production environment-associated) confirmed a high degree of sequence homology among four of the five strains. Phylogenetic analysis using microarray (MA) and WGS data showed that the ST83 strains were highly phylogenetically related and MA showed that between 5 and 38 genes differed from one another in these strains. All strains possessed the pESA3-like virulence plasmid and one strain possessed a pESA2-like plasmid. In addition, a pCS1-like plasmid was also found. In order to assess the potential in vivo pathogenicity of the ST83 strains, each strain was subjected to infection studies using the recently developed zebrafish embryo model. Our results showed a high (90–100%) zebrafish mortality rate for all of these strains, suggesting a high risk for infections and illness in neonates potentially exposed to PIF contaminated with ST83 C. sakazakii strains. In summary, virulent ST83, CC65, serotype CsakO:7 strains, though rarely found intrinsically in PIF, can persist within a PIF manufacturing facility for years and potentially pose significant quality assurance challenges to the PIF manufacturing industry

Nonsense-Mediated Decay Enables Intron Gain in Drosophila

Intron number varies considerably among genomes, but despite their fundamental importance, the mutational mechanisms and evolutionary processes underlying the expansion of intron number remain unknown. Here we show that Drosophila, in contrast to most eukaryotic lineages, is still undergoing a dramatic rate of intron gain. These novel introns carry significantly weaker splice sites that may impede their identification by the spliceosome. Novel introns are more likely to encode a premature termination codon (PTC), indicating that nonsense-mediated decay (NMD) functions as a backup for weak splicing of new introns. Our data suggest that new introns originate when genomic insertions with weak splice sites are hidden from selection by NMD. This mechanism reduces the sequence requirement imposed on novel introns and implies that the capacity of the spliceosome to recognize weak splice sites was a prerequisite for intron gain during eukaryotic evolution

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Comparison of ligation versus ligation with stripping of long Saphenous vein in varicose veins of lower limb

Aim: To compare the incident of complications after ligation versus ligation with stripping of long saphenous vein in the treatment of primary varicose veins of lower limb. Materials and methods: The prospective randomized study is based on analysis of 80 cases of primary varicose veins of long saphenous vein or without perforator in competence from the period of 1 year in department of general surgery, Patients are divided into Group-L 40 patients for simple ligation and group-LS 40 patients for ligation with stripping of long saphenous vein. Results: Similar significant difference with respect to the surgical time, amount of bleeding and duration of hospital stays was observed in Group-LS as compared to group-L (P &lt; 0.01). Hematoma and wound infection is not significant in group-L compared to group –LS . Limb edema is significant in group-LS compared to group –L . In the group that underwent ligation alone, thigh perforators were present in 62.5% cases whereas in the group that underwent ligation combined with stripping thigh perforators were present in 57.5 % of cases which is significant. After surgery residual long saphenous vein reflux was present in 37.5% cases in the group in which ligation alone was done and residual thigh perforators were identified in 33.3% of cases which is significant ,whereas in the group which underwent ligation combined with stripping of the thigh segment of long saphenous vein residual reflux was present in 21.4% cases and no residual thigh perforators were identified which is significant. Conclusion: Ligation with stripping for patients with varicosities of the lower extremity with a shorter surgical time, fewer bleeding, duration of hospital stays and residual perforator

Performance of two anchor systems of externally bonded fiber-reinforced polymer laminates

Fiber-reinforced polymer (FRP) composite materials have, been proposed and used for the upgrade of concrete-based transportation and civil infrastructure. This technology has been proven to be effective, but issues related to bond length, end anchorage, and premature peeling have been a concern when strengthening structures in flexure or shear. This study presents two different types of FRPbased anchor system, namely, a near surface mounted (NSM) end anchor and a spike anchor. Each has been tested in an attempt to prevent or delay the problems listed above. A total of 16 specimens were tested to failure to check the effectiveness of the end anchor system and 19 specimens were tested to check the effectiveness of the spike anchor system. The focus of the study is on the influence of 1) the location, groove size, and anchor bar size for the first system; and 2) the location and embedment of the spike anchors for the second system. Test results show that each of these systems is highly effective in increasing the capacity of the strengthened member by delaying delamination of the FRP material. Bonddependent coefficients of 0.90 (Km) and 0.25 (Kv) are recommended when using these anchors for flexural and shear strengthening applications, after comparing the experimental results with the design guidelines. Copyright © 2008, American Concrete Institute. All rights reserved,

Effectiveness of CFRP strengthening on curved soffit RC beams

The objective of this research program was to study carbon fiber reinforced polymer (CFRP) strengthening of reinforced concrete (RC) members which have concavely-curved soffits. In the presence of such curvature, the FRP laminates attempt to straighten under tension, leading to direct transverse tensile stress at the adhesive-concrete interface, which may cause premature peeling. This tensile stress could also lead to concrete cover delamination. Ten beams, each 6 m in length and having the same soffit curvature (i.e., 4 × 10 -5 mm-1 or in-1), extending over 1, 3 and 5 meters, were tested under three-point static flexure. Two different types of CFRP strengthening techniques, namely wet layup laminates and pultruded precured laminates, were used for external strengthening. One of the beams strengthened with CFRP wet layup laminates (with a curved soffit over an extent of 5 m), used glass FRP spike anchors to prevent peeling. Beams with soffit curvature limited to an extent of 1 m, failed at similar loads when compared with control specimens containing flat soffits. For beams having a soffit curvature extending over 3 and 5 m, reduction in strength was observed because of premature peeling. Inclusion of the GFRP spike anchors with the wet layup system led to a performance superior to the control specimen (flat soffit) confirming that application of GFRP spike anchors delays premature peeling