Development of ESTs from chickpea roots and their use in diversity analysis of the Cicer genus

BACKGROUND: Chickpea is a major crop in many drier regions of the world where it is an important protein-rich food and an increasingly valuable traded commodity. The wild annual Cicer species are known to possess unique sources of resistance to pests and diseases, and tolerance to environmental stresses. However, there has been limited utilization of these wild species by chickpea breeding programs due to interspecific crossing barriers and deleterious linkage drag. Molecular genetic diversity analysis may help predict which accessions are most likely to produce fertile progeny when crossed with chickpea cultivars. While, trait-markers may provide an effective tool for breaking linkage drag. Although SSR markers are the assay of choice for marker-assisted selection of specific traits in conventional breeding populations, they may not provide reliable estimates of interspecific diversity, and may lose selective power in backcross programs based on interspecific introgressions. Thus, we have pursued the development of gene-based markers to resolve these problems and to provide candidate gene markers for QTL mapping of important agronomic traits. RESULTS: An EST library was constructed after subtractive suppressive hybridization (SSH) of root tissue from two very closely related chickpea genotypes (Cicer arietinum). A total of 106 EST-based markers were designed from 477 sequences with functional annotations and these were tested on C. arietinum. Forty-four EST markers were polymorphic when screened across nine Cicer species (including the cultigen). Parsimony and PCoA analysis of the resultant EST-marker dataset indicated that most accessions cluster in accordance with the previously defined classification of primary (C. arietinum, C. echinospermum and C. reticulatum), secondary (C. pinnatifidum, C. bijugum and C. judaicum), and tertiary (C. yamashitae, C. chrossanicum and C. cuneatum) gene-pools. A large proportion of EST alleles (45%) were only present in one or two of the accessions tested whilst the others were represented in up to twelve of the accessions tested. CONCLUSION: Gene-based markers have proven to be effective tools for diversity analysis in Cicer and EST diversity analysis may be useful in identifying promising candidates for interspecific hybridization programs. The EST markers generated in this study have detected high levels of polymorphism amongst both common and rare alleles. This suggests that they would be useful for allele-mining of germplasm collections for identification of candidate accessions in the search for new sources of resistance to pests / diseases, and tolerance to abiotic stresses

Different Shades of Listeria monocytogenes: Strain, Serotype, and Lineage-Based Variability in Virulence and Stress Tolerance Profiles

Listeria monocytogenes is a public health and food safety challenge due to its virulence and natural stress resistance phenotypes. The variable distribution of L. monocytogenes molecular subtypes with respect to food products and processing environments and among human and animal clinical listeriosis cases is observed. Sixty-two clinical and food-associated L. monocytogenes isolates were examined through phenome and genome analysis. Virulence assessed using a zebrafish infection model revealed serotype and genotype-specific differences in pathogenicity. Strains of genetic lineage I serotype 4b and multilocus sequence type clonal complexes CC1, CC2, CC4, and CC6 grew and survived better and were more virulent than serotype 1/2a and 1/2c lineage II, CC8, and CC9 strains. Hemolysis, phospholipase activity, and lysozyme tolerance profiles were associated with the differences observed in virulence. Osmotic stress resistance evaluation revealed serotype 4b lineage I CC2 and CC4 strains as more osmotolerant, whereas serotype 1/2c lineage II CC9 strains were more osmo-sensitive than others. Variable tolerance to the widely used quaternary ammonium compound benzalkonium chloride (BC) was observed. Some outbreak and sporadic clinical case associated strains demonstrated BC tolerance, which might have contributed to their survival and transition in the food-processing environment facilitating food product contamination and ultimately outbreaks or sporadic listeriosis cases. Genome comparison uncovered various moderate differences in virulence and stress associated genes between the strains indicating that these differences in addition to gene expression regulation variations might largely be responsible for the observed virulence and stress sensitivity phenotypic differences. Overall, our study uncovered strain and genotype-dependent variation in virulence and stress resilience among clinical and food-associated L. monocytogenes isolates with potential public health risk implications. The extensive genome and phenotypic data generated provide a basis for developing improved Listeria control strategies and policies

A difficult diagnosis - constrictive pericarditis and its treatment: a case report

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Abstract The diagnosis of constrictive pericarditis requires a high degree of clinical suspicion, for the signs and symptoms of this disease can be falsely attributed to other causes. Herein, we present a case of a 70-year old retired farmer whose symptoms of right heart failure were initially attributed to co-existing pneumonia and pulmonary embolism. He was discharged. Three weeks later he presented with worsening breathlessness and ascites. Echocardiography, computed tomography and cardiac catheterization revealed the diagnosis of constrictive pericarditis. He underwent complete pericardectomy and to date has made a good recovery. This case exemplifies the difficulty in diagnosing this condition, the investigation required, and provides a discussion of the benefit and outcomes of prompt treatment.Peer Reviewe

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

The Analysis of Field Strains Isolated From Food, Animal and Clinical Sources Uncovers Natural Mutations in Listeria monocytogenes Nisin Resistance Genes

Nisin is a commonly used bacteriocin for controlling spoilage and pathogenic bacteria in food products. Strains possessing high natural nisin resistance that reduce or increase the potency of this bacteriocin against Listeria monocytogenes have been described. Our study sought to gather more insights into nisin resistance mechanisms in natural L. monocytogenes populations by examining a collection of 356 field strains that were isolated from different foods, food production environments, animals and human infections. A growth curve analysis-based approach was used to access nisin inhibition levels and assign the L. monocytogenes strains into three nisin response phenotypic categories; resistant (66%), intermediate (26%), and sensitive (8%). Using this categorization isolation source, serotype, genetic lineage, clonal complex (CC) and strain-dependent natural variation in nisin phenotypic resistance among L. monocytogenes field strains was revealed. Whole genome sequence analysis and comparison of high nisin resistant and sensitive strains led to the identification of new naturally occurring mutations in nisin response genes associated with increased nisin resistance and sensitivity in this bacterium. Increased nisin resistance was detected in strains harboring RsbUG77S and PBPB3V240F amino acid substitution mutations, which also showed increased detergent stress resistance as well as increased virulence in a zebra fish infection model. On the other hand, increased natural nisin sensitivity was detected among strains with mutations in sigB, vir, and dlt operons that also showed increased lysozyme sensitivity and lower virulence. Overall, our study identified naturally selected mutations involving pbpB3 (lm0441) as well as sigB, vir, and dlt operon genes that are associated with intrinsic nisin resistance in L. monocytogenes field strains recovered from various food and human associated sources. Finally, we show that combining growth parameter-based phenotypic analysis and genome sequencing is an effective approach that can be useful for the identification of novel nisin response associated genetic variants among L. monocytogenes field strains.Peer reviewe

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

Reactive Separation of Vanillic Acid Using Tri-n-butyl Phosphate

The aim of this study was to extract vanillic acid from an aqueous solution through reactive extraction. Vanillic acid is utilized in the food and beverage industry as a flavoring agent. Tri-n-butyl phosphate (TBP) served as the reactive extractant, while natural oils like sesame oil and groundnut oil acted as diluents. The recovery of vanillic acid from fermentation broth or wastewater poses challenges due to toxicity concerns. However, employing natural solvents in the extraction process can significantly reduce toxicity. Various parameters, such as extraction efficiency, loading ratio, distribution coefficient, and equilibrium complexation constant, were determined to evaluate the extent of extraction achieved. Groundnut oil emerged as more effective than sesame oil as a diluent, exhibiting a maximum distribution coefficient of 13.31 and an extraction efficiency of 93.01 % at 5.27 mmol L–1 of acid concentration and 40 % v/v of TBP. Additionally, the number of theoretical stages required for the reactive extraction system was calculated, resulting in five stages for groundnut oil and four stages for sesame oil

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

Analysis of the Oxidative Stress Regulon Identifies soxS as a Genetic Target for Resistance Reversal in Multidrug-Resistant Klebsiella pneumoniae

In bacteria, the defense system deployed to counter oxidative stress is orchestrated by three transcriptional factors, SoxS, SoxR, and OxyR. Although the regulon that these factors control is known in many bacteria, similar data are not available for Klebsiella pneumoniae. To address this data gap, oxidative stress was artificially induced in K. pneumoniae MGH78578 using paraquat and the corresponding oxidative stress regulon recorded using transcriptome sequencing (RNA-seq). The soxS gene was significantly induced during oxidative stress, and a knockout mutant was constructed to explore its functionality. The wild type and mutant were grown in the presence of paraquat and subjected to RNA-seq to elucidate the soxS regulon in K. pneumoniae MGH78578. Genes that are commonly regulated both in the oxidative stress and soxS regulons were identified and denoted as the oxidative SoxS regulon; these included a group of genes specifically regulated by SoxS. Efflux pump-encoding genes and global regulators were identified as part of this regulon. Consequently, the isogenic soxS mutant was found to exhibit a reduction in the minimum bactericidal concentration against tetracycline compared to that of the wild type. Impaired efflux activity, allowing tetracycline to be accumulated in the cytoplasm to bactericidal levels, was further evaluated using a tetraphenylphosphonium (TPP+) accumulation assay. The soxS mutant was also susceptible to tetracycline in vivo in a zebrafish embryo model. We conclude that the soxS gene could be considered a genetic target against which an inhibitor could be developed and used in combinatorial therapy to combat infections associated with multidrug-resistant K. pneumoniae. IMPORTANCE Antimicrobial resistance is a global health challenge. Few new antibiotics have been developed for use over the years, and preserving the efficacy of existing compounds is an important step to protect public health. This paper describes a study that examines the effects of exogenously induced oxidative stress on K. pneumoniae and uncovers a target that could be useful to harness as a strategy to mitigate resistance

Laboratory Information Management Software for genotyping workflows: applications in high throughput crop genotyping

BACKGROUND: With the advances in DNA sequencer-based technologies, it has become possible to automate several steps of the genotyping process leading to increased throughput. To efficiently handle the large amounts of genotypic data generated and help with quality control, there is a strong need for a software system that can help with the tracking of samples and capture and management of data at different steps of the process. Such systems, while serving to manage the workflow precisely, also encourage good laboratory practice by standardizing protocols, recording and annotating data from every step of the workflow. RESULTS: A laboratory information management system (LIMS) has been designed and implemented at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) that meets the requirements of a moderately high throughput molecular genotyping facility. The application is designed as modules and is simple to learn and use. The application leads the user through each step of the process from starting an experiment to the storing of output data from the genotype detection step with auto-binning of alleles; thus ensuring that every DNA sample is handled in an identical manner and all the necessary data are captured. The application keeps track of DNA samples and generated data. Data entry into the system is through the use of forms for file uploads. The LIMS provides functions to trace back to the electrophoresis gel files or sample source for any genotypic data and for repeating experiments. The LIMS is being presently used for the capture of high throughput SSR (simple-sequence repeat) genotyping data from the legume (chickpea, groundnut and pigeonpea) and cereal (sorghum and millets) crops of importance in the semi-arid tropics. CONCLUSION: A laboratory information management system is available that has been found useful in the management of microsatellite genotype data in a moderately high throughput genotyping laboratory. The application with source code is freely available for academic users and can be downloaded from