235 research outputs found
Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications.
Analysis of DNA methylation patterns relies increasingly on sequencing-based profiling methods. The four most frequently used sequencing-based technologies are the bisulfite-based methods MethylC-seq and reduced representation bisulfite sequencing (RRBS), and the enrichment-based techniques methylated DNA immunoprecipitation sequencing (MeDIP-seq) and methylated DNA binding domain sequencing (MBD-seq). We applied all four methods to biological replicates of human embryonic stem cells to assess their genome-wide CpG coverage, resolution, cost, concordance and the influence of CpG density and genomic context. The methylation levels assessed by the two bisulfite methods were concordant (their difference did not exceed a given threshold) for 82% for CpGs and 99% of the non-CpG cytosines. Using binary methylation calls, the two enrichment methods were 99% concordant and regions assessed by all four methods were 97% concordant. We combined MeDIP-seq with methylation-sensitive restriction enzyme (MRE-seq) sequencing for comprehensive methylome coverage at lower cost. This, along with RNA-seq and ChIP-seq of the ES cells enabled us to detect regions with allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression
Revisiting the Hetero-Fertilization Phenomenon in Maize
Development of a seed DNA-based genotyping system for marker-assisted selection (MAS) has provided a novel opportunity for understanding aberrant reproductive phenomena such as hetero-fertilization (HF) by observing the mismatch of endosperm and leaf genotypes in monocot species. In contrast to conventional approaches using specific morphological markers, this approach can be used for any population derived from diverse parental genotypes. A large-scale experiment was implemented using seven F2 populations and four three-way cross populations, each with 534 to 1024 individuals. The frequency of HF within these populations ranged from 0.14% to 3.12%, with an average of 1.46%. The highest frequency of HF in both types of population was contributed by the pollen gametes. Using three-way crosses allowed, for the first time, detection of the HF contributed by maternal gametes, albeit at very low frequency (0.14%–0.65%). Four HF events identified from each of two F2 populations were tested and confirmed using 1032 single nucleotide polymorphic markers. This analysis indicated that only 50% of polymorphic markers can detect a known HF event, and thus the real HF frequency can be inferred by doubling the estimate obtained from using only one polymorphic marker. As expected, 99% of the HF events can be detected by using seven independent markers in combination. Although seed DNA-based analysis may wrongly predict plant genotypes due to the mismatch of endosperm and leaf DNA caused by HF, the relatively low HF frequencies revealed with diverse germplasm in this study indicates that the effect on the accuracy of MAS is limited. In addition, comparative endosperm and leaf DNA analysis of specific genetic stocks could be useful for revealing the relationships among various aberrant fertilization phenomena including haploidy and apomixis
Host Plant Induced Variation in Gut Bacteria of Helicoverpa armigera
Helicoverpa are important polyphagous agricultural insect pests and they have a worldwide distribution. In this study, we report the bacterial community structure in the midgut of fifth instar larvae of Helicoverpa armigera, a species prevalent in the India, China, South Asia, South East Asia, Southern & Eastern Africa and Australia. Using culturable techniques, we isolated and identified members of Bacillus firmus, Bacillus niabense, Paenibacillus jamilae, Cellulomonas variformis, Acinetobacter schindleri, Micrococcus yunnanesis, Enterobacter sp., and Enterococcus cassiliflavus in insect samples collected from host plants grown in different parts of India. Besides these the presence of Sphingomonas, Ralstonia, Delftia, Paracoccus and Bacteriodetes was determined by culture independent molecular analysis. We found that Enterobacter and Enterococcus were universally present in all our Helicoverpa samples collected from different crops and in different parts of India. The bacterial diversity varied greatly among insects that were from different host plants than those from the same host plant of different locations. This result suggested that the type of host plant greatly influences the midgut bacterial diversity of H. armigera, more than the location of the host plant. On further analyzing the leaf from which the larva was collected, it was found that the H. armigera midgut bacterial community was similar to that of the leaf phyllosphere. This finding indicates that the bacterial flora of the larval midgut is influenced by the leaf surface bacterial community of the crop on which it feeds. Additionally, we found that laboratory made media or the artificial diet is a poor bacterial source for these insects compared to a natural diet of crop plant
Gamma estimator of Jarzynski equality for recovering binding energies from noisy dynamic data sets
A fundamental problem in thermodynamics is the recovery of macroscopic equilibrated interaction energies from experimentally measured single-molecular interactions. The Jarzynski equality forms a theoretical basis in recovering the free energy difference between two states from exponentially averaged work performed to switch the states. In practice, the exponentially averaged work value is estimated as the mean of finite samples. Numerical simulations have shown that samples having thousands of measurements are not large enough for the mean to converge when the fluctuation of external work is above 4 kBT, which is easily observable in biomolecular interactions. We report the first example of a statistical gamma work distribution applied to single molecule pulling experiments. The Gibbs free energy of surface adsorption can be accurately evaluated even for a small sample size. The values obtained are comparable to those derived from multi-parametric surface plasmon resonance measurements and molecular dynamics simulations
Spectrophotometric determination of tizanidine and orphenadrine via ion pair complex formation using eosin Y
A simple, sensitive and rapid spectrophotometric method was developed and validated for the determination of two skeletal muscle relaxants namely, tizanidine hydrochloride (I) and orphenadrine citrate (II) in pharmaceutical formulations. The proposed method is based on the formation of a binary complex between the studied drugs and eosin Y in aqueous buffered medium (pH 3.5). Under the optimum conditions, the binary complex showed absorption maxima at 545 nm for tizanidine and 542 nm for orphenadrine. The calibration plots were rectilinear over concentration range of 0.5-8 μg/mL and 1-12 μg/mL with limits of detection of 0.1 μg/mL and 0.3 μg/mL for tizanidine and orphenadrine respectively. The different experimental parameters affecting the development and stability of the complex were studied and optimized. The method was successfully applied for determination of the studied drugs in their dosage forms; and to the content uniformity test of tizanidine in tablets
Identification of Attractive Drug Targets in Neglected-Disease Pathogens Using an In Silico Approach
In cell-based drug development, researchers attempt to create drugs that kill a pathogen without necessarily understanding the details of how the drugs work. In contrast, target-based drug development entails the search for compounds that act on a specific intracellular target—often a protein known or suspected to be required for survival of the pathogen. The latter approach to drug development has been facilitated greatly by the sequencing of many pathogen genomes and the incorporation of genome data into user-friendly databases. The present paper shows how the database TDRtargets.org can identify proteins that might be considered good drug targets for diseases such as African sleeping sickness, Chagas disease, parasitic worm infections, tuberculosis, and malaria. These proteins may score highly in searches of the database because they are dissimilar to human proteins, are structurally similar to other “druggable” proteins, have functions that are easy to measure, and/or fulfill other criteria. Researchers can use the lists of high-scoring proteins as a basis for deciding which potential drug targets to pursue experimentally
Cluster randomised controlled trial of a peer-led lifestyle intervention program: study protocol for the Kerala diabetes prevention program.
BACKGROUND: India currently has more than 60 million people with Type 2 Diabetes Mellitus (T2DM) and this is predicted to increase by nearly two-thirds by 2030. While management of those with T2DM is important, preventing or delaying the onset of the disease, especially in those individuals at 'high risk' of developing T2DM, is urgently needed, particularly in resource-constrained settings. This paper describes the protocol for a cluster randomised controlled trial of a peer-led lifestyle intervention program to prevent diabetes in Kerala, India. METHODS/DESIGN: A total of 60 polling booths are randomised to the intervention arm or control arm in rural Kerala, India. Data collection is conducted in two steps. Step 1 (Home screening): Participants aged 30-60 years are administered a screening questionnaire. Those having no history of T2DM and other chronic illnesses with an Indian Diabetes Risk Score value of ≥60 are invited to attend a mobile clinic (Step 2). At the mobile clinic, participants complete questionnaires, undergo physical measurements, and provide blood samples for biochemical analysis. Participants identified with T2DM at Step 2 are excluded from further study participation. Participants in the control arm are provided with a health education booklet containing information on symptoms, complications, and risk factors of T2DM with the recommended levels for primary prevention. Participants in the intervention arm receive: (1) eleven peer-led small group sessions to motivate, guide and support in planning, initiation and maintenance of lifestyle changes; (2) two diabetes prevention education sessions led by experts to raise awareness on T2DM risk factors, prevention and management; (3) a participant handbook containing information primarily on peer support and its role in assisting with lifestyle modification; (4) a participant workbook to guide self-monitoring of lifestyle behaviours, goal setting and goal review; (5) the health education booklet that is given to the control arm. Follow-up assessments are conducted at 12 and 24 months. The primary outcome is incidence of T2DM. Secondary outcomes include behavioural, psychosocial, clinical, and biochemical measures. An economic evaluation is planned. DISCUSSION: Results from this trial will contribute to improved policy and practice regarding lifestyle intervention programs to prevent diabetes in India and other resource-constrained settings. TRIAL REGISTRATION: Australia and New Zealand Clinical Trials Registry: ACTRN12611000262909
Stoichiometric representation of geneproteinreaction associations leverages constraint-based analysis from reaction to gene-level phenotype prediction
Genome-scale metabolic reconstructions are currently available for hundreds of organisms. Constraint-based modeling enables the analysis of the phenotypic landscape of these organisms, predicting the response to genetic and environmental perturbations. However, since constraint-based models can only describe the metabolic phenotype at the reaction level, understanding the mechanistic link between genotype and phenotype is still hampered by the complexity of gene-protein-reaction associations. We implement a model transformation that enables constraint-based methods to be applied at the gene level by explicitly accounting for the individual fluxes of enzymes (and subunits) encoded by each gene. We show how this can be applied to different kinds of constraint-based analysis: flux distribution prediction, gene essentiality analysis, random flux sampling, elementary mode analysis, transcriptomics data integration, and rational strain design. In each case we demonstrate how this approach can lead to improved phenotype predictions and a deeper understanding of the genotype-to-phenotype link. In particular, we show that a large fraction of reaction-based designs obtained by current strain design methods are not actually feasible, and show how our approach allows using the same methods to obtain feasible gene-based designs. We also show, by extensive comparison with experimental 13C-flux data, how simple reformulations of different simulation methods with gene-wise objective functions result in improved prediction accuracy. The model transformation proposed in this work enables existing constraint-based methods to be used at the gene level without modification. This automatically leverages phenotype analysis from reaction to gene level, improving the biological insight that can be obtained from genome-scale models.DM was supported by the Portuguese Foundationfor Science and Technologythrough a post-doc fellowship (ref: SFRH/BPD/111519/ 2015). This study was supported by the PortugueseFoundationfor Science and Technology (FCT) under the scope of the strategic fundingof UID/BIO/04469/2013 unitand COMPETE2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145FEDER-000004) fundedby EuropeanRegional Development Fund under the scope of Norte2020Programa Operacional Regional do Norte. This project has received fundingfrom the European Union’s Horizon 2020 research and innovation programme under grant agreementNo 686070. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
Effect of dietary restriction and subsequent re-alimentation on the transcriptional profile of bovine ruminal epithelium
peer-reviewedCompensatory growth (CG) is utilised worldwide in beef production systems as a management approach to reduce feed costs. However the underlying biology regulating the expression of CG remains to be fully elucidated. The objective of this study was to examine the effect of dietary restriction and subsequent re-alimentation induced CG on the global gene expression profile of ruminal epithelial papillae. Holstein Friesian bulls (n = 60) were assigned to one of two groups: restricted feed allowance (RES; n = 30) for 125 days (Period 1) followed by ad libitum access to feed for 55 days (Period 2) or (ii) ad libitum access to feed throughout (ADLIB; n = 30). At the end of each period, 15 animals from each treatment were slaughtered and rumen papillae harvested. mRNA was isolated from all papillae samples collected. cDNA libraries were then prepared and sequenced. Resultant reads were subsequently analysed bioinformatically and differentially expressed genes (DEGs) are defined as having a Benjamini-Hochberg P value of <0.05. During re-alimentation in Period 2, RES animals displayed CG, growing at 1.8 times the rate of their ADLIB contemporary animals in Period 2 (P < 0.001). At the end of Period 1, 64 DEGs were identified between RES and ADLIB, with only one DEG identified at the end of Period 2. When analysed within RES treatment (RES, Period 2 v Period 1), 411 DEGs were evident. Genes identified as differentially expressed in response to both dietary restriction and subsequent CG included those involved in processes such as cellular interactions and transport, protein folding and gene expression, as well as immune response. This study provides an insight into the molecular mechanisms underlying the expression of CG in rumen papillae of cattle; however the results suggest that the role of the ruminal epithelium in supporting overall animal CG may have declined by day 55 of re-alimentation.SMW received financial assistance from
Science Foundation Ireland (SFI) contract no 09/
RFP/GEN2447
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