Peanut gene expression profiling in developing seeds at different reproduction stages during Aspergillus parasiticus infection

<p>Abstract</p> <p>Background</p> <p>Peanut (<it>Arachis hypogaea </it>L.) is an important crop economically and nutritionally, and is one of the most susceptible host crops to colonization of <it>Aspergillus parasiticus </it>and subsequent aflatoxin contamination. Knowledge from molecular genetic studies could help to devise strategies in alleviating this problem; however, few peanut DNA sequences are available in the public database. In order to understand the molecular basis of host resistance to aflatoxin contamination, a large-scale project was conducted to generate expressed sequence tags (ESTs) from developing seeds to identify resistance-related genes involved in defense response against <it>Aspergillus </it>infection and subsequent aflatoxin contamination.</p> <p>Results</p> <p>We constructed six different cDNA libraries derived from developing peanut seeds at three reproduction stages (R5, R6 and R7) from a resistant and a susceptible cultivated peanut genotypes, 'Tifrunner' (susceptible to <it>Aspergillus </it>infection with higher aflatoxin contamination and resistant to TSWV) and 'GT-C20' (resistant to <it>Aspergillus </it>with reduced aflatoxin contamination and susceptible to TSWV). The developing peanut seed tissues were challenged by <it>A. parasiticus </it>and drought stress in the field. A total of 24,192 randomly selected cDNA clones from six libraries were sequenced. After removing vector sequences and quality trimming, 21,777 high-quality EST sequences were generated. Sequence clustering and assembling resulted in 8,689 unique EST sequences with 1,741 tentative consensus EST sequences (TCs) and 6,948 singleton ESTs. Functional classification was performed according to MIPS functional catalogue criteria. The unique EST sequences were divided into twenty-two categories. A similarity search against the non-redundant protein database available from NCBI indicated that 84.78% of total ESTs showed significant similarity to known proteins, of which 165 genes had been previously reported in peanuts. There were differences in overall expression patterns in different libraries and genotypes. A number of sequences were expressed throughout all of the libraries, representing constitutive expressed sequences. In order to identify resistance-related genes with significantly differential expression, a statistical analysis to estimate the relative abundance (<it>R</it>) was used to compare the relative abundance of each gene transcripts in each cDNA library. Thirty six and forty seven unique EST sequences with threshold of <it>R </it>> 4 from libraries of 'GT-C20' and 'Tifrunner', respectively, were selected for examination of temporal gene expression patterns according to EST frequencies. Nine and eight resistance-related genes with significant up-regulation were obtained in 'GT-C20' and 'Tifrunner' libraries, respectively. Among them, three genes were common in both genotypes. Furthermore, a comparison of our EST sequences with other plant sequences in the TIGR Gene Indices libraries showed that the percentage of peanut EST matched to <it>Arabidopsis thaliana</it>, maize (<it>Zea mays</it>), <it>Medicago truncatula</it>, rapeseed (<it>Brassica napus</it>), rice (<it>Oryza sativa</it>), soybean (<it>Glycine max</it>) and wheat (<it>Triticum aestivum</it>) ESTs ranged from 33.84% to 79.46% with the sequence identity ≥ 80%. These results revealed that peanut ESTs are more closely related to legume species than to cereal crops, and more homologous to dicot than to monocot plant species.</p> <p>Conclusion</p> <p>The developed ESTs can be used to discover novel sequences or genes, to identify resistance-related genes and to detect the differences among alleles or markers between these resistant and susceptible peanut genotypes. Additionally, this large collection of cultivated peanut EST sequences will make it possible to construct microarrays for gene expression studies and for further characterization of host resistance mechanisms. It will be a valuable genomic resource for the peanut community. The 21,777 ESTs have been deposited to the NCBI GenBank database with accession numbers <ext-link ext-link-type="gen" ext-link-id="ES702769">ES702769</ext-link> to <ext-link ext-link-type="gen" ext-link-id="ES724546">ES724546</ext-link>.</p

Spatial Patterns of Aflatoxin Levels in Relation to Ear-Feeding Insect Damage in Pre-Harvest Corn

Key impediments to increased corn yield and quality in the southeastern US coastal plain region are damage by ear-feeding insects and aflatoxin contamination caused by infection of Aspergillus flavus. Key ear-feeding insects are corn earworm, Helicoverpa zea, fall armyworm, Spodoptera frugiperda, maize weevil, Sitophilus zeamais, and brown stink bug, Euschistus servus. In 2006 and 2007, aflatoxin contamination and insect damage were sampled before harvest in three 0.4-hectare corn fields using a grid sampling method. The feeding damage by each of ear/kernel-feeding insects (i.e., corn earworm/fall armyworm damage on the silk/cob, and discoloration of corn kernels by stink bugs), and maize weevil population were assessed at each grid point with five ears. The spatial distribution pattern of aflatoxin contamination was also assessed using the corn samples collected at each sampling point. Aflatoxin level was correlated to the number of maize weevils and stink bug-discolored kernels, but not closely correlated to either husk coverage or corn earworm damage. Contour maps of the maize weevil populations, stink bug-damaged kernels, and aflatoxin levels exhibited an aggregated distribution pattern with a strong edge effect on all three parameters. The separation of silk- and cob-feeding insects from kernel-feeding insects, as well as chewing (i.e., the corn earworm and maize weevil) and piercing-sucking insects (i.e., the stink bugs) and their damage in relation to aflatoxin accumulation is economically important. Both theoretic and applied ramifications of this study were discussed by proposing a hypothesis on the underlying mechanisms of the aggregated distribution patterns and strong edge effect of insect damage and aflatoxin contamination, and by discussing possible management tactics for aflatoxin reduction by proper management of kernel-feeding insects. Future directions on basic and applied research related to aflatoxin contamination are also discussed

Nucleosynthesis Constraints on a Massive Gravitino in Neutralino Dark Matter Scenarios

The decays of massive gravitinos into neutralino dark matter particles and Standard Model secondaries during or after Big-Bang nucleosynthesis (BBN) may alter the primordial light-element abundances. We present here details of a new suite of codes for evaluating such effects, including a new treatment based on PYTHIA of the evolution of showers induced by hadronic decays of massive, unstable particles such as a gravitino. We also develop an analytical treatment of non-thermal hadron propagation in the early universe, and use this to derive analytical estimates for light-element production and in turn on decaying particle lifetimes and abundances. We then consider specifically the case of an unstable massive gravitino within the constrained minimal supersymmetric extension of the Standard Model (CMSSM). We present upper limits on its possible primordial abundance before decay for different possible gravitino masses, with CMSSM parameters along strips where the lightest neutralino provides all the astrophysical cold dark matter density. We do not find any CMSSM solution to the cosmological Li7 problem for small m_{3/2}. Discounting this, for m_{1/2} ~ 500 GeV and tan beta = 10 the other light-element abundances impose an upper limit m_{3/2} n_{3/2}/n_\gamma < 3 \times 10^{-12} GeV to < 2 \times 10^{-13} GeV for m_{3/2} = 250 GeV to 1 TeV, which is similar in both the coannihilation and focus-point strips and somewhat weaker for tan beta = 50, particularly for larger m_{1/2}. The constraints also weaken in general for larger m_{3/2}, and for m_{3/2} > 3 TeV we find a narrow range of m_{3/2} n_{3/2}/n_\gamma, at values which increase with m_{3/2}, where the Li7 abundance is marginally compatible with the other light-element abundances.Comment: 74 pages, 40 Figure

Expression Analysis of Stress-Related Genes in Kernels of Different Maize (Zea mays L.) Inbred Lines with Different Resistance to Aflatoxin Contamination

This research examined the expression patterns of 94 stress-related genes in seven maize inbred lines with differential expressions of resistance to aflatoxin contamination. The objective was to develop a set of genes/probes associated with resistance to A. flavus and/or aflatoxin contamination. Ninety four genes were selected from previous gene expression studies with abiotic stress to test the differential expression in maize lines, A638, B73, Lo964, Lo1016, Mo17, Mp313E, and Tex6, using real-time RT-PCR. Based on the relative-expression levels, the seven maize inbred lines clustered into two different groups. One group included B73, Lo1016 and Mo17, which had higher levels of aflatoxin contamination and lower levels of overall gene expression. The second group which included Tex6, Mp313E, Lo964 and A638 had lower levels of aflatoxin contamination and higher overall levels of gene expressions. A total of six “cross-talking” genes were identified between the two groups, which are highly expressed in the resistant Group 2 but down-regulated in susceptible Group 1. When further subjected to drought stress, Tex6 expressed more genes up-regulated and B73 has fewer genes up-regulated. The transcript patterns and interactions measured in these experiments indicate that the resistant mechanism is an interconnected process involving many gene products and transcriptional regulators, as well as various host interactions with environmental factors, particularly, drought and high temperature

Reph, a Regulator of Eph Receptor Expression in the Drosophila melanogaster Optic Lobe

Receptors of the Eph family of tyrosine kinases and their Ephrin ligands are involved in developmental processes as diverse as angiogenesis, axon guidance and cell migration. However, our understanding of the Eph signaling pathway is incomplete, and could benefit from an analysis by genetic methods. To this end, we performed a genetic modifier screen for mutations that affect Eph signaling in Drosophila melanogaster. Several dozen loci were identified on the basis of their suppression or enhancement of an eye defect induced by the ectopic expression of Ephrin during development; many of these mutant loci were found to disrupt visual system development. One modifier locus, reph (regulator of eph expression), was characterized in molecular detail and found to encode a putative nuclear protein that interacts genetically with Eph signaling pathway mutations. Reph is an autonomous regulator of Eph receptor expression, required for the graded expression of Eph protein and the establishment of an optic lobe axonal topographic map. These results reveal a novel component of the regulatory pathway controlling expression of eph and identify reph as a novel factor in the developing visual system

Evaluation of unclassified variants in the breast cancer susceptibility genes BRCA1 and BRCA2 using five methods: results from a population-based study of young breast cancer patients

Introduction Efforts are ongoing to determine the significance of unclassified variants (UVs) in the breast cancer susceptibility genes BRCA1/BRCA2, but no study has systematically assessed whether women carrying the suspected deleterious UVs have characteristics commonly seen among women carrying known deleterious or disease-causing mutations in BRCA1/BRCA2. Methods We sequenced BRCA1/BRCA2 in 1,469 population-based female breast cancer patients diagnosed between the ages of 20 and 49 years. We used existing literature to classify variants into known deleterious mutations, polymorphic variants, and UVs. The UVs were further classified as high risk or low risk based on five methods: allele frequency, Polyphen algorithm, sequence conservation, Grantham matrix scores, and a combination of the Grantham matrix score and sequence conservation. Furthermore, we examined whether patients who carry the variants classified as high risk using these methods have risk characteristics similar to patients with known deleterious BRCA1/BRCA2 mutations (early age at diagnosis, family history of breast cancer or ovarian cancer, and negative estrogen receptor/progesterone receptor). Results We identified 262 distinct BRCA1/BRCA2 variants, including 147 UVs, in our study population. The BRCA1 UV carriers, but not the BRCA2 UV carriers, who were classified as high risk using each classification method were more similar to the deleterious mutation carriers with respect to family history than those carriers classified as low risk. For example, the odds ratio of having a first-degree family history for the high-risk women classified using Polyphen was 3.39 (95% confidence interval = 1.16 to 9.94) compared with normal/polymorphic BRCA1 carriers. The corresponding odds ratio of low-risk women was 1.53 (95% confidence interval = 1.07 to 2.18). The odds ratio for high-risk women defined by allele frequency was 2.00 (95% confidence interval = 1.14 to 3.51), and that of low-risk women was 1.30 (95% confidence interval = 0.87 to 1.93). Conclusion The results suggest that the five classification methods yielded similar results. Polyphen was particularly better at isolating BRCA1 UV carriers likely to have a family history of breast cancer or ovarian cancer, and may therefore help to classify BRCA1 UVs. Our study suggests that these methods may not be as successful in classifying BRCA2 UVs

High-coverage whole-genome analysis of 1220 cancers reveals hundreds of genes deregulated by rearrangement-mediated cis-regulatory alterations.

The impact of somatic structural variants (SVs) on gene expression in cancer is largely unknown. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole-genome sequencing data and RNA sequencing from a common set of 1220 cancer cases, we report hundreds of genes for which the presence within 100 kb of an SV breakpoint associates with altered expression. For the majority of these genes, expression increases rather than decreases with corresponding breakpoint events. Up-regulated cancer-associated genes impacted by this phenomenon include TERT, MDM2, CDK4, ERBB2, CD274, PDCD1LG2, and IGF2. TERT-associated breakpoints involve ~3% of cases, most frequently in liver biliary, melanoma, sarcoma, stomach, and kidney cancers. SVs associated with up-regulation of PD1 and PDL1 genes involve ~1% of non-amplified cases. For many genes, SVs are significantly associated with increased numbers or greater proximity of enhancer regulatory elements near the gene. DNA methylation near the promoter is often increased with nearby SV breakpoint, which may involve inactivation of repressor elements

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Worcester HS career aspiration replication study

A gender comparative study determining the career interests of juniors was performed in the Worcester Public High Schools for the Advisory Committee on the Status of Women. The primary goal was to determine if the results replicated the previous year's findings. The same approximate gender ratios of students by career interests, post-graduation plans, and career concerns were found to repeat. The research team concluded the findings and trends from the previous study by Handler and Hogan (2005) were still present among juniors. It was less clear whether acceptable sophomore data collected with the same survey instrument produced the same result