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Particulate matter (PM) exposure is associated with adverse health outcomes, including respiratory illness. A large fraction of exposure to airborne contaminants occurs in the home. This study, conducted over 5 months in a community with high asthma rates (Chelsea, MA, USA), investigated the use of portable air cleaners (PACs) to reduce indoor PM. Seven asthma-affected households participated, receiving a PAC (Austin Air Health Mate HEPA filter), a QuantAQ sensor to measure PM 1 , PM 2.5 , PM 10 (µg/m 3 ), and a HOBO plug-load data logger to track PAC usage. Results describe hourly and daily PM concentrations and PAC usage for each household. Hourly average PM concentrations decreased when PACs were turned on (vs. when they were turned off) across households during the study period: PM 1 decreased by 0.46 µg/m 3 , PM 2.5 decreased by 0.69 µg/m 3 , and PM 10 decreased by 3.22 µg/m 3 . PAC usage varied for each household, including constant usage in one household and only usage at certain times of day in others. Higher filtration settings led to lower PM, with significant reductions in some, but not all, homes. Our findings highlight some difficulties in implementing household PAC interventions, yet also provide evidence to support household-level interventions to reduce PM and other indoor sources of air pollution. We also highlight academic-community partnerships as contributing to evidence-based solutions.

Abstract Background Medulloblastoma (MB) patients with MYC oncogene amplification or overexpression exhibit extremely poor prognoses and therapy resistance. However, MYC itself has been one of the most challenging targets for cancer treatment. Here, we identify a novel marinopyrrole natural derivative, MP1, that shows desirable anti-MYC and anti-cancer activities in MB. Methods In this study, using MYC-amplified (Group 3) and non-MYC amplified MB cell lines in vitro and in vivo, we evaluated anti-cancer efficacies and molecular mechanism(s) of MP1. Results MP1 significantly suppressed MB cell growth and sphere counts and induced G2 cell cycle arrest and apoptosis in a MYC-dependent manner. Mechanistically, MP1 strongly downregulated the expression of MYC protein. Our results with RNA-seq revealed that MP1 significantly modulated global gene expression and inhibited MYC-associated transcriptional targets including translation/mTOR targets. In addition, MP1 inhibited MYC-target metabolism, leading to declined energy levels. The combination of MP1 with an FDA-approved mTOR inhibitor temsirolimus synergistically inhibited MB cell growth/survival by downregulating the expression of MYC and mTOR signaling components. Our results further showed that as single agents, both MP1 and temsirolimus, were able to significantly inhibit tumor growth and MYC expression in subcutaneously or orthotopically MYC-amplified MB bearing mice. In combination, there were further anti-MB effects on the tumor growth and MYC expression in mice. Conclusion These preclinical findings highlight the promise of marinopyrrole MP1 as a novel MYC inhibition approach for MYC-amplified MB.

Abstract Genome-wide association studies have identified numerous loci with allelic associations to Type 1 Diabetes (T1D) risk. Most disease-associated variants are enriched in regulatory sequences active in lymphoid cell types, suggesting that lymphocyte gene expression is altered in T1D. Here we assay gene expression between T1D cases and healthy controls in two autoimmunity-relevant lymphocyte cell types, memory CD4+/CD25+ regulatory T cells (Treg) and memory CD4+/CD25- T cells, using a splicing event-based approach to characterize tissue-specific transcriptomes. Limited differences in isoform usage between T1D cases and controls are observed in memory CD4+/CD25- T-cells. In Tregs, 402 genes demonstrate differences in isoform usage between cases and controls, particularly RNA recognition and splicing factor genes. Many of these genes are regulated by the variable inclusion of exons that can trigger nonsense mediated decay. Our results suggest that dysregulation of gene expression, through shifts in alternative splicing in Tregs, contributes to T1D pathophysiology.

Abstract Purpose The role of locoregional therapy compared to systemic chemotherapy (SYS) for unresectable intrahepatic cholangiocarcinoma (IHC) remains controversial. The importance of hepatic disease control, either as initial or salvage therapy, is also unclear. We compared overall survival (OS) in patients treated with resection, hepatic arterial infusion pump (HAIP) chemotherapy, or SYS as it relates to hepatic recurrence or progression. We also evaluated recurrence after resection to determine the efficacy of locoregional salvage therapy. Patients and Methods In this single‐institution retrospective analysis, patients with biopsy‐proven IHC treated with either curative‐intent resection, HAIP (with or without SYS), or SYS alone were analyzed. Propensity score matching (PSM) was used to compare patients with liver‐limited, advanced disease treated with HAIP versus SYS. The impact of locoregional salvage therapies in patients with liver‐limited recurrence was analyzed in the resection cohort. Results From 2000 to 2017, 714 patients with IHC were treated, 219 (30.7%) with resectable disease, 316 (44.3%) with locally advanced disease, and 179 (25.1%) with metastatic disease. Resected patients were less likely to recur or progress in the liver (hazard ratio [HR] 0.41, 95% CI 0.34–0.45) versus those that received HAIP or SYS (HR 0.58, 95% CI 0.50–0.65 vs. HR 0.63, 95% CI 0.57–0.69, respectively). In resected patients, 161 (64.4%) recurred, with 65 liver‐only recurrences. Thirty of these patients received subsequent locoregional therapy. On multivariable analysis, locoregional therapy was associated with improved OS after isolated liver recurrence (HR 0.46, 95% CI 0.29–0.75; p = 0.002). In patients with locally advanced unresectable or multifocal liver disease (with or without distant organ metastases), PSM demonstrated improved hepatic progression‐free survival in patients treated with HAIP versus SYS (HR 0.65; 95% CI 0.46–0.91; p = 0.01), which correlated with improved OS (HR 0.59, 95% CI 0.43–0.80; p

Summary: Traditionally, midbody remnants (MBRs) are isolated from cell culture medium using ultracentrifugation, which is expensive and time consuming. Here, we present a protocol for isolating MBRs or large extracellular vesicles (EVs) from mammalian cell culture using either 1.5% polyethylene glycol 6000 (PEG6000) or PEG5000-coated gold nanoparticles. We describe steps for growing cells, collecting media, and precipitating MBRs and EVs from cell culture medium. We then detail characterization of MBRs through immunofluorescent antibody staining and immunofluorescent imaging. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Abstract Background Genomic complexity is a growing field of evolution, with case studies for comparative evolutionary analyses in model and emerging non-model systems. Understanding complexity and the functional components of the genome is an untapped wealth of knowledge ripe for exploration. With the “remarkable lack of correspondence” between genome size and complexity, there needs to be a way to quantify complexity across organisms. In this study, we use a set of complexity metrics that allow for evaluating changes in complexity using TranD. Results We ascertain if complexity is increasing or decreasing across transcriptomes and at what structural level, as complexity varies. In this study, we define three metrics – TpG, EpT, and EpG- to quantify the transcriptome's complexity that encapsulates the dynamics of alternative splicing. Here we compare complexity metrics across 1) whole genome annotations, 2) a filtered subset of orthologs, and 3) novel genes to elucidate the impacts of orthologs and novel genes in transcript model analysis. Effective Exon Number (EEN) issued to compare the distribution of exon sizes within transcripts against random expectations of uniform exon placement. EEN accounts for differences in exon size, which is important because novel gene differences in complexity for orthologs and whole-transcriptome analyses are biased towards low-complexity genes with few exons and few alternative transcripts. Conclusions With our metric analyses, we are able to quantify changes in complexity across diverse lineages with greater precision and accuracy than previous cross-species comparisons under ortholog conditioning. These analyses represent a step toward whole-transcriptome analysis in the emerging field of non-model evolutionary genomics, with key insights for evolutionary inference of complexity changes on deep timescales across the tree of life. We suggest a means to quantify biases generated in ortholog calling and correct complexity analysis for lineage-specific effects. With these metrics, we directly assay the quantitative properties of newly formed lineage-specific genes as they lower complexity.

During April–July 2022, outbreaks of severe acute hepatitis of unknown etiology (SAHUE) were reported in 35 countries. Five percent of cases required liver transplantation, and 22 patients died. Viral metagenomic studies of clinical samples from SAHUE cases showed a correlation with human adenovirus F type 41 (HAdV-F41) and adeno-associated virus type 2 (AAV2). To explore the association between those DNA viruses and SAHUE in children in Ireland, we quantified HAdV-F41 and AAV2 in samples collected from a wastewater treatment plant serving 40% of Ireland’s population. We noted a high correlation between HAdV-F41 and AAV2 circulation in the community and SAHUE clinical cases. Next-generation sequencing of the adenovirus hexon in wastewater demonstrated HAdV-F41 was the predominant HAdV type circulating. Our environmental analysis showed increased HAdV-F41 and AAV2 prevalence in the community during the SAHUE outbreak. Our findings highlight how wastewater sampling could aid in surveillance for respiratory adenovirus species.

Abstract Elevated serum cytokine production in COVID-19 patients is associated with disease progression and severity. However, the stimuli that initiate cytokine production in patients remain to be fully revealed. Virus-infected cells release virus-associated exosomes, extracellular vesicles of endocytic origin, into the blood to deliver viral cargoes able to regulate immune responses. Here, we report that plasma exosomes of COVID-19 patients contain SARS-CoV-2 double stranded RNA (dsRNA) and stimulate robust production of interleukin-6 (IL-6), IL-8, tumor necrosis factor-α (TNF-α), and other inflammatory cytokines and chemokines by human peripheral mononuclear cells. Exosome depletion abolished these stimulated responses. COVID-19 plasma exosomes induced proinflammatory responses in CD4+ T cells, CD8+ T cells, and CD14+ monocytes but not significantly in regulatory T cells, Th17 T cells, or central memory T cells. COVID-19 plasma exosomes protect the SARS-CoV-2 dsRNA cargo from RNase and deliver the dsRNA into recipient cells. These exosomes significantly increase expression of endosomal toll-like receptor 3 (TLR3), TLR7, TLR8, and TLR9 in peripheral T cells and monocytes. A pharmacological inhibitor of TLR3 considerably reduced cytokine and chemokine production by CD4+ and CD8+ T cells but not by CD14+ monocytes, highlighting divergent signaling pathways of immune cells in response to COVID-19 plasma exosomes. Our results identify a novel model of intercellular crosstalk following SARS-CoV-2 infection that evoke immune responses positioned to contribute to elevated cytokine production associated with COVID-19 progression, severity, and long-haul symptoms.

Abstract The growing frequency, intensity, and duration of extreme heat events necessitates interventions to reduce heat exposures. Local opportunities for heat adaptation may be optimally identified through collection of both quantitative exposure metrics and qualitative data on perceptions of heat. In this study, we used mixed methods to characterize heat exposure among urban residents in the area of Boston, Massachusetts, US, in summer 2020. Repeated interviews of N = 24 study participants ascertained heat vulnerability and adaptation strategies. Participants also used low-cost sensors to collect temperature, location, sleep, and physical activity data. We saw significant differences across temperature metrics: median personal temperature exposures were 3.9 °C higher than median ambient weather station temperatures. Existing air conditioning (AC) units did not adequately control indoor temperatures to desired thermostat levels: even with AC use, indoor maximum temperatures increased by 0.24 °C per °C of maximum outdoor temperature. Sleep duration was not associated with indoor or outdoor temperature. On warmer days, we observed a range of changes in time-at-home, expected given our small study size. Interview results further indicated opportunities for heat adaptation interventions including AC upgrades, hydration education campaigns, and amelioration of energy costs during high heat periods. Our mixed methods design informs heat adaptation interventions tailored to the challenges faced by residents in the study area. The strength of our community-academic partnership was a large part of the success of the mixed methods approach.

Abstract Background Medulloblastoma (MB) patients with MYC oncogene amplification or overexpression exhibit extremely poor clinical outcomes and respond poorly to current therapies. Epigenetic deregulation is very common in MYC-driven MB. The bromodomain extra-terminal (BET) proteins and histone deacetylases (HDACs) are epigenetic regulators of MYC transcription and its associated tumorigenic programs. This study aimed to investigate the therapeutic potential of inhibiting the BET proteins and HDACs together in MB. Methods Using clinically relevant BET inhibitors (JQ1 or OTX015) and a pan-HDAC inhibitor (panobinostat), we evaluated the effects of combined inhibition on cell growth/survival in MYC-amplified MB cell lines and xenografts and examined underlying molecular mechanism(s). Results Co-treatment of JQ1 or OTX015 with panobinostat synergistically suppressed growth/survival of MYC-amplified MB cells by inducing G2 cell cycle arrest and apoptosis. Mechanistic investigation using RNA-seq revealed that co-treatment of JQ1 with panobinostat synergistically modulated global gene expression including MYC/HDAC targets. SYK and MSI1 oncogenes were among the top 50 genes synergistically downregulated by JQ1 and panobinostat. RT-PCR and western blot analyses confirmed that JQ1 and panobinostat synergistically inhibited the mRNA and protein expression of MSI1/SYK along with MYC expression. Reduced SYK/MSI expression after BET (specifically, BRD4) gene-knockdown further confirmed the epigenetic regulation of SYK and MSI1 genes. In addition, the combination of OTX015 and panobinostat significantly inhibited tumor growth in MYC-amplified MB xenografted mice by downregulating expression of MYC, compared to single-agent therapy. Conclusions Together, our findings demonstrated that dual-inhibition of BET and HDAC proteins of the epigenetic pathway can be a novel therapeutic approach against MYC-driven MB.

Abstract Parkinson’s disease (PD) is a disabling neurodegenerative disorder in which multiple cell types, including dopaminergic and cholinergic neurons, are affected. The mechanisms of neurodegeneration in PD are not fully understood, limiting the development of therapies directed at disease-relevant molecular targets. C. elegans is a genetically tractable model system that can be used to disentangle disease mechanisms in complex diseases such as PD. Such mechanisms can be studied combining high-throughput molecular profiling technologies such as transcriptomics and metabolomics. However, the integrative analysis of multi-omics data in order to unravel disease mechanisms is a challenging task without advanced bioinformatics training. Galaxy, a widely-used resource for enabling bioinformatics analysis by the broad scientific community, has poor representation of multi-omics integration pipelines. We present the integrative analysis of gene expression and metabolite levels of a C. elegans PD model using GAIT-GM, a new Galaxy tool for multi-omics data analysis. Using GAIT-GM, we discovered an association between branched-chain amino acid metabolism and cholinergic neurons in the C. elegans PD model. An independent follow-up experiment uncovered cholinergic neurodegeneration in the C. elegans model that is consistent with cholinergic cell loss observed in PD. GAIT-GM is an easy to use Galaxy-based tool for generating novel testable hypotheses of disease mechanisms involving gene-metabolite relationships.

Abstract Aim The aim of this study was to describe the overall sleep quality, sleep patterns and severity of sleep difficulties among nursing faculty using the Pittsburgh Sleep Quality Index (PSQI). Design Cross‐sectional descriptive and correlational study. Methods A survey of faculty recruited from Texas Board of Nursing approved colleges/schools was used. A purposive, convenience sample of 105 faculty completed the PSQI, a reliable and valid instrument, in an online setting. Data was collected from January 2019 through April 2019. Results Nursing faculty reported sleep difficulty across all components, with more problems in sleep latency, sleep disturbances and subjective sleep quality. Overall, 70.5% of participants were reportedly “poor” sleepers, a salient finding. Faculty experienced poor sleep quality across all academic ranks and age groups. Sleep latency and subjective sleep quality were components that presented higher in severity for the youngest faculty.

Abstract Objective Allelic imbalance (AI) is the differential expression of the two alleles in a diploid. AI can vary between tissues, treatments, and environments. Methods for testing AI exist, but methods are needed to estimate type I error and power for detecting AI and difference of AI between conditions. As the costs of the technology plummet, what is more important: reads or replicates? Results We find that a minimum of 2400, 480, and 240 allele specific reads divided equally among 12, 5, and 3 replicates is needed to detect a 10, 20, and 30%, respectively, deviation from allelic balance in a condition with power > 80%. A minimum of 960 and 240 allele specific reads divided equally among 8 replicates is needed to detect a 20 or 30% difference in AI between conditions with comparable power. Higher numbers of replicates increase power more than adding coverage without affecting type I error. We provide a Python package that enables simulation of AI scenarios and enables individuals to estimate type I error and power in detecting AI and differences in AI between conditions.

Abstract Background Neuroblastoma (NB) patients with MYCN amplification or overexpression respond poorly to current therapies and exhibit extremely poor clinical outcomes. PI3K-mTOR signaling-driven deregulation of protein synthesis is very common in NB and various other cancers that promote MYCN stabilization. In addition, both the MYCN and mTOR signaling axes can directly regulate a common translation pathway that leads to increased protein synthesis and cell proliferation. However, a strategy of concurrently targeting MYCN and mTOR signaling in NB remains unexplored. This study aimed to investigate the therapeutic potential of targeting dysregulated protein synthesis pathways by inhibiting the MYCN and mTOR pathways together in NB. Methods Using small molecule/pharmacologic approaches, we evaluated the effects of combined inhibition of MYCN transcription and mTOR signaling on NB cell growth/survival and associated molecular mechanism(s) in NB cell lines. We used two well-established BET (bromodomain extra-terminal) protein inhibitors (JQ1, OTX-015), and a clinically relevant mTOR inhibitor, temsirolimus, to target MYCN transcription and mTOR signaling, respectively. The single agent and combined efficacies of these inhibitors on NB cell growth, apoptosis, cell cycle and neurospheres were assessed using MTT, Annexin-V, propidium-iodide staining and sphere assays, respectively. Effects of inhibitors on global protein synthesis were quantified using a fluorescence-based (FamAzide)-based protein synthesis assay. Further, we investigated the specificities of these inhibitors in targeting the associated pathways/molecules using western blot analyses. Results Co-treatment of JQ1 or OTX-015 with temsirolimus synergistically suppressed NB cell growth/survival by inducing G1 cell cycle arrest and apoptosis with greatest efficacy in MYCN-amplified NB cells. Mechanistically, the co-treatment of JQ1 or OTX-015 with temsirolimus significantly downregulated the expression levels of phosphorylated 4EBP1/p70-S6K/eIF4E (mTOR components) and BRD4 (BET protein)/MYCN proteins. Further, this combination significantly inhibited global protein synthesis, compared to single agents. Our findings also demonstrated that both JQ1 and temsirolimus chemosensitized NB cells when tested in combination with cisplatin chemotherapy. Conclusions Together, our findings demonstrate synergistic efficacy of JQ1 or OTX-015 and temsirolimus against MYCN-driven NB, by dual-inhibition of MYCN (targeting transcription) and mTOR (targeting translation). Additional preclinical evaluation is warranted to determine the clinical utility of targeted therapy for high-risk NB patients.

Untargeted metabolomics studies are unbiased but identifying the same feature across studies is complicated by environmental variation, batch effects, and instrument variability. Ideally, several studies that assay the same set of metabolic features would be used to select recurring features to pursue for identification. Here, we developed an anchored experimental design. This generalizable approach enabled us to integrate three genetic studies consisting of 14 test strains of Caenorhabditis elegans prior to the compound identification process. An anchor strain, PD1074, was included in every sample collection, resulting in a large set of biological replicates of a genetically identical strain that anchored each study. This enables us to estimate treatment effects within each batch and apply straightforward meta-analytic approaches to combine treatment effects across batches without the need for estimation of batch effects and complex normalization strategies. We collected 104 test samples for three genetic studies across six batches to produce five analytical datasets from two complementary technologies commonly used in untargeted metabolomics. Here, we use the model system C. elegans to demonstrate that an augmented design combined with experimental blocks and other metabolomic QC approaches can be used to anchor studies and enable comparisons of stable spectral features across time without the need for compound identification. This approach is generalizable to systems where the same genotype can be assayed in multiple environments and provides biologically relevant features for downstream compound identification efforts. All methods are included in the newest release of the publicly available SECIMTools based on the open-source Galaxy platform.

Abstract Background Very little has been researched about the efficacy, effectiveness, feasibility, sustainability and impact of food-based approaches on the diets and nutritional status of populations at risk of hunger and food insecurity. This study contributes knowledge about the impact of food-based approaches on the diets of populations at risk of hunger and food insecurity in four of the poorest rural communities in South Africa. The study investigated the consumption and production patterns of rural households (278 in summer and 280 in winter) in four sites in the poorest municipalities in South Africa. Methods A multistage stratified random sampling technique was applied to identify the communities and sample households for the quantitative survey and qualitative assessments. Qualitative and quantitative data were collected between 2013 and 2015 through focus group discussions (FGDs), key informant interviews and the two-round panel survey to cover both the summer and winter seasons at each site. Results Home gardening led to a significant positive increase in the consumption of white roots and tubers, dark green leafy vegetables, orange-coloured fruit and other fruit in the 24 h prior to the survey. Participation in a community garden led to significant increases in the consumption of dark green leafy vegetables and other vegetables. School gardening did not demonstrate any statistical relationships with the consumption of foods from the crop-related food groups. Crop production improved dietary diversity. Selling produce and irrigation showed a stronger improvement in dietary diversity. Seasonality affected the availability of fresh fruit and vegetables for home consumption in winter. Conclusions Producing beyond that solely for home consumption has greater benefits for dietary diversity and a consumption-smoothing effect during the post-harvest period. Politicians and the scientific community should recognise the role that household and small-scale crop production plays in supporting household consumption and the provision of essential micronutrients despite constraints and disincentives. Production and education programmes should focus on strengthening existing good consumption patterns and promoting the consumption of foods that can improve dietary diversity.

Abstract Recent advances in long-read sequencing solve inaccuracies in alternative transcript identification of full-length transcripts in short-read RNA-Seq data, which encourages the development of methods for isoform-centered functional analysis. Here, we present tappAS, the first framework to enable a comprehensive Functional Iso-Transcriptomics (FIT) analysis, which is effective at revealing the functional impact of context-specific post-transcriptional regulation. tappAS uses isoform-resolved annotation of coding and non-coding functional domains, motifs, and sites, in combination with novel analysis methods to interrogate different aspects of the functional readout of transcript variants and isoform regulation. tappAS software and documentation are available at https://app.tappas.org .

Abstract Background The activity of MP1, a pyrrolomycin, was studied in MYCN amplified neuroblastoma (NB) alone and combined with temsirolimus (TEM). Methods Activity of MP1 was tested in MYCN amplified (BE-2c, IMR) and non amplified (SKN-AS) NB cells. The effect of MP1 on MYCN, MCL-1, cleaved PARP, LC3II/LC3I, bcl-2, BAX, and BRD-4 were determined by western blot and RNAseq. The effect of MP1 on metabolism, mitochondrial morphology, and cell cycle was determined. Toxicology and efficacy of MP1 plus TEM were evaluated. Results The IC50 of MP1 was 0.096 μM in BE-2c cells compared to 0.89 μM in IMR, and >50 μM in SKN-AS. The IC50 of MP1 plus TEM in BE-2c cells was 0.023 μM. MP1 inhibited metabolism leading to quiescence and produced a decline in cell cycle S-phase. Electron microscopy showed cristae loss and rounding up of mitochondria. Gene and protein expression for MYCN and MCL-1 declined while LCII and cleaved PARP increased. Protein expression of BAX, bcl-2, and BRD-4 were not significantly changed after MP1 treatment. The in-vivo concentrations of MP1 in blood and tumor were sufficient to produce the biologic effects seen in-vitro. MP1 plus TEM produced a complete response in 3 out of 5 tumor bearing mice. In a second mouse study, the combination of MP1 and TEM slowed tumor growth compared to control. Conclusions MP1 has a potent inhibitory effect on the viability of MYCN amplified NB. Inhibition of metabolism by MP1 induced quiescence and autophagy with a favorable toxicology and drug distribution profile. When combined with TEM anti-tumor activity was potentiated in-vitro and in-vivo.

AbstractAllelic imbalance (AI) occurs when alleles in a diploid individual are differentially expressed and indicates cisn2nnnhttps://github.com/McIntyre-Lab/BayesASE

Platelet-activating factor (PAF) is a potent inflammatory mediator that exerts its actions via the single PAF receptor (PAF-R). Cells that biosynthesize alkyl-PAF also make abundant amounts of the less potent PAF analogue acyl-PAF, which competes for PAF-R. Both PAF species are degraded by the plasma form of PAF acetylhydrolase (PAF-AH). We examined whether cogenerated acyl-PAF protects alkyl-PAF from systemic degradation by acting as a sacrificial substrate to enhance inflammatory stimulation or as an inhibitor to dampen PAF-R signaling. In ex vivo experiments both PAF species are prothrombotic in isolation, but acyl-PAF reduced the alkyl-PAF-induced stimulation of human platelets that express canonical PAF-R. In Swiss albino mice, alkyl-PAF causes sudden death, but this effect can also be suppressed by simultaneously administering boluses of acyl-PAF. When PAF-AH levels were incrementally elevated, the protective effect of acyl-PAF on alkyl-PAF-induced death was serially decreased. We conclude that, although acyl-PAF in isolation is mildly proinflammatory, in a pathophysiological setting abundant acyl-PAF suppresses the action of alkyl-PAF. These studies provide evidence for a previously unrecognized role for acyl-PAF as an inflammatory set-point modulator that regulates both PAF-R signaling and hydrolysis.

Alternative splicing leverages genomic content by allowing the synthesis of multiple transcripts and, by implication, protein isoforms, from a single gene. However, estimating the abundance of transcripts produced in a given tissue from short sequencing reads is difficult and can result in both the construction of transcripts that do not exist, and the failure to identify true transcripts. An alternative approach is to catalog the events that make up isoforms (splice junctions and exons). We present here the Event Analysis (EA) approach, where we project transcripts onto the genome and identify overlapping/unique regions and junctions. In addition, all possible logical junctions are assembled into a catalog. Transcripts are filtered before quantitation based on simple measures: the proportion of the events detected, and the coverage. We find that mapping to a junction catalog is more efficient at detecting novel junctions than mapping in a splice aware manner. We identify 99.8% of true transcripts while iReckon identifies 82% of the true transcripts and creates more transcripts not included in the simulation than were initially used in the simulation. Using PacBio Iso-seq data from a mouse neural progenitor cell model, EA detects 60% of the novel junctions that are combinations of existing exons while only 43% are detected by STAR. EA further detects ∼5,000 annotated junctions missed by STAR. Filtering transcripts based on the proportion of the transcript detected and the number of reads on average supporting that transcript captures 95% of the PacBio transcriptome. Filtering the reference transcriptome before quantitation, results in is a more stable estimate of isoform abundance, with improved correlation between replicates. This was particularly evident when EA is applied to an RNA-seq study of type 1 diabetes (T1D), where the coefficient of variation among subjects (n = 81) in the transcript abundance estimates was substantially reduced compared to the estimation using the full reference. EA focuses on individual transcriptional events. These events can be quantitate and analyzed directly or used to identify the probable set of expressed transcripts. Simple rules based on detected events and coverage used in filtering result in a dramatic improvement in isoform estimation without the use of ancillary data (e.g., ChIP, long reads) that may not be available for many studies.

Abstract Background Metabolomics has the promise to transform the area of personalized medicine with the rapid development of high throughput technology for untargeted analysis of metabolites. Open access, easy to use, analytic tools that are broadly accessible to the biological community need to be developed. While technology used in metabolomics varies, most metabolomics studies have a set of features identified. Galaxy is an open access platform that enables scientists at all levels to interact with big data. Galaxy promotes reproducibility by saving histories and enabling the sharing workflows among scientists. Results SECIMTools (SouthEast Center for Integrated Metabolomics) is a set of Python applications that are available both as standalone tools and wrapped for use in Galaxy. The suite includes a comprehensive set of quality control metrics (retention time window evaluation and various peak evaluation tools), visualization techniques (hierarchical cluster heatmap, principal component analysis, modular modularity clustering), basic statistical analysis methods (partial least squares - discriminant analysis, analysis of variance, t-test, Kruskal-Wallis non-parametric test), advanced classification methods (random forest, support vector machines), and advanced variable selection tools (least absolute shrinkage and selection operator LASSO and Elastic Net). Conclusions SECIMTools leverages the Galaxy platform and enables integrated workflows for metabolomics data analysis made from building blocks designed for easy use and interpretability. Standard data formats and a set of utilities allow arbitrary linkages between tools to encourage novel workflow designs. The Galaxy framework enables future data integration for metabolomics studies with other omics data.

Allelic imbalance (AI) indicates the presence of functional variation in cis regulatory regions. Detecting cis regulatory differences using AI is widespread, yet there is no formal statistical methodology that tests whether AI differs between conditions. Here, we present a novel model and formally test differences in AI across conditions using Bayesian credible intervals. The approach tests AI by environment (G×E) interactions, and can be used to test AI between environments, genotypes, sex, and any other condition. We incorporate bias into the modeling process. Bias is allowed to vary between conditions, making the formulation of the model general. As gene expression affects power for detection of AI, and, as expression may vary between conditions, the model explicitly takes coverage into account. The proposed model has low type I and II error under several scenarios, and is robust to large differences in coverage between conditions. We reanalyze RNA-seq data from a Drosophila melanogaster population panel, with F1 genotypes, to compare levels of AI between mated and virgin female flies, and we show that AI × genotype interactions can also be tested. To demonstrate the use of the model to test genetic differences and interactions, a formal test between two F1s was performed, showing the expected 20% difference in AI. The proposed model allows a formal test of G×E and G×G, and reaffirms a previous finding that cis regulation is robust between environments.

Platelet-activating factor (PAF) and related phospholipid oxidation products termed oxidized phospholipids (OxPLs) promote inflammation. PAF is made in response to bacterial endotoxin-lipopolysaccharide (LPS) that is recognized by Toll-like receptor-4 (TLR-4) whose activation leads to translocation of transcription factor NF-ΚB to the nucleus—a key regulator of multiple pro-inflammatory genes including COX-2 and IL-8. Paradoxically, PAF and OxPLs are claimed to inhibit LPS-mediated signaling, questioning the very pro-inflammatory roles of PAF and OxPLs and anti-inflammatory nature of PAF-acetylhydrolase (PAF-AH), an enzyme that attenuates both PAF and OxPLs signaling. We investigated the effect of PAF and representative OxPLs: 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphocholine (POVPC), 1-palmitoyl-2-glutaroyl-sn- glycero-3-phosphocholine (PGPC) and 1-alkyl-2-butanoyl-sn-glycero-3-phosphocholine PAF (C4 PAF) on LPS-induced expression of NF-ΚB mediated inflammation in isolated human myeloid cells: polymorphonuclear leukocyte (PMNs), monocytes and human umbilical vein endothelial cells (HUVECs). Using intracellular calcium transients, we show that POVPC and PGPC dose-dependently activate the PAF-receptor (PAF-R) in PMNs, that can beblocked by the PAF-R antagonist WEB-2086 and rPAF-AH pre-treatment. All the three cell types express minute or no detectable COX-2 when stimulated with either PAF (0.1 µM) or OxPLs (0.1 µM) alone. While LPS (100 ng/mL) induced expression of COX-2 in all the cell types, pre-activation of PAF-R with PAF (0.1 µM) or OxPLs (0.1 µM) did not suppress LPS (100 ng/mL)-induced COX-2 expression and in fact we obresved incereased PGE2 levels in an NS-398 sensitive manner. In addition, pre-activation of PAF-R significantly augmented LPS (100 ng/mL)-induced IL-8 production in PMNs. Thus, PAF and OXPLs do not suppress the ability of LPS to exert its pro-inflammatory effects in isolated human vascular cells.

mRNA variance has been proposed to play key roles in normal development, population fitness, adaptability, and disease. While variance in gene expression levels may be beneficial for certain cellular processes, for example in a cell’s ability to respond to external stimuli, variance may be detrimental for the development of some organs. In the bilaterally symmetric vertebrate limb buds, the amount of Sonic Hedgehog (SHH) protein present at specific stages of development is essential to ensure proper patterning of this structure. To our surprise, we found that SHH protein variance is present during the first 10 hr of limb development. The variance is virtually eliminated after the first 10 hr of limb development. By examining mutant animals, we determined that the ability of the limb bud apical ectodermal ridge (AER) to respond to SHH protein was required for reducing SHH variance during limb formation. One consequence of the failure to eliminate variance in SHH protein was the presence of polydactyly and an increase in digit length. These data suggest a potential novel mechanism in which alterations in SHH variance during evolution may have driven changes in limb patterning and digit length.