RESUMO
Microbiome studies have become routine in biomedical, agricultural and environmental sciences with diverse aims, including diversity profiling, functional characterization, and translational applications. The resulting complex, often multi-omics datasets demand powerful, yet user-friendly bioinformatics tools to reveal key patterns, important biomarkers, and potential activities. Here we introduce MicrobiomeAnalyst 2.0 to support comprehensive statistics, visualization, functional interpretation, and integrative analysis of data outputs commonly generated from microbiome studies. Compared to the previous version, MicrobiomeAnalyst 2.0 features three new modules: (i) a Raw Data Processing module for amplicon data processing and taxonomy annotation that connects directly with the Marker Data Profiling module for downstream statistical analysis; (ii) a Microbiome Metabolomics Profiling module to help dissect associations between community compositions and metabolic activities through joint analysis of paired microbiome and metabolomics datasets; and (iii) a Statistical Meta-Analysis module to help identify consistent signatures by integrating datasets across multiple studies. Other important improvements include added support for multi-factor differential analysis and interactive visualizations for popular graphical outputs, updated methods for functional prediction and correlation analysis, and expanded taxon set libraries based on the latest literature. These new features are demonstrated using a multi-omics dataset from a recent type 1 diabetes study. MicrobiomeAnalyst 2.0 is freely available at microbiomeanalyst.ca.
Assuntos
Biologia Computacional , Técnicas Microbiológicas , Microbiota , Biomarcadores , Biologia Computacional/métodos , Metabolômica/métodos , Técnicas Microbiológicas/instrumentação , Técnicas Microbiológicas/métodos , Internet , Interface Usuário-ComputadorRESUMO
We subjected seven P. aeruginosa isolates to a 10-day serial passaging against five antipseudomonal agents to evaluate resistance levels post-exposure and putative resistance mechanisms in terminal mutants were analyzed by whole-genome sequencing analysis. Meropenem (mean, 38-fold increase), cefepime (14.4-fold), and piperacillin-tazobactam (52.9-fold) terminal mutants displayed high minimum inhibitory concentration (MIC) values compared to those obtained after exposure to ceftolozane-tazobactam (11.4-fold) and ceftazidime-avibactam (5.7-fold). Fewer isolates developed elevated MIC values for other ß-lactams and agents belonging to other classes when exposed to meropenem in comparison to other agents. Alterations in nalC and nalD, involved in the upregulation of the efflux pump system MexAB-OprM, were common and observed more frequently in isolates exposed to ceftazidime-avibactam and meropenem. These alterations, along with ones in mexR and amrR, provided resistance to most ß-lactams and levofloxacin but not imipenem. The second most common gene altered was mpl, which is involved in the recycling of the cell wall peptidoglycan. These alterations were mainly noted in isolates exposed to ceftolozane-tazobactam and piperacillin-tazobactam but also in one cefepime-exposed isolate. Alterations in other genes known to be involved in ß-lactam resistance (ftsI, oprD, phoP, pepA, and cplA) and multiple genes involved in lipopolysaccharide biosynthesis were also present. The data generated here suggest that there is a difference in the mechanisms selected for high-level resistance between newer ß-lactam/ß-lactamase inhibitor combinations and older agents. Nevertheless, the isolates exposed to all agents displayed elevated MIC values for other ß-lactams (except imipenem) and quinolones tested mainly due to alterations in the MexAB-OprM regulators that extrude these agents.
Assuntos
Antibacterianos , Compostos Azabicíclicos , Ceftazidima , Meropeném , Testes de Sensibilidade Microbiana , Combinação Piperacilina e Tazobactam , Pseudomonas aeruginosa , Tazobactam , Inibidores de beta-Lactamases , beta-Lactamas , Antibacterianos/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Inibidores de beta-Lactamases/farmacologia , Compostos Azabicíclicos/farmacologia , Meropeném/farmacologia , Tazobactam/farmacologia , Ceftazidima/farmacologia , beta-Lactamas/farmacologia , Combinação Piperacilina e Tazobactam/farmacologia , Combinação de Medicamentos , Cefalosporinas/farmacologia , Cefepima/farmacologia , Humanos , Piperacilina/farmacologia , Sequenciamento Completo do Genoma , Farmacorresistência Bacteriana Múltipla/genéticaRESUMO
Computational time and cost remain a major bottleneck for RNA-seq data analysis of nonmodel organisms without reference genomes. To address this challenge, we have developed Seq2Fun, a novel, all-in-one, ultrafast tool to directly perform functional quantification of RNA-seq reads without transcriptome de novo assembly. The pipeline starts with raw read quality control: sequencing error correction, removing poly(A) tails, and joining overlapped paired-end reads. It then conducts a DNA-to-protein search by translating each read into all possible amino acid fragments and subsequently identifies possible homologous sequences in a well-curated protein database. Finally, the pipeline generates several informative outputs including gene abundance tables, pathway and species hit tables, an HTML report to visualize the results, and an output of clean reads annotated with mapped genes ready for downstream analysis. Seq2Fun does not have any intermediate steps of file writing and loading, making I/O very efficient. Seq2Fun is written in C++ and can run on a personal computer with a limited number of CPUs and memory. It can process >2,000,000 reads/min and is >120 times faster than conventional workflows based on de novo assembly, while maintaining high accuracy in our various test data sets.
Assuntos
Perfilação da Expressão Gênica , RNA-Seq , Transcriptoma , Fluxo de TrabalhoRESUMO
Researchers are increasingly seeking to interpret molecular data within a multi-omics context to gain a more comprehensive picture of their study system. OmicsNet (www.omicsnet.ca) is a web-based tool developed to allow users to easily build, visualize, and analyze multi-omics networks to study rich relationships among lists of 'omics features of interest. Three major improvements have been introduced in OmicsNet 2.0, which include: (i) enhanced network visual analytics with eleven 2D graph layout options and a novel 3D module layout; (ii) support for three new 'omics types: single nucleotide polymorphism (SNP) list from genetic variation studies; taxon list from microbiome profiling studies, as well as liquid chromatography-mass spectrometry (LC-MS) peaks from untargeted metabolomics; and (iii) measures to improve research reproducibility by coupling R command history with the release of the companion OmicsNetR package, and generation of persistent links to share interactive network views. We performed a case study using the multi-omics data obtained from a recent large-scale investigation on inflammatory bowel disease (IBD) and demonstrated that OmicsNet was able to quickly create meaningful multi-omics context to facilitate hypothesis generation and mechanistic insights.
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Metabolômica , Multiômica , Software , Internet , Espectrometria de Massas , Reprodutibilidade dos Testes , Cromatografia LíquidaRESUMO
Growth of organohalide-respiring bacteria such as Dehalococcoides mccartyi on halogenated organics (e.g., polychlorinated biphenyls (PCBs)) at contaminated sites or in enrichment culture requires interaction and support from other microbial community members. To evaluate naturally occurring interactions between Dehalococcoides and key supporting microorganisms (e.g., production of H2, acetate, and corrinoids) in PCB-contaminated sediments, metagenomic and metatranscriptomic sequencing was conducted on DNA and RNA extracted from sediment microcosms, showing evidence of both Dehalococcoides growth and PCB dechlorination. Using a genome-resolved approach, 160 metagenome-assembled genomes (MAGs), including three Dehalococcoides MAGs, were recovered. A novel reductive dehalogenase gene, distantly related to the chlorophenol dehalogenase gene cprA (pairwise amino acid identity: 23.75%), was significantly expressed. Using MAG gene expression data, 112 MAGs were assigned functional roles (e.g., corrinoid producers, acetate/H2 producers, etc.). A network coexpression analysis of all 160 MAGs revealed correlations between 39 MAGs and the Dehalococcoides MAGs. The network analysis also showed that MAGs assigned with functional roles that support Dehalococcoides growth (e.g., corrinoid assembly, and production of intermediates required for corrinoid synthesis) displayed significant coexpression correlations with Dehalococcoides MAGs. This work demonstrates the power of genome-resolved metagenomic and metatranscriptomic analyses, which unify taxonomy and function, in investigating the ecology of dehalogenating microbial communities.
Assuntos
Chloroflexi , Microbiota , Bifenilos Policlorados , Bifenilos Policlorados/análise , Bifenilos Policlorados/química , Bifenilos Policlorados/metabolismo , Chloroflexi/genética , Chloroflexi/química , Chloroflexi/metabolismo , Anaerobiose , Biodegradação Ambiental , Acetatos/metabolismo , Sedimentos Geológicos/análiseRESUMO
Data analysis and interpretation remain a critical bottleneck in current multi-omics studies. Here, we introduce OmicsAnalyst, a user-friendly, web-based platform that allows users to perform a wide range of well-established data-driven approaches for multi-omics integration, and visually explore their results in a clear and meaningful manner. To help navigate complex landscapes of multi-omics analysis, these approaches are organized into three visual analytics tracks: (i) the correlation network analysis track, where users choose among univariate and multivariate methods to identify important features and explore their relationships in 2D or 3D networks; (ii) the cluster heatmap analysis track, where users apply several cutting-edge multi-view clustering algorithms and explore their results via interactive heatmaps; and (iii) the dimension reduction analysis track, where users choose among several recent multivariate techniques to reveal global data structures, and explore corresponding scores, loadings and biplots in interactive 3D scatter plots. The three visual analytics tracks are equipped with comprehensive options for parameter customization, view customization and targeted analysis. OmicsAnalyst lowers the access barriers to many well-established methods for multi-omics integration via novel visual analytics. It is freely available at https://www.omicsanalyst.ca.
Assuntos
Metabolômica , Proteômica , Software , Animais , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica , Humanos , Internet , Camundongos , Análise Multivariada , GravidezRESUMO
MOTIVATION: Transcriptomics dose-response analysis is a promising new approach method for toxicity testing. While international regulatory agencies have spent substantial effort establishing a standardized statistical approach, existing software that follows this approach is computationally inefficient and must be locally installed. RESULTS: FastBMD is a web-based tool that implements standardized methods for transcriptomics benchmark dose-response analysis in R. It is >60 times faster than the current leading software, supports transcriptomics data from 13 species, and offers a comprehensive analytical pipeline that goes from processing and normalization of raw gene expression values to interactive exploration of pathway-level benchmark dose results. AVAILABILITY AND IMPLEMENTATION: FastBMD is freely available at www.fastbmd.ca. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Benchmarking , Biologia Computacional , Software , TranscriptomaRESUMO
Transcriptomics dose-response analysis (TDRA) has emerged as a promising approach for integrating toxicogenomics data into a risk assessment context; however, variability and uncertainty associated with experimental design are not well understood. Here, we evaluated n = 55 RNA-seq profiles derived from Japanese quail liver tissue following exposure to chlorpyrifos (0, 0.04, 0.1, 0.2, 0.4, 1, 2, 4, 10, 20, and 40 µg/g; n = 5 replicates per group) via egg injection. The full dataset was subsampled 637 times to generate smaller datasets with different dose ranges and spacing (designs A-E) and number of replicates (n = 2-5). TDRA of the 637 datasets revealed substantial variability in the gene and pathway benchmark doses, but relative stability in overall transcriptomic point-of-departure (tPOD) values when tPODs were calculated with the "pathway" and "mode" methods. Further, we found that tPOD values were more dependent on the dose range and spacing than on the number of replicates, suggesting that optimal experimental designs should use fewer replicates (n = 2 or 3) and more dose groups to reduce uncertainty in the results. Finally, tPOD values ranged by over ten times for all surveyed experimental designs and tPOD types, suggesting that tPODs should be interpreted as order-of-magnitude estimates.
Assuntos
Coturnix , Transcriptoma , Animais , Incerteza , Relação Dose-Resposta a Droga , Toxicogenética/métodos , Medição de Risco/métodosRESUMO
We conducted experiments to determine whether bioaugmentation with aerobic, polychlorinated biphenyl (PCB)-degrading microorganisms can mitigate polychlorinated biphenyl (PCB) emissions from contaminated sediment to air. Paraburkholderia xenovorans strain LB400 was added to bioreactors containing PCB-contaminated site sediment. PCB mass in both the headspace and aqueous bioreactor compartments was measured using passive samplers over 35 days. Time-series measurements of all 209 PCB congeners revealed a 57% decrease in total PCB mass accumulated in the vapor phase of bioaugmented treatments relative to non-bioaugmented controls, on average. A comparative congener-specific analysis revealed preferential biodegradation of lower-chlorinated PCBs (LC-PCBs) by LB400. Release of the most abundant congener (PCB 4 [2,2'-dichlorobiphenyl]) decreased by over 90%. Simulations with a PCB reactive transport model closely aligned with experimental observations. We also evaluated the effect of the phytogenic biosurfactant, saponin, on PCB bioavailability and biodegradation by LB400. Time-series qPCR measurements of biphenyl dioxygenase (bphA) genes showed that saponin better maintained bphA abundance, compared to the saponin-free treatment. These findings indicate that an active population of bioaugmented, aerobic PCB-degrading microorganisms can effectively lower PCB emissions and may therefore contribute to minimizing PCB inhalation exposure in communities surrounding PCB-contaminated sites.
Assuntos
Dioxigenases , Bifenilos Policlorados , Biodegradação Ambiental , Hidroxilaminas , Bifenilos Policlorados/metabolismoRESUMO
There is an urgent demand for more efficient and ethical approaches in ecological risk assessment. Using 17α-ethinylestradiol (EE2) as a model compound, this study established an embryo benchmark dose (BMD) assay for rainbow trout (RBT; Oncorhynchus mykiss) to derive transcriptomic points-of-departure (tPODs) as an alternative to live-animal tests. Embryos were exposed to graded concentrations of EE2 (measured: 0, 1.13, 1.57, 6.22, 16.3, 55.1, and 169 ng/L) from hatch to 4 and up to 60 days post-hatch (dph) to assess molecular and apical responses, respectively. Whole proteome analyses of alevins did not show clear estrogenic effects. In contrast, transcriptomics revealed responses that were in agreement with apical effects, including excessive accumulation of intravascular and hepatic proteinaceous fluid and significant increases in mortality at 55.1 and 169 ng/L EE2 at later time points. Transcriptomic BMD analysis estimated the median of the 20th lowest geneBMD to be 0.18 ng/L, the most sensitive tPOD. Other estimates (0.78, 3.64, and 1.63 ng/L for the 10th percentile geneBMD, first peak geneBMD distribution, and median geneBMD of the most sensitive over-represented pathway, respectively) were within the same order of magnitude as empirically derived apical PODs for EE2 in the literature. This 4-day alternative RBT embryonic assay was effective in deriving tPODs that are protective of chronic effects of EE2.
Assuntos
Oncorhynchus mykiss , Poluentes Químicos da Água , Animais , Benchmarking , Estrogênios , Etinilestradiol/toxicidade , Oncorhynchus mykiss/genética , Transcriptoma , Poluentes Químicos da Água/toxicidadeRESUMO
The growing application of gene expression profiling demands powerful yet user-friendly bioinformatics tools to support systems-level data understanding. NetworkAnalyst was first released in 2014 to address the key need for interpreting gene expression data within the context of protein-protein interaction (PPI) networks. It was soon updated for gene expression meta-analysis with improved workflow and performance. Over the years, NetworkAnalyst has been continuously updated based on community feedback and technology progresses. Users can now perform gene expression profiling for 17 different species. In addition to generic PPI networks, users can now create cell-type or tissue specific PPI networks, gene regulatory networks, gene co-expression networks as well as networks for toxicogenomics and pharmacogenomics studies. The resulting networks can be customized and explored in 2D, 3D as well as Virtual Reality (VR) space. For meta-analysis, users can now visually compare multiple gene lists through interactive heatmaps, enrichment networks, Venn diagrams or chord diagrams. In addition, users have the option to create their own data analysis projects, which can be saved and resumed at a later time. These new features are released together as NetworkAnalyst 3.0, freely available at https://www.networkanalyst.ca.
Assuntos
Biologia Computacional/métodos , Expressão Gênica/genética , Redes Reguladoras de Genes/genética , Mapas de Interação de Proteínas , Software , Perfilação da Expressão Gênica/métodos , Mapeamento de Interação de Proteínas/métodosRESUMO
Traditional results from toxicogenomics studies are complex lists of significantly impacted genes or gene sets, which are challenging to synthesize down to actionable results with a clear interpretation. Here, we defined two sets of 21 custom gene sets, called the functional and statistical EcoToxModules, in fathead minnow (Pimephales promelas) to (1) re-cast predefined molecular pathways into a toxicological framework and (2) provide a data-driven, unsupervised grouping of genes impacted by exposure to environmental contaminants. The functional EcoToxModules were identified by re-organizing KEGG pathways into biological processes that are more relevant to ecotoxicology based on the input from expert scientists and regulators. The statistical EcoToxModules were identified using co-expression analysis of publicly available microarray data (n = 303 profiles) measured in livers of fathead minnows after exposure to 38 different conditions. Potential applications of the EcoToxModules were demonstrated with two case studies that represent exposure to a pure chemical and to environmental wastewater samples. In comparisons to differential expression and gene set analysis, we found that EcoToxModule responses were consistent with these traditional results. Additionally, they were easier to visualize and quantitatively compare across different conditions, which facilitated drawing conclusions about the relative toxicity of the exposures within each case study.
Assuntos
Cyprinidae , Poluentes Químicos da Água , Animais , ToxicogenéticaRESUMO
Methylmercury (MeHg) exposure can cause adverse reproductive and neurodevelopmental health effects. Estuarine fish may be exposed to MeHg produced in rivers and their watersheds, benthic sediment, and the marine water column, but the relative importance of each source is poorly understood. We measured stable isotopes of mercury (δ202Hg, Δ199Hg, and Δ201Hg), carbon (δ13C), and nitrogen (δ15N) in fish with contrasting habitats from a large subarctic coastal ecosystem to better understand MeHg exposure sources. We identify two distinct food chains exposed to predominantly freshwater and marine MeHg sources but do not find evidence for a benthic marine MeHg signature. This is consistent with our previous research showing benthic sediment is a net sink for MeHg in the estuary. Marine fish display lower and less variable Δ199Hg values (0.78 to 1.77) than freshwater fish (0.72 to 3.14) and higher δ202Hg values (marine: 0.1 to 0.57; freshwater: -0.76 to 0.15). We observe a shift in the Hg isotopic composition of juvenile and adult rainbow smelt (Osmerus mordax) when they transition between the freshwater and marine environment as their dominant foraging territory. The Hg isotopic composition of Atlantic salmon (Salmo salar) indicates they receive most of their MeHg from the marine environment despite a similar or longer duration spent in freshwater regions. We conclude that stable Hg isotopes effectively track fish MeHg exposure sources across different ontogenic stages.
Assuntos
Isótopos de Mercúrio , Mercúrio , Adolescente , Animais , Monitoramento Ambiental , Humanos , Compostos de Metilmercúrio , Poluentes Químicos da ÁguaRESUMO
High-throughput image-based profiling platforms are powerful technologies capable of collecting data from billions of cells exposed to thousands of perturbations in a time- and cost-effective manner. Therefore, image-based profiling data has been increasingly used for diverse biological applications, such as predicting drug mechanism of action or gene function. However, batch effects severely limit community-wide efforts to integrate and interpret image-based profiling data collected across different laboratories and equipment. To address this problem, we benchmark ten high-performing single-cell RNA sequencing (scRNA-seq) batch correction techniques, representing diverse approaches, using a newly released Cell Painting dataset, JUMP. We focus on five scenarios with varying complexity, ranging from batches prepared in a single lab over time to batches imaged using different microscopes in multiple labs. We find that Harmony and Seurat RPCA are noteworthy, consistently ranking among the top three methods for all tested scenarios while maintaining computational efficiency. Our proposed framework, benchmark, and metrics can be used to assess new batch correction methods in the future. This work paves the way for improvements that enable the community to make the best use of public Cell Painting data for scientific discovery.
Assuntos
Análise de Célula Única , Análise de Célula Única/métodos , Humanos , Processamento de Imagem Assistida por Computador/métodos , Análise de Sequência de RNA/métodos , BenchmarkingRESUMO
The growing number of multi-omics studies demands clear conceptual workflows coupled with easy-to-use software tools to facilitate data analysis and interpretation. This protocol covers three key components involved in multi-omics analysis, including single-omics data analysis, knowledge-driven integration using biological networks and data-driven integration through joint dimensionality reduction. Using the dataset from a recent multi-omics study of human pancreatic islet tissue and plasma samples, the first section introduces how to perform transcriptomics/proteomics data analysis using ExpressAnalyst and lipidomics data analysis using MetaboAnalyst. On the basis of significant features detected in these workflows, the second section demonstrates how to perform knowledge-driven integration using OmicsNet. The last section illustrates how to perform data-driven integration from the normalized omics data and metadata using OmicsAnalyst. The complete protocol can be executed in ~2 h. Compared with other available options for multi-omics integration, the Analyst software suite described in this protocol enables researchers to perform a wide range of omics data analysis tasks via a user-friendly web interface.
Assuntos
Internet , Metabolômica , Proteômica , Software , Humanos , Metabolômica/métodos , Proteômica/métodos , Ilhotas Pancreáticas/metabolismo , Biologia Computacional/métodos , Lipidômica/métodos , Genômica/métodos , MultiômicaRESUMO
The developing brain is vulnerable to maternal bacterial and viral infections which induce strong inflammatory responses in the mother that are mimicked in the offspring brain, resulting in irreversible neurodevelopmental defects, and associated cognitive and behavioural impairments. In contrast, infection during pregnancy and lactation with the immunoregulatory murine intestinal nematode, Heligmosomoides bakeri, upregulates expression of genes associated with long-term potentiation (LTP) of synaptic networks in the brain of neonatal uninfected offspring, and enhances spatial memory in uninfected juvenile offspring. As the hippocampus is involved in spatial navigation and sensitive to immune events during development, here we assessed hippocampal gene expression, LTP, and neuroimmunity in 3-week-old uninfected offspring born to H. bakeri infected mothers. Further, as maternal immunity shapes the developing immune system, we assessed the impact of maternal H. bakeri infection on the ability of offspring to resist direct infection. In response to maternal infection, we found an enhanced propensity to induce LTP at Schaffer collateral synapses, consistent with RNA-seq data indicating accelerated development of glutamatergic synapses in uninfected offspring, relative to those from uninfected mothers. Hippocampal RNA-seq analysis of offspring of infected mothers revealed increased expression of genes associated with neurogenesis, gliogenesis, and myelination. Furthermore, maternal infection improved resistance to direct infection of H. bakeri in offspring, correlated with transfer of parasite-specific IgG1 to their serum. Hippocampal immunohistochemistry and gene expression suggest Th2/Treg biased neuroimmunity in offspring, recapitulating peripheral immunoregulation of H. bakeri infected mothers. These findings indicate maternal H. bakeri infection during pregnancy and lactation alters peripheral and neural immunity in uninfected offspring, in a manner that accelerates neural maturation to promote hippocampal LTP, and upregulates the expression of genes associated with neurogenesis, gliogenesis, and myelination.
Assuntos
Hipocampo , Plasticidade Neuronal , Animais , Feminino , Hipocampo/metabolismo , Hipocampo/parasitologia , Gravidez , Camundongos , Infecções por Nematoides/imunologia , Infecções por Nematoides/parasitologia , Potenciação de Longa Duração , Efeitos Tardios da Exposição Pré-Natal/imunologia , Infecções por Strongylida/imunologia , Infecções por Strongylida/parasitologia , Masculino , NeuroimunomodulaçãoRESUMO
Efforts to use transcriptomics for toxicity testing have classically relied on the assumption that chemicals consistently produce characteristic transcriptomic signatures that are reflective of their mechanism of action. However, the degree to which transcriptomic responses are conserved across different test methodologies has seldom been explored. With increasing regulatory demand for New Approach Methods (NAMs) that use alternatives to animal models and high-content approaches such as transcriptomics, this type of comparative analysis is needed. We examined whether common genes are dysregulated in Japanese quail (Coturnix japonica) liver following sublethal exposure to the flame retardant hexabromocyclododecane (HBCD), when life stage and test methodologies differ. The four exposure scenarios included one NAM: Study 1-early-life stage (ELS) exposure via a single egg injection, and three more traditional approaches; Study 2-adult exposure using a single oral gavage; Study 3-ELS exposure via maternal deposition after adults were exposed through their diet for 7 weeks; and Study 4-ELS exposure via maternal deposition and re-exposure of nestlings through their diet for 17 weeks. The total number of differentially expressed genes (DEGs) detected in each study was variable (Study 1, 550; Study 2, 192; Study 3, 1; Study 4, 3) with only 19 DEGs shared between Studies 1 and 2. Factors contributing to this lack of concordance are discussed and include differences in dose, but also quail strain, exposure route, sampling time, and HBCD stereoisomer composition. The results provide a detailed overview of the transcriptomic responses to HBCD at different life stages and routes of exposure in a model avian species and highlight certain challenges and limits of comparing transcriptomics across different test methodologies. Environ Toxicol Chem 2024;00:1-11. © 2024 The Author(s). Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.
RESUMO
Understanding species differences in sensitivity to toxicants is a critical issue in ecotoxicology. We recently established that double-crested cormorant (DCCO) embryos are more sensitive than Japanese quail (JQ) to the developmental effects of ethinylestradiol (EE2). We explored how this difference in sensitivity between species is reflected at a transcriptomic level. The EE2 was dissolved in dimethyl sulfoxide and injected into the air cell of eggs prior to incubation at nominal concentrations of 0, 3.33, and 33.3 µg/g egg weight. At midincubation (JQ 9 days; DCCO 16 days), livers were collected from five embryos/treatment group for RNA sequencing. Data were processed and analyzed using EcoOmicsAnalyst and ExpressAnalyst. The EE2 exposure dysregulated 238 and 1,987 genes in JQ and DCCO, respectively, with 78 genes in common between the two species. These included classic biomarkers of estrogen exposure such as vitellogenin and apovitellenin. We also report DCCO-specific dysregulation of Phase I/II enzyme-coding genes and species-specific transcriptional ontogeny of vitellogenin-2. Twelve Kyoto Encyclopedia of Genes and Genomes pathways and two EcoToxModules were dysregulated in common in both species including the peroxisome proliferator-activated receptor (PPAR) signaling pathway and fatty acid metabolism. Similar to previously reported differences at the organismal level, DCCO were more responsive to EE2 exposure than JQ at the gene expression level. Our description of differences in transcriptional responses to EE2 in early life stage birds may contribute to a better understanding of the molecular basis for species differences. Environ Toxicol Chem 2024;43:772-783. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.
Assuntos
Coturnix , Etinilestradiol , Animais , Etinilestradiol/toxicidade , Coturnix/genética , Vitelogeninas , Perfilação da Expressão Gênica , FígadoRESUMO
P21-activated kinase 2 (PAK2) is a serine/threonine kinase essential for a variety of cellular processes including signal transduction, cellular survival, proliferation, and migration. A recent report proposed monoallelic PAK2 variants cause Knobloch syndrome type 2 (KNO2)-a developmental disorder primarily characterized by ocular anomalies. Here, we identified a novel de novo heterozygous missense variant in PAK2, NM_002577.4:c.1273G>A, p.(D425N), by whole genome sequencing in an individual with features consistent with KNO2. Notable clinical phenotypes include global developmental delay, congenital retinal detachment, mild cerebral ventriculomegaly, hypotonia, FTT, pyloric stenosis, feeding intolerance, patent ductus arteriosus, and mild facial dysmorphism. The p.(D425N) variant lies within the protein kinase domain and is predicted to be functionally damaging by in silico analysis. Previous clinical genetic testing did not report this variant due to unknown relevance of PAK2 variants at the time of testing, highlighting the importance of reanalysis. Our findings also substantiate the candidacy of PAK2 variants in KNO2 and expand the KNO2 clinical spectrum.
RESUMO
Population level variation and molecular mechanisms behind insulin secretion in response to carbohydrate, protein, and fat remain uncharacterized despite ramifications for personalized nutrition. Here, we define prototypical insulin secretion dynamics in response to the three macronutrients in islets from 140 cadaveric donors, including those diagnosed with type 2 diabetes. While islets from the majority of donors exhibited the expected relative response magnitudes, with glucose being highest, amino acid moderate, and fatty acid small, 9% of islets stimulated with amino acid and 8% of islets stimulated with fatty acids had larger responses compared with high glucose. We leveraged this insulin response heterogeneity and used transcriptomics and proteomics to identify molecular correlates of specific nutrient responsiveness, as well as those proteins and mRNAs altered in type 2 diabetes. We also examine nutrient-responsiveness in stem cell-derived islet clusters and observe that they have dysregulated fuel sensitivity, which is a hallmark of functionally immature cells. Our study now represents the first comparison of dynamic responses to nutrients and multi-omics analysis in human insulin secreting cells. Responses of different people's islets to carbohydrate, protein, and fat lay the groundwork for personalized nutrition. ONE-SENTENCE SUMMARY: Deep phenotyping and multi-omics reveal individualized nutrient-specific insulin secretion propensity.