Project description:BackgroundEnvironmental health research has recently undergone a dramatic shift, with ongoing technological advancements allowing for broader coverage of exposure and molecular biology signatures. Approaches to integrate such measures are still needed to increase understanding between systems-level exposure and biology.ObjectivesWe address this gap by evaluating placental tissues to identify novel chemical-biological interactions associated with preeclampsia. This study tests the hypothesis that understudied chemicals are present in the human placenta and associated with preeclampsia-relevant disruptions, including overall case status (preeclamptic vs. normotensive patients) and underlying transcriptomic/epigenomic signatures.MethodsA non-targeted analysis based on high-resolution mass spectrometry was used to analyze placental tissues from a cohort of 35 patients with preeclampsia (n = 18) and normotensive (n = 17) pregnancies. Molecular feature data were prioritized for confirmation based on association with preeclampsia case status and confidence of chemical identification. All molecular features were evaluated for relationships to mRNA, microRNA, and CpG methylation (i.e., multi-omic) signature alterations involved in preeclampsia.ResultsA total of 183 molecular features were identified with significantly differentiated abundance in placental extracts of preeclamptic patients; these features clustered into distinct chemical groupings using unsupervised methods. Of these features, 53 were identified (mapping to 40 distinct chemicals) using chemical standards, fragmentation spectra, and chemical metadata. In general, human metabolites had the largest feature intensities and strongest associations with preeclampsia-relevant multi-omic changes. Exogenous drugs were second most abundant and had fewer associations with multi-omic changes. Other exogenous chemicals (non-drugs) were least abundant and had the fewest associations with multi-omic changes.ConclusionsThese global data trends suggest that human metabolites are heavily intertwined with biological processes involved in preeclampsia etiology, while exogenous chemicals may still impact select transcriptomic/epigenomic processes. This study serves as a demonstration of merging systems exposures with systems biology to better understand chemical-disease relationships.
Project description:Placental extravillous trophoblast (EVT) invasion is essential in establishing proper blood supply to the fetus during pregnancy. However, traditional 2D in vitro systems do not model the in vivo invasion process in an anatomically-relevant manner. Our objectives were to develop a 3D spheroid model that would allow better emulation of placental invasion in vitro and to characterize the transcriptomic and functional outcomes. HTR8/SVneo EVT cells were self-assembled into 3D spheroids using ultra-low attachment plates. Transcriptomic profiling followed by gene set enrichment and gene ontology analyses revealed major global transcriptomic differences, with significant up-regulations in EVTs cultured as 3D spheroids in canonical pathways and biological processes such as immune response, angiogenesis, response to stimulus, wound healing, and others. These findings were further validated by RT-qPCR, showing significant up-regulations in genes and/or proteins related to epithelial-mesenchymal transition, cell-cell contact, angiogenesis, and invasion/migration. A high-throughput, spheroid invasion assay was applied to reveal the dynamic invasion of EVTs away from the spheroid core into extracellular matrix. Lastly, lipopolysaccharide, dexamethasone, or Δ9-tetrahydrocannabinol exposure was found to impact the invasion of EVT spheroids. Altogether, we present a well-characterized, 3D spheroid model of EVT invasion and demonstrate its potential use in drug and toxin screening during pregnancy.
Project description:Archival formalin-fixed paraffin-embedded (FFPE) tissue samples offer a vast but largely untapped resource for genomic research. The primary technical issues limiting use of FFPE samples are RNA yield and quality. In this study, we evaluated methods to demodify RNA highly fragmented and crosslinked by formalin fixation. Primary endpoints were RNA recovery, RNA-sequencing quality metrics, and transcriptional responses to a reference chemical (phenobarbital, PB). Frozen mouse liver samples from control and PB groups (n = 6/group) were divided and preserved for 3 months as follows: frozen (FR); 70% ethanol (OH); 10% buffered formalin for 18 h followed by ethanol (18F); or 10% buffered formalin (3F). Samples from OH, 18F, and 3F groups were processed to FFPE blocks and sectioned for RNA isolation. Additional sections from 3F received the following demodification protocols to mitigate RNA damage: short heated incubation with Tris-Acetate-EDTA buffer; overnight heated incubation with an organocatalyst using 2 different isolation kits; or overnight heated incubation without organocatalyst. Ribo-depleted, stranded, total RNA libraries were built and sequenced using the Illumina HiSeq 2500 platform. Overnight incubation (± organocatalyst) increased RNA yield >3-fold and RNA integrity numbers and fragment analysis values by > 1.5- and >3.0-fold, respectively, versus 3F. Postsequencing metrics also showed reduced bias in gene coverage and deletion rates for overnight incubation groups. All demodification groups had increased overlap for differentially expressed genes (77%-84%) and enriched pathways (91%-97%) with FR, with the highest overlap in the organocatalyst groups. These results demonstrate simple changes in RNA isolation methods that can enhance genomic analyses of FFPE samples.
Project description:This study aimed to optimise techniques for whole transcriptome and small RNA analyses on clinical tissue samples from patients with cardiovascular disease. Clinical samples often represent a particular challenge to extracting RNA of sufficient quality for robust RNA sequencing analysis, and due to availability, it is rarely possible to optimise techniques on the samples themselves. Therefore, we have used equivalent samples from pigs undergoing cardiopulmonary bypass surgery to test different protocols for optimal RNA extraction, and then validated the protocols in human samples. Here we present an assessment of the quality and quantity of RNA obtained using a variety of commercially-available RNA extraction kits on both left ventricular biopsies and blood plasma. RNA extraction from these samples presents different difficulties; left ventricular biopsies are small and fibrous, while blood plasma has a low RNA content. We have validated our optimised extraction techniques on human clinical samples collected as part of the ARCADIA (Association of non-coding RNAs with Coronary Artery Disease and type 2 Diabetes) cohort study, resulting in successful whole transcriptome and small RNA sequencing of human left ventricular tissue.
Project description:The placenta is a transient organ that is repsonsible for multiple processes during pregnancy. Here we aim to study the development of the placenta using mouse models at embryonic day (e) e7.5, e8.5 and e9.5 by integrating RNA-seq data at the three timepoints to identify important genes, inferring their interaction networks, and predict novel regulators of placental development.
Project description:Exosomes, derived from endocytic membrane vesicles are thought to participate in cell-cell communication and protein and RNA delivery. They are ubiquitous in most body fluids (breast milk, saliva, blood, urine, malignant ascites, amniotic, bronchoalveolar lavage, and synovial fluids). In particular, exosomes secreted in human saliva contain proteins and nucleic acids that could be exploited for diagnostic purposes. To investigate this potential use, we isolated exosomes from human saliva and characterized their structural and transcriptome contents.Exosomes were purified by differential ultracentrifugation and identified by immunoelectron microscopy (EM), flow cytometry, and Western blot with CD63 and Alix antibodies. We then described the morphology, shape, size distribution, and density using atomic force microscopy (AFM). Microarray analysis revealed that 509 mRNA core transcripts are relatively stable and present in the exosomes. Exosomal mRNA stability was determined by detergent lysis with RNase A treatment. In vitro, fluorescently labeled saliva exosomes could communicate with human keratinocytes, transferring their genetic information to human oral keratinocytes to alter gene expression at a new location.Our findings are consistent with the hypothesis that exosomes shuttle RNA between cells and that the RNAs present in the exosomes may be a possible resource for disease diagnostics.
Project description:BackgroundThrough several observational and mechanistic studies, microbial infection is known to promote cardiovascular disease. Direct infection of the vessel wall, along with the cardiovascular risk factors, is hypothesized to play a key role in the atherogenesis by promoting an inflammatory response leading to endothelial dysfunction and generating a proatherogenic and prothrombotic environment ultimately leading to clinical manifestations of cardiovascular disease, e.g., acute myocardial infarction or stroke. There are many reports of microbial DNA isolation and even a few studies of viable microbes isolated from human atherosclerotic vessels. However, high-resolution investigation of microbial infectious agents from human vessels that may contribute to atherosclerosis is very limited. In spite of the progress in recent sequencing technologies, analyzing host-associated metagenomes remain a challenge.ResultsTo investigate microbiome diversity within human atherosclerotic tissue samples, we employed high-throughput metagenomic analysis on: (1) atherosclerotic plaques obtained from a group of patients who underwent endarterectomy due to recent transient cerebral ischemia or stroke. (2) Presumed stabile atherosclerotic plaques obtained from autopsy from a control group of patients who all died from causes not related to cardiovascular disease. Our data provides evidence that suggest a wide range of microbial agents in atherosclerotic plaques, and an intriguing new observation that shows these microbiota displayed differences between symptomatic and asymptomatic plaques as judged from the taxonomic profiles in these two groups of patients. Additionally, functional annotations reveal significant differences in basic metabolic and disease pathway signatures between these groups.ConclusionsWe demonstrate the feasibility of novel high-resolution techniques aimed at identification and characterization of microbial genomes in human atherosclerotic tissue samples. Our analysis suggests that distinct groups of microbial agents might play different roles during the development of atherosclerotic plaques. These findings may serve as a reference point for future studies in this area of research.
Project description:Cell lines are extensively used to study cancer biology. However, the use of highly passaged commercial cell lines has to be questioned, as they do not closely resemble the originating tumor. To understand the reliability of preclinical models for Malignant pleural mesothelioma (MPM) studies, we have performed whole transcriptome and whole exome analyses of fresh frozen MPM tumors and compared them to cell lines generated from these tumors, as well as commercial cell lines and a preclinical MPM mouse model. Patient-derived cell lines were generated from digested fresh tumors and whole exome sequencing was performed on DNA isolated from formalin-fixed, paraffin-embedded (FFPE) tumor samples, corresponding patient-derived cell lines, and normal tissue. RNA sequencing libraries were prepared from 10 fresh frozen tumor samples, the 10 corresponding patient-derived cell lines, and 7 commercial cell lines. Our results identified alterations in tumor suppressor genes such as FBXW7, CDKN2A, CDKN2B, and MTAP, all known to drive MPM tumorigenesis. Patient-derived cell lines correlate to a high degree with their originating tumor. Gene expressions involved in multiple pathways such as EMT, apoptosis, myogenesis, and angiogenesis are upregulated in tumor samples when compared to patient-derived cell lines; however, they are downregulated in commercial cell lines compared to patient-derived cell lines, indicating significant differences between the two model systems. Our results show that the genome and transcriptome of tumors correlate to a higher degree with patient-derived cell lines rather than commercial cell lines. These results are of major relevance for the scientific community in regard to using cell lines as an appropriate model, resembling the pathway of interest to avoid misleading results for clinical applications.
Project description:The goal of this study was to compare the transcriptome between wild type strain of Listeria monocytogenes and delete nmlR mutant strain of L. monocytogenes using NGS. Method: Duplicate samples of rRNA depleted RNA from wild type and mutants were used to study transcriptomes by ion torrent platform. Transcriptomes of wild type and nmlR mutant were compared by EDGE-pro program. Result: Differential expression by EDGE-pro showed 74 genes with differential expressions between wild type and nmlR null mutant (46 genes were negatively regluated and 28 genes were positively regulated by NmlR). rRNA-depleted RNA samples from stationary phase wilde type and nmlR null mutant cultures were used to compare transcriptomes. Some affected genes from RNAseq result were selected for confirmation by quantitative reverse transcriptase PCR.