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Cultured naïve pluripotent ESC differentiate into first lineage, XEN or second lineage, formative pluripotency. Hyperosmotic stress (sorbitol), like retinoic acid, decreases naive pluripotency and increases XEN in two ESC lines, as reported by bulk and scRNAseq, analyzed by UMAP. Sorbitol overrides pluripotency in two ESC lines as reported by bulk and scRNAseq, analyzed by UMAP. UMAP analyzed the effects of 5 stimuli - three stressed (200-300mM sorbitol with leukemia inhibitory factor +LIF) and two unstressed (+LIF, normal stemness-NS and -LIF, normal differentiation-ND). Sorbitol and RA decrease naive pluripotency and increase subpopulations of 2-cell embryo-like and XEN sub-lineages; primitive, parietal, and visceral endoderm (VE). Between the naïve pluripotency and primitive endoderm clusters is a stress-induced cluster with transient intermediate cells with higher LIF receptor signaling, with increased Stat3, Klf4, and Tbx3 expression. Sorbitol, like RA, also suppresses formative pluripotency, increasing lineage imbalance. Although bulk RNAseq and gene ontology group analyses suggest that stress induces head organizer and placental markers, scRNAseq reveals few cells. But VE and placental markers/cells were in adjacent clusters, like recent reports. UMAPs show that dose-dependent stress overrides stemness to force premature lineage imbalance. Hyperosmotic stress induces lineage imbalance, and other toxicological stresses, like drugs with RA, may cause lineage imbalance, resulting in miscarriages or birth defects.

Lowest observable adverse effects level (LOAEL) is a standard point-of-departure dose in toxicology. However, first observable adverse effects level (FOAEL) was recently reported and is used, in this study, as one criterion to detect a mutagenic stimulus in a live imager. Fluorescence ubiquitinated cell cycle indicator (FUCCI) embryonic stem cells (ESC) are green in the S-G2-M phase of the cell cycle and not green in G1-phase. Standard media change here is a mild stress that delays G1-phase and media change increases green 2.5- to 5-fold. Since stress is mild, media change rapidly increases green cell number, but higher stresses of environmental toxicants and positive control hyperosmotic stress suppress increased green after media change. Perfluoro-octanoic acid (PFOA) and diethyl phthalate (DEP) previously suppressed progression of nongreen to green cell cycle progression. Here, bisphenol A (BPA), cortisol, and positive control hyperosmotic sorbitol also suppress green fluorescence, but benzo(a)pyrene (BaP) at high doses (10 μM) increases green fluorescence throughout the 74-h exposure. Since any stress can affect many cell cycle phases, messenger RNA (mRNA) markers are best interpreted in ratios as dose-dependent mutagens increase in G2/G1 and nonmutagens increase G1/G2. After 74-h exposure, RNAseq detects G1 and G2 markers and increasing BaP doses increase G2/G1 ratios but increasing hyperosmotic sorbitol and PFOA doses increase G1/G2 marker ratios. BaP causes rapid green increase in FOAEL at 2 h of stimulus, whereas retinoic acid caused significant green fluorescence increases only late in culture. Using a live imager to establish FOAEL and G2 delay with FUCCI ESC is a new method to allow commercial and basic developmental biologists to detect drugs and environmental stimuli that are mutagenic. Furthermore, it can be used to test compounds that prevent mutations. In longitudinal studies, uniquely provided by this viable reporter and live imager protocol, follow-up can be done to test whether the preventative compound itself causes harm.

The link between post-operative adhesion development and epigenetic modifications is important in understanding the mechanism behind their formation. The purpose of this study was to determine whether epigenetic differences exist between primary fibroblasts of normal peritoneum and adhesion tissues isolated from the same patient(s).DNA from fibroblasts isolated from normal peritoneum and adhesion tissues was isolated using Qiagen's EZ1 Advanced Kit. Methylation patterns of genes were quantified and compared in both cell lines using the Infinium Human Methylation 27 Beadchip system.A total of 7364 genes had been found to manifest significantly different DNA methylation levels in adhesion fibroblasts as compared to normal peritoneal fibroblasts (p0.01). A total of 1685 genes were found to have increased DNA methylation by 50% in adhesion compared to peritoneal fibroblasts, and were enriched in Gene Ontology categories, Glycoprotein, and Defense Response. Furthermore, 1287 genes were found to have decreased DNA methylation patterns with enriched Gene Ontology categories, "Homeobox", and Transcription Factor Activity in adhesion fibroblasts.Epigenetic differences in fibroblasts isolated from normal peritoneum and adhesion tissues were observed. Future studies focusing on the precise role of these genes in the development of post operative adhesions will allow us to more fully appreciate regulatory mechanisms leading to adhesion development, thereby establishing targets for therapeutic interventions to prevent or limit adhesion development.

A problem in developmental toxicology is the massive loss of life from fertilization through gastrulation, and the surprising lack of knowledge of causes of miscarriage. Half to two-thirds of embryos are lost, and environmental and genetic causes are nearly equal. Simply put, it can be inferred that this is a difficult period for normal embryos, but that environmental stresses may cause homeostatic responses that move from adaptive to maladaptive with increasing exposures. At the lower 50% estimate, miscarriage causes greater loss-of-life than all cancers combined or of all cardio- and cerebral-vascular accidents combined. Surprisingly, we do not know if miscarriage rates are increasing or decreasing. Overshadowed by the magnitude of miscarriages, are insufficient data on teratogenic or epigenetic imbalances in surviving embryos and their stem cells. Superimposed on the difficult normal trajectory for peri-gastrulation embryos are added malnutrition, hormonal, and environmental stresses. An overarching hypothesis is that high throughput screens (HTS) using cultured viable reporter embryonic and placental stem cells (e.g., embryonic stem cells [ESC] and trophoblast stem cells [TSC] that report status using fluorescent reporters in living cells) from the pre-gastrulation embryo will most rapidly test a range of hormonal, environmental, nutritional, drug, and diet supplement stresses that decrease stem cell proliferation and imbalance stemness/differentiation. A second hypothesis is that TSC respond with greater sensitivity in magnitude to stress that would cause miscarriage, but ESC are stress-resistant to irreversible stemness loss and are best used to predict long-term health defects. DevTox testing needs more ESC and TSC HTS to model environmental stresses leading to miscarriage or teratogenesis and more research on epidemiology of stress and miscarriage. This endeavor also requires a shift in emphasis on pre- and early gastrulation events during the difficult period of maximum loss by miscarriage.

The Frontiers Media family has over 200 journals, which are each headed by usually one Field Chief Editor, and several thousand specialty sections, which are each headed by one or more Specialty Chief Editors. The year 2021 was the 10th anniversary of the founding of the Frontiers in Genetics journal and the Frontiers in Toxicogenomics specialty section of this journal. In 2021, we also announce one of the newest of the Frontiers journals–Frontiers in Toxicology which is part of the Frontiers Media family of journals but independent of Frontiers in Genetics. Dr. Ruden is the founding, and currently sole, Specialty Chief Editor of Frontiers in Toxicogenomics and one of 9 Specialty Chief Editors of Frontiers in Toxicology. As of 2021, Frontiers in Toxicogenomics has published over 138 articles and has over 370 Editors including 90 Associate Editors and 280 Review Editors. The Frontiers in Genetics impact factor was initially approximately 2.5 when it was first listed in PubMed in 2015 and has risen steadily to its current value of 4.8, which is typical for the majority of the over 200 Frontiers journals that have established impact factors. In this overview of the first decade of Frontiers in Toxicogenomics, we discuss the top 5 articles with the highest Scopus citations, which were all written in the first few years of the journal. The article with the highest number of citations, with 353 Scopus over 600 Google Scholar citations, and the highest average number of citations (67) that steadily increased from 10 citations in 2013 to 119 citations in 2021, was written in 2012 by Dr. Ruden’s laboratory and titled, “Using Drosophila melanogaster as a model for genotoxic chemical mutational studies with a new program, SnpSift.” The five most influential authors who published in the journal in the past 10 years based on Scopus citations of a particular paper are Dr. Ruden’s laboratory, with 353 Scopus citations for the SnpSift paper mentioned above; Drs. Brock Christensen and Carmen J. Marsit, with 86 Scopus citations for their review, “Epigenomics in environmental health”; Dr. Michael Aschner and colleagues, with 61 Scopus citations for their paper “Genetic factors and manganese-induced neurotoxicity”; and Dr. Sandra C. dos Santos and colleagues, with 59 Scopus citations for their paper, “Yeast toxicogenomics: genome-wide responses to chemical stresses with impact in environmental health, pharmacology, and biotechnology.” While the top 5 papers were published in the early years of the journal, we will also discuss a more recent article published in 2018 on a comparison of RNA-seq and microarray methods by Dr. Michael Liguori’s laboratory, “Comparison of RNA-Seq and Microarray Gene Expression Platforms for the Toxicogenomic Evaluation of Liver From Short-Term Rat Toxicity Studies,” that far exceeds the number of downloads and views of all the other articles published in the first 10 years of the journal and will likely be a top cited paper in the second decade highlights of this journal. Finally, we discuss where the Frontiers in Toxicogenomics specialty journal and the Frontiers in Toxicology journal will go to advance the field of toxicogenomics, and more generally, toxicology, in the future.

Fetal inflammatory response syndrome (FIRS) is strongly associated with neonatal morbidity and mortality and can be classified as type I or type II. Clinically, FIRS type I and type II are considered as distinct syndromes, yet the molecular underpinnings of these fetal inflammatory responses are not well understood because of their low prevalence and the difficulty of postdelivery diagnosis. In this study, we performed RNA sequencing of human cord blood samples from preterm neonates diagnosed with FIRS type I or FIRS type II. We found that FIRS type I was characterized by an upregulation of host immune responses, including neutrophil and monocyte functions, together with a proinflammatory cytokine storm and a downregulation of T cell processes. In contrast, FIRS type II comprised a mild chronic inflammatory response involving perturbation of HLA transcripts, suggestive of fetal semiallograft rejection. Integrating single-cell RNA sequencing–derived signatures with bulk transcriptomic data confirmed that FIRS type I immune responses were mainly driven by monocytes, macrophages, and neutrophils. Last, tissue- and cell-specific signatures derived from the BioGPS Gene Atlas further corroborated the role of myeloid cells originating from the bone marrow in FIRS type I. Collectively, these data provide evidence that FIRS type I and FIRS type II are driven by distinct immune mechanisms; whereas the former involves the innate limb of immunity consistent with host defense, the latter resembles a process of semiallograft rejection. These findings shed light on the fetal immune responses caused by infection or alloreactivity that can lead to deleterious consequences in neonatal life.

Objective: As the number of refugees continues to rise, there is growing concern about the impact from trauma exposures on their mental health. However, there is a limited understanding of possible biological mechanisms contributing to the substantial inter-individual differences in trauma-related outcomes, especially as it relates to positive mental health. Only sparse work has focused on the biology of positive mental health, including energy and sleep, in trauma-exposed persons. In this study, we analyzed cytokines in blood from newly arrived refugees with differential trauma exposures in relationship to self-reported energy, as a key marker of positive mental health. Methods: Within the first month of arrival in the USA, 64 refugees from Iraq and Syria were interviewed. Refugees completed the clinical DSM-IV PTSD-Checklist Civilian Version (PCL-C), the Beck Anxiety Inventory (BAI), and the Hospital Anxiety and Depression Scale (HADS). Ten psychiatrically healthy non-refugee persons were used as healthy controls to compare levels of cytokines. Blood samples were collected at the time of the interview and subsequently analyzed for IL-1β, IL-6, IL-8, IL-10, and TNF-α concentrations. Results: Energy correlated positively with current concentration ability and sleep quality, and negatively with stress, PCL-C, BAI and HADS scores (Spearman correlations, all p

Traumatic brain injuries, a leading cause of death and disability worldwide, are caused by a severe impact to the head that impairs physiological and psychological function. In addition to severity, type and brain area affected, brain injury outcome is also influenced by the biological sex of the patient. Traumatic brain injury triggers accumulation of Tau protein and the subsequent development of Tauopathies, including Alzheimer’s disease and Chronic traumatic encephalopathy. Recent studies report differences in Tau network connections between healthy males and females, but the possible role of Tau in sex-dependent outcome to brain injury is unclear. Thus, we aimed to determine if Tau ablation would alleviate sex dependent outcomes in injured flies. We first assessed motor function and survival in tau knock-out flies and observed sex-differences in climbing ability, but no change in locomotor activity in either sex post-injury. Sex differences in survival time were also observed in injured tau deficient flies with a dramatically higher percent of female death within 24 h than males. Additionally, 3′mRNA-Seq studies in isolated fly brains found that tau deficient males show more gene transcript changes than females post-injury. Our results suggest that sex differences in TBI outcome and recovery are not dependent on the presence of Tau in Drosophila.

Stress-induced changes in viral receptor and susceptibility gene expression were measured in embryonic stem cells (ESC) and differentiated progeny. Rex1 promoter-Red Fluorescence Protein reporter ESC were tested by RNAseq after 72hr exposures to control stress hyperosmotic sorbitol under stemness culture (NS) to quantify stress-forced differentiation (SFD) transcriptomic programs. Control ESC cultured with stemness factor removal produced normal differentiation (ND). Bulk RNAseq transcriptomic analysis showed significant upregulation of two genes involved in Covid-19 cell uptake, Vimentin (VIM) and Transmembrane Serine Protease 2 (TMPRSS2). SFD increased the hepatitis A virus receptor (Havcr1) and the transplacental Herpes simplex 1 (HSV1) virus receptor (Pvrl1) compared with ESC undergoing ND. Several other coronavirus receptors, Glutamyl Aminopeptidase (ENPEP) and Dipeptidyl Peptidase 4 (DPP4) were upregulated significantly in SFD>ND. Although stressed ESC are more susceptible to infection due to increased expression of viral receptors and decreased resistance, the necessary Covid-19 receptor, angiotensin converting enzyme (ACE)2, was not expressed in our experiments. TMPRSS2, ENPEP, and DPP4 mediate Coronavirus uptake, but are also markers of extra-embryonic endoderm (XEN), which arise from ESC undergoing ND or SFD. Mouse and human ESCs differentiated to XEN increase TMPRSS2 and other Covid-19 uptake-mediating gene expression, but only some lines express ACE2. Covid-19 susceptibility appears to be genotype-specific and not ubiquitous. Of the 30 gene ontology (GO) groups for viral susceptibility, 15 underwent significant stress-forced changes. Of these, 4 GO groups mediated negative viral regulation and most genes in these increase in ND and decrease with SFD, thus suggesting that stress increases ESC viral susceptibility. Taken together, the data suggest that a control hyperosmotic stress can increase Covid-19 susceptibility and decrease viral host resistance in mouse ESC. However, this limited pilot study should be followed with studies in human ESC, tests of environmental, hormonal, and pharmaceutical stressors and direct tests for infection of stressed, cultured ESC and embryos by Covid-19.

Introduction B-cells have been strongly implicated in cardiac allograft rejection (CAR). Recently, however, the CTOT-11 trial showed that depleting mature CD20+ B-cells did not reduce rates of rejection in cardiac allograft recipients and unexpectedly increased the severity of allograft vasculopathy. Therefore, it can be hypothesized that differing phenotypic subtypes of B-cells correspond with different biological mechanisms relating to CAR. Though, current applications to quantify these subtypes of immune cells, i.e with immunohistochemistry or flow cytometry, are often restricted by limited cell markers and cost-burden; therefore, we demonstrate a novel deconvolution method, FARDEEP, that has been validated to accurately enumerate peripheral blood mononuclear cell-subtypes (PBMCs) in a quicker and more cost-effective manner. Purpose To better understand the association of different B-cell subtypes in CAR by identifying the B-cell subtype most predictive for pathologically defined rejection. Methods The machine learning tool, FARDEEP, was trained with the transcriptomic signatures of 29 PBMC subtypes, characterized by previous single-cell RNA experiments. FARDEEP then was used to deconvolute data-mined RNA from 259 blood samples from 98 cardiac allograft recipients enrolled in the CARGO study (GSE2445). Random forest tree (RF) was then used to analyze the levels of deconvoluted subtypes to predict the severity of rejection assessed by endomyocardial biopsy. Finally, RF was used to identify the subtypes of PBMCs most valuable in predicting rejection. Results Out of the 259 samples with consensus pathological readings, 140 had a consensus International Society of Heart and Lung Transplant grade of 0, 63 with grade 1a, 31 with grade 1b, and 25 with grade 3a or higher. We grouped biopsy samples with grade 0, 1a, and 1b as “low-risk” rejection (n=234). 3a or higher samples were grouped as “high-risk” (n=25). There were no grade 2s in the dataset. According to the dataset, blood was extracted from patients on average 72.5 days post-transplant. The RF had good performance in predicting rejection severity. (Figure 1a) CD20- plasmablast cells were stronger predictors for differentiating high-risk from low-risk compared to CD20+ B-cell populations (i.e B Naive and B Memory cells). (Figure 1b) Overall, however, dendritic cells (DCs), neutrophils, monocytes, and basophils were the strongest predictors for rejection. Conclusion Our findings support the results from the CTOT-11 trial showing that CD20+ B-cells may not contribute to CAR as significantly as seen with other PBMC subtypes. Instead, we showed that among B-cells, CD20- plasmablasts were more likely associated with CAR, possibly explaining why targeting CD20 was ineffective in preventing rejection. Thus, targeting plasmablast-associated markers could potentially be more useful to prevent CAR. Model Performance with Variables Funding Acknowledgement Type of funding source: Private grant(s) and/or Sponsorship. Main funding source(s): 1) Society of Academic Emergency Medicine Foundation; 2) The Jewish Fund

Objective: Determine whether various stresses change the expression in embryonic stem cells (ESC) and their differentiated lineages, of a large set of viral receptors and susceptibility genes Design: Experimental Materials and Methods: ESC were tested by bulk or single cell (sc) RNAseq after 72 hr exposures to 0-300 mM hyperosmotic sorbitol (with stemness-maintaining Leukemia Inhibitory Factor, LIF) to quantify stress-forced differentiation Controls for normal stemness were LIF+ and normal differentiation were LIF- RNA was isolated by RNAeasy lysis or 10XGenomics Dropseq, RNA quality was checked by Agilent TapeStation, cDNA was synthesized using Lexogen’s QuantSeq library kit, and barcoded, multiplexed and sequenced by Illumina NovaSeq 6000 Data were demultiplexed by CASAVA software and FC expression was compared between conditions In triplicate experiments, significant fold change (FC) genes (FC≥2;FDR≤0 05, P

Background Prenatal exposure to environmental chemicals has been associated with Autism Spectrum Disorder (ASD) symptoms in some, but not all, studies, but most research has not accounted for other childhood behavior problems. Objectives To evaluate the specific associations of prenatal phthalate exposures with ASD symptoms in children (ages 3–6) accounting for other behavior problems, and to assess sex differences in these associations. Methods We measured phthalate metabolites in prenatal urine samples. Mothers completed the Social Responsiveness Scale-2nd edition (SRS-2) to assess child ASD symptoms and the Child Behavior Checklist (CBCL) to assess general behavior problems. We assessed associations of the sum of di-(2-ethylhexyl) phthalate metabolites, monobutyl phthalate, mono-isobutyl phthalate, and monoethyl phthalate (mEP) with ASD symptoms, adjusting for other behavior problems, using linear regression models (n=77). Results Most associations were null, and the sample size limited power to detect associations, particularly in the stratified analyses. After adjusting for internalizing and externalizing problems from the CBCL, ASD symptoms increased for each doubling of prenatal mEP concentration among boys only. Conclusions Further investigation of maternal prenatal urinary phthalate metabolite concentrations and ASD symptoms while adjusting for other behavioral problems is warranted.

Plant and animal life forms evolved mechanisms for sensing and responding to gravity on Earth where homeostatic needs require responses. The lack of gravity, such as in the International Space Station (ISS), causes acute, intra-generational changes in the quality of life. These include maintaining calcium levels in bone, maintaining muscle tone, and disturbances in the vestibular apparatus in the ears. These problems decrease work efficiency and quality of life of humans not only during microgravity exposures but also after return to higher gravity on Earth or destinations such as Mars or the Moon. It has been hypothesized that lack of gravity during mammalian development may cause prenatal, postnatal and transgenerational effects that conflict with the environment, especially if the developing organism and its progeny are returned, or introduced de novo, into the varied gravity environments mentioned above. Although chicken and frog pregastrulation development, and plant root development, have profound effects due to orientation of cues by gravity-sensing mechanisms and responses, mammalian development is not typically characterized as gravity-sensing. Although no effects of microgravity simulation (MGS) on mouse fertilization were observed in two reports, negative effects of MGS on early mammalian development after fertilization and before gastrulation are presented in four reports that vary with the modality of MGS. This review will analyze the positive and negative mammalian early developmental outcomes, and enzymatic and epigenetic mechanisms known to mediate developmental responses to simulated microgravity on Earth and microgravity during spaceflight experiments. We will update experimental techniques that have already been developed or need to be developed for zero gravity molecular, cellular, and developmental biology experiments.

Acquired hearing loss is caused by complex interactions of multiple environmental risk factors, such as elevated levels of lead and noise, which are prevalent in urban communities. This study delineates the mechanism underlying lead-induced auditory dysfunction and its potential interaction with noise exposure. Young-adult C57BL/6 mice were exposed to: 1) control conditions; 2) 2 mM lead acetate in drinking water for 28 days; 3) 90 dB broadband noise 2 h/day for two weeks; and 4) both lead and noise. Blood lead levels were measured by inductively coupled plasma mass spectrometry analysis (ICP-MS) lead-induced cochlear oxidative stress signaling was assessed using targeted gene arrays, and the hearing thresholds were assessed by recording auditory brainstem responses. Chronic lead exposure downregulated cochlear Sod1, Gpx1, and Gstk1, which encode critical antioxidant enzymes, and upregulated ApoE, Hspa1a, Ercc2, Prnp, Ccl5, and Sqstm1, which are indicative of cellular apoptosis. Isolated exposure to lead or noise induced 8–12 dB and 11–25 dB shifts in hearing thresholds, respectively. Combined exposure induced 18–30 dB shifts, which was significantly higher than that observed with isolated exposures. This study suggests that chronic exposure to lead induces cochlear oxidative stress and potentiates noise-induced hearing impairment, possibly through parallel pathways.

Exposure to violence (ETV) has been linked to epigenomics mechanisms such as DNA methylation (DNAm). We used epigenetic profiling of blood collected from 32 African American young adult males who lived in Washington DC to determine if changes in DNAm at CpG sites affiliated with nervous and immune system were associated with exposure to violence. Pathway analysis of differentially methylated regions comparing high and low ETV groups revealed an enrichment of gene sets annotated to nervous system and immune ontologies. Many of these genes are known to interact with each other which suggests DNAm alters gene function in the nervous and immune system in response to ETV. Using data from a unique age group, young African American adult males, we provide evidence that lifetime ETV could impact DNA methylation in genes impacted at Central Nervous System and Immune Function sites. Method Methylation analysis was performed on DNA collected from the blood of participants classified with either high or low lifetime ETV. Illumina®MethylationEPIC Beadchips (~850k CpG sites) were processed on the iScan System to examine whole-genome methylation differences. Differentially methylated CpG-sites between high (n ​= ​19) and low (n ​= ​13) groups were identified using linear regression with violence and substance abuse as model covariates. Gene ontology analysis was used to identify enrichment categories from probes annotated to the nearest gene. Results A total of 595 probes (279 hypermethylated; 316 hypomethylated) annotated to 383 genes were considered differentially methylated in association with ETV. Males with high ETV showed elevated methylation in several signaling pathways but were most impacted at Central Nervous System and Immune Function affiliated sites. Eight candidate genes were identified that play important biological roles in stress response to violence with HDAC4 (10%), NR4A3 (11%), NR4A2 (12%), DSCAML1(12%), and ELAVL3 (13%) exhibiting higher levels in the low ETV group and DLGAP1 (10%), SHANK2 (10%), and NRG1(11%) having increased methylation in the high ETV group. These findings suggest that individuals subjected to high ETV may be at risk for poor health outcomes that have not been reported previously., Highlights • Exposure to violence has been linked to DNA methylation. • Total of 595 probes annotated to 383 genes were differentially methylated. • Differential methylation in central nervous system and immune signaling pathway. • Methylation at NRG1 and NR4A2 possibly associated with stress response to violence.

Cardiovascular disease encompasses a wide range of conditions, resulting in the highest number of deaths worldwide. The underlying pathologies surrounding cardiovascular disease include a vast and complicated network of both cellular and molecular mechanisms. Unique phenotypic alterations in specific cell types, visualized as varying RNA expression-levels (both coding and non-coding), have been identified as crucial factors in the pathology underlying conditions such as heart failure and atherosclerosis. Recent advances in single-cell RNA sequencing (scRNA-seq) have elucidated a new realm of cell subpopulations and transcriptional variations that are associated with normal and pathological physiology in a wide variety of diseases. This breakthrough in the phenotypical understanding of our cells has brought novel insight into cardiovascular basic science. scRNA-seq allows for separation of widely distinct cell subpopulations which were, until recently, simply averaged together with bulk-tissue RNA-seq. scRNA-seq has been used to identify novel cell types in the heart and vasculature that could be implicated in a variety of disease pathologies. Furthermore, scRNA-seq has been able to identify significant heterogeneity of phenotypes within individual cell subtype populations. The ability to characterize single cells based on transcriptional phenotypes allows researchers the ability to map development of cells and identify changes in specific subpopulations due to diseases at a very high throughput. This review looks at recent scRNA-seq studies of various aspects of the cardiovascular system and discusses their potential value to our understanding of the cardiovascular system and pathology.

Preterm birth is the leading cause of infant morbidity and mortality. Necrotizing enterocolitis (NEC) is an inflammatory bowel disease affecting primarily premature infants, which can be lethal. Microbial intestinal colonization may alter epigenetic signatures of the immature gut establishing inflammatory and barrier properties predisposing to the development of NEC. We hypothesize that a crosstalk exists between the epigenome of the host and the initial intestinal colonizing microbiota at critical neonatal stages. By exposing immature enterocytes to probiotic and pathogenic bacteria, we showed over 200 regions of differential DNA modification, which were specific for each exposure. Reciprocally, using a mouse model of prenatal exposure to dexamethasone we demonstrated that antenatal treatment with glucocorticoids alters the epigenome of the host. We investigated the effects on the expression profiles of genes associated with inflammatory responses and intestinal barrier by qPCR-based gene expression array and verified the DNA modification changes in 5 candidate genes by quantitative methylation specific PCR (qMSP). Importantly, by 16S RNA sequencing-based phylogenetic analysis of intestinal bacteria in mice at 2 weeks of life, we showed that epigenome changes conditioned early microbiota colonization leading to differential bacterial colonization at different taxonomic levels. Our findings support a novel conceptual framework in which epigenetic changes induced by intrauterine influences affect early microbial colonization and intestinal development, which may alter disease susceptibility.

Volatile organic compounds (VOCs) are a group of aromatic or chlorinated organic chemicals commonly found in manufactured products that have high vapor pressure, and thus vaporize readily at room temperature. While airshed VOCs are well studied and have provided insights into public health issues, we suggest that belowground VOCs and the related vapor intrusion process could be equally or even more relevant to public health. The persistence, movement, remediation, and human health implications of subsurface VOCs in urban landscapes remain relatively understudied despite evidence of widespread contamination. This review explores the state of the science of subsurface movement and remediation of VOCs through groundwater and soils, the linkages between these poorly understood contaminant exposure pathways and health outcomes based on research in various animal models, and describes the role of these contaminants in human health, focusing on birth outcomes, notably low birth weight and preterm birth. Finally, this review provides recommendations for future research to address knowledge gaps that are essential for not only tackling health disparities and environmental injustice in post-industrial cities, but also protecting and preserving critical freshwater resources.

Lead exposure has long been one of the most important topics in global public health because it is a potent developmental neurotoxin. Here, an eQTL analysis, which is the genome-wide association analysis of genetic variants with gene expression, was performed. In this analysis, the male heads of 79 Drosophila melanogaster inbred lines from Drosophila Synthetic Population Resource (DSPR) were treated with or without developmental exposure, from hatching to adults, to 250 μM lead acetate [Pb(C2H3O2)2]. The goal was to identify genomic intervals that influence the gene-expression response to lead. After detecting 1798 cis-eQTLs and performing an initial trans-eQTL analysis, we focused our analysis on lead-sensitive “trans-eQTL hotspots,” defined as genomic regions that are associated with a cluster of genes in a lead-dependent manner. We noticed that the genes associated with one of the 14 detected trans-eQTL hotspots, Chr 2L: 6,250,000 could be roughly divided into two groups based on their differential expression profile patterns and different categories of function. This trans-eQTL hotspot validates one identified in a previous study using different recombinant inbred lines. The expression of all the associated genes in the trans-eQTL hotspot was visualized with hierarchical clustering analysis. Besides the overall expression profile patterns, the heatmap displayed the segregation of differential parental genetic contributions. This suggested that trans-regulatory regions with different genetic contributions from the parental lines have significantly different expression changes after lead exposure. We believe this study confirms our earlier study, and provides important insights to unravel the genetic variation in lead susceptibility in Drosophila model.

This study explores the use of human embryonic stem cells (hESCs) for assessing nanotoxicology, specifically, the effect of gold nanoparticles (AuNPs) of different core sizes (1.5 nm, 4 nm, and 14 nm) on the viability, pluripotency, neuronal differentiation, and DNA methylation of hESCs. The hESCs exposed to 1.5 nm thiolate-capped AuNPs exhibited loss of cohesiveness and detachment suggesting ongoing cell death at concentrations as low as 0.1 µg/mL. The cells exposed to 1.5 nm AuNPs at this concentration did not form embryoid bodies but rather disintegrated into single cells within 48 hours. Cell death caused by 1.5 nm AuNPs also occurred in hESC-derived neural progenitor cells. None of the other nanoparticles exhibited toxic effects on the hESCs at concentrations as high as 10 µg/mL during a 19 day neural differentiation period. Thiolate-capped 4 nm AuNPs at 10 µg/mL caused a dramatic decrease in global DNA methylation (5mC) and a corresponding increase in global DNA hydroxymethylation (5hmC) of the hESC’s DNA in only 24 hours. This work identifies a type of AuNPs highly toxic to hESCs and demonstrates the potential of hESCs in predicting nanotoxicity and characterizing their ability to alter the DNA methylation and hydroxymethylation patterns in the cells.

Traumatic brain injury (TBI) can cause persistent pathological alteration of neurons. This may lead to cognitive dysfunction, depression and increased susceptibility to life threatening diseases, such as epilepsy and Alzheimer's disease. To investigate the underlying genetic and molecular basis of TBI, we subjected w1118 Drosophila melanogaster to mild closed head trauma and found that mitochondrial activity is reduced in the brains of these flies 24 h after inflicting trauma. To determine the transcriptomic changes after mild TBI, we collected fly heads 24 h after inflicting trauma, and performed RNA-seq analyses. Classification of alternative splicing changes showed selective retention (RI) of long introns (>81 bps), with a mean size of ~3,000 nucleotides. Some of the genes containing RI showed a significant reduction in transcript abundance and are involved in mitochondrial metabolism such as Isocitrate dehydrogenase (Idh), which makes α-KG, a co-factor needed for both DNA and histone demethylase enzymes. The long introns are enriched in CA-rich motifs known to bind to Smooth (Sm), a heterogeneous nuclear ribonucleoprotein L (hnRNP-L) class of splicing factor, which has been shown to interact with the H3K36 histone methyltransferase, SET2, and to be involved in intron retention in human cells. H3K36me3 is a histone mark that demarcates exons in genes by interacting with the mRNA splicing machinery. Mutating sm (sm4/Df) resulted in loss of both basal and induced levels of RI in many of the same long-intron containing genes. Reducing the levels of Kdm4A, the H3K36me3 histone demethylase, also resulted in loss of basal levels of RI in many of the same long-intron containing genes. Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) for H3K36me3 revealed increased levels of this histone modification in retained introns post-trauma at CA-rich motifs. Based on these results, we propose a model in which TBI temporarily decreases mitochondrial activity in the brain 24 h after inflicting trauma, which decreases α-KG levels, and increases H3K36me3 levels and intron retention of long introns by decreasing Kdm4A activity. The consequent reduction in mature mRNA levels in metabolism genes, such as Idh, further reduces α-KG levels in a negative feedback loop. We further propose that decreasing metabolism after TBI in such a manner is a protective mechanism that gives the brain time to repair cellular damage induced by TBI.