SARS-CoV-2 Covid-19 Infection During Pregnancy and Differential DNA Methylation in Human Cord Blood Cells From Term Neonates.

Background: The global pandemic of coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). About 18.4% of total Covid-19 cases were reported in children. Even though vertical transmission from mother to infant is likely to occur at a low rate, exposure to COVID-19 during fetal life may alter DNA methylation patterns with potential long-term effects. Objective: To determine if COVID-19 infection during pregnancy alters the DNA methylation patterns in umbilical cord blood cells from term infants and to identify potential pathways and genes affected by exposure to COVID-19 infection. Methods: Umbilical cord blood was collected from 8 infants exposed to COVID-19 during pregnancy and 8 control infants with no COVID-19 exposure. Genomic DNA was isolated from umbilical cord blood cells and genome-wide DNA methylation was performed using Illumina Methylation EPIC Array. Results: 119 differentially methylated loci were identified at the FDR level of 0.20 (64 hypermethylated loci and 55 hypomethylated loci) in umbilical cord blood cells of COVID-19 exposed neonates compared to the control group. Important canonical pathways identified by Ingenuity Pathway Analysis (IPA) were related to stress response (corticotropin releasing hormone signaling, glucocorticoid receptor signaling, and oxytocin in brain signaling pathway), and cardiovascular disease and development (nitric oxide signaling in the cardiovascular system, apelin cardiomyocyte signaling pathways, factors promoting cardiogenesis, and renin-angiotensin signaling). The genes affected by the differential methylations were associated with cardiac, renal, hepatic, neurological diseases, developmental and immunological disorders. Conclusions: COVID-19 induces differential DNA methylation in umbilical cord blood cells. The differentially methylated genes may contribute to hepatic, renal, cardiac, developmental and immunological disorders in offspring born to mothers with COVID-19 infection during pregnancy, and their developmental regulation.

The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses.

Prostate cancer (PCa), the second leading cause of death in American men, includes distinct genetic subtypes with distinct therapeutic vulnerabilities. The DACH1 gene encodes a winged helix/Forkhead DNA-binding protein that competes for binding to FOXM1 sites. Herein, DACH1 gene deletion within the 13q21.31-q21.33 region occurs in up to 18% of human PCa and was associated with increased AR activity and poor prognosis. In prostate OncoMice, prostate-specific deletion of the Dach1 gene enhanced prostatic intraepithelial neoplasia (PIN), and was associated with increased TGFß activity and DNA damage. Reduced Dach1 increased DNA damage in response to genotoxic stresses. DACH1 was recruited to sites of DNA damage, augmenting recruitment of Ku70/Ku80. Reduced Dach1 expression was associated with increased homology directed repair and resistance to PARP inhibitors and TGFß kinase inhibitors. Reduced Dach1 expression may define a subclass of PCa that warrants specific therapies.

The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses.

Prostate cancer (PCa), the second leading cause of death in American men, includes distinct genetic subtypes with distinct therapeutic vulnerabilities. The DACH1 gene encodes a winged helix/Forkhead DNA-binding protein that competes for binding to FOXM1 sites. Herein, DACH1 gene deletion within the 13q21.31-q21.33 region occurs in up to 18% of human PCa and was associated with increased AR activity and poor prognosis. In prostate OncoMice, prostate-specific deletion of the Dach1 gene enhanced prostatic intraepithelial neoplasia (PIN), and was associated with increased TGFb activity and DNA damage. Reduced Dach1 increased DNA damage in response to genotoxic stresses. DACH1 was recruited to sites of DNA damage, augmenting recruitment of Ku70/Ku80. Reduced Dach1 expression was associated with increased homology directed repair and resistance to PARP inhibitors and TGFb kinase inhibitors. Reduced Dach1 expression may define a subclass of PCa that warrants specific therapies.

Targeted Inhibition of Select Extracellular Signal-regulated Kinases 1 and 2 Functions Mitigates Pathological Features of Asthma in Mice.

ERK1/2 (extracellular signal-regulated kinases 1 and 2) regulate the activity of various transcription factors that contribute to asthma pathogenesis. Although an attractive drug target, broadly inhibiting ERK1/2 is challenging because of unwanted cellular toxicities. We have identified small molecule inhibitors with a benzenesulfonate scaffold that selectively inhibit ERK1/2-mediated activation of AP-1 (activator protein-1). Herein, we describe the findings of targeting ERK1/2-mediated substrate-specific signaling with the small molecule inhibitor SF-3-030 in a murine model of house dust mite (HDM)-induced asthma. In 8- to 10-week-old BALB/c mice, allergic asthma was established by repeated intranasal HDM (25 µg/mouse) instillation for 3 weeks (5 days/week). A subgroup of mice was prophylactically dosed with 10 mg/kg SF-3-030/DMSO intranasally 30 minutes before the HDM challenge. Following the dosing schedule, mice were evaluated for alterations in airway mechanics, inflammation, and markers of airway remodeling. SF-3-030 treatment significantly attenuated HDM-induced elevation of distinct inflammatory cell types and cytokine concentrations in BAL and IgE concentrations in the lungs. Histopathological analysis of lung tissue sections revealed diminished HDM-induced pleocellular peribronchial inflammation, mucus cell metaplasia, collagen accumulation, thickening of airway smooth muscle mass, and expression of markers of cell proliferation (Ki-67 and cyclin D1) in mice treated with SF-3-030. Furthermore, SF-3-030 treatment attenuated HDM-induced airway hyperresponsiveness in mice. Finally, mechanistic studies using transcriptome and proteome analyses suggest inhibition of HDM-induced genes involved in inflammation, cell proliferation, and tissue remodeling by SF-3-030. These preclinical findings demonstrate that function-selective inhibition of ERK1/2 signaling mitigates multiple features of asthma in a murine model.

Astronauts Plasma-Derived Exosomes Induced Aberrant EZH2-Mediated H3K27me3 Epigenetic Regulation of the Vitamin D Receptor.

There are unique stressors in the spaceflight environment. Exposure to such stressors may be associated with adverse effects on astronauts' health, including increased cancer and cardiovascular disease risks. Small extracellular vesicles (sEVs, i.e., exosomes) play a vital role in intercellular communication and regulate various biological processes contributing to their role in disease pathogenesis. To assess whether spaceflight alters sEVs transcriptome profile, sEVs were isolated from the blood plasma of 3 astronauts at two different time points: 10 days before launch (L-10) and 3 days after return (R+3) from the Shuttle mission. AC16 cells (human cardiomyocyte cell line) were treated with L-10 and R+3 astronauts-derived exosomes for 24 h. Total RNA was isolated and analyzed for gene expression profiling using Affymetrix microarrays. Enrichment analysis was performed using Enrichr. Transcription factor (TF) enrichment analysis using the ENCODE/ChEA Consensus TF database identified gene sets related to the polycomb repressive complex 2 (PRC2) and Vitamin D receptor (VDR) in AC16 cells treated with R+3 compared to cells treated with L-10 astronauts-derived exosomes. Further analysis of the histone modifications using datasets from the Roadmap Epigenomics Project confirmed enrichment in gene sets related to the H3K27me3 repressive mark. Interestingly, analysis of previously published H3K27me3-chromatin immunoprecipitation sequencing (ChIP-Seq) ENCODE datasets showed enrichment of H3K27me3 in the VDR promoter. Collectively, our results suggest that astronaut-derived sEVs may epigenetically repress the expression of the VDR in human adult cardiomyocytes by promoting the activation of the PRC2 complex and H3K27me3 levels.

COVID-19 Infection During Pregnancy Induces Differential Gene Expression in Human Cord Blood Cells From Term Neonates.

Background: The COVID-19 pandemic continues worldwide with fluctuating case numbers in the United States. This pandemic has affected every segment of the population with more recent hospitalizations in the pediatric population. Vertical transmission of COVID-19 is uncommon, but reports show that there are thrombotic, vascular, and inflammatory changes in the placenta to which neonates are prenatally exposed. Individuals exposed in utero to influenza during the 1918 pandemic had increased risk for heart disease, kidney disease, diabetes, stomach disease and hypertension. Early exposure of COVID-19 during fetal life may lead to altered gene expression with potential long-term consequences. Objective: To determine if gene expression is altered in cord blood cells from term neonates who were exposed to COVID-19 during pregnancy and to identify potential gene pathways impacted by maternal COVID-19. Methods: Cord blood was collected from 16 term neonates (8 exposed to COVID-19 during pregnancy and 8 controls without exposure to COVID-19). Genome-wide gene expression screening was performed using Human Clariom S gene chips on total RNA extracted from cord blood cells. Results: We identified 510 differentially expressed genes (374 genes up-regulated, 136 genes down-regulated, fold change ≥1.5, p-value ≤ 0.05) in cord blood cells associated with exposure to COVID-19 during pregnancy. Ingenuity Pathway Analysis identified important canonical pathways associated with diseases such as cardiovascular disease, hematological disease, embryonic cancer and cellular development. Tox functions related to cardiotoxicity, hepatotoxicity and nephrotoxicity were also altered after exposure to COVID-19 during pregnancy. Conclusions: Exposure to COVID-19 during pregnancy induces differential gene expression in cord blood cells. The differentially expressed genes may potentially contribute to cardiac, hepatic, renal and immunological disorders in offspring exposed to COVID-19 during pregnancy. These findings lead to a further understanding of the effects of COVID-19 exposure at an early stage of life and its potential long-term consequences as well as therapeutic targets.

HuR Plays a Role in Double-Strand Break Repair in Pancreatic Cancer Cells and Regulates Functional BRCA1-Associated-Ring-Domain-1(BARD1) Isoforms.

Human Antigen R (HuR/ELAVL1) is known to regulate stability of mRNAs involved in pancreatic ductal adenocarcinoma (PDAC) cell survival. Although several HuR targets are established, it is likely that many remain currently unknown. Here, we identified BARD1 mRNA as a novel target of HuR. Silencing HuR caused a >70% decrease in homologous recombination repair (HRR) efficiency as measured by the double-strand break repair (pDR-GFP reporter) assay. HuR-bound mRNAs extracted from RNP-immunoprecipitation and probed on a microarray, revealed a subset of HRR genes as putative HuR targets, including the BRCA1-Associated-Ring-Domain-1 (BARD1) (p < 0.005). BARD1 genetic alterations are infrequent in PDAC, and its context-dependent upregulation is poorly understood. Genetic silencing (siRNA and CRISPR knock-out) and pharmacological targeting of HuR inhibited both full length (FL) BARD1 and its functional isoforms (α, δ, Φ). Silencing BARD1 sensitized cells to olaparib and oxaliplatin; caused G2-M cell cycle arrest; and increased DNA-damage while decreasing HRR efficiency in cells. Exogenous overexpression of BARD1 in HuR-deficient cells partially rescued the HRR dysfunction, independent of an HuR pro-oncogenic function. Collectively, our findings demonstrate for the first time that BARD1 is a bona fide HuR target, which serves as an important regulatory point of the transient DNA-repair response in PDAC cells.

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in murine ß-coronavirus-induced neuroinflammation.

Mouse hepatitis virus (MHV; m-ß-CoV) serves as a useful model for studying the cellular factors involved in neuroinflammation. To understand the role of matrix metalloproteinases (MMPs) in neuroinflammation, brain tissues from m-ß-CoV-infected mice were harvested at different days post-infection (d.p.i) and investigated for Mmp expression by RT-qPCR. Mmp-2, -3, -8, -12 showed significant mRNA upregulation peaking with viral replication between 5 and 6 d.p.i. Elevated levels of MMP regulator TIMP-1 are suggestive of a TIMP-1 mediated host antiviral response. Biological network assessment suggested a direct involvement of MMP-3, -8, -14 in facilitating peripheral leukocyte infiltrations. Flow cytometry confirmed the increased presence of NK cells, CD4+ and CD8+ T cells, neutrophils, and MHCII expressing cells in the m-ß-CoV infected mice brain. Our study revealed that m-ß-CoV upregulated Park7, RelA, Nrf2, and Hmox1 transcripts involved in ROS production and antioxidant pathways, describing the possible nexus between oxidative pathways, MMPs, and TIMP in m-ß-CoV-induced neuroinflammation.

Global gene expression analysis of systemic sclerosis myofibroblasts demonstrates a marked increase in the expression of multiple NBPF genes.

Myofibroblasts are the key effector cells responsible for the exaggerated tissue fibrosis in Systemic Sclerosis (SSc). Despite their importance to SSc pathogenesis, the specific transcriptome of SSc myofibroblasts has not been described. The purpose of this study was to identify transcriptome differences between SSc myofibroblasts and non-myofibroblastic cells. Alpha smooth muscle actin (α-SMA) expressing myofibroblasts and α-SMA negative cells were isolated employing laser capture microdissection from dermal cell cultures from four patients with diffuse SSc of recent onset. Total mRNA was extracted from both cell populations, amplified and analyzed employing microarrays. Results for specific genes were validated by Western blots and by immunohistochemistry. Transcriptome analysis revealed 97 differentially expressed transcripts in SSc myofibroblasts compared with non-myofibroblasts. Annotation clustering of the SSc myofibroblast-specific transcripts failed to show a TGF-ß signature. The most represented transcripts corresponded to several different genes from the Neuroblastoma Breakpoint Family (NBPF) of genes. NBPF genes are highly expanded in humans but are not present in murine or rat genomes. In vitro studies employing cultured SSc dermal fibroblasts and immunohistochemistry of affected SSc skin confirmed increased NBPF expression in SSc. These results indicate that SSc myofibroblasts represent a unique cell lineage expressing a specific transcriptome that includes very high levels of transcripts corresponding to numerous NBPF genes. Elevated expression of NBPF genes in SSc myofibroblasts suggests that NBPF gene products may play a role in SSc pathogenesis and may represent a novel therapeutic target.

Potential Immunomodulatory Properties of Biologically Active Components of Spices Against SARS-CoV-2 and Pan ß-Coronaviruses.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced COVID-19 has emerged as a defining global health crisis in current times. Data from the World Health Organization shows demographic variations in COVID-19 severity and lethality. Diet may play a significant role in providing beneficial host cell factors contributing to immunity against deadly SARS-CoV-2 pathogenesis. Spices are essential components of the diet that possess anti-inflammatory, antioxidant, and antiviral properties. Hyperinflammation, an aberrant systemic inflammation associated with pneumonia, acute respiratory failure, and multiorgan dysfunction, is a major clinical outcome in COVID-19. Knowing the beneficial properties of spices, we hypothesize that spice-derived bioactive components can modulate host immune responses to provide protective immunity in COVID-19. This study emphasizes that biologically active components of spices might alleviate the sustained pro-inflammatory condition by inhibiting the activity of tumor necrosis factor-alpha (TNF-α), interleukins (IL6, IL8), and chemokine (CCL2) known to be elevated in COVID-19. Spices may potentially prevent the tissue damage induced by oxidative stress and pro-inflammatory mediators during SARS-CoV-2 infection. The current study also highlights the effects of spices on the antioxidant pathways mediated by Nrf2 (nuclear factor erythroid 2-related factor 2) and Hmox1 (heme oxygenase 1) to restore oxidative homeostasis and protect from aberrant tissue damage. Taken together, the anti-inflammatory and antioxidant activities of bioactive components of spices may hold a promise to target the cellular pathways for developing antivirals against SARS-CoV-2 and pan ß-coronaviruses.

Lack of Interferon (IFN) Regulatory Factor 8 Associated with Restricted IFN-γ Response Augmented Japanese Encephalitis Virus Replication in the Mouse Brain.

Interferon regulatory factor 8 (IRF8), a myeloid lineage transcription factor, emerges as an essential regulator for microglial activation. However, the precise role of IRF8 during Japanese encephalitis virus (JEV) infection in the brain remains elusive. Here, we report that JEV infection enhances IRF8 expression in the infected mouse brain. Comparative transcriptional profiling of whole-brain RNA analysis and validation by quantitative reverse transcription-PCR (qRT-PCR) reveals an impaired interferon gamma (IFN-γ) and related gene expression in Irf8 knockout (Irf8-/-)-infected mice. Further, Ifnγ knockout (Ifnγ-/-) mice exhibit a reduced level of Irf8. Both Ifnγ-/- and Irf8-/- mice exhibit significantly reduced levels of activated (CD11b+ CD45hi, CD11b+ CD45lo, Cd68, and CD86) and infiltrating immune cells (Ly6C+, CD4, and CD8) in the infected brain compared to those of wild-type (WT) mice. However, a higher level of granulocyte cell (Ly6G+) infiltration is evident in Irf8-/- mice as well as the increased concentration of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant protein 1 (MCP1) levels in the brain. Interestingly, neither the Irf8-/- nor the Ifnγ-/- conferred protection against lethal JEV challenge to mice and exhibit augmentation in JEV replication in the brain. The gain of function of Irf8 by overexpressing functional IRF8 in an IRF8-deficient cell line attenuates viral replication and enhances IFN-γ production. Overall, we summarize that in the murine model of JEV encephalitis, IRF8 modulation affects JEV replication. We also show that lack of Irf8 affects immune cell abundance in circulation and the infected brain, leading to a reduction in IFN-γ level and increased viral load in the brain. IMPORTANCE Microglial cells, the resident macrophages in the brain, play a vital role in Japanese encephalitis virus (JEV) pathogenesis. The deregulated activity of microglia can be lethal for the brain. Therefore, it is crucial to understand the regulators that drive microglia phenotype changes and induce inflammation in the brain. Interferon regulatory factor 8 (IRF8) is a myeloid lineage transcription factor involved in microglial activation. However, the impact of IRF8 modulation on JEV replication remains elusive. Moreover, the pathways regulated by IRF8 to initiate and amplify pathological neuroinflammation are not well understood. Here, we demonstrated the effect of IRF8 modulation on JEV replication, microglial activation, and immune cells infiltration in the brain.

Long-Term Effects of Very Low Dose Particle Radiation on Gene Expression in the Heart: Degenerative Disease Risks.

Compared to low doses of gamma irradiation (γ-IR), high-charge-and-energy (HZE) particle IR may have different biological response thresholds in cardiac tissue at lower doses, and these effects may be IR type and dose dependent. Three- to four-month-old female CB6F1/Hsd mice were exposed once to one of four different doses of the following types of radiation: γ-IR 137Cs (40-160 cGy, 0.662 MeV), 14Si-IR (4-32 cGy, 260 MeV/n), or 22Ti-IR (3-26 cGy, 1 GeV/n). At 16 months post-exposure, animals were sacrificed and hearts were harvested and archived as part of the NASA Space Radiation Tissue Sharing Forum. These heart tissue samples were used in our study for RNA isolation and microarray hybridization. Functional annotation of twofold up/down differentially expressed genes (DEGs) and bioinformatics analyses revealed the following: (i) there were no clear lower IR thresholds for HZE- or γ-IR; (ii) there were 12 common DEGs across all 3 IR types; (iii) these 12 overlapping genes predicted various degrees of cardiovascular, pulmonary, and metabolic diseases, cancer, and aging; and (iv) these 12 genes revealed an exclusive non-linear DEG pattern in 14Si- and 22Ti-IR-exposed hearts, whereas two-thirds of γ-IR-exposed hearts revealed a linear pattern of DEGs. Thus, our study may provide experimental evidence of excess relative risk (ERR) quantification of low/very low doses of full-body space-type IR-associated degenerative disease development.

miR-21 Plays a Dual Role in Tumor Formation and Cytotoxic Response in Breast Tumors.

Breast cancer (BrCa) relies on specific microRNAs to drive disease progression. Oncogenic miR-21 is upregulated in many cancers, including BrCa, and is associated with poor survival and treatment resistance. We sought to determine the role of miR-21 in BrCa tumor initiation, progression and treatment response. In a triple-negative BrCa model, radiation exposure increased miR-21 in both primary tumor and metastases. In vitro, miR-21 knockdown decreased survival in all BrCa subtypes in the presence of radiation. The role of miR-21 in BrCa initiation was evaluated by implanting wild-type miR-21 BrCa cells into genetically engineered mouse models where miR-21 was intact, heterozygous or globally ablated. Tumors were unable to grow in the mammary fat pads of miR-21-/- mice, and grew in ~50% of miR-21+/- and 100% in miR-21+/+ mice. The contribution of miR-21 to progression and metastases was tested by crossing miR-21-/- mice with mice that spontaneously develop BrCa. The global ablation of miR-21 significantly decreased the tumorigenesis and metastases of BrCa, while sensitizing tumors to radio- and chemotherapeutic agents via Fas/FasL-dependent apoptosis. Therefore, targeting miR-21 alone or in combination with various radio or cytotoxic therapies may represent novel and efficacious therapeutic modalities for the future treatment of BrCa patients.

Pulmonary immune cell transcriptome changes in double-hit model of BPD induced by chorioamnionitis and postnatal hyperoxia.

BACKGROUND: Preterm infants with bronchopulmonary dysplasia (BPD) have lifelong increased risk of respiratory morbidities associated with environmental pathogen exposure and underlying mechanisms are poorly understood. The resident immune cells of the lung play vital roles in host defense. However, the effect of perinatal events associated with BPD on pulmonary-specific immune cells is not well understood. METHODS: We used a double-hit model of BPD induced by prenatal chorioamnionitis followed by postnatal hyperoxia, and performed a global transcriptome analysis of all resident pulmonary immune cells. RESULTS: We show significant up-regulation of genes involved in chemokine-mediated signaling and immune cell chemotaxis, and down-regulation of genes involved in multiple T lymphocyte functions. Multiple genes involved in T cell receptor signaling are downregulated and Cd8a gene expression remains downregulated at 2 months of age in spite of recovery in normoxia for 6 weeks. Furthermore, the proportion of CD8a+CD3+ pulmonary immune cells is decreased. CONCLUSIONS: Our study has highlighted that perinatal lung inflammation in a double-hit model of BPD results in short- and long-term dysregulation of genes associated with the pulmonary T cell receptor signaling pathway, which may contribute to increased environmental pathogen-associated respiratory morbidities seen in children and adults with BPD. IMPACT: In a translationally relevant double-hit model of BPD induced by chorioamnionitis and postnatal hyperoxia, we identified pulmonary immune cell-specific transcriptomic changes and showed that T cell receptor signaling genes are downregulated in short term and long term. This is the first comprehensive report delineating transcriptomic changes in resident immune cells of the lung in a translationally relevant double-hit model of BPD. Our study identifies novel resident pulmonary immune cell-specific targets for potential therapeutic modulation to improve short- and long-term respiratory health of preterm infants with BPD.

Xist attenuates acute inflammatory response by female cells.

Biological sex influences inflammatory response, as there is a greater incidence of acute inflammation in men and chronic inflammation in women. Here, we report that acute inflammation is attenuated by X-inactive specific transcript (Xist), a female cell-specific nuclear long noncoding RNA crucial for X-chromosome inactivation. Lipopolysaccharide-mediated acute inflammation increased Xist levels in the cytoplasm of female mouse J774A.1 macrophages and human AML193 monocytes. In both cell types, cytoplasmic Xist colocalizes with the p65 subunit of NF-κB. This interaction was associated with reduced NF-κB nuclear migration, suggesting a novel mechanism to suppress acute inflammation. Further supporting this hypothesis, expression of 5' XIST in male cells significantly reduced IL-6 and NF-κB activity. Adoptive transfer of male splenocytes expressing Xist reduced acute paw swelling in male mice indicating that Xist can have a protective anti-inflammatory effect. These findings show that XIST has functions beyond X chromosome inactivation and suggest that XIST can contribute to sex-specific differences underlying inflammatory response by attenuating acute inflammation in women.

Increased expression of interferon regulated and antiviral response genes in CD31+/CD102+ lung microvascular endothelial cells from systemic sclerosis patients with end-stage interstitial lung disease.

OBJECTIVES: Systemic sclerosis (SSc) is characterised by severe fibroproliferative vasculopathy, fibrosis in skin and multiple internal organs, and humoral, cellular and innate immunity abnormalities. Vascular alterations are the earliest and most severe SSc manifestations, however, the mechanisms responsible have remained elusive. To investigate the molecular abnormalities involved in SSc-vasculopathy we examined global gene expression differences between highly purified lung microvascular endothelial cells (MVECs) from patients with SSc-interstitial lung disease (SSc-ILD) and normal lung MVECs. METHODS: MVECs were isolated from fresh transplanted lungs from patients with SSc-ILD. Sequential CD31 and CD102 immunopurification was performed to obtain highly purified CD31+/CD102+ lung MVECs. Global gene expression analysis was successfully performed in CD31+/CD102+ MVEC from two SSc-ILD patients and from two normal lungs. RT-PCR, Western blots, and indirect immunofluorescence validated the gene expression results. RESULTS: Numerous interferon-regulated genes (IRGs) including IFI44, IFI44L, IFI6, IFIH1, IFIT1, ISG-15, BST-2/Tetherin, and RSAD2/Viperin, genes encoding innate immunity antiviral responses (OAS1, OAS2, OAS3, OASL) and antiviral MX1 and MX2 proteins, and mesenchymal cell-specific genes were significantly overexpressed in CD31+/CD102+ SSc-ILD lung MVECs. CONCLUSIONS: Highly purified CD31+/CD102+ MVECs from lungs from SSc patients with end stage SSc-ILD displayed remarkable overexpression of numerous IRGs and of genes encoding antiviral innate immune response and antiviral proteins. These observations suggest that interferon-induced and antiviral response proteins may participate in the pathogenesis of SSc vasculopathy and SSc-ILD. The CD31+/CD102+ lung MVECs from SSc-ILD also showed elevated expression of mesenchymal cell-specific genes confirming the presence of endothelial to mesenchymal transition in SSc-ILD.

Integrative genomics and pathway analysis identified prevalent FA-BRCA pathway alterations in arsenic-associated urinary bladder carcinoma: Chronic arsenic accumulation in cancer tissues hampers the FA-BRCA pathway.

Arsenic in drinking water is one of the major etiological factors in urinary bladder carcinoma (BlCa). Here, high-resolution CGH-SNP microarray analysis in arsenic accumulated BlCa tissues showed significant (p < 0.05) association of chromosomal alterations with high arsenic (≥112 ng/g) accumulation, further corroborated by high γH2AX nuclear expression. Cytobands 5q11-35, 9p24.3-21.5, 18q11.1-25, etc. showed deletion, whereas 12q was amplified in high arsenic samples (AsH). Consecutively, IPA® found FA-BRCA pathway to be exclusively altered in AsH group. Validation of several key regulatory genes (RAD50, BRIP1, UIMC1, FANCD2, BRCA2 and BRCA1) of the pathway, were performed in independent BlCa cases (n = 81). UIMC1, RAD50 and BRIP1 were differentially deleted and associated with poor survival of AsH samples. Moreover, reduced nuclear expression with diffused cytoplasmic expression of FANCD2 was higher in AsH samples. Collectively, frequent deregulation of RAD50, UIMC1 and BRIP1 may result in reduced nuclear translocation of FANCD2, which may cause more chromosomal aberrations among AsH samples.

DNA Methylation Profile in Human Cord Blood Mononuclear Leukocytes From Term Neonates: Effects of Histological Chorioamnionitis.

Background: Histological chorioamnionitis (HCA) is an infection/inflammation of fetal membranes and complicates 5.2-28.5% of all live births. Exposure to HCA can have long-term consequences including abnormal neurodevelopment and an increased risk for allergic disorders and asthma later in childhood. HCA may incite epigenetic changes, which have the potential to modulate both the immune and neurological systems as well as increase the risk of related disorders later in life. However, there is limited data on the impact of HCA on epigenetics, in particular DNA methylation, and changes to immune and neurological systems in full-term human neonates. Objective: To determine differential DNA methylation in cord blood mononuclear leukocytes from neonates exposed to HCA. Methods: Cord blood was collected from 10 term neonates (5 with HCA and 5 controls without HCA) and mononuclear leukocytes were isolated. Genome-wide DNA methylation screening was performed on Genomic DNA extracted from mononuclear leukocytes. Results: Mononuclear leukocytes from cord blood of HCA-exposed neonates showed differential DNA methylation of 68 probe sets compared to the control group (44 hypermethylated, 24 hypomethylated) with a p ≤ 0.0001. Several genes involved in immune modulation and nervous system development were found to be differentially methylated. Important canonical pathways as revealed by Ingenuity Pathway Analysis (IPA) were CREB Signaling in Neurons, FcγRIIB Signaling in B Lymphocytes, Cell Cycle: G1/S Checkpoint Regulation, Interleukin-1, 2, 3, 6, 8, 10, 17, and 17A signaling, p53 signaling, dopamine degradation, and serotonin degradation. The diseases and disorders picked up by IPA were nervous system development and function, neurological disease, respiratory disease, immune cell trafficking, inflammatory response, and immunological disease. Conclusions: HCA induces differential DNA methylation in cord blood mononuclear leukocytes. The differentially methylated genes may contribute to inflammatory, immunological and neurodevelopmental disorders in neonates exposed to HCA.

Comparison of inbred mouse strains shows diverse phenotypic outcomes of intervertebral disc aging.

Intervertebral disc degeneration presents a wide spectrum of clinically degenerative disc phenotypes; however, the contribution of genetic background to the degenerative outcomes has not been established. We characterized the spinal phenotype of 3 mouse strains with varying cartilage-regenerative potential at 6 and 23 months: C57BL/6, LG/J and SM/J. All strains showed different aging phenotypes. Importantly, LG/J mice showed an increased prevalence of dystrophic disc calcification in caudal discs with aging. Quantitative-histological analyses of LG/J and SM/J caudal discs evidenced accelerated degeneration compared to BL6, with cellular disorganization and cell loss together with fibrosis of the NP, respectively. Along with the higher grades of disc degeneration, SM/J, at 6M, also differed the most in terms of NP gene expression compared to other strains. Moreover, although we found common DEGs between BL6 and LG/J aging, most of them were divergent between the strains. Noteworthy, the common DEGs altered in both LG/J and BL6 aging were associated with inflammatory processes, response to stress, cell differentiation, cell metabolism and cell division. Results suggested that disc calcification in LG/J resulted from a dystrophic calcification process likely aggravated by cell death, matrix remodelling, changes in calcium/phosphate homeostasis and cell transformation. Lastly, we report 7 distinct phenotypes of human disc degeneration based on transcriptomic profiles, that presented similar pathways and DEGs found in aging mouse strains. Together, our results suggest that disc aging and degeneration depends on the genetic background and involves changes in various molecular pathways, which might help to explain the diverse phenotypes seen during disc disease.