Identification of global transcriptional variations in human peripheral blood mononuclear cells two months postheat injury helps categorization heat-tolerant or heat-intolerant phenotypes.

Thermal intolerance may limit activity in hostile environments. After heat illness, two physiologically distinct phenotypes evolve: heat tolerant (HT) and heat intolerant (HI). The recognition that heat illness alters gene expression justified revisiting the established physiological concept of HI. We used a DNA microarray to examine the global transcriptional response in peripheral blood mononuclear cells (PMBCs) from HI and HT phenotypes, categorized 2-mo postheat injury using a functional physiological heat-tolerance test (HTT, 40°C)-Recovery (R, 24°C) protocol. The impact of recurrent heat stress was studied in vitro using peripheral blood mononuclear cells (PBMCs) from controls (participants with no history of heat injury), HI, and HT (categorized by functional HTT) with a customized NanoString array. There were significant differences under basal conditions between the HI and HT. HI were more immunological alerted. Almost no shared genes were found between end-HTT and recovery phases, suggesting vast cellular plasticity. In HI, mitochondrial function was dysregulated, canonical pathways associated with exercise endurance-NRF2 and insulin were downregulated, whereas AMPK and peroxisome proliferator-activated receptor (PPAR) were upregulated. HT exhibited reciprocal responses, suggesting that energy dysregulation found in HI interfered with performance in the heat. The endoplasmic-reticulum stress response was also suppressed in HI. In vitro HTT (43°C) abolished differences between HI and HT PBMCs including the HSPs genes, whereas controls showed profound HSPs upregulation.

APOBEC3G protects the genome of human cultured cells and mice from radiation-induced damage.

Cytosine deaminases AID/APOBEC proteins act as potent nucleic acid editors, playing important roles in innate and adaptive immunity. However, the mutagenic effects of some of these proteins compromise genomic integrity and may promote tumorigenesis. Here, we demonstrate that human APOBEC3G (A3G), in addition to its role in innate immunity, promotes repair of double-strand breaks (DSBs) in vitro and in vivo. Transgenic mice expressing A3G successfully survived lethal irradiation, whereas wild-type controls quickly succumbed to radiation syndrome. Mass spectrometric analyses identified the differential upregulation of a plethora of proteins involved in DSB repair pathways in A3G-expressing cells early following irradiation to facilitate repair. Importantly, we find that A3G not only accelerates DSB repair but also promotes deamination-dependent error-free rejoining. These findings have two implications: (a) strategies aimed at inhibiting A3G may improve the efficacy of genotoxic therapies used to cure malignant tumours; and (b) enhancing A3G activity may reduce acute radiation syndrome in individuals exposed to ionizing radiation.

Laminin111-based defined culture promoting self-renewing human pluripotent stem cells with properties of the early post-implantation epiblast.

In the mammalian embryo, a formative pluripotent phase is proposed to exist at the early post-implantation period, during the transition from the pre-implantation naive-to the post-implantation primed-epiblast. By recapitulating a laminin component of the extracellular matrix niche during embryonic formative transition, and defined culture conditions, we generated cultures highly enriched for self-renewing human pluripotent stem cells (hPSCs), exhibiting properties of early post-implantation epiblast cells. These hPSCs display post-implantation-epiblast gene expression profiles. FGF and TGF-ß signaling maintain their self-renewal for multiple passages. They have inactive canonical Wnt signaling, do not express primitive streak markers, and are competent to initiate differentiation toward germline and somatic fates. hPSCs exhibiting early post-implantation epiblast properties may shed light on human embryonic PSCs development and may serve for initiating somatic and germ cell specification.

Splice-Variant Knock-Out of TGFß Receptors Perturbates the Proteome of Ovarian Carcinoma Cells.

The aim of this study was to analyze the biological role of different transforming growth factor-ß (TGFß) receptor splice variants in ovarian carcinoma (OC). Specific receptor variant knockouts (KO) were prepared using the CRISPR/Cas9 genome editing system in two OC cell lines, TßRI variant 1 (TßRIv1) KO in ES-2 cells and TßRII variant 1 (TßRIIv1) KO in OVCAR-8 cells. Control and KO cells were compared by proteomic analysis, functional tests, analysis of epithelial-mesenchymal transition (EMT) drivers, and Western blot of signaling proteins. Proteomic analysis revealed significant changes in protein pathways in the KO cells. TßRIv1 KO resulted in a significant reduction in both cellular motility and invasion, while TßRIIv1 KO significantly reduced cellular motility and increased Reactive Oxygen Species (ROS) production. Both receptor variant KOs reduced MET protein levels. Of the EMT drivers, a significant decrease in TWIST protein expression, and increase in SNAIL protein and MALAT1 mRNA levels were observed in the TßRIIv1 KO compared to control. A significant decrease in JNK1 and JNK2 activation was found in the TßRIv1 KO compared to control cells. These findings provide new insight regarding the biological role of the TGFß receptor variants in the biology and potentially the progression of OC.

The Role Played by Transcription Factor E3 in Modulating Cardiac Hypertrophy.

Transcription factor E3 (TFE3), which is a key regulator of cellular adaptation, is expressed in most tissues, including the heart, and is reportedly overexpressed during cardiac hypertrophy. In this study, TFE3's role in cardiac hypertrophy was investigated. To understand TFE3's physiological importance in cardiac hypertrophy, pressure-overload cardiac hypertrophy was induced through transverse aortic constriction (TAC) in both wild-type (WT) and TFE3 knockout mice (TFE3-/-). Eleven weeks after TAC induction, cardiac hypertrophy was observed in both WT and TFE3-/- mice. However, significant reductions in ejection fraction and fractional shortening were observed in WT mice compared to TFE3-/- mice. To understand the mechanism, we found that myosin heavy chain (Myh7), which increases during hemodynamic overload, was lower in TFE3-/- TAC mice than in WT TAC mice, whereas extracellular signal-regulated protein kinases (ERK) phosphorylation, which confers cardioprotection, was lower in the left ventricles of WT mice than in TFE3-/- mice. We also found high expressions of TFE3, histone, and MYH7 and low expression of pERK in the normal human heart compared to the hypertensive heart. In the H9c2 cell line, we found that ERK inhibition caused TFE3 nuclear localization. In addition, we found that MYH7 was associated with TFE3, and during TFE3 knockdown, MYH7 and histone were downregulated. Therefore, we showed that TFE3 expression was increased in the mouse model of cardiac hypertrophy and tissues from human hypertensive hearts, whereas pERK was decreased reversibly, which suggested that TFE3 is involved in cardiac hypertrophy through TFE3-histone-MYH7-pERK signaling.

APOBEC3G rescues cells from the deleterious effects of DNA damage.

Human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G (hA3G), a member of the APOBEC family, was described as an anti-HIV-1 restriction factor, deaminating reverse transcripts of the HIV-1 genome. Several types of cancer cells that express high levels of A3G, such as diffuse large B-cell lymphoma cells and glioblastomas, show enhanced cell survival after ionizing radiation and chemotherapy treatments. Previously, we showed that hA3G promotes (DNA) double-strand breaks repair in cultured cells and rescues transgenic mice from a lethal dose of ionizing radiation. Here, we show that A3G rescues cells from the detrimental effects of DNA damage induced by ultraviolet irradiation and by combined bromodeoxyuridine and ultraviolet treatments. The combined treatments stimulate the synthesis of cellular proteins, which are exclusively associated with A3G expression. These proteins participate mainly in nucleotide excision repair and homologous recombination DNA repair pathways. Our results implicate A3G inhibition as a potential strategy for increasing tumor cell sensitivity to genotoxic treatments.

Differential functions of TLE1 and TLE3 depending on a specific phosphorylation site.

Mammalian Transducin-like enhancer of split (TLE) confer global repression of numerous target genes in conjunction with a myriad of DNA-binding repressors. These factors have a major role in the regulation of multiple signal transduction pathways. Evidence have been obtained regarding the possible role of some of these proteins in cancer. TLE3 was suggested as a marker for increased chemosensitivity from pathological studies. Here we demonstrate, using the TCGA data base, differences in expression of this gene compared to TLE1 in several cancers. In-vitro transduction of a retrovirus encoding TLE3 to A549 lung cancer cells increased paclitaxel effectivity while TLE1 introduction to these cells decreased it. While TLE1 and TLE3 share ∼80% amino acid identity, we show that mutating or reconstituting an amino-terminal phosphorylation site, which is present only in TLE1 but absent from TLE3, and is evolutionary conserved, converts the activity of TLE1 to that of TLE3 like and vice versa. We repeated these results in an adipocytes differentiation system. Our results reveal how a single phosphorylation site can confer distinct qualitative or quantitative activities on highly homologous transcriptional regulators.

Aberrant corin and PCSK6 in placentas of the maternal hyperinsulinemia IUGR rat model.

OBJECTIVES: Corin is a protease that converts pro-atrial natriuretic peptide (pro-ANP) to ANP. While the involvement of ANP in the cardiovascular regulation is well established, there is increasing evidence that the pregnant uterus produces ANP, which promotes spiral artery remodeling. The present study examines the alterations in corin and PCSK6, a key enzyme in the conversion of pro-corin to corin, in the placenta of hyperinsulinemic dams (HD) featuring pregnancy-induced hypertension (PIH). MATERIALS AND METHODS: The study was conducted on female Wistar rats. Rats were rendered hyperinsulinemic by subcutaneous insulin pellet, mated and followed to the twenty-first day of pregnancy. Normal pregnant dams (NPD) served as controls. Both groups were sacrificed on day 21 of gestation and their placentas were dissected along with the mesometrial triangle (MT). The tissue was then sectioned from the maternal surface to the base of the MT, and processed for histological and molecular biology analysis of Corin, PCSK6 and ANP expression/immunoreactivity. RESULTS: Hyperinsulinemic dams developed PIH, along lower placental and fetal weights. Corin expression and immunoreactivity were significantly decreased in the placenta by ~40-50%, but not in the MT. Similarly, placental but not MT PCSK6 immunoreactivity was lower in HD. Concomitantly with the downregulation of corin/PCSK6, proANP levels increased in the placenta of HD. CONCLUSIONS: Corin and PCSK6 are expressed in the placenta and MT. The decline in these two enzymes in the placenta of HD suggests a role of corin/PCSK6 machinery in the development of PIH and intrauterine growth restriction characterizing hyperinsulinemia.

Pleiotropic tumor suppressor functions of WWOX antagonize metastasis.

Tumor progression and metastasis are the major causes of death among cancer associated mortality. Metastatic cells acquire features of migration and invasion and usually undergo epithelia-mesenchymal transition (EMT). Acquirement of these various hallmarks rely on different cellular pathways, including TGF-ß and Wnt signaling. Recently, we reported that WW domain-containing oxidoreductase (WWOX) acts as a tumor suppressor and has anti-metastatic activities involving regulation of several key microRNAs (miRNAs) in triple-negative breast cancer (TNBC). Here, we report that WWOX restoration in highly metastatic MDA-MB435S cancer cells alters mRNA expression profiles; further, WWOX interacts with various proteins to exert its tumor suppressor function. Careful alignment and analysis of gene and miRNA expression in these cells revealed profound changes in cellular pathways mediating adhesion, invasion and motility. We further demonstrate that WWOX, through regulation of miR-146a levels, regulates SMAD3, which is a member of the TGF-ß signaling pathway. Moreover, proteomic analysis of WWOX partners revealed regulation of the Wnt-signaling activation through physical interaction with Disheveled. Altogether, these findings underscore a significant role for WWOX in antagonizing metastasis, further highlighting its role and therapeutic potential in suppressing tumor progression.

Guanine Content of MicroRNAs is Associated with their Tumor- Suppressive and Oncogenic Roles in Lung and Breast Cancers.

BACKGROUND: microRNAs (miRNAs, miRs) are small noncoding RNAs that negatively regulate gene expression at the post-transcriptional level and fine-tune gene functions. Global repression of miRNAs expression in different types of human tumors, after exposure to cigarette smoke, or to the hormone estrogen, have been shown to be associated with guanine (G) enrichment in the terminal Loops (TLs) of their precursors. METHODS: We integrated the G content of miRNA mature forms and precursor miRNA TLs with their described function in the literature, using the PubMed database. Gene Ontology term analysis was used to describe the pathways in which the G-enriched miRNA targets are involved. RESULTS: Herein, we show an association between the relative G enrichment of precursor miRNAs' TLs and their tendency to act as tumor suppressor miRs in human lung and breast cancers. Another association was observed between the high G content of the miRNAs 5-mature forms and their tendency to act as oncomiRs. CONCLUSION: The results support previous findings showing that the G sequence content is an important feature determining miRNA expression and function, and opens the way for future cancer investigations in this direction.

Ferritin is regulated by a neuro-intestinal axis in the nematode Caenorhabditis elegans.

Iron is vital for the life of most organisms. However, when dysregulated, iron can catalyze the formation of oxygen (O2) radicals that can destroy any biological molecule and thus lead to oxidative injury and death. Therefore, iron metabolism must be tightly regulated at all times, as well as coordinated with the metabolism of O2. However, how is this achieved at the whole animal level is not well understood. Here, we explore this question using the nematode Caenorhabditis elegans. Exposure of worms to O2 starvation conditions (i.e. hypoxia) induces a major upregulation in levels of the conserved iron-cage protein ferritin 1 (ftn-1) in the intestine, while exposure to 21% O2 decreases ftn-1 level. This O2-dependent inhibition is mediated by O2-sensing neurons that communicate with the intestine through neurotransmitter and neuropeptide signalling, and requires the activity of hydroxylated HIF-1. By contrast, the induction of ftn-1 in hypoxia appears to be HIF-1-independent. This upregulation provides protection against Pseudomonas aeruginosa bacteria and oxidative injury. Taken together, our studies uncover a neuro-intestine axis that coordinates O2 and iron responses at the whole animal level.

Modulation of mitochondrial functions by xenobiotic-induced microRNA: From environmental sentinel organisms to mammals.

Mitochondria play a crucial role in energetic metabolism, signaling pathways, and overall cell viability. They are in the first line in facing cellular energy requirements in stress conditions, such as in response to xenobiotic exposure. Recently, a novel regulatory key role of microRNAs (miRNAs) in important signaling pathways in mitochondria has been proposed. Consequently, alteration in miRNAs expression by xenobiotics could outcome into mitochondrial dysfunction, reactive oxygen species overexpression, and liberation of apoptosis or necrosis activating proteins. The aim of this review is to show the highlights about mitochondria-associated miRNAs in cellular processes exposed to xenobiotic stress in different cell types involved in detoxification processes or sensitive to environmental hazards in marine sentinel organisms and mammals.

Expression levels of selected genes can predict individual rheumatoid arthritis patient response to tumor necrosis factor alpha blocker treatment.

OBJECTIVES: Rheumatoid arthritis (RA) patients have many therapeutic options; however, tools to predict individual patient response are limited. The Genefron personal diagnostic kit, developed by analyzing large datasets, utilizes selected interferon stimulated gene expressions to predict treatment response. This study evaluates the kit's prediction accuracy of individual RA patients' response to tumor necrosis alpha (TNFα) blockers. METHODS: A retrospective analysis was performed on RA patients reported in published datasets. A prospective analysis assessed RA patients, before and 3 months after starting a TNFα blocker. Clinical response was evaluated according to EULAR response criteria. Blood samples were obtained before starting treatment and were analyzed utilizing the kit which measures expression levels of selected genes by quantitative real time polymerase chain reaction (PCR). ROC analysis was applied to the published datasets and the prospective data. RESULTS: The Genefron kit analysis of retrospective data predicted the response to a TNFα blocker in 53 of 61 RA patients (86.8% accuracy). In the prospective analysis, the kit predicted the response in 16 of 18 patients (89% accuracy) achieving a EULAR moderate response, and in 15 of 18 patients achieving a EULAR good response (83.3% accuracy). ROC analysis applied to the two published datasets yielded an AUC of 0.89. ROC analysis applied to the prospective data yielded an AUC of 0.83 (sensitivity - 100%, specificity - 75%) The statistical power obtained in the prospective study was .9. CONCLUSION: The diagnostic kit predicted the response to TNFα blockers in a high percentage of patients assessed retrospectively or prospectively. This personal kit may guide selection of a suitable biological drug for the individual RA patient.

Expression and clinical role of long non-coding RNA in high-grade serous carcinoma.

OBJECTIVE: To profile long non-coding RNA (lncRNA) expression at the various anatomic sites of high-grades serous carcinoma (HGSC) and in effusion-derived exosomes. METHODS: LncRNA profiling was performed on 60 HGSC specimens, including 10 ovarian tumors, 10 solid metastases and 10 malignant effusions, as well as exosomes from 30 effusion supernatants. Anatomic site-related expression of ESRG, Link-A, GAS5, MEG3, GATS, PVT1 H19, Linc-RoR, HOTAIR and MALAT1 was validated by quantitative PCR and assessed for clinical relevance in a series of 77 HGSC effusions, 40 ovarian carcinomas, 21 solid metastases and 42 supernatant exosomes. RESULTS: Significantly different (p<0.05) expression of 241, 406 and 3634 lncRNAs was found in comparative analysis of the ovarian tumors to solid metastases, effusions and exosomes, respectively. Cut-off at two-fold change in lncRNA expression identified 54 lncRNAs present at the 3 anatomic sites and in exosomes. Validation analysis showed significantly different expression of 5 of 10 lncRNAs in the 4 specimen groups (ESRG, Link-A, MEG3, GATS and PVT1, all p<0.001). Higher ESRG levels in HGSC effusions were associated with longer overall survival in the entire effusion cohort (p=0.023) and in patients with pre-chemotherapy effusions tapped at diagnosis (p=0.048). Higher Link-A levels were associated with better overall (p=0.015) and progression-free (p=0.023) survival for patients with post-chemotherapy effusions. Link-A was an independent prognostic marker in Cox multivariate analysis in the latter group (p=0.045). CONCLUSIONS: We present the first evidence of differential LncRNA expression as function of anatomic site in HGSC. LncRNA levels in HGSC effusions are candidate prognostic markers.

A potential microRNA regulation of immune-related genes in invertebrate haemocytes.

Bivalve mollusks have been employed as sentinel organisms in environmental health programs due to their sedentary lifestyle, filter-feeding behavior and their ability to accumulate pathogens or toxin molecules inside tissues. Endocrine disrupting chemicals (EDCs) can be up taken and bioaccumulated, and due to sensibility of mollusks to these EDCs, being able to cause immune alterations. Recently, microRNAs (miRNAs) were shown to be involved in modulation and buffering developmental processes against the effects of environmental alterations and pathogenic microorganisms. Moreover, it is suggested that this miRNAs are incorporated into the estrogen-controlled immune network, regulating mechanism of immune gene expression at the posttranscriptional level, modulating immune responses as phagocytosis, redox reaction and apoptosis in bivalve haemocytes. Thus, miRNAs can be used as biomarkers that specifically elucidate immunotoxic effects caused by exogenous biotic or abiotic factors, and can act as useful tools in integrated monitoring environmental health programs. In this review, we aim to describe the investigations that have been carried out on miRNAs in bivalve mollusks, especially those associated with immune responses against infectious agents and xenobiotic exposure.

microRNAs Downregulation in Cancer is Associated with Guanine Enrichment in the Terminal Loop Sequences of their Precursors.

BACKGROUND: microRNAs (miRNAs) are small noncoding segments of RNA that negatively regulate gene expression at the post-transcriptional level and fine-tune gene functions. A global repression in miRNA expression is a phenomenon observed in different types of cancer. In this study we aimed to reveal a possible association of miRNAs downregulation in cancer, with the guanine (G) content in the terminal loop (TL) sequences of their precursors. METHODS: Lists of most significantly downregulated miRNAs in different tumor types, obtained from previously published microarray experiments, were selected for bioinformatics analysis. The complete precursor, TL, and mature miRNA sequences, were analyzed for evaluation of nucleotide composition and motif enrichment. RESULTS: Herein, we show an association of miRNAs downregulation in cancer, with G enrichment in the TL sequences of their precursors. High G (and GG) content was mostly found in repressed miRNAs of breast, lung and ovary cancers, predominantly in poorly differentiated tumors. The mature sequences of repressed miRNAs had significantly low G content and were enriched with an ACA motif. CONCLUSION: This study suggests a new link between G enrichment of precursor miRNAs TLs and carcinogenesis, and the possible association of specific sequence motifs with the regulation of their expression.

Estrogen Repression of MicroRNAs Is Associated with High Guanine Content in the Terminal Loop Sequences of Their Precursors.

Widespread microRNA (miRNA) repression is a phenomenon observed in mammals after exposure to cigarette smoke and in many types of cancer. A comprehensive reduction in miRNA expression after treatment with the hormone estrogen has also previously been described. Here, we reveal a conserved association of miRNA downregulation after estrogen exposure in zebrafish, mouse, and human breast cancer cell line, with a high guanine content in the terminal loop sequences of their precursors, and offer a possible link between estrogen-related miRNA-adducts formation and carcinogenesis. We also show common gene expression patterns shared by breast cancer tumors and estrogen-treated zebrafish, suggesting that this organism can be used as a powerful model system for the study of human breast cancer.

Characterization of gene expression associated with the adaptation of the nematode C. elegans to hypoxia and reoxygenation stress reveals an unexpected function of the neuroglobin GLB-5 in innate immunity.

Oxygen (O2) is a double-edged sword to cells, for while it is vital for energy production in all aerobic animals and insufficient O2 (hypoxia) can lead to cell death, the reoxygenation of hypoxic tissues may trigger the generation of reactive oxygen species (ROS) that can destroy any biological molecule. Indeed, both hypoxia and hypoxia-reoxygenation (H/R) stress are harmful, and may play a critical role in the pathophysiology of many human diseases, such as myocardial ischemia and stroke. Therefore, understanding how animals adapt to hypoxia and H/R stress is critical for developing better treatments for these diseases. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from H/R stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to Pseudomonas aeruginosa pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.

Distinctive gene expression profile in women with history of postpartum depression.

Postpartum depression (PPD) is a disease which incorporates a variety of depressive states differing in nature and severity. To assist in the understanding of the pathogenesis of the disease, we aimed to ascertain a molecular mechanism underlying PPD development. We applied microarray technology to characterize gene expression of euthymic women with a history of PPD and compared the results with healthy controls. Our study demonstrated that women who considered euthymic on a clinical level, in fact, had an altered molecular profile when compared to participants with no PPD history. We identified nine genes significantly distinguished expression in post- depressive women; they may serve as a diagnostic tool for the detection of a predisposition to PPD. Our findings contribute significantly to the understanding of PPD etiology and its pathogenesis, offer a plausible explanation for the risk of the PPD recurrence, and may also contribute to clinical treatment.