Transcriptomics modeling of the late-gestation fetal pituitary response to transient hypoxia

Background The late-gestation fetal sheep responds to hypoxia with physiological, neuroendocrine, and cellular responses that aid in fetal survival. The response of the fetus to hypoxia represents a coordinated effort to maximize oxygen transfer from the mother and minimize wasteful oxygen consumption by the fetus. While there have been many studies aimed at investigating the coordinated physiological and endocrine responses to hypoxia, and while immunohistochemical or in situ hybridization studies have revealed pathways supporting the endocrine function of the pituitary, there is little known about the coordinated cellular response of the pituitary to the hypoxia. Results Thirty min hypoxia (from 17.0±1.7 to 8.0±0.8 mm Hg, followed by 30 min normoxia) upregulated 595 and downregulated 790 genes in fetal pituitary (123-132 days' gestation; term = 147 days). Network inference of up- and down- regulated genes revealed a high degree of functional relatedness amongst the gene sets. Gene ontology analysis revealed upregulation of cellular metabolic processes (e.g., RNA synthesis, response to estrogens) and downregulation Conclusions The multiple analytical approaches used in this study suggests that the acute response to 30 min of transient hypoxia in the late-gestation fetus results in reduced cellular metabolism and a pattern of gene expression that is consistent with cellular oxygen and ATP starvation. In this early time point, we see a vigorous gene response. But, like the hypothalamus, the transcriptomic response is not consistent with mediation by HIF-1. If HIF-1 is a significant controller of gene expression in the fetal pituitary after hypoxia, it must be at a later time.Fil: Wood, Charles E.. University of Florida; Estados UnidosFil: Chang, Eileen I.. University of Florida; Estados UnidosFil: Richards, Elaine M.. University of Florida; Estados UnidosFil: Rabaglino, Maria Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Keller Wood, Maureen. University of Florida; Estados Unido

Ketamine decreases inflammatory and immune pathways after transient hypoxia in late gestation fetal cerebral cortex

Transient hypoxia in pregnancy stimulates a physiological reflex response that redistributes blood flow and defends oxygen delivery to the fetal brain. We designed the present experiment to test the hypotheses that transient hypoxia produces damage of the cerebral cortex and that ketamine, an antagonist of NMDA receptors and a known anti-inflammatory agent, reduces the damage. Late gestation, chronically catheterized fetal sheep were subjected to a 30-min period of ventilatory hypoxia that decreased fetal PaO2 from 17 ± 1 to 10 ± 1 mmHg, or normoxia (PaO2 17 ± 1 mmHg), with or without pretreatment (10 min before hypoxia/normoxia) with ketamine (3 mg/kg, i.v.). One day (24 h) after hypoxia/normoxia, fetal cerebral cortex was removed and mRNA extracted for transcriptomics and systems biology analysis (n = 3-5 per group). Hypoxia stimulated a transcriptomic response consistent with a reduction in cellular metabolism and an increase in inflammation. Ketamine pretreatment reduced both of these responses. The inflammation response modeled with transcriptomic systems biology was validated by immunohistochemistry and showed increased abundance of microglia/macrophages after hypoxia in the cerebral cortical tissue that ketamine significantly reduced. We conclude that transient hypoxia produces inflammation of the fetal cerebral cortex and that ketamine, in a standard clinical dose, reduces the inflammation response.Fil: Chang, Eileen I.. University of Florida; Estados UnidosFil: Zárate, Miguel A.. University of Florida; Estados UnidosFil: Rabaglino, Maria Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina. Provincia de Córdoba. Ministerio de Ciencia y Técnica. Centro de Excelencia en Productos y Procesos de Córdoba; ArgentinaFil: Richards, Elaine M.. University of Florida; Estados UnidosFil: Arndt, Thomas J.. University of Florida; Estados UnidosFil: Keller Wood, Maureen. University of Florida; Estados UnidosFil: Wood, Charles E.. University of Florida; Estados Unido

Ketamine reduces inflammation pathways in the hypothalamus and hippocampus following transient hypoxia in the late-gestation fetal sheep

The physiological response to hypoxia in the fetus has been extensively studied with regard to redistribution of fetal combined ventricular output and sparing of oxygen delivery to fetal brain and heart. Previously, we have shown that the fetal brain is capable of mounting changes in gene expression that are consistent with tissue inflammation. The present study was designed to use transcriptomics and systems biology modeling to test the hypothesis that ketamine reduces or prevents the upregulation of inflammation-related pathways in hypothalamus and hippocampus after transient hypoxic hypoxia. Chronically catheterized fetal sheep (122 ± 5 days gestation) were subjected to 30 min hypoxia (relative reduction in PaO2∼50%) caused by infusion of nitrogen into the inspired gas of the pregnant ewe. RNA was isolated from fetal hypothalamus and hippocampus collected 24 h after hypoxia, and was analyzed for gene expression using the Agilent 15.5 k ovine microarray. Ketamine, injected 10 min prior to hypoxia, reduced the cerebral immune response activation to the hypoxia in both brain regions. Genes both upregulated by hypoxia and downregulated by ketamine after hypoxia were significantly associated with gene ontology terms and KEGG pathways that are, themselves, associated with the tissue response to exposure to bacteria. We conclude that the results are consistent with interruption of the cellular response to bacteria by ketamine.Fil: Chang, Eileen I.. University of Florida; Estados UnidosFil: Zarate, Miguel A.. University of Florida; Estados UnidosFil: Arndt, Thomas J.. University of Florida; Estados UnidosFil: Richards, Elaine M.. University of Florida; Estados UnidosFil: Rabaglino, Maria Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Keller Wood, Maureen. University of Florida; Estados UnidosFil: Wood, Charles E.. University of Florida; Estados Unido

Advanced EFL learners' beliefs about language learning and teaching: a comparison between grammar, pronunciation, and vocabulary

This paper reports on the results of a study exploring learners’ beliefs on the learning and teaching of English grammar, pronunciation and vocabulary at tertiary level. While the importance of learners’ beliefs on the acquisition process is generally recognized, few studies have focussed on and compared learners’ views on different components of the language system. A questionnaire containing semantic scale and Likert scale items probing learners’ views on grammar, pronunciation and vocabulary was designed and completed by 117 native speakers of Dutch in Flanders, who were studying English at university. The analysis of the responses revealed that (i) vocabulary was considered to be different from grammar and pronunciation, both in the extent to which an incorrect use could lead to communication breakdown and with respect to the learners’ language learning strategies, (ii) learners believed in the feasibility of achieving a native-like proficiency in all three components, and (iii) in-class grammar, pronunciation and vocabulary exercises were considered to be useful, even at tertiary level. The results are discussed in light of pedagogical approaches to language teaching

Family satisfaction with critical care in the UK: a multicentre cohort study.

OBJECTIVE: To assess family satisfaction with intensive care units (ICUs) in the UK using the Family Satisfaction in the Intensive Care Unit 24-item (FS-ICU-24) questionnaire, and to investigate how characteristics of patients and their family members impact on family satisfaction. DESIGN: Prospective cohort study nested within a national clinical audit database. SETTING: Stratified, random sample of 20 adult general ICUs participating in the Intensive Care National Audit & Research Centre Case Mix Programme. PARTICIPANTS: Family members of patients staying at least 24 hours in ICU were recruited between May 2013 and June 2014. INTERVENTIONS: Consenting family members were sent a postal questionnaire 3 weeks after the patient died or was discharged from ICU. Up to four family members were recruited per patient. MAIN OUTCOME MEASURES: Family satisfaction was measured using the FS-ICU-24 questionnaire. MAIN RESULTS: A total of 12 346 family members of 6380 patients were recruited and 7173 (58%) family members of 4615 patients returned a completed questionnaire. Overall and domain-specific family satisfaction scores were high (mean overall family satisfaction 80, satisfaction with care 83, satisfaction with information 76 and satisfaction with decision-making 73 out of 100) but varied significantly across adult general ICUs studied and by whether the patient survived ICU. For family members of ICU survivors, characteristics of both the family member (age, ethnicity, relationship to patient (next-of-kin and/or lived with patient) and visit frequency) and the patient (acute severity of illness and receipt of invasive mechanical ventilation) were significant determinants of family satisfaction, whereas, for family members of ICU non-survivors, only patient characteristics (age, acute severity of illness and duration of stay) were significant. CONCLUSIONS: Overall family satisfaction in UK adult general ICUs was high but varied significantly. Adjustment for differences in family member/patient characteristics is important to avoid falsely identifying ICUs as statistical outliers. TRIAL REGISTRATION NUMBER: ISRCTN47363549

CTCF genetic alterations in endometrial carcinoma are pro-tumorigenic

CTCF is a haploinsufficient tumour suppressor gene with diverse normal functions in genome structure and gene regulation. However the mechanism by which CTCF haploinsufficiency contributes to cancer development is not well understood. CTCF is frequently mutated in endometrial cancer. Here we show that most CTCF mutations effectively result in CTCF haploinsufficiency through nonsense-mediated decay of mutant transcripts, or loss-of-function missense mutation. Conversely, we identified a recurrent CTCF mutation K365T, which alters a DNA binding residue, and acts as a gain-of-function mutation enhancing cell survival. CTCF genetic deletion occurs predominantly in poor prognosis serous subtype tumours, and this genetic deletion is associated with poor overall survival. In addition, we have shown that CTCF haploinsufficiency also occurs in poor prognosis endometrial clear cell carcinomas and has some association with endometrial cancer relapse and metastasis. Using shRNA targeting CTCF to recapitulate CTCF haploinsufficiency, we have identified a novel role for CTCF in the regulation of cellular polarity of endometrial glandular epithelium. Overall, we have identified two novel pro-tumorigenic roles (promoting cell survival and altering cell polarity) for genetic alterations of CTCF in endometrial cancer

Cumulate causes for the low contents of sulfide-loving elements in the continental crust

Despite the economic importance of chalcophile (sulfide-loving) and siderophile (metal-loving) elements (CSEs), it is unclear how they become enriched or depleted in the continental crust, compared with the oceanic crust. This is due in part to our limited understanding of the partitioning behaviour of the CSEs. Here I compile compositional data for mid-ocean ridge basalts and subduction-related volcanic rocks. I show that the mantle-derived melts that contribute to oceanic and continental crust formation rarely avoid sulfide saturation during cooling in the crust and, on average, subduction-zone magmas fractionate sulfide at the base of the continental crust prior to ascent. Differentiation of mantle-derived melts enriches lower crustal sulfide- and silicate-bearing cumulates in some CSEs compared with the upper crust. This storage predisposes the cumulate-hosted compatible CSEs (such as Cu and Au) to be recycled back into the mantle during subduction and delamination, resulting in their low contents in the bulk continental crust and potentially contributing to the scarcity of ore deposits in the upper continental crust. By contrast, differentiation causes the upper oceanic and continental crust to become enriched in incompatible CSEs (such as W) compared with the lower oceanic and continental crust. Consequently, incompatible CSEs are predisposed to become enriched in subduction-zone magmas that contribute to continental crust formation and are less susceptible to removal from the continental crust via delamination compared with the compatible CSEs

Angiotensin Converting Enzyme 2 in Cardiopulmonary Diseases: Ramifications for the Control of SARS-CoV-2

Discovery of angiotensin converting enzyme 2 (ACE2) revealed that the renin angiotensin system (RAS) has two counterbalancing arms. ACE2 is a major player in the protective arm, highly expressed in lungs and gut with the ability to mitigate cardiopulmonary diseases such as inflammatory lung disease. ACE2 also exhibits activities involving gut microbiome, nutrition, and as a chaperone stabilizing the neutral amino acid transporter, B0AT1, in gut. But the current interest in ACE2 arises because it is the cell surface receptor for the novel coronavirus, SARS-CoV-2, to infect host cells, similar to SARS-CoV. This suggests that ACE2 be considered harmful, however because of its important other roles, it is paradoxically a potential therapeutic target for cardiopulmonary diseases including COVID-19, caused by SARS-CoV-2. This review describes the discovery of ACE2, its physiological functions, and its place in the RAS. It illustrates new analyses of the structure of ACE2 that provides better understanding of its actions particularly in lung and gut, shedding of ACE2 by ADAM17 and role of TMPRSS2 in SARS-CoV-2 entry into host cells. Cardiopulmonary diseases are associated with decreased ACE2 activity and the mitigation by increasing ACE2 activity along with its therapeutic relevance are addressed. Finally, the potential use of ACE2 as a treatment target in COVID-19, despite its role to allow viral entry into host cells, is suggested

Complete loss of ATM function augments replication catastrophe induced by ATR inhibition and gemcitabine in pancreatic cancer models

Abstract: Background: Personalised medicine strategies may improve outcomes in pancreatic ductal adenocarcinoma (PDAC), but validation of predictive biomarkers is required. Having developed a clinical trial to assess the ATR inhibitor, AZD6738, in combination with gemcitabine (ATRi/gem), we investigated ATM loss as a predictive biomarker of response to ATRi/gem in PDAC. Methods: Through kinase inhibition, siRNA depletion and CRISPR knockout of ATM, we assessed how ATM targeting affected the sensitivity of PDAC cells to ATRi/gem. Using flow cytometry, immunofluorescence and immunoblotting, we investigated how ATRi/gem synergise in ATM-proficient and ATM-deficient cells, before assessing the impact of ATM loss on ATRi/gem sensitivity in vivo. Results: Complete loss of ATM function (through pharmacological inhibition or CRISPR knockout), but not siRNA depletion, sensitised to ATRi/gem. In ATM-deficient cells, ATRi/gem-induced replication catastrophe was augmented, while phospho-Chk2-T68 and phospho-KAP1-S824 persisted via DNA-PK activity. ATRi/gem caused growth delay in ATM-WT xenografts in NSG mice and induced regression in ATM-KO xenografts. Conclusions: ATM loss augments replication catastrophe-mediated cell death induced by ATRi/gem and may predict clinical responsiveness to this combination. ATM status should be carefully assessed in tumours from patients with PDAC, since distinction between ATM-low and ATM-null could be critical in maximising the success of clinical trials using ATM expression as a predictive biomarker

Complete loss of ATM function augments replication catastrophe induced by ATR inhibition and gemcitabine in pancreatic cancer models

Abstract: Background: Personalised medicine strategies may improve outcomes in pancreatic ductal adenocarcinoma (PDAC), but validation of predictive biomarkers is required. Having developed a clinical trial to assess the ATR inhibitor, AZD6738, in combination with gemcitabine (ATRi/gem), we investigated ATM loss as a predictive biomarker of response to ATRi/gem in PDAC. Methods: Through kinase inhibition, siRNA depletion and CRISPR knockout of ATM, we assessed how ATM targeting affected the sensitivity of PDAC cells to ATRi/gem. Using flow cytometry, immunofluorescence and immunoblotting, we investigated how ATRi/gem synergise in ATM-proficient and ATM-deficient cells, before assessing the impact of ATM loss on ATRi/gem sensitivity in vivo. Results: Complete loss of ATM function (through pharmacological inhibition or CRISPR knockout), but not siRNA depletion, sensitised to ATRi/gem. In ATM-deficient cells, ATRi/gem-induced replication catastrophe was augmented, while phospho-Chk2-T68 and phospho-KAP1-S824 persisted via DNA-PK activity. ATRi/gem caused growth delay in ATM-WT xenografts in NSG mice and induced regression in ATM-KO xenografts. Conclusions: ATM loss augments replication catastrophe-mediated cell death induced by ATRi/gem and may predict clinical responsiveness to this combination. ATM status should be carefully assessed in tumours from patients with PDAC, since distinction between ATM-low and ATM-null could be critical in maximising the success of clinical trials using ATM expression as a predictive biomarker