Role of Akt signaling pathway regulation in the speckled mousebird (Colius striatus) during torpor displays tissue specific responses

Pronounced heterothermic responses are relatively rare among birds. Along with taxa such as hummingbirds and caprimulgids, the order Coliiformes (mousebirds) is known to possess the physiological capacity for torpor. During torpor, body temperature is greatly reduced and a bird becomes unresponsive to external stimuli until ambient temperatures return to more favorable conditions. Under such conditions, these birds are forced to rely only on their internal fuel storage for energy and show great reduction in metabolic rates by decreasing energy-expensive processes. This study investigated the role of the key insulin-Akt signaling kinase pathway involved in regulating energy metabolism and protein translation in the liver, kidney, heart, skeletal muscle, and brain of the speckled mousebird (Colius striatus). The degree of phosphorylation of well-conserved target residues with important regulatory function was examined in both the euthermic control and torpid birds. The results demonstrated marked differences in responses between the tissues with decreases in RPS6 S235/236 phosphorylation in the kidney (0.52 fold of euthermic) and muscle (0.29 fold of euthermic) as well as decreases in GS3K3β S9 in muscle (0.60 fold of euthermic) and GSK3α S21 (0.71 fold of euthermic) phosphorylation in kidney during torpor, suggesting a downregulation of this pathway. Interestingly, the liver demonstrated an increase in RPS6 S235/236 (2.89 fold increase) and P70S6K T412 (1.44 fold increase) phosphorylation in the torpor group suggesting that protein translation is maintained in this tissue. This study demonstrates that avian torpor is a complex phenomenon and alterations in this signaling pathway follow a tissue specific pattern.Supplementary material 1: Western blots for quantifying the relative levels of phosphorylated S209 eIF-4E and total mTOR levels in euthermic and torpid C. striatus tissuesSupplementary material 2: Sequence alignment of mousebird protein targets involved in the Akt signaling kinase pathway and protein translation in comparison to human sequencesThe Natural Sciences and Engineering Research Council of Canada (NSERC), the Canada Research Chair in Molecular Physiology, an NSERC funded Alexander Graham Bell Canada Graduate Scholarship, an Ontario Graduate Scholarship and the National Research Foundation of South Africa.https://www.elsevier.com/locate/cellsig2021-11-01hj2020Zoology and Entomolog

Phosphorylation status of pyruvate dehydrogenase in the mousebird Colius striatus undergoing torpor

DATA AVAILABILITY STATEMENT : Data available on request from the authors.Torpor is a heterothermic response that occurs in some animals to reduce metabolic expenditure. The speckled mousebird (Colius striatus) belongs to one of the few avian taxa possessing the capacity for pronounced torpor, entering a hypometabolic state with concomitant decreases in body temperature in response to reduced food access or elevated thermoregulatory energy requirements. The pyruvate dehydrogenase complex (PDC) is a crucial site regulating metabolism by bridging glycolysis and the Krebs cycle. Three highly conserved phosphorylation sites are found within the E1 enzyme of the complex that inhibit PDC activity and reduce the flow of carbohydrate substrates into the mitochondria. The current study demonstrates a marked increase in S232 phosphorylation during torpor in liver, heart, and skeletal muscle of C. striatus. The increase in S232 phosphorylation during torpor was particularly notable in skeletal muscle where levels were ~49-fold higher in torpid birds compared to controls. This was in contrast to the other two phosphorylation sites (S293 and S300) which remained consistently phosphorylated regardless of tissue. The relevant PDH kinase (PDHK1) known to phosphorylate S232 was found to be substantially upregulated (~5-fold change) in the muscle during torpor as well as increasing moderately in the liver (~2.2-fold increase). Additionally, in the heart, a slight (~23%) decrease in total PDH levels was noted. Taken together the phosphorylation changes in PDH suggest that inhibition of the complex is a common feature across several tissues in the mousebird during torpor and that this regulation is mediated at a specific residue.Natural Sciences and Engineering Research Council of Canada and National Research Foundation of South Africa.http://wileyonlinelibrary.com/journal/jezhj2023Zoology and Entomolog

Desmopressin for prevention of bleeding for thrombocytopenic, critically ill patients undergoing invasive procedures: A randomised, double‐blind, placebo‐controlled feasibility trial

Thrombocytopenic patients have an increased risk of bleeding when undergoing invasive procedures. In a multicentre, phase II, blinded, randomised, controlled feasibility trial, critically ill patients with platelet count 100 × 109/L or less were randomised 1:1 to intravenous desmopressin (0.3 µg/kg) or placebo before an invasive procedure. Forty‐three participants (18.8% of those eligible) were recruited, with 41 eligible for analysis. Post‐procedure bleeding occurred in one of 22 (4.5%) in the placebo arm and zero of 19 in the desmopressin arm. Despite liberal inclusion criteria, there were significant feasibility challenges recruiting patients in the critical care setting prior to invasive procedures

Whole-genome sequencing reveals host factors underlying critical COVID-19

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2–4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease

Genetic mechanisms of critical illness in COVID-19.

Host-mediated lung inflammation is present1, and drives mortality2, in the critical illness caused by coronavirus disease 2019 (COVID-19). Host genetic variants associated with critical illness may identify mechanistic targets for therapeutic development3. Here we report the results of the GenOMICC (Genetics Of Mortality In Critical Care) genome-wide association study in 2,244 critically ill patients with COVID-19 from 208 UK intensive care units. We have identified and replicated the following new genome-wide significant associations: on chromosome 12q24.13 (rs10735079, P = 1.65 × 10-8) in a gene cluster that encodes antiviral restriction enzyme activators (OAS1, OAS2 and OAS3); on chromosome 19p13.2 (rs74956615, P = 2.3 × 10-8) near the gene that encodes tyrosine kinase 2 (TYK2); on chromosome 19p13.3 (rs2109069, P = 3.98 ×  10-12) within the gene that encodes dipeptidyl peptidase 9 (DPP9); and on chromosome 21q22.1 (rs2236757, P = 4.99 × 10-8) in the interferon receptor gene IFNAR2. We identified potential targets for repurposing of licensed medications: using Mendelian randomization, we found evidence that low expression of IFNAR2, or high expression of TYK2, are associated with life-threatening disease; and transcriptome-wide association in lung tissue revealed that high expression of the monocyte-macrophage chemotactic receptor CCR2 is associated with severe COVID-19. Our results identify robust genetic signals relating to key host antiviral defence mechanisms and mediators of inflammatory organ damage in COVID-19. Both mechanisms may be amenable to targeted treatment with existing drugs. However, large-scale randomized clinical trials will be essential before any change to clinical practice

Common, low-frequency, rare, and ultra-rare coding variants contribute to COVID-19 severity

The combined impact of common and rare exonic variants in COVID-19 host genetics is currently insufficiently understood. Here, common and rare variants from whole-exome sequencing data of about 4000 SARS-CoV-2-positive individuals were used to define an interpretable machine-learning model for predicting COVID-19 severity. First, variants were converted into separate sets of Boolean features, depending on the absence or the presence of variants in each gene. An ensemble of LASSO logistic regression models was used to identify the most informative Boolean features with respect to the genetic bases of severity. The Boolean features selected by these logistic models were combined into an Integrated PolyGenic Score that offers a synthetic and interpretable index for describing the contribution of host genetics in COVID-19 severity, as demonstrated through testing in several independent cohorts. Selected features belong to ultra-rare, rare, low-frequency, and common variants, including those in linkage disequilibrium with known GWAS loci. Noteworthily, around one quarter of the selected genes are sex-specific. Pathway analysis of the selected genes associated with COVID-19 severity reflected the multi-organ nature of the disease. The proposed model might provide useful information for developing diagnostics and therapeutics, while also being able to guide bedside disease management. © 2021, The Author(s)

Hyperoxemia and excess oxygen use in early acute respiratory distress syndrome : Insights from the LUNG SAFE study

Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Concerns exist regarding the prevalence and impact of unnecessary oxygen use in patients with acute respiratory distress syndrome (ARDS). We examined this issue in patients with ARDS enrolled in the Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) study. Methods: In this secondary analysis of the LUNG SAFE study, we wished to determine the prevalence and the outcomes associated with hyperoxemia on day 1, sustained hyperoxemia, and excessive oxygen use in patients with early ARDS. Patients who fulfilled criteria of ARDS on day 1 and day 2 of acute hypoxemic respiratory failure were categorized based on the presence of hyperoxemia (PaO2 > 100 mmHg) on day 1, sustained (i.e., present on day 1 and day 2) hyperoxemia, or excessive oxygen use (FIO2 ≥ 0.60 during hyperoxemia). Results: Of 2005 patients that met the inclusion criteria, 131 (6.5%) were hypoxemic (PaO2 < 55 mmHg), 607 (30%) had hyperoxemia on day 1, and 250 (12%) had sustained hyperoxemia. Excess FIO2 use occurred in 400 (66%) out of 607 patients with hyperoxemia. Excess FIO2 use decreased from day 1 to day 2 of ARDS, with most hyperoxemic patients on day 2 receiving relatively low FIO2. Multivariate analyses found no independent relationship between day 1 hyperoxemia, sustained hyperoxemia, or excess FIO2 use and adverse clinical outcomes. Mortality was 42% in patients with excess FIO2 use, compared to 39% in a propensity-matched sample of normoxemic (PaO2 55-100 mmHg) patients (P = 0.47). Conclusions: Hyperoxemia and excess oxygen use are both prevalent in early ARDS but are most often non-sustained. No relationship was found between hyperoxemia or excessive oxygen use and patient outcome in this cohort. Trial registration: LUNG-SAFE is registered with ClinicalTrials.gov, NCT02010073publishersversionPeer reviewe

Whole-genome sequencing reveals host factors underlying critical COVID-19

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2,3,4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease

The roles of hyperoxia and mechanical deformation in alveolar epithelial injury and repair

The alveolar epithelium is a key functional component of the air-blood barrier in the lung. Comprised of two morphologically distinct cell types, alveolar epithelial type I (ATI) and type II (ATII) cells, effective repair of the alveolar epithelial barrier following injury appears to be an important determinant of clinical outcome. The prevailing view suggests this repair is achieved by the proliferation of ATII cells and the transdifferentiation of ATII cells into ATI cells. Supplemental oxygen and mechanical ventilation are key therapeutic interventions in the supportive treatment of respiratory failure following lung injury, but the effects of hyperoxia and mechanical deformation in the injured lung, and on alveolar epithelial repair in particular, are largely unknown. The clinical impression however, is that poor outcome is associated with exposure of injured (repairing) epithelium to such iatrogenic ‘hits’. This thesis describes studies investigating the hypothesis that hyperoxia & mechanical deformation inhibit normal epithelial repair. The in vitro data presented demonstrate that hyperoxia reversibly inhibits the transdifferentiation of ATII-like cells into ATI-like cells with time in culture. Whilst confirming that hyperoxia is injurious to alveolar epithelial cells, these data further suggest the ATII cell population harbours a subpopulation of cells resistant to hyperoxia-induced injury. This subpopulation of cells appears to generate fewer reactive oxygen species and express lower levels of the zonula adherens protein E-cadherin. Using a panel of antibodies to ATI (RTI40) and ATII (MMC4 & RTII70) cell-selective proteins, the effect of hyperoxia on the phenotype of the alveolar epithelium in a rat model of resolving S. aureus-induced lung injury was investigated. These in vivo studies support the view that, under normoxic conditions, alveolar epithelial repair occurs through ATII cell proliferation & transdifferentiation of ATII cells into ATI cells, with transdifferentiation occurring via a novel intermediate (MMC4/RTI40-coexpressing) immunophenotype. However, in S. aureus-injured lungs exposed to hyperoxia, the resolution of ATII cell hyperplasia was impaired, with an increase in ATII cell-staining membrane and a reduction in intermediate cell-staining membrane compared to injured lungs exposed to normoxia alone. As hyperoxia is pro-apoptotic and known to inhibit ATII cell proliferation, these data support the hypothesis that hyperoxia impairs normal epithelial repair by inhibiting the transdifferentiation of ATII cells into ATI cells in vivo. The effect of mechanical deformation on alveolar epithelial cells in culture was investigated by examining changes in cell viability following exposure of epithelial cell monolayers to quantified levels of cyclic equibiaxial mechanical strain. In the central region of monolayers, deformation-induced injury was a non-linear function of deformation magnitude, with significant injury occurring only following exposure to strains greater than those associated with inflation of the intact lung to total lung capacity. However, these studies demonstrate for the first time that different epithelial cell phenotypes within the same culture system have different sensitivities to deformation-induced injury, with spreading RTI40-expressing cells in the peripheral region of epithelial cell monolayers and in the region of ‘repairing’ wounds being injured even at physiological levels of mechanical strain. These findings are consistent with the hypothesis that alveolar epithelial cells in regions of epithelial repair are highly susceptible to deformation-induced injury

The roles of hyperoxia and mechanical deformation in alveolar epithelial injury and repair

The alveolar epithelium is a key functional component of the air-blood barrier in the lung. Comprised of two morphologically distinct cell types, alveolar epithelial type I (ATI) and type II (ATII) cells, effective repair of the alveolar epithelial barrier following injury appears to be an important determinant of clinical outcome. The prevailing view suggests this repair is achieved by the proliferation of ATII cells and the transdifferentiation of ATII cells into ATI cells. Supplemental oxygen and mechanical ventilation are key therapeutic interventions in the supportive treatment of respiratory failure following lung injury, but the effects of hyperoxia and mechanical deformation in the injured lung, and on alveolar epithelial repair in particular, are largely unknown. The clinical impression however, is that poor outcome is associated with exposure of injured (repairing) epithelium to such iatrogenic ‘hits’. This thesis describes studies investigating the hypothesis that hyperoxia & mechanical deformation inhibit normal epithelial repair. The in vitro data presented demonstrate that hyperoxia reversibly inhibits the transdifferentiation of ATII-like cells into ATI-like cells with time in culture. Whilst confirming that hyperoxia is injurious to alveolar epithelial cells, these data further suggest the ATII cell population harbours a subpopulation of cells resistant to hyperoxia-induced injury. This subpopulation of cells appears to generate fewer reactive oxygen species and express lower levels of the zonula adherens protein E-cadherin. Using a panel of antibodies to ATI (RTI40) and ATII (MMC4 & RTII70) cell-selective proteins, the effect of hyperoxia on the phenotype of the alveolar epithelium in a rat model of resolving S. aureus-induced lung injury was investigated. These in vivo studies support the view that, under normoxic conditions, alveolar epithelial repair occurs through ATII cell proliferation & transdifferentiation of ATII cells into ATI cells, with transdifferentiation occurring via a novel intermediate (MMC4/RTI40-coexpressing) immunophenotype. However, in S. aureus-injured lungs exposed to hyperoxia, the resolution of ATII cell hyperplasia was impaired, with an increase in ATII cell-staining membrane and a reduction in intermediate cell-staining membrane compared to injured lungs exposed to normoxia alone. As hyperoxia is pro-apoptotic and known to inhibit ATII cell proliferation, these data support the hypothesis that hyperoxia impairs normal epithelial repair by inhibiting the transdifferentiation of ATII cells into ATI cells in vivo. The effect of mechanical deformation on alveolar epithelial cells in culture was investigated by examining changes in cell viability following exposure of epithelial cell monolayers to quantified levels of cyclic equibiaxial mechanical strain. In the central region of monolayers, deformation-induced injury was a non-linear function of deformation magnitude, with significant injury occurring only following exposure to strains greater than those associated with inflation of the intact lung to total lung capacity. However, these studies demonstrate for the first time that different epithelial cell phenotypes within the same culture system have different sensitivities to deformation-induced injury, with spreading RTI40-expressing cells in the peripheral region of epithelial cell monolayers and in the region of ‘repairing’ wounds being injured even at physiological levels of mechanical strain. These findings are consistent with the hypothesis that alveolar epithelial cells in regions of epithelial repair are highly susceptible to deformation-induced injury.EThOS - Electronic Theses Online ServiceGBUnited Kingdo