Genetic Screens Identify Host Factors for SARS-CoV-2 and Common Cold Coronaviruses

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Genbank accession #: AF111812

Plant Molecular Biology -     

Measurement of the Infection and Dissemination of Bluetongue Virus in Culicoides Biting Midges Using a Semi-Quantitative RT-PCR Assay and Isolation of Infectious Virus

Background

Culicoides biting midges (Diptera: Ceratopogonidae) are the biological vectors of globally significant arboviruses of livestock including bluetongue virus (BTV), African horse sickness virus (AHSV) and the recently emerging Schmallenberg virus (SBV). From 2006–2009 outbreaks of BTV in northern Europe inflicted major disruption and economic losses to farmers and several attempts were made to implicate Palaearctic Culicoides species as vectors. Results from these studies were difficult to interpret as they used semi-quantitative RT-PCR (sqPCR) assays as the major diagnostic tool, a technique that had not been validated for use in this role. In this study we validate the use of these assays by carrying out time-series detection of BTV RNA in two colony species of Culicoides and compare the results with the more traditional isolation of infectious BTV on cell culture.

Methodology/Principal Findings

A BTV serotype 1 strain mixed with horse blood was fed to several hundred individuals of Culicoides sonorensis (Wirth & Jones) and C. nubeculosus (Mg.) using a membrane-based assay and replete individuals were then incubated at 25°C. At daily intervals 25 Culicoides of each species were removed from incubation, homogenised and BTV quantified in each individual using sqPCR (C_q values) and virus isolation on a KC-C. sonorensis embryonic cell line, followed by antigen enzyme-linked immunosorbent assay (ELISA). In addition, comparisons were also drawn between the results obtained with whole C. sonorensis and with individually dissected individuals to determine the level of BTV dissemination.

Conclusions/Significance

C_q values generated from time-series infection experiments in both C. sonorensis and C. nubeculosus confirmed previous studies that relied upon the isolation and detection of infectious BTV. Implications on the testing of field-collected Culicoides as potential virus vectors by PCR assays and the use of such assays as front-line tools for use in diagnostic laboratories in this role are discussed.     

Down-regulation of aldose reductase renders J774A.1 cells more susceptible to acrolein- or hydrogen peroxide-induced cell death

Aldose reductase (AR) is abundantly expressed in a variety of cell lineages and has been implicated in the cellular response against oxidative stress. However, the exact functional role of AR against oxidative stress remains relatively unclear. This study investigated the role of AR in acrolein- or hydrogen peroxide-induced apoptosis using the J774.A.1 macrophage cell line. Ablation of AR with a small interference RNA or inhibition of AR activity significantly enhanced the acrolein- or hydrogen peroxide-induced generation of reactive oxygen species and aldehydes, leading to increased apoptotic cell death. Blockade of AR activity in J774A.1 cells markedly augmented the acrolein- or hydrogen peroxide-induced translocation of Bax to mitochondria along with reduced Bcl-2 and increased release of cytochrome c from the mitochodria. Taken together, these findings indicate that AR plays an important role in the cellular response against oxidative stress, by sequestering the reactive molecules generated in cells exposed to toxic substances.     

Mitochondrial reactive oxygen species originating from Romo1 exert an important role in normal cell cycle progression by regulating p27Kip1 expression

Reactive oxygen species (ROS) steady-state levels are required for entry into the S phase of the cell cycle in normal cells, as well as in tumour cells. However, the contribution of mitochondrial ROS to normal cell proliferation has not been well investigated thus far. A previous report showed that Romo1 was responsible for the high ROS levels in tumour cells. Here, we show that endogenous ROS generated by Romo1 are indispensable for cell cycle transition from G1 to S phase in normal WI-38 human lung fibroblasts. The ROS level in these cells was down-regulated by Romo1 knockdown, resulting in cell cycle arrest in the G1 phase. This arrest was associated with an increase in the level of p27^Kip1. These results demonstrate that mitochondrial ROS generated by Romo1 expression is required for normal cell proliferation and it is suggested that Romo1 plays an important role in redox signalling during normal cell proliferation.     

Individual variation in early‐life telomere length and survival in a wild mammal

Individual variation in survival probability due to differential responses to early‐life environmental conditions is important in the evolution of life histories and senescence. A biomarker allowing quantification of such individual variation, and which links early‐life environmental conditions with survival by providing a measure of conditions experienced, is telomere length. Here, we examined telomere dynamics among 24 cohorts of European badgers (Meles meles). We found a complex cross‐sectional relationship between telomere length and age, with no apparent loss over the first 29 months, but with both decreases and increases in telomere length at older ages. Overall, we found low within‐individual consistency in telomere length across individual lifetimes. Importantly, we also observed increases in telomere length within individuals, which could not be explained by measurement error alone. We found no significant sex differences in telomere length, and provide evidence that early‐life telomere length predicts lifespan. However, while early‐life telomere length predicted survival to adulthood (≥1 year old), early‐life telomere length did not predict adult survival probability. Furthermore, adult telomere length did not predict survival to the subsequent year. These results show that the relationship between early‐life telomere length and lifespan was driven by conditions in early‐life, where early‐life telomere length varied strongly among cohorts. Our data provide evidence for associations between early‐life telomere length and individual life history, and highlight the dynamics of telomere length across individual lifetimes due to individuals experiencing different early‐life environments.     

Hypothermia Augments Neuroprotective Activity of Mesenchymal Stem Cells for Neonatal Hypoxic-Ischemic Encephalopathy

Though hypothermia is the only clinically available treatment for neonatal hypoxic-ischemic encephalopathy (HIE), it is not completely effective in severe cases. We hypothesized that combined treatment with hypothermia and transplantation of human umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) would synergistically attenuate severe HIE compared to stand-alone therapy. To induce hypoxia-ischemia (HI), male Sprague-Dawley rats were subjected to 8% oxygen for 120 min after unilateral carotid artery ligation on postnatal day (P) 7. After confirmation of severe HIE involving >50% of the ipsilateral hemisphere volume as determined by diffusion-weighted brain magnetic resonance imaging (MRI) within 2 h after HI, intraventricular MSC transplantation (1 × 105 cells) and/or hypothermia with target temperature at 32°C for 24 h were administered 6 h after induction of HI. Follow-up brain MRI at P12 and P42, sensorimotor function tests at P40–42, evaluation of cytokines in the cerebrospinal fluid (CSF) at P42, and histologic analysis of peri-infarct tissues at P42 were performed. Severe HI resulted in progressively increased brain infarction over time as assessed by serial MRI, increased number of cells positive for terminal deoxynucleotidyl transferase nick-end labeling, microgliosis and astrocytosis, increased CSF cytokine levels, and impaired function in behavioral tests such as rotarod and cylinder tests. All of the abnormalities observed in severe HIE showed greater improvement after combined treatment with hypothermia and MSC transplantation than with either therapy alone. Overall, these findings suggest that combined treatment with hypothermia and human UCB-derived MSC transplantation might be a novel therapeutic modality to improve the prognosis of severe HIE, an intractable disease that currently has no effective treatment.     

Optimal Route for Human Umbilical Cord Blood-Derived Mesenchymal Stem Cell Transplantation to Protect Against Neonatal Hyperoxic Lung Injury: Gene Expression Profiles and Histopathology

The aim of this study was to determine the optimal route of mesenchymal stem cell (MSC) transplantation. To this end, gene expression profiling was performed to compare the effects of intratracheal (IT) versus intravenous (IV) MSC administration. Furthermore, the therapeutic efficacy of each route to protect against neonatal hyperoxic lung injury was also determined. Newborn Sprague-Dawley rats were exposed to hyperoxia (90% oxygen) from birth for 14 days. Human umbilical cord blood-derived MSCs labeling with PKH26 were transplanted through either the IT (5×10⁵) or IV (2×10⁶) route at postnatal day (P) 5. At P14, lungs were harvested for histological, biochemical and microarray analyses. Hyperoxic conditions induced an increase in the mean linear intercept and mean alveolar volume (MAV), indicative of impaired alveolarization. The number of ED-1 positive cells was significantly decreased by both IT and IV transplantations. However, IT administration of MSCs resulted in a greater decrease in MAV and ED-1 positive cells compared to IV administration. Moreover, the number of TUNEL-positive cells was significantly decreased in the IT group, but not in the IV group. Although the IT group received only one fourth of the number of MSCs that the IV group did, a significantly higher number of donor cell-derived red PKH 26 positivity were recovered in the IT group. Hyperoxic conditions induced the up regulation of genes associated with the inflammatory response, such as macrophage inflammatory protein-1 α, tumor necrosis factor-α and inter leukin-6; genes associated with cell death, such as p53 and caspases; and genes associated with fibrosis, such as connective tissue growth factor. In contrast, hyperoxic conditions induced the dwon-regulation of vascular endothelial growth factor and hepatocyte growth factor. These hyperoxia-induced changes in gene expression were decreased in the IT group, but not in the IV group. Thus, local IT MSC transplantation was more effective than systemic IV MSC administration in protecting against neonatal hyperoxic lung injury.     

IDENTIFICATION OF THE HIGH‐TEMPERATURE RESPONSE GENES FROM PORPHYRA SERIATA (RHODOPHYTA) EXPRESSION SEQUENCE TAGS AND ENHANCEMENT OF HEAT TOLERANCE OF CHLAMYDOMONAS (CHLOROPHYTA) BY EXPRESSION OF THE PORPHYRA HTR2 GENE1

Temperature is one of the major environmental factors that affect the distribution, growth rate, and life cycle of intertidal organisms, including red algae. In an effort to identify the genes involved in the high‐temperature tolerance of Porphyra, we generated 3,979 expression sequence tags (ESTs) from gametophyte thalli of P. seriata Kjellm. under normal growth conditions and high‐temperature conditions. A comparison of the ESTs from two cDNA libraries allowed us to identify the high temperature response (HTR) genes, which are induced or up‐regulated as the result of high‐temperature treatment. Among the HTRs, HTR2 encodes for a small polypeptide consisting of 144 amino acids, which is a noble nuclear protein. Chlamydomonas expressing the Porphyra HTR2 gene shows higher survival and growth rates than the wild‐type strain after high‐temperature treatment. These results suggest that HTR2 may be relevant to the tolerance of high‐temperature stress conditions, and this Porphyra EST data set will provide important genetic information for studies of the molecular basis of high‐temperature tolerance in marine algae, as well as in Porphyra.