Background: SARS-CoV-2 is a recently emerged respiratory pathogen that has significantly impacted global human health. We wanted to rapidly characterise the transcriptomic, proteomic and phosphoproteomic landscape of this novel coronavirus to provide a fundamental description of the virus's genomic and proteomic potential. Methods: We used direct RNA sequencing to determine the transcriptome of SARS-CoV-2 grown in Vero E6 cells which is widely used to propagate the novel coronavirus. The viral transcriptome was analysed using a recently developed ORF-centric pipeline. Allied to this, we used tandem mass spectrometry to investigate the proteome and phosphoproteome of the same virally infected cells. Results: Our integrated analysis revealed that the viral transcripts (i.e. subgenomic mRNAs) generally fitted the expected transcription model for coronaviruses. Importantly, a 24 nt in-frame deletion was detected in over half of the subgenomic mRNAs encoding the spike (S) glycoprotein and was predicted to remove a proposed furin cleavage site from the S glycoprotein. Tandem mass spectrometry identified over 500 viral peptides and 44 phosphopeptides in virus-infected cells, covering almost all proteins predicted to be encoded by the SARS-CoV-2 genome, including peptides unique to the deleted variant of the S glycoprotein. Conclusions: Detection of an apparently viable deletion in the furin cleavage site of the S glycoprotein, a leading vaccine target, shows that this and other regions of SARS-CoV-2 proteins may readily mutate. The furin site directs cleavage of the S glycoprotein into functional subunits during virus entry or exit and likely contributes strongly to the pathogenesis and zoonosis of this virus. Our data emphasises that the viral genome sequence should be carefully monitored during the growth of viral stocks for research, animal challenge models and, potentially, in clinical samples. Such variations may result in different levels of virulence, morbidity and mortality.

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Mechanisms underpinning chronic sustained hypoxia (CH)-induced structural and functional adaptations in respiratory muscles are unclear despite the clinical relevance to respiratory diseases. The objectives of the present study were to thoroughly assess the putative role of CH-induced redox remodeling in murine diaphragm muscle over time and the subsequent effects on metabolic enzyme activities, catabolic signaling and catabolic processes, and diaphragm muscle contractile function. C57Bl6/J mice were exposed to normoxia or normobaric CH (fraction of inspired oxygen = 0.1) for 1, 3, or 6 weeks. A second cohort was exposed to CH for 6 weeks with and without antioxidant supplementation (tempol or N-acetyl cysteine). After CH exposure, we performed two-dimensional redox proteomics with mass spectrometry, enzyme activity assays, and cell-signaling assays on diaphragm homogenates. We also assessed diaphragm isotonic contractile and endurance properties ex vivo. Global protein redox changes in the diaphragm after CH are indicative of oxidation. Remodeling of proteins key to contractile, metabolic, and homeostatic functions was observed. Several oxidative and glycolytic enzyme activities were decreased by CH. Redox-sensitive chymotrypsin-like proteasome activity of the diaphragm was increased. CH decreased phospho-forkhead box O3a (FOXO3a) and phospho-mammalian target of rapamycin content. Hypoxia-inducible factor-1α and phospho-p38 mitogen-activated protein kinase content was increased in CH diaphragm, and this was attenuated by antioxidant treatment. CH exposure decreased force- and power-generating capacity of the diaphragm, and this was prevented by antioxidant supplementation with N-acetyl cysteine but not tempol. Redox remodeling is pivotal for diaphragm adaptation to CH, affecting metabolic activity, atrophy signaling, and functional performance. Antioxidant supplementation may be useful as an adjunctive therapy in respiratory-related diseases characterized by hypoxic stress.

The maximum number and diversity of macroinvertebrates from limestone-filled basket samplers were obtained by placing the samplers at a depth of 1 ft (0-3 m) below the water's surface for 8 weeks. The letigth ofthe exposure period had a greater effect on the number of organisms than depth of placement. The effects of longer exposure times (8 weeks, compared to 6 and 4 weeks) on Ihe number and diversity of organisms inhabiting baskets were more pronounced during cold weather. In rivers during the summer months, samples contain a variety of aquatic insects, which contributed as much as 95 % of the total number of individuals. The occurrence of some macroinvertebrate species was related to the depth of sampler placement, e.g. the caddisfly Cyrnellus fraternus was more abundant at depths exceeding 4ft {I•2 m); whereas the mayfly Stenonema interpunctatum was more abundant in samplers near the surface.The community diversity index (i) of collections on the same date varied by as much as two units depending on the depth of basket placement and length of exposure period, indicating that consistent practices in sampler installation and analysis are required for comparison of collections in water quality investigations.Baskets touching bottom accumulated greater amounts of sediments than surface satnplers and generally showed greater variability within pairs.In the Ohio River, three replicate baskets can be expected {F = 0 95) to provide an estimate ofthe true mean number of macroinvertebrates within ±20% ofthe sample mean and contain 71 % of the taxa in 10 replicates. However, estimates of sampling precision based on replicate series may vary considerably depending on time, place, number of replicates and natural history ofthe organisms.Baskets filled with 2-in. (5-1-cm) diameter porcelain spheres collect samples comparable in total abundance and diversity to baskets filled with 2-in. (5-1-cm) diameter limestone. However, the taxonomic groups occur in different proportions on the two types of substrates.Hardboard multiplates of l-ft^ (Ol-m^) surface area with -\-(0-32) or ^-in. (0-64-cm) spacings collect significantly greater numbers of individuals than porcelain multiplates with the same spacings. However, differences between samples from the same substrate type are not significant. Multiplates of the same surface area and plate spacing collect substantially the same number of taxa regardless of substrate type.Basket samplers (3-2 ft^ -0-3 m^ surface area) collect a greater abundance and diversity of organisms than the standard 1 ft^ = 0-1 m^ hardboard multiplates. The performance ofthe samplers in terms of abundance and diversity is equalized, however, by increasing the surface area of the multiplate to that of the basket. IntroductionField studies of effects of water quality on benthic macroinvertebrates are often handicapped by the problem of sampling similar, natural substrates suitable for colonization at all sampling stations. The problem is especially difficult in rivers, where fluctuations in hydrologic condition...

BackgroundCircadian system time cues (zeitgebers) acting synergistically at the right times can foster chronobiological homeostasis and ultimately health. Modern 24/7 societies are challenging chronobiological homeostasis and public health. Exercise has been discussed as a potential zeitgeber for the human circadian system. Thus, if timed correctly, exercise may help in maintenance of chronobiological homeostasis and foster public health amidst increasingly challenging 24/7 lifestyles.ObjectiveTo test, using a systematic review of the literature, the following hypothesis: exercise is a zeitgeber for the human circadian system.Data sourcesThe PubMed database was systematically searched on 19 October 2017 for relevant scientific studies and reports concerning chronobiology and exercise. Eligibility criteria were defined to include articles considering exercise as a potential zeitgeber for human circadian rhythmicity or chronobiological effects of exercise on health and/or physical performance. Cognitive effects and effects on children were excluded from the synthesis.ResultsOur systematic literature search and synthesis is compatible with the validity of the hypothesis. We report that potential exercise-zeitgeber properties may be used to improve health and performance.ConclusionsInformed timing of exercise, specific to the circadian rhythm phase and zeitgeber exposure of the individual, must be advocated in performance and disease contexts as an adjunct therapeutic or preventative strategy and physical enhancer.

Ten 48‐h static acute toxicity tests were conducted with Daphnia magna Straus and Daphnia pulex Leydig with each of the three reference toxicants — sodium dodecyl sulfate (SDS), sodium pentachlorophenate (NaPCP), and cadmium (Cd) (as CdCl2) — at 20 and 26°C to investigate the effect of temperature on test results at the temperature extremes usually encountered during field studies. The mean 48‐h LC50 value for SDS with Daphnia pulex at 26°C was lower (10.2 mg/L) than at 20°C (12.6 mg/L), but the difference was not significant (α = 0.05). For Daphnia magna the mean LC50 was significantly (α = 0.05) lower at 26°C (10.8 mg/L) than at 20°C (13.5 mg/L). Sensitivity of either species to NaPCP was not significantly different (α = 0.05) at the two temperatures. The data show a fourfold increase in sensitivity to Cd at the higher temperature with Daphnia magna (mean LC50 = 0.038 mg/L at 20°C and 0.009 mg/L at 26°C) and a sevenfold increase with Daphnia pulex (mean LC50 = 0.042 mg/L at 20°C and 0.006 mg/L at 26°C). These data indicate that toxicity tests conducted at 20 and 26°C may give significantly different results with Daphnia. Because test precision, control survival, and the likelihood of obtaining valid tests are better at 20°C, that temperature is recommended for acute toxicity tests with Daphnia.

Chronic sustained hypoxia (CH) induces structural and functional adaptations in respiratory muscles of animal models, however the underlying molecular mechanisms are unclear. This study explores the putative role of CH-induced redox remodeling in a translational mouse model, with a focus on the sternohyoid—a representative upper airway dilator muscle involved in the control of pharyngeal airway caliber. We hypothesized that exposure to CH induces redox disturbance in mouse sternohyoid muscle in a time-dependent manner affecting metabolic capacity and contractile performance. C57Bl6/J mice were exposed to normoxia or normobaric CH (FiO2 = 0.1) for 1, 3, or 6 weeks. A second cohort of animals was exposed to CH for 6 weeks with and without antioxidant supplementation (tempol or N-acetyl cysteine in the drinking water). Following CH exposure, we performed 2D redox proteomics with mass spectrometry, metabolic enzyme activity assays, and cell-signaling assays. Additionally, we assessed isotonic contractile and endurance properties ex vivo. Temporal changes in protein oxidation and glycolytic enzyme activities were observed. Redox modulation of sternohyoid muscle proteins key to contraction, metabolism and cellular homeostasis was identified. There was no change in redox-sensitive proteasome activity or HIF-1α content, but CH decreased phospho-JNK content independent of antioxidant supplementation. CH was detrimental to sternohyoid force- and power-generating capacity and this was prevented by chronic antioxidant supplementation. We conclude that CH causes upper airway dilator muscle dysfunction due to redox modulation of proteins key to function and homeostasis. Such changes could serve to further disrupt respiratory homeostasis in diseases characterized by CH such as chronic obstructive pulmonary disease. Antioxidants may have potential use as an adjunctive therapy in hypoxic respiratory disease.

Chronic intermittent hypoxia (CIH) causes upper airway muscle dysfunction. We hypothesized that the superoxide generating NADPH oxidase (NOX) is upregulated in CIH-exposed muscle causing oxidative stress. Adult male Wistar rats were exposed to intermittent hypoxia (5% O2 at the nadir for 90 s followed by 210 s of normoxia), for 8 h per day for 14 days. The effect of CIH exposure on the expression of NOX subunits, total myosin and 4-hydroxynonenal (4-HNE) protein adducts in sternohyoid muscle was determined by western blotting and densitometry. Sternohyoid protein free thiol and carbonyl group contents were determined by 1D electrophoresis using specific fluorophore probes. Aconitase and glutathione reductase activities were measured as indices of oxidative stress. HIF-1α content and key oxidative and glycolytic enzyme activities were determined. Contractile properties of sternohyoid muscle were determined ex vivo in the absence and presence of apocynin (putative NOX inhibitor). We observed an increase in NOX 2 and p47 phox expression in CIH-exposed sternohyoid muscle with decreased aconitase and glutathione reductase activities. There was no evidence, however, of increased lipid peroxidation or protein oxidation in CIH-exposed muscle. CIH exposure did not affect sternohyoid HIF-1α content or aldolase, lactate dehydrogenase, or glyceraldehyde-3-phosphate dehydrogenase activities. Citrate synthase activity was also unaffected by CIH exposure. Apocynin significantly increased sternohyoid force and power. We conclude that CIH exposure upregulates NOX expression in rat sternohyoid muscle with concomitant modest oxidative stress but it does not result in a HIF-1α-dependent increase in glycolytic enzyme activity. Constitutive NOX activity decreases sternohyoid force and power. Our results implicate NOX-dependent reactive oxygen species in CIH-induced upper airway muscle dysfunction which likely relates to redox modulation of key regulatory proteins in excitation-contraction coupling.