Characterisation of the transcriptome and proteome of SARS-CoV-2 reveals a cell passage induced in-frame deletion of the furin-like cleavage site from the spike glycoprotein

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

Ultrasound modulates neuronal potassium currents via ionotropic glutamate receptors

Background Focused ultrasound stimulation (FUS) has the potential to provide non-invasive neuromodulation of deep brain regions with unparalleled spatial precision. However, the cellular and molecular consequences of ultrasound stimulation on neurons remains poorly understood. We previously reported that ultrasound stimulation induces increases in neuronal excitability that persist for hours following stimulation in vitro. In the present study we sought to further elucidate the molecular mechanisms by which ultrasound regulates neuronal excitability and synaptic function. Objectives To determine the effect of ultrasound stimulation on voltage-gated ion channel function and synaptic plasticity. Methods Primary rat cortical neurons were exposed to a 40 s, 200 kHz pulsed ultrasound stimulus or sham-stimulus. Whole-cell patch clamp electrophysiology, quantitative proteomics and high-resolution confocal microscopy were employed to determine the effects of ultrasound stimulation on molecular regulators of neuronal excitability and synaptic function. Results We find that ultrasound exposure elicits sustained but reversible increases in whole-cell potassium currents. In addition, we find that ultrasound exposure activates synaptic signalling cascades that result in marked increases in excitatory synaptic transmission. Finally, we demonstrate the requirement of ionotropic glutamate receptor (AMPAR/NMDAR) activation for ultrasound-induced modulation of neuronal potassium currents. Conclusion These results suggest specific patterns of pulsed ultrasound can induce contemporaneous enhancement of both neuronal excitability and synaptic function, with implications for the application of FUS in experimental and therapeutic settings. Further study is now required to deduce the precise molecular mechanisms through which these changes occur

Phasic contractions of isolated human myometrium are associated with Rho-kinase (ROCK)-dependent phosphorylation of myosin phosphatase-targeting subunit (MYPT1)

Force generation in smooth muscle is driven by phosphorylation of myosin light chains (MYL), which is regulated by the equilibrium between the activities of myosin light chain kinase (MYLK) and myosin phosphatase (MYLP). MYLK is activated by Ca2+-calmodulin whereas MYLP is inhibited by phosphorylation of its myosin-binding subunit (MYPT1) by Ca2+-independent mechanisms, leading to generation of increased MYL phosphorylation and force for a given intracellular Ca2+ concentration, a phenomenon known as ‘calcium-sensitization’. The regulation of MYPT1 phosphorylation in human myometrium, which shows increasing phasic contractility at the onset of labour, has yet to be fully investigated. Here, we explore phosphorylation of MYPT1 at Thr696 and Thr853, alongside phosphorylation of MYL, in fresh human myometrial tissue and cultured myometrial cells. We report that pMYPT1 (Thr853) levels are dependent on the activity of Rho-associated kinase (ROCK), determined using the ROCK inhibitor g-H-1152 and siRNA-mediated knockdown of ROCK1/2, and are highly correlated to ppMYL (Thr18/Ser19) levels. Pharmacological inhibition of ROCK was associated with a decrease in oxytocin (OXT)-stimulated contractility of myometrial strips in vitro. Moreover, we have measured pMYPT1 and pMYL levels between and during spontaneous and OXT-stimulated phasic contractions by rapidly freezing contracting muscle, and demonstrate for the first time functional coupling between increases in pMYPT1 (Thr853), ppMYL (Thr18/Ser19) and phasic contractility that is ROCK-dependent. The combined approach of measuring contractility and phosphorylation has demonstrated that the phosphorylation of MYPT1 (Thr853) changes dynamically with each contraction and has elucidated a defined role for ROCK in regulating myometrial contractility through MYLP, providing new insights into uterine physiology which will stimulate further research into treatments for preterm labour

Genetic deficiency of aldose reductase counteracts the development of diabetic nephropathy in C57BL/6 mice

National Science Foundation of China [30770490]; 973 Program of China [2009CB941601]; Science Planning Program of Fujian Province [2009J1010]; Natural Science Foundation of Fujian Province [2009J01180]; Fujian Provincial Department of Science and TechnoloThe aim of the study was to investigate the effects of genetic deficiency of aldose reductase in mice on the development of key endpoints of diabetic nephropathy. A line of Ar (also known as Akr1b3)-knockout (KO) mice, a line of Ar-bitransgenic mice and control C57BL/6 mice were used in the study. The KO and bitransgenic mice were deficient for Ar in the renal glomeruli and all other tissues, with the exception of, in the bitransgenic mice, a human AR cDNA knockin-transgene that directed collecting-tubule epithelial-cell-specific AR expression. Diabetes was induced in 8-week-old male mice with streptozotocin. Mice were further maintained for 17 weeks then killed. A number of serum and urinary variables were determined for these 25-week-old mice. Periodic acid-Schiff staining, western blots, immunohistochemistry and protein kinase C (PKC) activity assays were performed for histological analyses, and to determine the levels of collagen IV and TGF-beta 1 and PKC activities in renal cortical tissues. Diabetes-induced extracellular matrix accumulation and collagen IV overproduction were completely prevented in diabetic Ar-KO and bitransgenic mice. Ar deficiency also completely or partially prevented diabetes-induced activation of renal cortical PKC, TGF-beta 1 and glomerular hypertrophy. Loss of Ar results in a 43% reduction in urine albumin excretion in the diabetic Ar-KO mice and a 48% reduction in the diabetic bitransgenic mice (p < 0.01). Genetic deficiency of Ar significantly ameliorated development of key endpoints linked with early diabetic nephropathy in vivo. Robust and specific inhibition of aldose reductase might be an effective strategy for the prevention and treatment of diabetic nephropathy