A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research.

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.

Frequent loss-of-function mutations in the AMPK-α2 catalytic subunit suggest a tumour suppressor role in human skin cancers.

The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status activated by increases in AMP or ADP relative to ATP. Once activated, it phosphorylates targets that promote ATP-generating catabolic pathways or inhibit ATP-consuming anabolic pathways, helping to restore cellular energy balance. Analysis of human cancer genome studies reveals that the PRKAA2 gene (encoding the α2 isoform of the catalytic subunit) is often subject to mis-sense mutations in cancer, particularly in melanoma and non-melanoma skin cancers, where up to 70 mis-sense mutations have been documented, often accompanied by loss of the tumour suppressor NF1. Recently it has been reported that knockout of PRKAA2 in NF1-deficient melanoma cells promoted anchorage-independent growth in vitro, as well as growth as xenografts in immunodeficient mice in vivo, suggesting that AMPK-α2 can act as a tumour suppressor in that context. However, very few of the mis-sense mutations in PRKAA2 that occur in human skin cancer and melanoma have been tested to see whether they cause loss-of-function. We have addressed this by making most of the reported mutations and testing their activity when expressed in AMPK knockout cells. Of 55 different mis-sense mutations (representing 75 cases), 9 (12%) appeared to cause a total loss of activity, 18 (24%) a partial loss, 11 (15%) an increase in phenformin-stimulated kinase activity, while just 37 (49%) had no clear effect on kinase activity. This supports the idea that AMPK-α2 acts as a tumour suppressor in the context of human skin cancer.

A conserved ATG2-GABARAP family interaction is critical for phagophore formation.

The intracellular trafficking pathway, macroautophagy, is a recycling and disposal service that can be upregulated during periods of stress to maintain cellular homeostasis. An essential phase is the elongation and closure of the phagophore to seal and isolate unwanted cargo prior to lysosomal degradation. Human ATG2A and ATG2B proteins, through their interaction with WIPI proteins, are thought to be key players during phagophore elongation and closure, but little mechanistic detail is known about their function. We have identified a highly conserved motif driving the interaction between human ATG2 and GABARAP proteins that is in close proximity to the ATG2-WIPI4 interaction site. We show that the ATG2A-GABARAP interaction mutants are unable to form and close phagophores resulting in blocked autophagy, similar to ATG2A/ATG2B double-knockout cells. In contrast, the ATG2A-WIPI4 interaction mutant fully restored phagophore formation and autophagy flux, similar to wild-type ATG2A. Taken together, we provide new mechanistic insights into the requirements for ATG2 function at the phagophore and suggest that an ATG2-GABARAP/GABARAP-L1 interaction is essential for phagophore formation, whereas ATG2-WIPI4 interaction is dispensable.

Evaluation of patient-reported outcomes and quality of life with the I-neb AAD system in patients with chronic obstructive pulmonary disease.

BACKGROUND: The I-neb Adaptive Aerosol Delivery (AAD) System is a novel portable mesh nebulizer that provides patient feedback regarding adherence to prescribed treatment and compliance with the correct use of the device. METHODS: This multicenter study was composed of 98 patients aged 53-80 years with Chronic Obstructive Pulmonary Disease (COPD). The primary variables were ease of use and satisfaction, which were assessed after 3 months of use of the I-neb AAD System (assessed at visit 2) and after 3 months of use of the patient's previous nebulizer system (assessed at visit 1) using matched questions from pre- and poststudy questionnaires. Quality of life was also assessed at visits 1 and 2 using the validated Chronic Respiratory Questionnaire (CRQ), which consists of dyspnea, emotional function, fatigue, and mastery domains. Differences in the distribution of responses between the pre- and poststudy ease of use and satisfaction questions were analyzed using the Marginal Homogeneity test. Differences in mean CRQ scores between the pre- and poststudy assessments were analyzed using the Wilcoxon Signed-Rank test. RESULTS: Patient responses on the ease of use and satisfaction questions significantly (p < or = 0.001) favored the I-neb AAD System compared with the patient's previous nebulizer system. In addition, significant (p < or = 0.015) improvements in the CRQ dimensions of dyspnea and fatigue were reported with the I-neb AAD System compared with the patients' previous nebulizer systems. CONCLUSIONS: The results from this study demonstrated that patients were more satisfied with the I-neb AAD System and found it easier to use than their previous nebulizer systems. In addition, the I-neb AAD System significantly improved dyspnea and fatigue compared with the patients' previous nebulizer systems, which may reflect improved adherence or more correct use of the nebulizer system with the I-neb AAD System.