MicroRNAs (miRNAs) were shown to be important for pancreas development, yet their roles in differentiated b-cells remain unclear. Here, we show that miRNA inactivation in b-cells of adult mice results in a striking diabetic phenotype. While islet architecture is intact and differentiation markers are maintained, Dicer1-deficient b-cells show a dramatic decrease in insulin content and insulin mRNA. As a consequence of the change in insulin content, the animals become diabetic. We provide evidence for involvement of a set of miRNAs in regulating insulin synthesis. The specific knockdown of miR-24, miR-26, miR-182 or miR-148 in cultured b-cells or in isolated primary islets downregulates insulin promoter activity and insulin mRNA levels. Further, miRNA-dependent regulation of insulin expression is associated with upregulation of transcriptional repressors, including Bhlhe22 and Sox6. Thus, miRNAs in the adult pancreas act in a new network that reinforces insulin expression by reducing the expression of insulin transcriptional repressors.

The COVID-19 pandemic caused by the SARS-CoV-2 virus remains a global public health crisis. Although widespread vaccination campaigns are underway, their efficacy is reduced owing to emerging variants of concern1,2. Development of host-directed therapeutics and prophylactics could limit such resistance and offer urgently needed protection against variants of concern3,4. Attractive pharmacological targets to impede viral entry include type-II transmembrane serine proteases (TTSPs) such as TMPRSS2; these proteases cleave the viral spike protein to expose the fusion peptide for cell entry, and thus have an essential role in the virus lifecycle5,6. Here we identify and characterize a small-molecule compound, N-0385, which exhibits low nanomolar potency and a selectivity index of higher than 106 in inhibiting SARS-CoV-2 infection in human lung cells and in donor-derived colonoids7. In Calu-3 cells it inhibits the entry of the SARS-CoV-2 variants of concern B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta). Notably, in the K18-human ACE2 transgenic mouse model of severe COVID-19, we found that N-0385 affords a high level of prophylactic and therapeutic benefit after multiple administrations or even after a single administration. Together, our findings show that TTSP-mediated proteolytic maturation of the spike protein is critical for SARS-CoV-2 infection in vivo, and suggest that N-0385 provides an effective early treatment option against COVID-19 and emerging SARS-CoV-2 variants of concern.

a b s t r a c tThe p53 tumor suppressor coordinates a multitude of cellular and organismal processes and exerts its activities mainly by activation of gene transcription. Here we describe the transcriptional activation of ectodysplasin A2 receptor (EDA2R) by p53 in a variety of cell types and tissues. We demonstrate that treatment of cancer cells with the ligand EDA-A2, known to specifically activate EDA2R, results in p53-dependent cell death. Moreover, we show that EDA2R is transactivated by p53 during chemotherapy-induced hair-loss, although its presence is not necessary for this process. These data shed new light on the role of EDA2R in exerting p53 function.

A screen of a eukaryotic kinase inhibitor library in an established intracellular infection model identified a set of drug candidates enabling intracellular killing of Mycobacterium tuberculosis (M.tb). Screen validity was confirmed internally by a Z = 0.5 and externally by detecting previously reported host-targeting anti-M.tb compounds. Inhibitors of the CHK kinase family, specifically checkpoint kinase 2 (CHK2), showed the highest inhibition and lowest toxicity of all kinase families. The screen identified and validated DDUG, a CHK2 inhibitor, as a novel bactericidal anti-M.tb compound. CHK2 inhibition by RNAi phenocopied the intracellular inhibitory effect of DDUG. DDUG was active intracellularly against M.tb, but not other mycobacteria. DDUG also had extracellular activity against 4 of 12 bacteria tested, including M.tb. Combined, these observations suggest DDUG acts in tandem against both host and pathogen. Importantly, DDUG's validation highlights the screening and analysis methodology developed for this screen, which identified novel host-directed anti-M.tb compounds.

!!NCR1!! presents a great challenge to antimycobacterial therapy due to its innate resistance against most antibiotics. M. abscessus is able to grow intracellularly in human macrophages, suggesting that intracellular models can facilitate drug discovery. Thus, we have developed two host cell models: human macrophages for use in a new high-content screening method for M. abscessus growth and a Dictyostelium discoideum infection model with the potential to simplify downstream genetic analysis of host cell factors. A screen of 568 antibiotics for activity against intracellular M. abscessus led to the identification of two hit compounds with distinct growth inhibition. A collection of 317 human kinase inhibitors was analyzed, with the results yielding three compounds with an inhibitory effect on mycobacterial growth, strengthening the notion that host-directed therapy can be applied for M. abscessus.

4-N,N-Dimethylamino-1,8-naphthalimide conjugate of trehalose (DMN-Tre) is a fluorogenic dye recently developed as a diagnostic tool for tuberculosis. DMN-Tre selectively labels the mycobacterial cell wall through the Ag85 enzymes. In this work, we disclose a protocol describing the total synthesis of DMN-Tre with more than 99% purity. We further developed a protocol for in vitro and intercellular labeling of various mycobacterial strains. DMN-Tre labeling was found to be a useful tool to study in vitro and intracellular Mycobacterium tuberculosis (Mtb) physiology and as an end-point readout system in high-content image-based screening (HCS) of drug molecules. Such uses of DMN-Tre labeling provide a simple, fast, and cheap alternative to the existing, time-consuming approach that requires Mtb strains to be genetically transformed with fluorescent reporter genes.

There has been a recent surge of interest in computer-aided rapid data acquisition to increase the potential throughput and reduce the labour costs of large scale Caenorhabditis elegans studies. We present Automated WormScan, a low-cost, high-throughput automated system using commercial photo scanners, which is extremely easy to implement and use, capable of scoring tens of thousands of organisms per hour with minimal operator input, and is scalable. The method does not rely on software training for image recognition, but uses the generation of difference images from sequential scans to identify moving objects. This approach results in robust identification of worms with little computational demand. We demonstrate the utility of the system by conducting toxicity, growth and fecundity assays, which demonstrate the consistency of our automated system, the quality of the data relative to manual scoring methods and congruity with previously published results.

SummaryThe COVID-19 pandemic caused by the SARS-CoV-2 virus remains a global public health crisis. Although widespread vaccination campaigns are underway, their efficacy is reduced against emerging variants of concern (VOCs) 1,2. Development of host-directed therapeutics and prophylactics could limit such resistance and offer urgently needed protection against VOCs 3,4. Attractive pharmacological targets to impede viral entry include type-II transmembrane serine proteases (TTSPs), such as TMPRSS2, whose essential role in the virus lifecycle is responsible for the cleavage and priming of the viral spike protein 5–7. Here, we identify and characterize a small-molecule compound, N-0385, as the most potent inhibitor of TMPRSS2 reported to date. N-0385 exhibited low nanomolar potency and a selectivity index of >106 at inhibiting SARS-CoV-2 infection in human lung cells and in donor-derived colonoids 8. Importantly, N-0385 acted as a broad-spectrum coronavirus inhibitor of two SARS-CoV-2 VOCs, B.1.1.7 and B.1.351. Strikingly, single daily intranasal administration of N-0385 early in infection significantly improved weight loss and clinical outcomes, and yielded 100% survival in the severe K18-human ACE2 transgenic mouse model of SARS-CoV-2 disease. This demonstrates that TTSP-mediated proteolytic maturation of spike is critical for SARS-CoV-2 infection in vivo and suggests that N-0385 provides a novel effective early treatment option against COVID-19 and emerging SARS-CoV-2 VOCs.