Human infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19) and there is no cure currently. The 3CL protease (3CLpro) is a highly conserved protease which is indispensable for CoVs replication, and is a promising target for development of broad-spectrum antiviral drugs. In this study we investigated the anti-SARS-CoV-2 potential of Shuanghuanglian preparation, a Chinese traditional patent medicine with a long history for treating respiratory tract infection in China. We showed that either the oral liquid of Shuanghuanglian, the lyophilized powder of Shuanghuanglian for injection or their bioactive components dose-dependently inhibited SARS-CoV-2 3CLpro as well as the replication of SARS-CoV-2 in Vero E6 cells. Baicalin and baicalein, two ingredients of Shuanghuanglian, were characterized as the first noncovalent, nonpeptidomimetic inhibitors of SARS-CoV-2 3CLpro and exhibited potent antiviral activities in a cell-based system. Remarkably, the binding mode of baicalein with SARS-CoV-2 3CLpro determined by X-ray protein crystallography was distinctly different from those of known 3CLpro inhibitors. Baicalein was productively ensconced in the core of the substrate-binding pocket by interacting with two catalytic residues, the crucial S1/S2 subsites and the oxyanion loop, acting as a “shield” in front of the catalytic dyad to effectively prevent substrate access to the catalytic dyad within the active site. Overall, this study provides an example for exploring the in vitro potency of Chinese traditional patent medicines and effectively identifying bioactive ingredients toward a specific target, and gains evidence supporting the in vivo studies of Shuanghuanglian oral liquid as well as two natural products for COVID-19 treatment.

Influenza virus and coronavirus epidemics or pandemics have occurred in succession worldwide throughout the early 21st century. These epidemics or pandemics pose a major threat to human health. Here, we outline a critical role of the host cell protease TMPRSS2 in influenza virus and coronavirus infections and highlight an antiviral therapeutic strategy targeting TMPRSS2.

Cytokine storm and multi-organ failure are the main causes of SARS-CoV-2-related death. However, the origin of excessive damages caused by SARS-CoV-2 remains largely unknown. Here we show that the SARS-CoV-2 envelope (2-E) protein alone is able to cause acute respiratory distress syndrome (ARDS)-like damages in vitro and in vivo. 2-E proteins were found to form a type of pH-sensitive cation channels in bilayer lipid membranes. As observed in SARS-CoV-2-infected cells, heterologous expression of 2-E channels induced rapid cell death in various susceptible cell types and robust secretion of cytokines and chemokines in macrophages. Intravenous administration of purified 2-E protein into mice caused ARDS-like pathological damages in lung and spleen. A dominant negative mutation lowering 2-E channel activity attenuated cell death and SARS-CoV-2 production. Newly identified channel inhibitors exhibited potent anti-SARS-CoV-2 activity and excellent cell protective activity in vitro and these activities were positively correlated with inhibition of 2-E channel. Importantly, prophylactic and therapeutic administration of the channel inhibitor effectively reduced both the viral load and secretion of inflammation cytokines in lungs of SARS-CoV-2-infected transgenic mice expressing human angiotensin-converting enzyme 2 (hACE-2). Our study supports that 2-E is a promising drug target against SARS-CoV-2.

Emerging and re-emerging RNA viruses occasionally cause epidemics and pandemics worldwide, such as the on-going outbreak of the novel coronavirus SARS-CoV-2. Herein, we identified two potent inhibitors of human DHODH, S312 and S416, with favorable drug-likeness and pharmacokinetic profiles, which all showed broad-spectrum antiviral effects against various RNA viruses, including influenza A virus, Zika virus, Ebola virus, and particularly against SARS-CoV-2. Notably, S416 is reported to be the most potent inhibitor so far with an EC50 of 17 nmol/L and an SI value of 10,505.88 in infected cells. Our results are the first to validate that DHODH is an attractive host target through high antiviral efficacy in vivo and low virus replication in DHODH knock-out cells. This work demonstrates that both S312/S416 and old drugs (Leflunomide/Teriflunomide) with dual actions of antiviral and immuno-regulation may have clinical potentials to cure SARS-CoV-2 or other RNA viruses circulating worldwide, no matter such viruses are mutated or not.

Three‐dimensional (3D) printing, also called additive manufacturing (AM) or rapid prototyping (RP), is a layer by layer manufacturing method and now has been widely used in many areas such as organ printing, aerospace and industrial design. Now 3D printed microfluidics attract more and more interests for its rapid printing in the lab. In this review, we focused on the advances of 3D printed microfluidic chips especially the use in the chemistry and biology (vascularization and organs on chips). Based on the brief review of different 3D printing methods, we discussed how to choose the suitable 3D printing methods to print the desired microfluidics. We predict that microfluidics will be evolved from 2D chips to 3D cubes, printed hydrogel‐based microfluidics will be reported and widely used, sensors & actuators could be integrated in the microfluidics during printing, and rapid assembling chips with printed microfluidic modules will be popular in the near future.

PTEN/MMAC is a phosphatase that is mutated in multiple human tumors. PTEN/MMAC dephosphorylates 3-phosphorylated phosphatidylinositol phosphates that activate AKT/protein kinase B (PKB) kinase activity. AKT/PKB is implicated in the inhibition of apoptosis, and cell lines and tumors with mutated PTEN/MMAC show increased AKT/PKB kinase activity and resistance to apoptosis. PTEN/MMAC contains a PDZ domain-binding site, and we show here that the phosphatase binds to a PDZ domain of membrane-associated guanylate kinase with inverted orientation (MAGI) 3, a novel inverted membrane-associated guanylate kinase that localizes to epithelial cell tight junctions. Importantly, MAGI3 and PTEN/MMAC cooperate to modulate the kinase activity of AKT/PKB. These data suggest that MAGI3 allows for the juxtaposition of PTEN/MMAC to phospholipid signaling pathways involved with cell survival.Tumor progression is often accompanied by the loss of heterozygosity at a diversity of genetic loci. A notable example of this phenomenon is encountered in advanced gliomas, where homozygous deletions at 10q23 are commonly detected (1). Loss of heterozygosity at an identical chromosomal site is also observed in Cowden's and Bannayan-Zonana syndromes, two autosomal dominant diseases that result in a predisposition to formation of a variety of malignant tumors (2). Together, these genetic data suggested that a tumor suppressor was likely to be localized at 10q23, and the isolation from this chromosomal region of a gene encoding a phosphatase termed PTEN/MMAC has lent strong support to this idea (3-5). For example, examination of glial, prostate, and endometrial tumors revealed that a high percentage of these cancers contained homozygously mutated PTEN/MMAC loci (6 -8). In addition, in vitro analyses of the potential of PTEN/MMAC as a cell growth regulator demonstrated that overexpression of the catalytically active protein in cells that lacked the phosphatase suppressed cell proliferation (9 -13). Importantly, recent studies have demonstrated that mice with heterozygous deletions of the PTEN/ MMAC gene show gastrointestinal and prostatic hyperplasia/ dysplasia as well as a predilection for the development of colon carcinomas that show loss of heterozygosity of the PTEN/ MMAC locus (14, 15). These results are consistent with the suggestion that PTEN/MMAC is an important tumor suppressor in various human malignancies.Although initial studies suggested that the PTEN/MMAC phosphatase acted upon protein substrates and might be involved with the control of integrin-mediated adhesion (4, 16 -18), subsequent analyses demonstrated that the enzyme was a catalyst for the removal of the 3-phosphate from phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) 1 (19,20). This phospholipid is a product of the catalytic activity of phosphatidylinositol 3-kinase, and it is involved with a number of signaling pathways, including the activation of the protooncogene AKT/PKB (21). AKT/PKB is a pleckstrin homology domaincontaining serine/threonine kinase with a var...

Wiskott-Aldrich syndrome is an X-linked hematopoietic disease that manifests itself in platelet deficiency and a compromised immune system. Analysis of hematopoietic cells from affected individuals reveals that mutations in the Wiskott-Aldrich syndrome protein (WASP) result in structural and functional abnormalities in the cell cortex, consistent with the suggestion that WASP is involved with regulation of the actin-rich cortical cytoskeleton. Here we report that WASP interacts with a recently described cytoskeletal-associated protein, PST-PIP, a molecule that is related to the Schizosaccharomyces pombe cleavage furrow regulatory protein, CDC15p. This association is mediated by an interaction between the PSTPIP SH3 domain and two polyproline-rich regions in WASP. Co-expression of PSTPIP with WASP in vivo results in a loss of WASP-induced actin bundling activity and co-localization of the two proteins, which requires the PSTPIP SH3 domain. Analysis of tyrosine phosphorylation of PSTPIP reveals that two sites are modified in response to v-Src co-transfection or pervanadate incubation. One of these tyrosines is found in the SH3 domain poly-proline recognition site, and mutation of this tyrosine to aspartate or glutamate to mimic this phosphorylation state results in a loss of WASP binding in vitro and a dissolution of co-localization in vivo. In addition, PSTPIP that is tyrosine phosphorylated in the SH3 domain interacts poorly with WASP in vitro. These data suggest that the PSTPIP and WASP interaction is regulated by tyrosine phosphorylation of the PSTPIP SH3 domain, and this binding event may control aspects of the actin cytoskeleton. PSTPIP is a recently described coiled-coil and SH3 domaincontaining protein that is homologous to CDC15p, a Schizosaccharomyces pombe phosphoprotein involved with the assembly of the cytokinetic cleavage furrow (1-3). PSTPIP is associated with protein-tyrosine phosphatase (PTP) 1 HSCF, a member of the PEST family of PTPs (4), via a critical tryptophan in the PSTPIP coiled-coil domain (2), and this association mediates the dephosphorylation of tyrosine residues in PSTPIP that are modified either by co-expression of the v-Src tyrosine kinase or in the presence of the PTP inhibitor pervanadate. Throughout interphase, the coiled-coil domain localizes PST-PIP to actin-rich regions of the cell, including the cortical cytoskeleton and lammelipodia, and the protein migrates to the cleavage furrow during cytokinesis. Overexpression of PSTPIP in mammalian cells induces filapodial extension and cell rounding, and induced expression of the mammalian protein in S. pombe results in a dominant negative inhibition of cytokinesis (1). These data suggest that PSTPIP is a tyrosine phosphorylated cytoskeletal regulatory protein that may be involved with the control of cytokinesis.Wiskott-Aldrich syndrome is an X-linked hematopoietic disease that presents with platelet abnormalities and immunodeficiency (5, 6), and mutations in the Wiskott-Aldrich syndrome protein (WASP) cause this syndrome (7-9). WAS...

The let-60 ras gene of Caenorhabditis elegans is one of the key players in a signal transduction pathway that controls the choice between vulval and epidermal differentiation in response to extracellular signals. To identify components acting downstream of let-60 ras in the vulval signaling pathway, we have identified a reduction-of-function mutation in the sur-1 gene that completely suppresses the multivulva phenotype of a hyperactive let-60 ras mutation. About 10% of animals homozygous for the sur-1 mutation also display a specific and intriguing vulval cell lineage defect. In addition, the sur-1 mutation results in a cold-sensitive egg-laying defective phenotype and a partial larval lethal phenotype. We have cloned the sur-1 gene by DNA-mediated transformation and have shown that it encodes a protein similar in overall structure to mammalian MAP kinases (ERKs). The functional homology between Sur-1 MAP kinase and mammalian MAP kinases was also demonstrated by the ability of a rat ERK2 kinase to rescue the sur-1 mutant phenotypes. Genetic double-mutant analyses place sur-1 downstream of let-60 ras but upstream of lin-1 in the vulval signaling pathway. Our results provide further evidence for the extreme conservation of Ras-mediated signaling pathway between worms and humans and for the function of MAP kinases in cell signaling processes that control cell differentiation and animal development.