The pandemic of coronavirus disease 2019 (COVID-19) is changing the world like never before. This crisis is unlikely contained in the absence of effective therapeutics or vaccine. The papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays essential roles in virus replication and immune evasion, presenting a charming drug target. Given the PLpro proteases of SARS-CoV-2 and SARS-CoV share significant homology, inhibitor developed for SARS-CoV PLpro is a promising starting point of therapeutic development. In this study, we sought to provide structural frameworks for PLpro inhibitor design. We determined the unliganded structure of SARS-CoV-2 PLpro mutant C111S, which shares many structural features of SARS-CoV PLpro. This crystal form has unique packing, high solvent content and reasonable resolution 2.5 Å, hence provides a good possibility for fragment-based screening using crystallographic approach. We characterized the protease activity of PLpro in cleaving synthetic peptide harboring nsp2/nsp3 juncture. We demonstrate that a potent SARS-CoV PLpro inhibitor GRL0617 is highly effective in inhibiting protease activity of SARS-CoV-2 with the IC 50 of 2.2 ± 0.3 μmol/L. We then determined the structure of SARS-CoV-2 PLpro complexed by GRL0617 to 2.6 Å, showing the inhibitor accommodates the S3–S4 pockets of the substrate binding cleft. The binding of GRL0617 induces closure of the BL2 loop and narrows the substrate binding cleft, whereas the binding of a tetrapeptide substrate enlarges the cleft. Hence, our results suggest a mechanism of GRL0617 inhibition, that GRL0617 not only occupies the substrate pockets, but also seals the entrance to the substrate binding cleft hence prevents the binding of the LXGG motif of the substrate.
Expression of the trabecular meshwork inducible glucocorticoid response (TIGR) gene progressively increases from barely detectable levels to greater than 2% of total cellular mRNA over 10 days exposure of trabecular meshwork (TM) cells to dexamethasone. Cycloheximide blocked most of the TIGR mRNA induction, suggesting a requirement for ongoing protein synthesis. The genomic structure of TIGR (ϳ20 kilobases) consists of 3 exons, and a 5-kilobase promoter region that contains 13 predicted hormone response elements, including several glucocorticoid regulatory elements, and other potentially important regulatory motifs. TIGR cDNA encodes an olfactomedin-related glycoprotein of 504 amino acids with motifs for N-and O-linked glycosylation, glycosaminoglycan initiation, hyaluronic acid binding, and leucine zippers. Recombinant TIGR (rTIGR) showed oligomerization and specific binding to TM cells. Anti-rTIGR antibody detected multiple translational/post-translational forms of TIGR produced by the cells (including secreted 66 kDa/55 kDa glycoproteins/proteins in the media and 55 kDa cellular proteins), whereas Northern blot showed a single mRNA species. The findings suggest potential mechanisms by which TIGR could obstruct the aqueous humor fluid flow and participate in the pathogenesis of glaucoma.The trabecular meshwork inducible glucocorticoid response (TIGR) 1 protein, which has significant homology in its C-terminal domain with olfactomedins, was initially cloned in our laboratories as a candidate gene for glaucoma using differential library screening in a trabecular meshwork cell culture model (1, 2). Mutations were recently found in this gene that co-segregated with both juvenile and adult forms of the disease (3).Glaucoma is a major cause of blindness, with its most prevalent form thought to involve the specialized endothelial cells lining the outflow pathway of the eye, termed the trabecular meshwork (TM) (4, 5). The synthesis and/or degradation of a variety of extracellular molecules in the meshwork are thought to be regulated by the TM cells, and alterations in the type or amount of connective tissue elements have been postulated to explain the increased outflow resistance seen in glaucoma cases (6). However, an understanding of the biochemical changes that actually contribute to this process has remained elusive.Previously, we described a highly expressed protein and related glycoprotein (55 and 66 kDa, respectively) found in the media of TM cell culture, but not in other cell types examined, after a prolonged exposure to dexamethasone (DEX) (2). We used this observation to define a cell culture model for "steroidinduced glaucoma" and elevated intraocular pressure due to corticosteroids. The extracellular induced proteins appeared as reasonable candidates for being involved in steroid glaucoma since the time course and dose response of their induction mimicked the intraocular pressure elevation and increased outflow resistance seen in patients receiving glucocorticoid (GC) therapy (7,8).Coincident with our res...
Studies of the effects of glucocorticoid (GC) and oxidative stress stimuli in differentiated cultures of human trabecular meshwork (HTM) cells have provided the rationale for our studies of a major new gene termed TIGR (trabecular meshwork inducible GC response). The TIGR clone was isolated by differential library screening using selection criteria based on the induction pattern of a new protein/glycoprotein found in HTM cultures after prolonged but not brief exposure to GCs. This GC induction patter matched the time course and dose response required for intraocular pressure elevation in patients receiving corticosteroids. The very large, progressive induction of TIGR combined with specific structural features of its cDNA suggested that TIGR should be considered a candidate gene for outflow obstruction in glaucoma. Among the properties of TIGR cDNA were a signal sequence for secretion, several structural features for interactions with glycosaminoglycans and other glycoproteins and putative sites for cell surface interactions. In addition, the leucine zippers in the structure were related to TIGR-TIGR oligomerization that was shown to occur with native and recombinant TIGR protein. The verification that TIGR was a major stress response protein in HTM cells following hydrogen peroxide (or phorbol esters) exposure provided a potential link between GC and oxidative mechanisms thought to be involved in glaucoma pathogenesis. Pharmacological evaluation showed that basic fíbroblast growth factory and transforming growth factor β decreased the GC induction of TIGR, and certain nonsteroidal anti-inflammatory drugs protected against both GC- and oxidation-induced stress responses in HTM cells. Our recent studies of TIGR’s genomic structure have shown motifs in the promoter region that suggest a basis by which multiple hormonal/environmental stimuli can regulate TIGR production and by which putative genetic alterations could lead to an overexpression of the protein. Further application of cell biology/biochemistry, molecular biology, genetic and histological approaches will be helpful in understanding the role of TIGR in different glaucoma syndromes.
Aqueous metal batteries routinely suffer from the dendritic growth at the anode, leading to significant capacity fading and ultimately, battery failure from short‐circuit. Herein, we utilize polyethylene glycol to regulate dendrite growth and improve the long‐term cycling stability of an aqueous rechargeable lithium/zinc battery. PEG200 in the electrolyte decreases the corrosion and chronoamperometric current densities of the zinc electrode up to four‐fold. Batteries with pre‐grown dendrites also perform significantly better when PEG is present in the electrolyte (41.4 mAh g−1 vs. 7.9 mAh g−1 after 1000 cycles). X‐ray diffraction and electron microscopy studies show that dendrites in the PEG‐containing electrolyte have been inhibited, leading to much smaller/smoother surface features than those of the control. The facile preparation process of the aqueous electrolyte combined with low cost and vast performance improvement in batteries of all sizes indicates high upscaling viability.
Regenerative medicine and tissue engineering have seen unprecedented growth in the past decade, driving the field of artificial tissue models towards a revolution in future medicine. Major progress has been achieved through the development of innovative biomanufacturing strategies to pattern and assemble cells and extracellular matrix (ECM) in three-dimensions (3D) to create functional tissue constructs. Bioprinting has emerged as a promising 3D biomanufacturing technology, enabling precise control over spatial and temporal distribution of cells and ECM. Bioprinting technology can be used to engineer artificial tissues and organs by producing scaffolds with controlled spatial heterogeneity of physical properties, cellular composition, and ECM organization. This innovative approach is increasingly utilized in biomedicine, and has potential to create artificial functional constructs for drug screening and toxicology research, as well as tissue and organ transplantation. Herein, we review the recent advances in bioprinting technologies and discuss current markets, approaches, and biomedical applications. We also present current challenges and provide future directions for bioprinting research.
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