EGCG, a Green Tea Catechin, as a Potential Therapeutic Agent for Symptomatic and Asymptomatic SARS-CoV-2 Infection.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has emerged to be the greatest threat to humanity in the modern world and has claimed nearly 2.2 million lives worldwide. The United States alone accounts for more than one fourth of 100 million COVID-19 cases across the globe. Although vaccination against SARS-CoV-2 has begun, its efficacy in preventing a new or repeat COVID-19 infection in immunized individuals is yet to be determined. Calls for repurposing of existing, approved, drugs that target the inflammatory condition in COVID-19 are growing. Our initial gene ontology analysis predicts a similarity between SARS-CoV-2 induced inflammatory and immune dysregulation and the pathophysiology of rheumatoid arthritis. Interestingly, many of the drugs related to rheumatoid arthritis have been found to be lifesaving and contribute to lower COVID-19 morbidity. We also performed in silico investigation of binding of epigallocatechin gallate (EGCG), a well-known catechin, and other catechins on viral proteins and identified papain-like protease protein (PLPro) as a binding partner. Catechins bind to the S1 ubiquitin-binding site of PLPro, which might inhibit its protease function and abrogate SARS-CoV-2 inhibitory function on ubiquitin proteasome system and interferon stimulated gene system. In the realms of addressing inflammation and how to effectively target SARS-CoV-2 mediated respiratory distress syndrome, we review in this article the available knowledge on the strategic placement of EGCG in curbing inflammatory signals and how it may serve as a broad spectrum therapeutic in asymptomatic and symptomatic COVID-19 patients.

Lead identification for the K-Ras protein: virtual screening and combinatorial fragment-based approaches.

OBJECTIVE: Kirsten rat sarcoma (K-Ras) protein is a member of Ras family belonging to the small guanosine triphosphatases superfamily. The members of this family share a conserved structure and biochemical properties, acting as binary molecular switches. The guanosine triphosphate-bound active K-Ras interacts with a range of effectors, resulting in the stimulation of downstream signaling pathways regulating cell proliferation, differentiation, and apoptosis. Efforts to target K-Ras have been unsuccessful until now, placing it among high-value molecules against which developing a therapy would have an enormous impact. K-Ras transduces signals when it binds to guanosine triphosphate by directly binding to downstream effector proteins, but in case of guanosine diphosphate-bound conformation, these interactions get disrupted. METHODS: In the present study, we targeted the nucleotide-binding site in the "on" and "off" state conformations of the K-Ras protein to find out suitable lead compounds. A structure-based virtual screening approach has been used to screen compounds from different databases, followed by a combinatorial fragment-based approach to design the apposite lead for the K-Ras protein. RESULTS: Interestingly, the designed compounds exhibit a binding preference for the "off" state over "on" state conformation of K-Ras protein. Moreover, the designed compounds' interactions are similar to guanosine diphosphate and, thus, could presumably act as a potential lead for K-Ras. The predicted drug-likeness properties of these compounds suggest that these compounds follow the Lipinski's rule of five and have tolerable absorption, distribution, metabolism, excretion and toxicity values. CONCLUSION: Thus, through the current study, we propose targeting only "off" state conformations as a promising strategy for the design of reversible inhibitors to pharmacologically inhibit distinct conformations of K-Ras protein.

Regulation of cardiac expression of the diabetic marker microRNA miR-29.

Diabetes mellitus (DM) is an independent risk factor for heart disease and its underlying mechanisms are unclear. Increased expression of diabetic marker miR-29 family miRNAs (miR-29a, b and c) that suppress the pro-survival protein Myeloid Cell Leukemia 1(MCL-1) is reported in pancreatic ß-cells in Type 1 DM. Whether an up-regulation of miR-29 family miRNAs and suppression of MCL-1 (dysregulation of miR-29-MCL-1 axis) occurs in diabetic heart is not known. This study tested the hypothesis that insulin regulates cardiac miR-29-MCL-1 axis and its dysregulation correlates with DM progression. In vitro studies with mouse cardiomyocyte HL-1 cells showed that insulin suppressed the expression of miR-29a, b and c and increased MCL-1 mRNA. Conversely, Rapamycin (Rap), a drug implicated in the new onset DM, increased the expression of miR-29a, b and c and suppressed MCL-1 and this effect was reversed by transfection with miR-29 inhibitors. Rap inhibited mammalian target of rapamycin complex 1 (mTORC1) signaling in HL-1 cells. Moreover, inhibition of either mTORC1 substrate S6K1 by PF-4708671, or eIF4E-induced translation by 4E1RCat suppressed MCL-1. We used Zucker diabetic fatty (ZDF) rat, a rodent model for DM, to test whether dysregulation of cardiac miR-29-MCL-1 axis correlates with DM progression. 11-week old ZDF rats exhibited significantly increased body weight, plasma glucose, insulin, cholesterol, triglycerides, body fat, heart weight, and decreased lean muscle mass compared to age-matched lean rats. Rap treatment (1.2 mg/kg/day, from 9-weeks to 15-weeks) significantly reduced plasma insulin, body weight and heart weight, and severely dysregulated cardiac miR-29-MCL1 axis in ZDF rats. Importantly, dysregulation of cardiac miR-29-MCL-1 axis in ZDF rat heart correlated with cardiac structural damage (disorganization or loss of myofibril bundles). We conclude that insulin and mTORC1 regulate cardiac miR-29-MCL-1 axis and its dysregulation caused by reduced insulin and mTORC1 inhibition increases the vulnerability of a diabetic heart to structural damage.

Unstimulated diagnostic marrow tap--a minimally invasive and reliable source for mesenchymal stem cells.

BMMNCs (bone marrow mononuclear cells) were isolated by density gradient centrifugation from unstimulated diagnostic marrow tap to propagate and characterize hBMSCs (human bone marrow stromal cells) and to explore their plasticity towards neuronal and other lineages. hBMSCs were characterized by flow cytometry for established markers, serially passaged and differentiated into adipo, osteo, chondro and neuronal lineages. Neural differentiation was analysed by RT-PCR (reverse transcriptase-PCR), ICC (immunocytochemistry) and Western blotting. The hBMSCs (n = 39) were spindle-shaped and immunoreactive for mesenchymal markers such as CD71, CD106, CD105, CD90 and Vimentin and negative for haematopoietic markers such as CD11c, CD34 and CD45. These cells showed differentiation into adipocytes, osteocytes and chondrocytes. Upon neuronal differentiation, hBMSCs expressed neuronal markers, i.e. beta-III tubulin, GAP43 (growth-associated proteins), neurofilament by ICC, RT-PCR and Western blotting. Our study demonstrates that minimal volumes of unstimulated diagnostic marrow tap forms a minimally invasive and reliable source for isolation of BMMNCs to establish cultures of mesenchymal stem cells and expand them. The plasticity observed in these cells towards mesenchymal (adipogenic, osteogenic and chondrocytic) and non-mesenchymal lineage (neural) substantiates the nature of mesenchymal stem cells and warrants further studies to evaluate their functional role.

Histocompatibility testing of cultivated human bone marrow stromal cells - a promising step towards pre-clinical screening for allogeneic stem cell therapy.

Mesenchymal stem cells (MSCs) lack major histocompatibility complex (MHC)-II and only show minimal MHC-I expression. Despite MSCs demonstrating T-cell anergy, there are no established methods to evaluate their suitability. It is crucial to evaluate the complete mismatch of MHC compatibility in view of the hypo-immunogenic nature and immunomodulatory properties of MSCs with respect to their proliferation potential (PP) and utility in terms of passage number. With bone marrow (BM) being the major source of MSCs, the use of these cells becomes even more complicated, due to many other receptors coming to fore and triggering alternative pathways. This prospective study included five BM aspirates for MSC cultures and five allogeneic peripheral blood mono nuclear cells (PBMNCs) from healthy volunteers. MHC compatibility was assessed by polymerase chain reaction-sequence specific primer (PCR-SSP). The PP and a T-cell response to MSCs was addressed in mixed cultures and evaluated on the basis of their stimulation index (SI). Allogeneic circulatory antibodies against the donor MSCs was performed by cytotoxicity assay. The PP of MSCs during interactions with PBMNCs (T-cells) demonstrated T-cell anergy and the response to circulatory antibodies was minimal, in consonance with other published reports. Although, the results are encouraging for potential clinical application of MSC transplantation, autologous is always preferable to allogeneic, at least until the long-term safety of these cells is established in clinical trials.