Factors regulating dynamics of angiotensin-converting enzyme-2 (ACE2), the gateway of SARS-CoV-2: Epigenetic modifications and therapeutic interventions by epidrugs.

Angiotensin-converting enzyme-2 (ACE2) is one of the major components of the renin-angiotensin system (RAS) and participates in the physiological functions of the cardiovascular system and lungs. Recent studies identified ACE2 as the receptor for the S-protein of the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and thus acts as the gateway for viral entry into the human body. Virus infection causes an imbalance in the RAS axis and induces acute lungs injury and fibrosis. Various factors regulate ACE2 expression patterns as well as control its epigenetic status at both transcription and translational levels. This review is mainly focused on the impact of environmental toxicants, drugs, endocrine disruptors, and hypoxia as controlling parameters for ACE2 expression and its possible modulation by epigenetic changes which are marked by DNA methylation, histone modifications, and micro-RNAs (miRNAs) profile. Furthermore, we have emphasized on interventions of various phytochemicals and bioactive compounds as epidrugs that regulate ACE2-S-protein interaction and thereby curb viral infection. Since ACE2 is an important component of the RAAS axis and a crucial entry point of SARS-CoV-2, the dynamics of ACE2 expression in response to various extrinsic and intrinsic factors are of contemporary relevance. We have collated updated information on ACE2 expression modulated by epidrugs, and urge to take over further studies on these important physiological regulators to unravel many more systemic linkages related to both metabolic and infectious diseases, in general and SARS-CoV-2 in particular for further development of targeted interventions.

Using Immunohistochemistry Without Linkers to Determine the Optimum Concentrations of Primary Antibodies for Immunofluorescence Staining of Formalin-fixed Paraffin-embedded Tissue Sections.

The use of immunofluorescence (IF) technique to detect and evaluate expression levels and localization of cellular proteins and other antigens of interest through the antibodies in their cellular or tissue context has become a standard approach among researchers. Optimizing primary antibody concentrations/dilutions is an essential step in the fluorescent antibody staining protocol. The steps in IF staining are similar to those of the immunohistochemistry (IHC) technique. The use of IHC technique to determine the optimal working dilutions of primary antibodies for IF staining of formalin-fixed paraffin-embedded (FFPE) tissues sections can minimize time wasting and cumbersome approach of using direct IF single labeling using variable dilutions of both primary and secondary antibodies. We used IHC staining technique to determine the working dilutions of the respective primary antibodies by staining 3-µm sections of recommended positive FFPE tissue sections using 3 different dilutions of the primary antibodies and an isotype control (used at the highest concentration). Digital images of sections stained were reviewed in ImageScope by a Consultant Pathologist for positivity, intensity, and histologic distribution. We adopted the IHC predetermined optimal dilutions of primary antibodies to CD4, CD8, CD16, CD21, CD56, CD68, CD163, FOXP3, and PD1 to carry out IF staining of FFPE tissue sections. This approach has helped to remove the complexities associated with grappling with 2 unknown to optimize for both the primary and secondary antibodies using IF technique.