Biology subjects

Shukla, R. K.

Publications and source records attributed to Shukla, R. K..

3 recordsLinked to original sources

bioRxiv2022

A NOVEL METHOD FOR BLOOD DETECTION USING FLUORESCENT DYE

Detection of the body fluid present at a crime scene is essential for any forensic investigation. Amongst all the body fluids (sweat, semen, vaginal fluid, saliva, etc.,) blood is the most frequently encountered evidence at the crime scene. Currently, reagents like benzidine, orthotoluidine, tetramethylbenzidine, phenolphthalein, leucomalachite green, luminol, and fluorescein are used to screen the presence of blood on different surfaces (porous/nonporous). Majority of these tests are based on colorimetric changes owing to the nature of hemoglobin to catalyze the oxidation of chromogenic compounds. Apart from aiding the investigation, these reagents show toxic behavior (DNA damage, carcinogenic, etc.) and false-positive results. Hence, to circumvent this issue, the present study attempts to develop a state-of-art methodology for preliminary blood detection and screening using fluorescein-derived 2,7-dichlorofluorescein di-acetate (DCFDA) dye. It is hereby proposed that the fluorescein-based dye can successfully detect blood and bloodstains aged up to 20 days. Moreover, supplemental experiments have suggested that the dye doesnt interfere with DNA integritycausing any damage to DNA. Parallelly, no false-positive reactions have been observed as tested against similar-looking substances.

biochemistry↗

bioRxiv2022

CD3e-immunotoxin spares CD62L(lo) Tregs and reshapes organ-specific T-cell composition by preferentially depleting CD3e(hi) T cells.

CD3-epsilon(CD3e) immunotoxins (IT), a promising precision reagent for various clinical conditions requiring effective depletion of T cells, often shows limited treatment efficacy for largely unknown reasons. Tissue-resident T cells that persist in peripheral tissues have been shown to play pivotal roles in local and systemic immunity, as well as transplant rejection, autoimmunity and cancers. The impact of CD3e-IT treatment on these local cells, however, remains poorly understood. Here, using a new murine testing model, we demonstrate a substantial enrichment of tissue-resident Foxp3+ Tregs following CD3e-IT treatment. Differential surface expression of CD3e among T-cell subsets appears to be a main driver of Treg enrichment in CD3e-IT treatment. The surviving Tregs in CD3e-IT-treated mice were mostly the CD3edimCD62Llo effector phenotype, but the levels of this phenotype markedly varied among different lymphoid and nonlymphoid organs. We also found notable variations in surface CD3e levels among tissue-resident T cells of different organs, and these variations drive CD3e-IT to uniquely reshape T-cell compositions in local organs. The functions of organs and anatomic locations (lymph nodes) also affected the efficacy of CD3e-IT. The multi-organ pharmacodynamics of CD3e-IT and potential treatment resistance mechanisms identified in this study may generate new opportunities to further improve this promising treatment.

immunology↗

bioRxiv2021

Conformation of HIV-1 Envelope governs rhesus CD4 usage and simian-human immunodeficiency virus replication

Infection of rhesus macaques with simian-human immunodeficiency viruses (SHIVs) is the preferred model system for vaccine development because SHIVs encode HIV-1 envelope glycoproteins (Env) - a key target of HIV-1 neutralizing antibodies. Since the goal of vaccines is to prevent new infections, SHIVs encoding circulating HIV-1 Env are desired as challenge viruses. Development of such biologically relevant SHIVs has been challenging as they fail to infect rhesus macaques, mainly because most circulating HIV-1 Env do not use rhesus CD4 (rhCD4) receptor for viral entry. Most primary HIV-1 Env exist in a closed conformation and occasionally transit to downstream, open conformation through an obligate intermediate conformation. Here, we provide genetic evidence that open Env conformations can overcome the rhCD4 entry barrier and increase replication of SHIVs in rhesus lymphocytes. Consistent with prior studies, we found that circulating HIV-1 Env do not use rhCD4 efficiently for viral entry. However, using HIV-1 Env with single amino acid substitutions that alter their conformational state, we found that transitions to intermediate and open Env conformation allow usage of physiological levels of rhCD4 for viral entry. We engineered these single amino acid substitutions in the transmitted/founder HIV-1BG505 Env encoded by SHIV-BG505 and found that open Env conformation enhances SHIV replication in rhesus lymphocytes. Lastly, CD4-mediated SHIV pull-down, sensitivity to soluble CD4, and fusogenicity assays indicated that open Env conformation promotes efficient rhCD4 binding and viral-host membrane fusion. These findings identify conformational state of HIV-1 Env as a major determinant for rhCD4 usage, viral fusion, and SHIV replication. ImportanceRhesus macaques are critical animal model for preclinical testing of HIV-1 vaccine and prevention approaches. However, HIV-1 does not replicate in rhesus macaques, and thus chimeric simian-human immunodeficiency viruses (SHIVs), which encode HIV-1 envelope glycoproteins, are used as surrogate challenge viruses to infect rhesus macaques for modeling HIV-1 infection. Development of SHIVs encoding envelope from clinically relevant, circulating HIV-1 variants has been extremely challenging as such SHIVs replicate poorly, if at all, in rhesus lymphocytes. This is because most circulating HIV-1 envelope do not use rhesus CD4 efficiently for viral entry. In this study, we identify conformational state of HIV-1 envelope as a key determinant for rhesus CD4 usage, viral-host membrane fusion, and SHIV replication in rhesus lymphocytes.

microbiology↗