35 research outputs found

    ROS is the major player in regulating altered autophagy and lifespan in <i>sin-3</i> mutants of <i>C. elegans</i>

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    <p>SIN3, a transcriptional corepressor has been implicated in varied functions both as transcription activator and repressor. Recent studies associated Sin3 with the macroautophagic/autophagic process as a negative regulator of Atg8 and Atg32. Though the role of SIN3 in autophagy is being explored, little is known about the overall effect of <i>SIN3</i> deletion on the survival of an organism. In this study using a <i>Caenorhabditis elegans sin-3(tm1279);him-5(e1490)</i> strain, we demonstrate that under <i>in vivo</i> conditions SIN-3 differentially modulates autophagy and lifespan. We provide evidence that the enhanced autophagy and decreased lifespan observed in <i>sin-3</i> deletion mutants is dependent on ROS and intracellular oxidative stress. Inability of the mutant worms to maintain redox balance along with dysregulation of enzymatic antioxidants, depletion of GSH and NADP reserves and elevation of ROS markers compromises the longevity of the worms. It is possible that the enhanced autophagic process observed in <i>sin-3(tm1279);him-5(e1490)</i> worms is required to compensate for oxidative stress generated in these worms.</p> <p><b>Abbreviations:</b><i>cat</i>: catalase; DCFDA: 2╩╣,7╩╣-dichlorodihydrofluoroscein diacetate; GSH: reduced glutathione; GSSG: oxidized glutathione; H<sub>2</sub>O<sub>2</sub>: hydrogen peroxide; HDAC: Histone deacetylase; HID: HDAC interacting domain; <i>him-5</i>: high incidence of males; HLH-30: Helix Loop Helix-30; HNE: 4-hydroxyl-2-noneal; LIPL: LIPase Like; MDA: malondialdehyde; NGM: nematode growth medium; PAH: paired amphipathic ╬▒-helix; PE: phosphatidylethanolamine; RFU: relative fluorescence unit; ROS: reactive oxygen species; <i>sin-3/SIN3</i>: yeast Switch Independent; SOD: superoxide dismutase; NADP: nicotinamide adenine dinucleotide phosphate; SQST-1: SeQueSTosome related-1; ATG: AuTophaGy related</p

    Structure-based drug discovery to identify SARS-CoV2 spike proteinÔÇôACE2 interaction inhibitors

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    After the emergence of the COVID-19 pandemic in late 2019, the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has undergone a dynamic evolution driven by the acquisition of genetic modifications, resulting in several variants that are further classified as variants of interest (VOIs), variants under monitoring (VUM) and variants of concern (VOC) by World Health Organization (WHO). Currently, there are five SARS-CoV-2 VOCs (Alpha, Beta, Delta, Gamma and Omicron), two VOIs (Lambda and Mu) and several other VOIs that have been reported globally. In this study, we report a natural compound, Curcumin, as the potential inhibitor to the interactions between receptor binding domain (RBD(S1)) and human angiotensin-converting enzyme 2 (hACE2) domains and showcased its inhibitory potential for the Delta and Omicron variants through a computational approach by implementing state of the art methods. The study for the first time revealed a higher efficiency of Curcumin, especially for hindering the interaction between RBD(S1) and hACE-2 domains of Delta and Omicron variants as compared to other lead compounds. We investigated that the mutations in the RBD(S1) of VOC especially Delta and Omicron variants affect its structure compared to that of the wild type and other variants and therefore altered its binding to the hACE2 receptor. Molecular docking and molecular dynamics (MD) simulation analyses substantially supported the findings in terms of the stability of the docked complexes. This study offers compelling evidence, warranting a more in-depth exploration into the impact of these alterations on the binding of identified drug molecules with the Spike protein. Further investigation into their potential therapeutic effects in vivo is highly recommended. Communicated by Ramaswamy H. Sarma</p

    qRT-PCR analysis of Heat shock protein70 gene expressions at mRNA level and indirect ELISA analysis at protein level.

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    <p>(A) represents the scattered plot of levels of HSP70 gene expression at mRNA level in the individual study subjects by qRT-PCR. The relative HSP70 gene expression in patients and controls is expressed in delta-CT values. Results were expressed as densitometric ratio (HSP70 to 18S rRNA gene) in patients and controls group ┬▒ SD. P < 0.001. Higher delta-CT value deflects the lower gene expression. (B) represents the scattered plot of individual data of HSP70 expression at protein level by indirect ELISA technique in the study subjects. P < 0.0001.</p

    Heat shock protein70 mRNA expression in study subjects.

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    <p>Fig 3 represents the expression of HSP 70 at mRNA levels in study subjects. Results were expressed as average delta-CT value in patients and controls group ┬▒ SD. P < 0.0001 is considered to be significant.</p

    Multivariate logistic regression analysis of different parameters in the study subjects.

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    <p>Multivariate logistic regression analysis of different parameters in the study subjects.</p

    Correlation study between different parameters in the study subjects.

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    <p>Correlation study between different parameters in the study subjects.</p

    Plasma levels of inflammatory markers.

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    <p>(A) represents the scattered plot of levels of interleukin-6 in the individual study subjects. (B) represents the scattered plot of individual data of tumor necrosis factor-alpha in the study subjects. (C) represents the scattered plot of individual data of C-reactive protein in the study subjects. (D) represents the scattered plot of individual data of p53 in the study subjects.</p

    A Novel Methodology for Enhanced and Consistent Heterologous Expression of Unmodified Human Cytochrome P450 1B1 (CYP1B1)

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    <div><p>Cytochrome P450 1B1 (CYP1B1) is a universal cancer marker and is implicated in many other disorders. Mutations in CYP1B1 are also associated with childhood blindness due to primary congenital glaucoma (PCG). To understand the CYP1B1 mediated etiopathology of PCG and pathomechanism of various cancers, it is important to carry out its functional studies. Heterologous expression of CYP1B1 in prokaryotes is imperative because bacteria yield a higher amount of heterologous proteins in lesser time and so the expressed protein is ideal for functional studies. In such expression system there is no interference by other eukaryotic proteins. But the story is not that simple as expression of heterologous CYP1B1 poses many technical difficulties. Investigators have employed various modifications/deletions of CYP N-terminus to improve CYP1B1 expression. However, the drawback of these studies is that it changes the original protein and, as a result, invalidates functional studies. The present study examines the role of various conditions and reagents in successful and consistent expression of sufficient quantities of unmodified/native human CYP1B1 in <i>E. coli</i>. We aimed at expressing CYP1B1 in various strains of <i>E. coli</i> and in the course developed a protocol that results in high expression of unmodified protein sufficient for functional/biophysical studies. We examined CYP1B1 expression with respect to different expression vectors, bacterial strains, types of culture media, time, Isopropyl ╬▓-D-1-thiogalactopyranoside concentrations, temperatures, rotations per minute, conditioning reagents and the efficacy of a newly described technique called double colony selection. We report a protocol that is simple, easy and can be carried out in any laboratory without the requirement of a fermentor. Though employed for CYP1B1 expression, this protocol can ideally be used to express any eukaryotic membrane protein.</p></div
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