Computational and physicochemical insight into 4-hydroxy-2-nonenal induced structural and functional perturbations in human low-density lipoprotein.

Lipid peroxidation (LPO) is a biological process that frequently occurs under physiological conditions. Undue oxidative stress increases the level of LPO; which may further contribute to the development of cancer. 4-Hydroxy-2-nonenal (HNE), one of the principal by-products of LPO, is present in high concentrations in oxidatively stressed cells. HNE rapidly reacts with various biological components, including DNA and proteins; however, the extent of protein degradation by lipid electrophiles is not well understood. The influence of HNE on protein structures will likely have a considerable therapeutic value. This research elucidates the potential of HNE, one of the most researched phospholipid peroxidation products, in modifying low-density lipoprotein (LDL). In this study, we tracked the structural alterations in LDL by HNE using various physicochemical techniques. To comprehend the stability, binding mechanism and conformational dynamics of the HNE-LDL complex, computational investigations were carried out. LDL was altered in vitro by HNE, and the secondary and tertiary structural alterations were examined using spectroscopic methods, such as UV-visible, fluorescence, circular dichroism and fourier transform infrared spectroscopy. Carbonyl content, thiobarbituric acid-reactive-substance (TBARS) and nitroblue tetrazolium (NBT) reduction assays were used to examine changes in the oxidation status of LDL. Thioflavin T (ThT), 1-anilinonaphthalene-8-sulfonic (ANS) binding assay and electron microscopy were used to investigate aggregates formation. According to our research, LDL modified by HNE results in changes in structural dynamics, oxidative stress and the formation of LDL aggregates. The current investigation must characterize HNE's interactions with LDL and comprehend how it can change their physiological or pathological functions.Communicated by Ramaswamy H. Sarma.

4-Chloro-1,2-phenylenediamine induced structural perturbation and genotoxic aggregation in human serum albumin.

4-Chloro-1,2-phenylenediamine (4-Cl-OPD) is a halogenated aromatic diamine used as a precursor in permanent hair color production. Despite its well-documented mutagenic and carcinogenic effects in various in vitro and in vivo models, its role in fibrillar aggregate formation and their genotoxic effect in therapeutic proteins has received less attention. The significance of human serum albumin (HSA) arises from its involvement in bio-regulatory and transport processes. HSA misfolding and aggregation are responsible for some of the most frequent neurodegenerative disorders. We used various complementary approaches to track the formation of amyloid fibrils and their genotoxic effect. Molecular dynamics study demonstrated the complex stability. The impact of 4-Cl-OPD on the structural dynamics of HSA was confirmed by Raman spectroscopy, X-ray diffraction, HPLC and SDS-PAGE. Fibrilllar aggregates were investigated using Congo red assay, DLS, and SEM. The genotoxic nature of 4-Cl-OPD was confirmed using plasmid nicking assay and DAPI staining, which revealed DNA damage and cell apoptosis. 4-Cl-OPD provides a model system for studying fibrillar aggregation and their genotoxic potential in the current investigation. Future studies should investigate the inhibition of the aggregation/fibrillation process, which may yield valuable clinical insights.

Withania somnifera (L.) Dunal as Add-On Therapy for COPD Patients: A Randomized, Placebo-Controlled, Double-Blind Study.

Background: The current gold-standard therapies for chronic obstructive pulmonary disease (COPD) lack disease-modifying potential and exert adverse side effects. Moreover, COPD patients are at a higher risk of severe outcomes if they get infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, the cause of the current epidemic. This is the first study to document clinical research on an adaptogenic and steroidal activity-containing herb as a complementary medicine for COPD treatment. Objective: We aimed to evaluate the efficacy of Withania somnifera (L.) Dunal [Solanaceae] (WS) as an add-on therapy for COPD patients. Methods: A randomized, placebo-controlled, and double-blind clinical study was conducted. A total of 150 patients were randomly assigned to three groups: control, placebo, and WS group. In addition to conventional medicines, WS root capsules or starch capsules were given twice a day to the WS group and the placebo group, respectively. Their lung functioning, quality of life, exercise tolerance, systemic oxidative stress (OS), and systemic inflammation were assessed before and after 12 weeks of intervention. WS root phytochemicals were identified by LC-ESI-MS. The inhibitory activity of these phytochemicals against angiotensin-converting enzyme 2 (ACE-2); the SARS-CoV-2 receptor; myeloperoxidase (MPO); and interleukin-6 (IL-6) was evaluated by in silico docking to investigate the mechanism of action of WS. Results: The pulmonary functioning, quality of life, and exercise tolerance improved, and inflammation reduced notably the most in the WS group. Systemic oxidative stress subsided significantly only in the WS group. Although a minor placebo effect was observed in the SGRQ test, but it was not present in other tests. Withanolides found in the WS roots demonstrated substantial inhibitory activity against the proteins ACE-2, MPO, and IL-6, compared to that of a standard drug or known inhibitor. Moreover, FEV1% predicted had significant correlation with systemic antioxidative status (positive correlation) and malondialdehyde (MDA, negative correlation), suggesting that the antioxidative potential of WS has significant contribution to improving lung functioning. Conclusion: Our study clinically demonstrated that WS root when given along with conventional drugs ameliorated COPD significantly more in comparison to the conventional drugs alone, in GOLD 2 and 3 categories of COPD patients. In silico, it has potent inhibitory activity against SARS-CoV-2 receptor, ACE-2, MPO, and IL-6.

Hydroxyl radical induced structural perturbations make insulin highly immunogenic and generate an auto-immune response in type 2 diabetes mellitus.

Reactive oxygen species (ROS) cause oxidative damage to proteins and generate deleterious by-products which induce a breakdown of immune tolerance and produce antibodies against host macromolecules with implication in human diseases. This study characterizes the hydroxyl radical (OH) modifications of insulin, evaluates its cytotoxicity and immunogenicity, and probes its role in type 2 diabetes (T2DM) autoimmunity. The results demonstrate susceptibility of insulin to modifications induced by OH, causing exposure of its chromophoric aromatic amino acid residues, quenching of tyrosine fluorescence intensity, loss of α-helix and gain in ß content. Modification causes re-arrangement of native interactions of the aromatic residues in insulin. It enhanced the carbonyl content in insulin, exposed its hydrophobic patches and generated non-fibrillar, amorphous type of aggregates that are cytotoxic in nature. Native insulin induced low titre antibodies in immunized rabbits, whereas OH modified insulin generated a strong immune response. Competitive ELISA studies showed high specificity of antibodies generated against OH modified insulin towards the modified protein. Cross reaction studies showed the presence of common antigenic determinants on various oxidised proteins. Since T2DM patients show increased ROS production, oxidation of insulin is expected to occur, which might amplify autoimmune reactions against insulin. True to the assumption, direct binding ELISA showed the presence of anti-OH insulin circulating antibodies in T2DM patients which are specific for the oxidized insulin. In conclusion, insulin loses structural integrity to OH, forms cytotoxic amorphous aggregates, turns highly immunogenic and elicits humoral response in T2DM patients.