Agitation does not induce fibrillation in reduced hen egg-white lysozyme at physiological temperature and pH.

Several proteins and peptides tend to form an amyloid fibril, causing a range of unrelated diseases, from neurodegenerative to certain types of cancer. In the native state, these proteins are folded and soluble. However, these proteins acquired ß-sheet amyloid fibril due to unfolding and aggregation. The conversion mechanism from well-folded soluble into amorphous or amyloid fibril is not well understood yet. Here, we induced unfolding and aggregation of hen egg-white lysozyme (HEWL) by reducing agent dithiothreitol and applied mechanical sheering force by constant shaking (1000 rpm) on the thermostat for 7 days. Our turbidity results showed that reduced HEWL rapidly formed aggregates, and a plateau was attained in nearly 5 h of incubation in both shaking and non-shaking conditions. The turbidity was lower in the shaking condition than in the non-shaking condition. The thioflavin T binding and transmission electron micrographs showed that reduced HEWL formed amorphous aggregates in both conditions. Far-UV circular dichroism results showed that reduced HEWL lost nearly all alpha-helical structure, and ß-sheet secondary structure was not formed in both conditions. All the spectroscopic and microscopic results showed that reduced HEWL formed amorphous aggregates under both conditions.

Potential of green-synthesized ZnO NPs against human ovarian teratocarcinoma: an in vitro study.

BACKGROUND: Ovarian cancer leads to devastating outcomes, and its treatment is highly challenging. At present, there is a lack of clinical symptoms, well-known sensitivity biomarkers, and patients are diagnosed at an advanced stage. Currently, available therapeutics against ovarian cancer are inefficient, costly, and associated with severe side effects. The present study evaluated the anticancer potential of zinc oxide nanoparticles (ZnO NPs) that were successfully biosynthesized in an ecofriendly mode using pumpkin seed extracts. METHODS AND RESULTS: The anticancer potential of the biosynthesized ZnO NPs was assessed using an in vitro human ovarian teratocarcinoma cell line (PA-1) by well-known assays such as MTT assay, morphological alterations, induction of apoptosis, measurement of reactive oxygen species (ROS) production, and inhibition of cell adhesion/migration. The biogenic ZnO NPs exerted a high level of cytotoxicity against PA-1 cells. Furthermore, the ZnO NPs inhibited cellular adhesion and migration but induced ROS production and cell death through programmed cell death. CONCLUSION: The aforementioned anticancer properties highlight the therapeutic utility of ZnO NPs in ovarian cancer treatment. However, further research is recommended to envisage their mechanism of action in different cancer models and validation in a suitable in vivo system.

Redox Status, Immune Alterations, Histopathology, and Micronuclei Induction in Labeo rohita Dwelling in Polluted River Water.

In this study, we measured various parameters of oxidative stress, immune response, and abnormalities in the erythrocyte nucleus of Labeo rohita inhabiting the polluted Kshipra River, India. The river water contains heavy metals in this order: Ni > Fe > Cd > Cr > Mn > Zn > Cu. Fe showed the highest accumulation in gills, liver, and gut, whereas Ni (gills and gut) and Cd (liver) were lowest accumulated. The superoxide dismutase (SOD) and catalase (CAT) were found to be increased significantly (p < 0.05) in the gills (SOD: 211%; CAT: 150%), liver (SOD: 447%; CAT: 304%), and gut (SOD: 98.11%; CAT: 58.69%) in comparison with the reference fish. However, glutathione S transferase (GST) showed significantly (p < 0.05) higher activity in the gills (25.5%) but lower activity in the liver (- 49.22%) and the gut (- 30.57%). Moreover, reduced glutathione (GSH) decreased significantly (p < 0.05) in the gills (- 46.66%), liver (- 33.20%), and gut (- 39.87%). Despite the active response of the antioxidant enzymes, the highest lipid peroxidation was observed in the liver (463%). The effect of heavy metals was also observed on the immunity of the fish, causing immunosuppression as evident by significantly (p < 0.05) lower values of acid phosphatase (- 50%), myeloperoxidase (- 48.33%), and nitric oxide synthase (- 50%) in serum. Histopathological findings showed gill lamellae shortening, hyperplasia, club-shaped lamellar tip in exposed gills and necrosis, vacuolization, and pyknosis in the exposed liver. Furthermore, polluted river water was also found to induce micronuclei (2.1%) and lobed nuclei (0.72%) in erythrocytes (0.65%). These results indicate the potential of heavy metal-induced oxidative stress and other forms of stress in inhabiting fish, highlighting the need to control the pollution of this river water.

Effect of Antihypertensive Drug (Chlorothiazide) on Fibrillation of Lysozyme: A Combined Spectroscopy, Microscopy, and Computational Study.

Amyloid fibrils abnormally accumulate together in the human body under certain conditions, which can result in lethal conditions. Thus, blocking this aggregation may prevent or treat this disease. Chlorothiazide (CTZ) is a diuretic and is used to treat hypertension. Several previous studies suggest that diuretics prevent amyloid-related diseases and reduce amyloid aggregation. Thus, in this study we examine the effects of CTZ on hen egg white lysozyme (HEWL) aggregation using spectroscopic, docking, and microscopic approaches. Our results showed that under protein misfolding conditions of 55 °C, pH 2.0, and 600 rpm agitation, HEWL aggregated as evidenced by the increased turbidity and Rayleigh light scattering (RLS). Furthermore, thioflavin-T, as well as trans electron microscope (TEM) analysis confirmed the formation of amyloid structures. An anti-aggregation effect of CTZ is observed on HEWL aggregations. Circular dichroism (CD), TEM, and Thioflavin-T fluorescence show that both CTZ concentrations reduce the formation of amyloid fibrils as compared to fibrillated. The turbidity, RLS, and ANS fluorescence increase with CTZ increasing. This increase is attributed to the formation of a soluble aggregation. As evidenced by CD analysis, there was no significant difference in α-helix content and ß-sheet content between at 10 µM CTZ and 100 µM. A TEM analysis of HEWL coincubated with CTZ at different concentrations validated all the above-mentioned results. The TEM results show that CTZ induces morphological changes in the typical structure of amyloid fibrils. The steady-state quenching study demonstrated that CTZ and HEWL bind spontaneously via hydrophobic interactions. HEWL-CTZ also interacts dynamically with changes in the environment surrounding tryptophan. Computational results revealed the binding of CTZ to ILE98, GLN57, ASP52, TRP108, TRP63, TRP63, ILE58, and ALA107 residues in HEWL via hydrophobic interactions and hydrogen bonds with a binding energy of -6.58 kcal mol-1. We suggest that at 10 µM and 100 µM, CTZ binds to the aggregation-prone region (APR) of HEWL and stabilizes it, thus preventing aggregation. Based on these findings, we can conclude that CTZ has antiamyloidogenic activity and can prevent fibril aggregation.

Anticancer Potential of Sulfonamide Moieties via In-Vitro and In-Silico Approaches: Comparative Investigations for Future Drug Development.

Several kinds of anticancer drugs are presently commercially accessible, but low efficacy, solubility, and toxicity have reduced the overall therapeutic indices. Thus, the search for promising anticancer drugs continues. The interactions of numerous essential anticancer drugs with DNA are crucial to their biological functions. Here, the anticancer effects of N-ethyl toluene-4-sulphonamide (8a) and 2,5-Dichlorothiophene-3-sulphonamide (8b) on cell lines from breast and cervical cancer were investigated. The study also compared how these substances interacted with the hearing sperm DNA. The most promising anticancer drug was identified as 2,5-Dichlorothiophene-3-sulfonamide (8b), which showed GI50 of 7.2 ± 1.12 µM, 4.62 ± 0.13 µM and 7.13 ± 0.13 µM against HeLa, MDA-MB231 and MCF-7 cells, respectively. Moreover, it also exhibited significant electrostatic and non-electrostatic contributions to the binding free energy. The work utilized computational techniques, such as molecular docking and molecular dynamic (MD) simulations, to demonstrate the strong cytotoxicity of 2,5-Dichlorothiophene-3-sulfamide (8b) in comparison to standard Doxorubicin and cisplatin, respectively. Molecular docking experiments provided additional support for a role for the minor groove in the binding of the 2,5-Dichlorothiophene-3-sulfamide (8b)-DNA complex. The molecular docking studies and MD simulation showed that both compounds revealed comparable inhibitory potential against standard Doxorubicin and cisplatin. This study has the potential to lead to the discovery of new bioactive compounds for use in cancer treatment, including metallic and non-metallic derivatives of 2,5-Dichlorothiophene-3-sulfonamide (8b). It also emphasizes the worth of computational approaches in the development of new drugs and lays the groundwork for future research.

Toxicity Study and Binding Analysis of Newly Synthesized Antifungal N-(4-aryl/cyclohexyl)-2-(pyridine-4-yl carbonyl) hydrazinecarbothioamide Derivative with Bovine Serum Albumin.

The presence of the p-aryl/cyclohexyl ring in the N-(4-aryl/cyclohexyl)-2-(pyridine-4-yl carbonyl) hydrazine carbothioamide derivative (2C) is reported to enhance the antifungal properties when compared to those of itraconazole. Serum albumins present in plasma bind and transport ligands, including pharmaceuticals. This study explored 2C interactions with BSA using spectroscopic methods such as fluorescence and UV-visible spectroscopy. In order to acquire a deeper comprehension of how BSA interacts with binding pockets, a molecular docking study was carried out. The fluorescence of BSA was quenched by 2C via a static quenching mechanism since a decrease in quenching constants was observed from 1.27 × 105 to 1.14 × 105. Thermodynamic parameters indicated hydrogen and van der Waals forces responsible for the BSA-2C complex formation with binding constants ranging between 2.91 × 105 and 1.29 × 105, which suggest a strong binding interaction. Site marker studies displayed that 2C binds to BSA's subdomains IIA and IIIA. Molecular docking studies were conducted to further comprehend the molecular mechanism of the BSA-2C interaction. The toxicity of 2C was predicted by Derek Nexus software. Human and mammalian carcinogenicity and skin sensitivity predictions were associated with a reasoning level of equivocal, inferring 2C to be a potential drug candidate.

Evaluation of the Possible Pathways Involved in the Protective Effects of Quercetin, Naringenin, and Rutin at the Gene, Protein and miRNA Levels Using In-Silico Multidimensional Data Analysis.

Flavonoids are secondary metabolites that are non-essential for plant growth or survival, and they also provide numerous health benefits to humans. They are antioxidants that shield plants from the ill effects of ultraviolet light, pests, and diseases. They are beneficial to health for several reasons, including lowering inflammation, boosting cardiovascular health, and lowering cancer risk. This study looked into the physicochemical features of these substances to determine the potential pharmacological pathways involved in their protective actions. Potential targets responsible for the protective effects of quercetin, naringenin, and rutin were identified with SwissADME. The associated biological processes and protein-protein networks were analyzed by using the GeneMANIA, Metascape, and STRING servers. All the flavonoids were predicted to be orally bioavailable, with more than 90% targets as enzymes, including kinases and lyases, and with common targets such as NOS2, CASP3, CASP9, CAT, BCL2, TNF, and HMOX1. TNF was shown to be a major target in over 250 interactions. To extract the "biological meanings" from the MCODE networks' constituent parts, a GO enrichment analysis was performed on each one. The most important transcription factors in gene regulation were RELA, NFKB1, PPARG, and SP1. Treatment with quercetin, naringenin, or rutin increased the expression and interaction of the microRNAs' hsa-miR-34a-5p, hsa-miR-30b-5p, hsa-let-7a-5p, and hsa-miR-26a-1-3p. The anticancer effects of hsa-miR-34a-5p have been experimentally confirmed. It also plays a critical role in controlling other cancer-related processes such as cell proliferation, apoptosis, EMT, and metastasis. This study's findings might lead to a deeper comprehension of the mechanisms responsible for flavonoids' protective effects and could present new avenues for exploration.

Acetophenone-Based 3,4-Dihydropyrimidine-2(1H)-Thione as Potential Inhibitor of Tyrosinase and Ribonucleotide Reductase: Facile Synthesis, Crystal Structure, In-Vitro and In-Silico Investigations.

The acetophenone-based 3,4-dihydropyrimidine-2(1H)-thione was synthesized by the reaction of 4-methylpent-3-en-2-one (1), 4-acetyl aniline (2) and potassium thiocyanate. The spectroscopic analysis including: FTIR, 1H-NMR, and single crystal analysis proved the structure of synthesized compound (4), with the six-membered nonplanar ring in envelope conformation. In crystal structure, the intermolecular N-H ⋯ S and C-H ⋯ O hydrogen bonds link the molecule in a two-dimensional manner which is parallel to (010) the plane enclosing R22 (8) and R22 (10) ring motifs. After that, the Hirshfeld surfaces and their related two-dimensional fingerprint plots were used for thorough investigation of intermolecular interactions. According to Hirshfeld surface analysis, the most substantial contributions to the crystal packing are from H ⋯ H (59.5%), H ⋯ S/S ⋯ H (16.1%), and H ⋯ C/C ⋯ H (13.1%) interactions. The electronic properties and stability of the compound were investigated through density functional theory (DFT) studies using B3LYP functional and 6-31G* as a basis set. The compound 4 displayed the high chemical reactivity with chemical softness of 2.48. In comparison to the already reported known tyrosinase inhibitor, the newly synthesized derivatives exhibited almost seven-fold better inhibition of tyrosinase (IC50 = 1.97 µM), which was further supported by molecular docking studies. The compound 4 inside the active pocket of ribonucleotide reductase (RNR) exhibited a binding energy of -19.68 kJ/mol, and with mammalian deoxy ribonucleic acid (DNA) it acts as an effective DNA groove binder with a binding energy of -21.32 kJ/mol. The results suggested further exploration of this compound at molecular level to synthesize more potential leads for the treatment of cancer.

Binding Studies of Caffeic and p-Coumaric Acid with α-Amylase: Multispectroscopic and Computational Approaches Deciphering the Effect on Advanced Glycation End Products (AGEs).

Alpha-amylase (α-amylase) is a key player in the management of diabetes and its related complications. This study was intended to have an insight into the binding of caffeic acid and coumaric acid with α-amylase and analyze the effect of these compounds on the formation of advanced glycation end-products (AGEs). Fluorescence quenching studies suggested that both the compounds showed an appreciable binding affinity towards α-amylase. The evaluation of thermodynamic parameters (ΔH and ΔS) suggested that the α-amylase-caffeic/coumaric acid complex formation is driven by van der Waals force and hydrogen bonding, and thus complexation process is seemingly specific. Moreover, glycation and oxidation studies were also performed to explore the multitarget to manage diabetes complications. Caffeic and coumaric acid both inhibited fructosamine content and AGE fluorescence, suggesting their role in the inhibition of early and advanced glycation end-products (AGEs). However, the glycation inhibitory potential of caffeic acid was more in comparison to p-coumaric acid. This high antiglycative potential can be attributed to its additional -OH group and high antioxidant activity. There was a significant recovery of 84.5% in free thiol groups in the presence of caffeic acid, while coumaric attenuated the slow recovery of 29.4% of thiol groups. In vitro studies were further entrenched by in silico studies. Molecular docking studies revealed that caffeic acid formed six hydrogen bonds (Trp 59, Gln 63, Arg 195, Arg 195, Asp 197 and Asp 197) while coumaric acid formed four H-bonds with Trp 59, Gln 63, Arg 195 and Asp 300. Our studies highlighted the role of hydrogen bonding, and the ligands such as caffeic or coumaric acid could be exploited to design antidiabetic drugs.

Binding and drug displacement study of colchicine and bovine serum albumin in presence of azithromycin using multispectroscopic techniques and molecular dynamic simulation.

The binding and displacement interaction of colchicine and azithromycin to the model transport protein bovine serum albumin (BSA) was evaluated in this study. Azithromycin, a macrolide antibiotic, has antiviral properties and hence, has been used concomitantly with hydroxychloroquine against SARS-CoV-2. Colchicine, a natural plant product is used to treat and prevent acute gout flares. Some macrolide antibiotics are reported to have fatal drug-drug interactions with colchicine. The displacement interaction between colchicine and azithromycin on binding to BSA was evaluated using spectroscopic techniques, molecular docking and molecular dynamic simulation studies. The binding constant recorded for the binary system BSA-colchicine was 7.44 × 104 whereas, the binding constant for the ternary system BSA-colchicine in presence of azithromycin was 7.38 × 104 and were similar. Azithromycin didn't bind to BSA neither did it interfere in binding of colchicine. The results from molecular docking studies also led to a similar conclusion that azithromycin didn't interfere in the binding of colchicine to BSA. These findings are important since there is possibility of serious adverse event with co-administration of colchicine and azithromycin in patients with underlying gouty arthritis and these patients need to be continuously monitored for colchicine toxicity.