Catharanthine, an anticancer vinca alkaloid: an in silico and in vitro analysis of the autophagic system as the major mechanism of cell death in liver HepG2 cells.

Catharanthine, a component of the anticancer drug vinblastine along with vindoline, disrupts the cell cycle by interfering with mitotic spindle formation. Apart from their antioxidant properties, vinca alkaloids like catharanthine inhibit phosphodiesterase activity and elevate intracellular cAMP levels. The aim of this study was to investigate how catharantine affects apoptosis and autophagy. This study conducted experiments on HepG2 liver carcinoma cells with varying doses of catharanthine to evaluate cell death rates and viability and determine the IC50 concentration via MTT assays. The apoptotic and autophagic effects of catharanthine were assessed using flow cytometry with annexin V and PI staining, while the expression of autophagy-related genes was analyzed through quantitative PCR. Additionally, molecular docking and molecular dynamics simulations were employed to further investigate catharanthine's impact on autophagy mechanisms. The study showed that catharanthine reduced oxidative stress and triggered apoptosis in HepG2 cells in a dose-dependent manner. Catharanthine also upregulated the expression of autophagy-related genes like LC3, Beclin1, and ULK1. Notably, catharanthine increased sirtuin-1 levels, a known autophagy inducer, while decreasing Akt expression compared to untreated cells. Molecular docking results indicated rapamycin had a stronger binding affinity with FRB (-10.7 KJ/mol-1) than catharanthine (-7.3 KJ/mol-1). Additionally, molecular dynamics simulations revealed that catharanthine interacted effectively with the FRB domain of mTOR, displaying stability and a strong binding affinity, although not as potent as rapamycin. In summary, besides its cytotoxic and pro-apoptotic effects, catharanthine activates autophagy signaling pathways and induces autophagic necrosis by inhibiting mTOR.

Implications of ALS-Associated Mutations on Biochemical and Biophysical Features of hSOD1 and Aggregation Formation.

One of the recognized motor neuron degenerative disorders is amyotrophic lateral sclerosis (ALS). By now, several mutations have been reported and linked to ALS patients, some of which are induced by mutations in the human superoxide dismutase (hSOD1) gene. The ALS-provoking mutations are located throughout the structure of hSOD1 and promote the propensity to aggregate. Despite numerous investigations, the underlying mechanism related to the toxicity of mutant hSOD1 through the gain of a toxic function is still vague. We surveyed two mutant forms of hSOD1 by removing and adding cysteine at positions 146 and 72, respectively, to investigate the biochemical characterization and amyloid formation. Our findings predicted the harmful and destabilizing impact of two SOD1 mutants using multiple programs. The specific activity of the wild-type form was about 1.42- and 1.92-fold higher than that of C146R and G72C mutants, respectively. Comparative structural studies using CD spectropolarimetry, and intrinsic and ANS fluorescence showed alterations in secondary structure content, exposure of hydrophobic patches, and structural compactness of WT-hSOD1 vs. mutants. We demonstrated that two mutants were able to promote amyloid-like aggregates under amyloid induction circumstances (50-mM Tris-HCl pH 7.4, 0.2-M KSCN, 50-mM DTT, 37 °C, 190 rpm). Monitoring aggregates were done using an enhancement in thioflavin T fluorescence and alterations in Congo red absorption. The mutants accelerated fibrillation with subsequently greater fluorescence amplitude and a shorter lag time compared to WT-SOD1. These findings support the aggregation of ALS-associated SOD1 mutants as an integral part of ALS pathology.

Polyphenolic flavonoid compounds act as the inhibitory potential of aggregation process: Implications for the prevention and therapeutics against FALS-associated D101G SOD1 mutant.

Aggregation of proteins is a biological phenomenon caused by misfolded proteins. Human superoxide dismutase (hSOD1) misfolding and aggregation underlie the neurological illness amyotrophic lateral sclerosis (ALS). The most significant contributing factor to ALS is genetic point mutations in SOD1. particularly, D101G mutant is the most harmful because it significantly reduces the life expectancy of patients. Subsequently, the use of natural polyphenolic flavonoids is strongly recommended to reduce the amyloidogenic behavior of protopathic proteins. In this study, using computational parameters such as protein-ligand interaction and molecular dynamics (MD) simulation analyses, we are trying to identify a pharmacodynamically promising flavonoid compound that can effectively inhibit the pathogenic behavior of the D101G mutant. Epigallocatechin-gallate (EGCG), Hesperidin, Isorhamnetin, and Diosmetin were identified as potential leads in a preliminary screening of flavonoids to anti-amyloid action. The results of MD showed that the binding of flavonoids to D101G mutant caused changes in stability, hydrophobicity of protein, and flexibility, as well as significantly led to the restoration of lost hydrogen bonds. Secondary structure analysis showed that protein destabilization and the increased propensity of ß-sheet caused by the mutation were restored to the wild-type state upon binding of flavonoids. Besides, to differentiate aggregation, we elucidated alterations in the free energy landscape (FEL) and dynamic cross-correlation matrix (DCCM) of WT-SOD1 and mutant (unbound /bound) states. Among flavonoids, Epigallocatechin-gallate and Hesperidin had the most therapeutic efficacy against the D101G mutant. Therefore, Epigallocatechin-gallate and Hesperidin promise considerable therapeutic potential to develop highly effective inhibitors in reducing fatal and irreversible ALS.

Role of charged residues of the "electrostatic loop" of hSOD1 in promotion of aggregation: Implications for the mechanism of ALS-associated mutations under amyloidogenic conditions.

Protein misfolding and amyloid formation are hallmarks of numerous diseases, including amyotrophic lateral sclerosis (ALS), in which hSOD1 aggregation is involved in pathogenesis. We used two point mutations in the electrostatic loop, G138E and T137R, to analyze charge distribution under destabilizing circumstances to gain more about how ALS-linked mutations affect SOD1 protein stability or net repulsive charge. We show that protein charge is important in the ALS disease process using bioinformatics and experiments. The MD simulation findings demonstrate that the mutant protein differs significantly from WT SOD1, which is consistent with the experimental evidence. The specific activity of the wild type was 1.61 and 1.48 times higher than that of the G138E and T137R mutants, respectively. Under amyloid induction conditions, the intensity of intrinsic and ANS fluorescence in both mutants reduced. Increasing the content of ß-sheet structures in mutants can be attributed to aggregation propensity, which was confirmed using CD polarimetry and FTIR spectroscopy. Our findings show that two ALS-related mutations promote the formation of amyloid-like aggregates at near physiological pH under destabilizing conditions, which were detected using spectroscopic probes such as Congo red and ThT fluorescence, and also further confirmation of amyloid-like species by TEM. Overall, our results provide evidence supporting the notion that negative charge changes combined with other destabilizing factors play an important role in increasing protein aggregation by reducing repulsive negative charges.

Computational insight into in silico analysis and molecular dynamics simulation of the dimer interface residues of ALS-linked hSOD1 forms in apo/holo states: a combined experimental and bioinformatic perspective.

The aggregation of misfolded SOD1 proteins in neurodegenerative illnesses is a key pathological hallmark in amyotrophic lateral sclerosis (ALS). SOD1 is stabilized and enzymatically activated after binding to Cu/Zn and forming intramolecular disulfide. SOD1 aggregation/oligomerization is triggered by the dissociation of Cu and/or Zn ions. Therefore, we compared the possible effects of ALS-associated point mutations of the holo/apo forms of WT/I149T/V148G SOD1 variants located at the dimer interface to determine structural characterization using spectroscopic methods, computational approaches as well as molecular dynamics (MD) simulations. Predictive results of computational analysis of single-nucleotide polymorphisms (SNPs) suggested that mutant SOD1 has a deleterious effect on activity and structure destabilization. MD data analysis indicated that changes in flexibility, stability, hydrophobicity of the protein as well as increased intramolecular interactions of apo-SOD1 were more than holo-SOD1. Furthermore, a decrease in enzymatic activity in apo-SOD1 was observed compared to holo-SOD1. Comparative intrinsic and ANS fluorescence results of holo/apo-WT-hSOD1 and mutants indicated structural alterations in the local environment of tryptophan residue and hydrophobic patches, respectively. Experimental and MD data supported that substitution effect and metal deficiency of mutants (apo forms) in the dimer interface may promote the tendency to protein mis-folding and aggregation, consequently disrupting the dimer-monomer equilibrium and increased propensity to dissociation dimer into SOD-monomer ultimately leading to loss of stability and function. Overall, data analysis of apo/holo SOD1 forms on protein structure and function using computational and experimental studies will contribute to a better understanding of ALS pathogenicity.

A double point mutation of SOD1 targeting net charge promotes aggregation under destabilizing conditions: Correlation of charge distribution and ALS-provoking mutation.

A progressive neurodegenerative illness such as amyotrophic lateral sclerosis (ALS) is characterized by the misfolding and aggregation of human CuZn superoxide dismutase (hSOD1) into amyloid aggregates. Thus, designing strategies for the choice of WT-SOD1 and double mutant (G12D/G138E) with an increased net negative charge can be a good idea to elucidate the pathological mechanism of SOD1 in ALS under some destabilizing conditions. Consequently, we show evidence that protein charge, together with other destabilizing conditions, plays an important role in ALS disease. To achieve this purpose, we use methods, such as spectroscopy and transmission electron microscopy (TEM) to monitor the formation of amyloid aggregation. The specific activity of WT-SOD1 was approximately 1.72 times higher than that of the double mutant. Under amyloidogenic circumstances, structural properties such as local, secondary, oligomeric, and fibrillar structures were explored. The double mutant's far-UV CD spectra displayed a broad minimum peak in the region 213 to 218 nm, suggesting the production of ß-rich amyloid fibrils. FTIR spectra of the double mutant samples at different incubation times showed a low-frequency peak around 1630-1640 cm-1, attributed to a parallel ß-sheet. Moreover, CR-binding assay and TEM analysis revealed and confirmed that mutation with an increased repulsive charge promotes the formation of fibrous aggregates. Consequently, ALS mutations with a higher repulsive charge are the apparent exceptions that validate the rule. This findings revealed that the double mutant increases protein aggregation through a novel mechanism, likely involving destabilization of structure and a change in the net negative charge.

Zinc binding loop mutations of hSOD1 promote amyloid fibrils under physiological conditions: Implications for initiation of amyotrophic lateral sclerosis.

Neurotoxic species of misfolded hSOD1 are involved in the process of causing amyotrophic lateral sclerosis (ALS), which is a devastating neurodegenerative disease. Considerable evidence exists on that hSOD1 mutants-mediated toxicity is resulted from gain-of-function, while the mechanism of this toxicity is unknown yet. In the present study, we focused on the possible mechanism of two point-mutations (namely L67P and D76Y) on metal-binding sites and their possible consequent effects on ALS progress. For this purpose, the exposed hydrophobic patches were detected using ANS fluorescence and the formation of hSOD1 aggregates was monitored using the ThT fluorescence and absorption spectra of Congo red (CR). Moreover, to assess aggregate's morphology, transmission electron microscopy (TEM) was used. The specific activity of wild-type, as well as L67P and D76Y mutants was obtained as 12345, 8625, and 7066 U/mg, respectively. The existence of ß-sheet-dominated structures was observed under amyloidogenic conditions using far UV CD and FTIR spectroscopy. As well, comparative study of wild-type and mutants by intrinsic and extrinsic fluorescence revealed structural alterations and the increased hydrophobic surface pockets, respectively. The formation of the amyloid fibrils was monitored under destabilizing. The results of ThT and CR showed the process formation of amyloid aggregates and moreover, the presence of morphological forms was confirmed by the TEM image. Overall, our findings supported that mutation in the zinc-binding loop could significantly increase the tendency to mediate amyloid aggregation and it may possibly trigger misfolding and fibrillar aggregation, which are pathological changes in familial forms of ALS.

Human DT-diaphorase expression in Escherichia coli: optimization, purification and structural stability.

Expression and purification of human DT-diaphorase, also referred to as NAD(P)H quinone oxidoreductase 1 (NQO1; EC. 1.6.99.2), which is a flavoprotein belongs to the family of oxidoreductases are optimized. The DT-diaphorase plays an important role in biosensor design for laboratory analysis and also developing biosensor for measurement of glucose level in blood. The aim of this study was to investigate various parameters regarding the expression of DT-diaphorase in Escherichia coli BL21 (DE3) and thermal stability of DT-diaphorase activity at different temperatures in the presence of sucrose. Expression conditions of DT-diaphorase in E. coli were optimized with an induction time (22.00 hr), induction temperature (18.00 ˚C) and also lactose (5.00 mM) and isopropyl ß-D-1-thiogalactopyranoside (1.00 mM) concentrations as inducers. The Km, Vmax and kcat values for NADH as a substrate were 25.50 µM, 357 µM per min and 446.40 µM mg-1 per min, respectively. Results of our research revealed that different concentrations of sucrose at 40.00 ˚C did not have any significant effect on enzyme structure; while, relatively significant changes, especially in the presence of sucrose (0.75 M) at 50.00 ˚C were observed. The results presented show that sucrose causes DT-diaphorase inactivation rate reduction and relatively little increases in thermal stability and thus, sustains its conformation against thermal unfolding.

Development of In Silico Analysis and Molecular Dynamics Simulation on L67P and D76Y Mutants of the Human Superoxide Dismutase 1(hSOD1) Related to Amyotrophic Lateral Sclerosis.

Background: One neurodegenerative disorder that is caused by a mutation in the hSOD1 gene is Amyotrophic lateral sclerosis (ALS). Objectives: The current study was developed in order to evaluate the effect exerted by two ALS-associated point mutations, L67P and D76Y are located in the metal-binding loop, on structural characterization of hSOD1 protein using molecular dynamics (MD) simulations and computational predictions. Materials and Methods: In this study, GROMACS was utilized to perform molecular dynamics simulations along with 9 different algorithms such as Predict SNP, PhD-SNP, MAPP, PolyPhen-1, Polyphen-2, SNP, SIFT, SNP&GO, and PMUT for predicting and also evaluating the mutational effect on the structural and conformational characterization of hSOD1. Results: Our study was done by several programs predicting the destabilizing and harmful effect exerted by mutant hSOD1. The deleterious effect of L67P mutation was predicted by MAPP and PhD-SNP algorithms, and D76Y mutation was predicted by 9 algorithms. Comparative studies that were conducted on mutants and wild-type indicated the altar in flexibility and protein conformational stability influenced the metal-binding loop's conformation. The outcomes of the MD exhibited an increase and decrease of flexibility for D76Y and L67P mutants compared to the wild type, respectively. On the other hand, analysis of the gyration radius indicated lower and higher compactness for D76Y and L67P, respectively, suggesting that replacing amino acid at the metal-binding loop can alter the protein compactness compared with the protein the wild type. Conclusions: Overall, these findings provided insight into the effect of mutations on the hSOD1, which leads to neurodegeneration disorders in humans. The results show that the mutations of L67P and D76Y influence the stability of protein conformational and flexibility associated with ALS disease. Thus, results of such mutations are can be a prerequisite to achieve a thorough understanding of ALS pathogenicity.

Lipase immobilization on a novel class of Zr-MOF/electrospun nanofibrous polymers: Biochemical characterization and efficient biodiesel production.

In this study, due to the favorable properties of MOF compounds and fibrous materials, new nanostructures of Zr-MOF/PVP nanofibrous composites were synthesized by electrospinning procedure. The related features of these samples were characterized by relevant analyzes, including SEM, BET surface area analysis, XRD, and FTIR spectroscopy. The final product showed significant properties such as small particle size distribution, large surface area, and high crystallinity. This strategy for producing these nanostructures could lead to new compounds as novel alternative materials for biological applications. Lipase MG10 was successfully immobilized on the mentioned nanofibrous composites and biochemically characterized. The lipase activity of free and immobilized lipases was considered by measuring the absorbance of pNPP (500 µM in 40 mM Tris/HCl buffer, pH 7.8, and 0.01% Triton X100) at 37 °C for 30 min. Different concentrations of glutaraldehyde, different crosslinking times, different times of immobilization, different enzyme loading, and different pH values have been optimized. Results showed that the optimized immobilization condition was achieved in 2.5% glutaraldehyde, after 2 h of crosslinking time, after 6 h immobilization time, using 180 mg protein/g support at pH 9.0. The immobilized enzyme was also totally stable after 180 min incubation at 60 °C. The free enzyme showed the maximum activity at pH 9.0, but the optimal pH of the immobilized lipase was shifted about 1.5 pH units to the alkaline area. The immobilized lipase showed about 2.7 folds (78%) higher stability than the free enzyme at 50 °C. Some divalent metal ions, including Cu2+ (22%), Co2+ (37%), Mg2+ (12%), Hg2+ (11%), and Mn2+ (17%) enhanced the enzyme activity of immobilized enzyme. The maximum biodiesel production (27%) from R. communis oil was obtained after 18 h of incubation by lipase MG10. The immobilized lipase displayed high potency in biodiesel production, about 83% after 12 h of incubation. These results indicated the high potency of Zr-MOF/PVP nanofibrous composites for efficient lipase immobilization.

Antioxidant, histopathological and biochemical outcomes of short-term exposure to acetamiprid in liver and brain of rat: The protective role of N-acetylcysteine and S-methylcysteine.

The present study was conducted to investigate the protective effects of N-Acetyl-L-cysteine (NAC) and S-methyl- L-cysteine (SMC) against hepatic oxidative stress and brain damage induced by acetamiprid (ACP) in rats, which were evaluated by histopathological changes, measuring serum biomarkers and antioxidant defense systems. In this study, 42 rats were randomly divided into 6 groups and administered by intraperitoneally for one week: the control group, the sham group (normal saline), ACP alone (5 mg/kg) (group1), NAC alone (160 mg/kg) (group2), ACP + SMC (100 mg/kg) (group3), ACP + NAC (group 4) and ACP + NAC + SMC (group 5). Our results showed that acetamiprid induces liver injures including infiltration of inflammatory cells, congestion and altered histo-architecture and brain damages including gliosis, hyperemia and necrosis. The biochemical analyses showed that acetamiprid significantly altered the structural and biochemical profiles of liver which may be due to the loss of integrity of cell membranes. Furthermore, antioxidant parameters results of ACP group revealed that glutathione (GSH) and total antioxidant capacity (TAC) levels decreased significantly, while lipid peroxidation (LPO) content and glutathione-S-transferase (GST) and catalase (CAT) activities increased in both tissues (P < 0.05), suggesting tissue oxidative damage, which was also confirmed histopathological. Conversely, administration of NAC and SMC ameliorated LPO, GSH content and antioxidant enzymes system considerably (P < 0.05) in both tissues. Moreover, NAC and SMC administration also improved liver and brain malfunction. These results indicate that both NAC and in to a lesser amount SMC have a potent antioxidant protection in both tissues of rat against ACP-induced oxidative stress.

Biochemical, Toxicological, and Histopathological outcome in rat brain following treatment with NiO and NiO nanoparticles.

Nickel oxide nanoparticle (NiO NPs) has been widely used in various fields such as catalysts, radiotherapy, and nanomedicine. The aim of this study was to compare the effects of nickel oxide (NiO) and NiO NPs on oxidative stress biomarkers and histopathological changes in brain tissue of rats. In this study, 49 male rats were randomly divided into one control group and 6 experimental groups (n = 7). The control group received normal saline and the treatment groups received NiO and NiO NPs at doses of 10, 25, and 50 mg/kg intraperitoneally for 8 days. After 8 days, animal was sacrificed, brain excised, homogenized, centrifuged, and then supernatant was collected for antioxidant assays. The results showed that activity of GST in NiO NPs groups with doses of 10, 25, and 50 mg/kg (79.42 ± 4.24, p = 0.035; 78.77 ± 8.49, p = 0.041; 81.38 ± 12.39, p = 0.042 to 47.26 ± 7.17) and catalase in NiO NPs groups with concentrations of 25 and 50 mg/kg (69.95 ± 8.65 to 39.75 ± 5.11, p = 0.02) and (68.80 ± 4.18 to 39.75 ± 5.11 p = 0.027) were significantly increased compared with the control, respectively. Total antioxidant capacity in NiONPs group with doses of 50 mg/kg was significantly decreased (345.00 ± 23.62, p = 0.015 to 496.66 ± 25.77) compared with control. The GSH level in all doses NiO and NiONPs was significantly decreased compared with the control (p = 0.002). MDA level in NiONPs and NiO groups with doses of 50 mg/kg was significantly increased (13.03 ± 1.29, p = < 0.01; 15.61 ± 1.08, p = < 0.001 to 7.32 ± 0.51) compared with the control, respectively. Our results revealed a range of histopathological changes, including necrosis, hyperemia, gliosis, and spongy changes in brain tissue. Thus, increasing level of MDA, GST, and CAT enzymes and decreasing GSH and TAC and also histopathological changes confirmed NiONPs and NiO toxicity.

Copper nanoparticles promote rapid wound healing in acute full thickness defect via acceleration of skin cell migration, proliferation, and neovascularization.

Worldwide, impaired wound healing leads to a large burden of morbidity and mortality. Current treatments have several limitations. Recently, nanomaterials such as copper nanoparticles (CuNPs) have attracted considerable research interest. Here, we investigated the potential therapeutic effect of various CuNPs concentrations (1 µM, 10 µM, 100 µM, 1 mM, and 10 mM) and sizes (20 nm, 40 nm, 80 nm) in wound healing. Our results revealed that the 10 µM concentration of 40 nm CuNPs and the 1 µM concentration of 80 nm CuNPs were not toxic to the cultured fibroblast, endothelial, and keratinocyte cells, and also 1 µM concentration of 80 nm CuNPs enhanced endothelial cell migration and proliferation. Extensive assessment of in vivo wound healing demonstrated that the 1 µM concentration of 80 nm CuNPs accelerated wound healing over a shorter time via formation of granulation tissue and higher new blood vessels. Importantly, serum biochemical analysis confirmed that the 40 nm CuNP (10 µM) and 80 nm CuNP (1 µM) did not show any accumulation in the liver during wound healing. Overall, our results have indicated that the 1 µM concentration of 80 nm CuNPs is a promising NP for wound healing applications without adverse side effects.