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The budding yeast, Saccharomyces cerevisiae, is an efficient model for studying oxidative stress, programmed cell death and aging. The present study was carried out to investigate antioxidant, the anti-apoptotic and anti-aging activity of a natural compound, astaxanthin, in S. cerevisiae model. The survivability of yeast antioxidant-deficient strains (sod1Δ, sod2Δ, cta1Δ, ctt1Δ and tsa1Δ) increased by 20%–40% when cells were pre-treated with astaxanthin, compared to hydrogen peroxide alone, as demonstrated in spot and colony forming unit assays. Reduced reactive oxygen species (ROS) levels, increased glutathione, decreased lipid peroxidation and induced superoxide dismutase activity in astaxanthin-treated cells indicate that astaxanthin protected the cells from oxidative-stress-induced cell death. In addition, astaxanthin protected anti-apoptotic-deficient strains (pep4Δ and fis1Δ) against acetic acid and hydrogen peroxide-induced cell death that suggests anti-apoptotic property of astaxanthin, and it was further confirmed by acridine orange/ethidium bromide, annexin V and 4',6-diamidino-2-phenylindole staining. The yeast chronological lifespan assay results showed that astaxanthin extends the lifespan of antioxidant-deficient strains by scavenging ROS, and anti-apoptotic-deficient mutants by protecting from apoptotic cell death compared to their respective untreated cells and wild type. Our results suggest that astaxanthin enhances the longevity of yeast S. cerevisiae by reducing oxidative stress and apoptosis. [ABSTRACT FROM AUTHOR] more...

Adults older than 45 years old are at higher risk of developing venous blood clotting known as venous thrombosis/thromboembolism than a cohort <45 years old. Complement activation, which can be mediated by oxidative stress, plays a central role in venous thrombosis. Yet, whether RBCs contribute to complement activation triggering thrombosis in aging and in patients with venous thrombosis/thromboembolism remains an open question. RBCs from healthy Mid-life stage (55-68 years old) adults and patients with venous thrombosis/thromboembolism showed higher deposition of the complement C3 and the anaphylatoxin C3a, and NADPH oxidase (Nox)1 expression than a younger cohort (21-30 years old). Increased C3/C3a deposition on RBCs from mid-life stage adults and patients with venous thrombosis/thromboembolism triggered prothrombin activation via Nox1-dependent reactive oxygen species (ROS) generation, and G protein-coupled receptor kinase 2 (GRK2) activation. Interaction of C3/C3a positive RBCs from mid-life stage adults with endothelial cells led to increased endothelial ROS production. TGF-β1-stimulated GRK2 and Nox1 activation in RBCs from the younger and older adults exacerbated RBC C3/C3a deposition and C3/C3a-mediated prothrombotic activation, which appears to result from ROS-mediated increased RBC phosphatidylserine exposure. Using human RBCs, and Rpl13a snoRNA knockout aged mice, we show that Rpl13a snoRNAs, the master regulators of ROS levels and oxidative stress response, regulate human and murine RBC C3a deposition and prothrombic activation in aging by modulating Nox1 mRNA expression. In vivo Rpl13a snoRNA knockout in aged mice decreased thrombi size by blunting RBC C3a deposition, and RBCs-triggering prothrombin activation. These findings point out to a novel role of RBC Rpl13a snoRNAs in dysregulating RBC ROS-induced C3a deposition promoting venous thrombosis in aging., Competing Interests: Declaration of interest statement I hereby declare that the disclosed information is correct and that no other situation of real, potential or apparent conflict of interest is known to me., (Copyright © 2025. Published by Elsevier Inc.) more...

Betulinic acid (BA), a pentacyclic triterpenoid found in certain plant species, has been reported to have several health benefits including antioxidant and anti-apoptotic properties. However, the mechanism by which BA confers these properties is currently unknown. Saccharomyces cerevisiae , a budding yeast with a short life cycle and conserved cellular mechanism with high homology to humans, was used as a model for determining the role of BA in aging and programmed cell death (PCD). Treatment with hydrogen peroxide (H 2 O 2 ) exhibited significantly increased (30-35%) survivability of antioxidant ( sod1Δ, sod2Δ, cta1Δ, ctt1Δ, and tsa1Δ ) and anti-apoptotic ( pep4Δ and fis1Δ ) mutant strains when cells were pretreated with BA (30 µM) as demonstrated in spot and CFU (Colony forming units) assays. Measurement of intracellular oxidation level using the ROS-specific dye H 2 DCF-DA showed that all tested BA-pretreated mutants exhibited decreased ROS than the control when exposed to H 2 O 2 . Similarly, when mutant strains were pretreated with BA and then exposed to H 2 O 2 , there was reduced lipid peroxidation as revealed by the reduced malondialdehyde content. Furthermore, BA-pretreated mutant cells showed significantly lower apoptotic activity by decreasing DNA/nuclear fragmentation and chromatin condensation under H 2 O 2 -induced stress as determined by DAPI and acridine orange/ethidium bromide staining. In addition, BA treatment also extended the life span of antioxidant and anti-apoptotic mutants by ∼10-25% by scavenging ROS and preventing apoptotic cell death. Our overall results suggest that BA extends the chronological life span of mutant strains lacking antioxidant and anti-apoptotic genes by lowering the impact of oxidative stress, ROS levels, and apoptotic activity. These properties of BA could be further explored for its use as a valuable nutraceutical. more...

The medicinal properties of Ashwagandha (Withania somnifera) are attributed to triterpenoid steroidal lactones, withanolides, which are proposed to be derived from phytosterol pathway, through the action of cytochrome P450 (CYP450) enzymes. Here, we report the characterization of three transcriptome-mined CYP450 genes (WsCYP749B1, WsCYP76 and WsCYP71B10), which exhibited induced expression in response to methyl jasmonate treatment indicating their role in secondary metabolism. All three WsCYP450s had the highest expression in leaf compared to other tissues. In planta characterization of WsCYP450s through virus induced gene silencing (VIGS) and transient overexpression approaches and subsequent metabolite analysis indicated differential modulation in the accumulation of certain withanolides in W. somnifera leaves. While WsCYP749B1-vigs significantly enhanced withaferin A (~ 450%) and reduced withanolide A (~ 50%), its overexpression drastically led to enhanced withanolide A (> 250%) and withanolide B (> 200%) levels and reduced 12-deoxywithastramonolide (~ 60%). Whereas WsCYP76-vigs led to reduced withanolide A (~ 60%) and its overexpression increased withanolide A (~ 150%) and reduced 12-deoxywithastramonolide (~ 60%). Silencing and overexpression of WsCYP71B10 resulted in significant reduction of withanolide B (~ 50%) and withanolide A (~ 60%), respectively. Further, while VIGS of WsCYP450s negatively affected the expression of pathogenesis-related (PR) genes and compromised tolerance to bacteria P. syringae DC3000, their overexpression in W. somnifera and transgenic tobacco led to improved tolerance to the bacteria. Overall, these results showed that the identified WsCYP450s have a role in one or several steps of withanolides biosynthetic pathway and are involved in conferring tolerance to biotic stress., (© 2022. The Author(s).) more...

In the present study, galactan exopolysaccharide (EPS) from Weissella confusa KR780676 was evaluated for its potential to alleviate oxidative stress using in vitro assays and in vivo studies in Saccharomyces cerevisiae (wild type) and its antioxidant (sod1∆, sod2∆, tsa1∆, cta2∆ and ctt1∆), anti-apoptotic (pep4∆ and fis1∆) and anti-aging (sod2∆, tsa1∆ and ctt1∆)) isogenic gene deletion mutants. Galactan exhibited strong DPPH and nitric oxide scavenging activity with an IC 50 value of 450 and 138 µg/mL respectively. In the yeast mutant model, oxidative stress generated by H 2 O 2 was extensively scavenged by galactan in the medium as confirmed using spot assays followed by fluorescencent DCF-DA staining and microscopic studies. Galactan treatment resulted in reduction in the ROS generated in the yeast mutant cells as demonstrated by decreased fluorescence intensity. Furthermore, galactan exhibited protection against oxidative damage through H 2 O 2 -induced apoptosis inhibition in the yeast mutant strains (pep4∆ and fis1∆) leading to increased survival rate by neutralizing the oxidative stress. In the chronological life span assay, WT cells treated with galactan EPS showed 8% increase in viability whereas sod2∆ mutant showed 10-15% increase indicating pronounced anti-aging effects. Galactan from W. confusa KR780676 has immense potential to be used as a natural antioxidant for nutraceutical, pharmaceutical and food technological applications. As per our knowledge, this is the first report on in-depth assessment of in vivo antioxidant properties of a bacterial EPS in a yeast deletion model system., (© 2022. The Author(s).) more...

Background: Papaverine is a benzylisoquinoline alkaloid from the plant Papaver somniferum (Opium poppy). It is approved as an antispasmodic drug by the US FDA and is also reported to have anti-cancer properties. Here, Papaverine's activity in chronic myeloid leukemia (CML) is explored using Saccharomyces cerevisiae, mammalian cancer cell lines, and in silico studies., Methods: The sensitivity of wild-type and mutant (anti-oxidant defense, apoptosis) strains of S. cerevisiae to the drug Papaverine was tested by colony formation, spot assays, and AO/EB staining. In vitro cytotoxic effect was investigated on HCT15 (colon), A549 (lung), HeLa (cervical), and K562 (Bcr-Abl positive CML), and RAW 264.7 cell lines; cell cycle, mitochondrial membrane potential, ROS detection analyzed in K562 cells using flow cytometry and apoptotic markers, Bcr-Abl signaling pathways examined by western blotting. Molecular docking and molecular dynamics simulation of Papaverine against the target Bcr-Abl were also carried out., Results: Investigation in S. cerevisiae evidenced Papaverine induces ROS-mediated apoptosis. Subsequent in vitro examination showed that CML cell line K562 was more sensitive to the drug Papaverine. Papaverine induces ROS generation, promotes apoptosis, and inhibits Bcr-Abl downstream signaling. Papaverine acts synergistically with the drug Imatinib. Furthermore, the docking and molecular dynamic simulation studies supported that Papaverine binds to the allosteric site of Bcr-Abl., Conclusion: The data presented here have added support to the concept of polypharmacology of existing drugs and natural compounds to interact with more than one target. This study provides a proof-of-concept for repositioning Papaverine as an anti-CML drug. more...

Reactive oxygen species (ROS) have long been found to play an important role in oxidative mediated DNA damage. Fortunately, cells possess an antioxidant system that can neutralize ROS. However, oxidative stress occurs when antioxidants are overwhelmed by ROS or impaired antioxidant pathways. This study was carried out to find the protective effect of astaxanthin on the yeast DNA repair-deficient mutant cells under hydrogen peroxide stress. The results showed that astaxanthin enhances the percent cell growth of rad1∆, rad51∆, apn1∆, apn2∆ and ogg1∆ cells. Further, the spot test and colony-forming unit count results confirmed that astaxanthin protects DNA repair mutant cells from oxidative stress. The DNA binding property of astaxanthin studied by in silico and in vitro methods indicated that astaxanthin binds to the DNA in the major and minor groove, and that might protect DNA against oxidative stress induced by Fenton's reagent. The intracellular ROS, 8-OHdG level and the DNA fragmentation as measured by comet tail was reduced by astaxanthin under oxidative stress. Similarly, reduced nuclear fragmentation and chromatin condensation results suggest that astaxanthin might reduce apoptosis. Finally, we show that astaxanthin decreases the accumulation of mutation rate and enhances the longevity of DNA repair-deficient mutants' cells during a chronological lifespan. more...

Perfluorooctanoic acid (PFA) has been identified as an environmental contaminant of high concern for human health. In this study, we demonstrated that PFA induces a dose (0 to 1.5 mM) dependent cytotoxicity in S. cerevisiae cells which can be rescued by astaxanthin. The percent sensitivity induced by PFA and the cell protection offered by astaxanthin (30 μM) were demonstrated by CFU counts and spots. The increase in intracellular ROS, superoxide dismutase (SOD), glutathione and lipid peroxidation levels in PFA treated cells suggested that increased oxidative stress resulted in yeast cell death. In contrast, decreased ROS level, increased SOD activity, reduced glutathione and decreased lipid peroxidation by astaxanthin supplementation suggest that the cells are protected from the PFA induced oxidative stress mediated cytotoxicity. Reduced chromatin condensation and nuclear fragmentation in astaxanthin pre-treated cells indicate that astaxanthin rescued the cells from PFA induced apoptosis. Our overall results suggest that PFA induces oxidative stress-mediated cytotoxicity in yeast cells, which were rescued by astaxanthin treatment., (This journal is © The Royal Society of Chemistry 2019.) more...

This study evaluates the protective effect of astaxanthin against dichlorvos cytotoxicity in yeast Saccharomyces cerevisiae . Dichlorvos induce a dose-dependent cytotoxicity in yeast cells, which is mediated by oxidative stress. Our experimental results showed pre-treatment with astaxanthin enhances cell viability by 20-30% in yeast cells exposed to dichlorvos. A decrease in DCF fluorescence intensity and lipid peroxidation, increased SOD activity, and glutathione levels in astaxanthin-treated cells indicate that astaxanthin protected the cells against dichlorvos-induced oxidative stress. Reduced chromatin condensation and nuclear fragmentation in astaxanthin pre-treated cells also indicate that astaxanthin rescued the cells from dichlorvos-induced apoptosis. Our overall results suggest that dichlorvos induces oxidative stress-mediated cytotoxicity in yeast cells, and that was rescued by astaxanthin pre-treatment., Competing Interests: Compliance with ethical standardsThe authors declare that they have no competing interest. more...

Excessive production and restricted elimination of free radicals like superoxide, hydroxyl radical ( · OH), anion radical (O 2 ·- ), and non-radical hydrogen peroxide (H 2 O 2 ) are related to the development of cancer, arteriosclerosis, arthritis and neurodegenerative diseases. According to a report of World Health Organisation, about 80% of the population living in the developing countries predominantly depends on the traditional medicine for their primary healthcare. Plants possess innate ability to synthesize a wide variety of enzymatic and non-enzymatic antioxidants capable of attenuating ROS-induced oxidative damage. The ethanolic leaf extracts of Syzygium jambos L. and Terminalia citrina Roxb. exhibited a significant in vitro antioxidant activity when compared with natural antioxidant, ascorbic acid. The extracts also provided strong cellular protection against the damaging effects of H 2 O 2 induced oxidative stress in the mutant strains ( tsa1Δ and sod1Δ ) of Saccharomyces cerevisiae . The GC-MS analysis of the leaf extracts revealed the presence of phytoconstituents majorly constituting of terpenes, vitamin and fatty acids contributing to the antioxidant property. The plant extracts may serve as a potential source of exogenous antioxidants to combat the undesirable effects of oxidative stress., Competing Interests: Compliance with ethical standardsThe authors declare no conflict of interest in this work. more...

Background: The current research aims to isolate pyruvate kinase (Pyk) gene from Mycobacterium tuberculosis and expression of the gene (Rv1617) to obtain a purified enzyme. The enzyme activity and secondary structural features were assessed through biochemical assays and circular dichroism (CD) spectroscopy, respectively., Methods: The Pyk-encoding gene from the complete genome of M. tuberculosis was cloned, sequenced, and expressed in Escherichia coli BL21 (DE3). The enzyme was purified by nickel-nitrilotriacetic acid affinity chromatography and enzyme activity was determined by a lactose dehydrogenase-coupled assay system. Further, far ultraviolet CD spectra of the enzyme and the substrate bound enzyme were analyzed using a Jasco J712 spectrophotometer., Results: A single protein with an approximate molecular mass of 54 kDa was purified and a specific activity of 5.31 units/mg was determined from purified M. tuberculosis Pyk. The activity of the enzyme indicating a protein is defined by separate domain for each catalytic function. The secondary structure analysis of CD spectra of the recombinant Pyk has revealed a content of 17% α-helix, 34% β-sheet, and 49% turns in the enzyme., Conclusion: The growing evidence has impacted M. tuberculosis central carbon metabolism as a key determinant of the survival and pathogenicity in the host. The purified Pyk was observed to have increased enzyme activity in all steps of purification. Retention of Pyk activity indicates a possible catalytic role for the lower part of the glycolytic pathway. The overall results of the spectra obtained from the CD suggest that the substrate phosphoenolpyruvate and adenosine diphosphate binding to the enzyme can cause conformational changes resulting in the exposure or shielding of residues susceptible to modification., Competing Interests: None more...

Wild edible mushrooms are one of the most fascinating nutraceuticals because of their pleasant texture, peculiar aroma, and tremendous therapeutic potential; they have been used since ancient times. In this study we evaluated the antioxidant potential of 4 wild edible mushrooms-Cantharellus tropicalis, C. cibarius, Lentinus edodes, and Russula delica-collected from Mizoram, India. We performed in vitro and in vivo studies using a Saccharomyces cerevisiae stress response mechanism as the model system. Among the 4 mushrooms, C. cibarius exhibited the most significant antioxidant activity both in vitro and in vivo, followed by L. edodes, R. delica, and C. tropicalis. Gas chromatography-mass spectrometry analysis of methanolic extracts of the mushrooms revealed the presence of substantial amounts of fatty acids, fatty acid esters, and other bioactive constituents. The in vitro antioxidant activity was corroborated by in vivo studies using an S. cerevisiae oxidative stress response mechanism. In spot assays, the C. cibarius methanolic extract showed the highest scavenging potential in wild and mutant (sodlΔ and tsalΔ) strains of S. cerevisiae. These results were confirmed further by determining the level of reactive oxygen species through the use of fluorescent microscopy and intensity studies. The results suggested the efficacy of wild edible mushrooms as prominent therapeutic agents and that they have tremendous nutraceutical properties. more...

Background: From ancient times, plants and plant-derived products have been used as folkloric medicines for a variety of health disorders owing to their tremendous therapeutic potential. The present study aimed to determine the antioxidant efficacy of crude Acacia nilotica extract in the oxidative stress response system of Saccharomyces cerevisiae as a model organism., Results: Acacia nilotica showed significant antioxidant activity, with IC 50 values of 75.157 and 159.57 µg mL -1 for 2,2-diphenyl-1-picrylhydrazyl radical and hydroxyl radical scavenging activities respectively at a concentration of 500 µg mL -1 . The total antioxidant activity of A. nilotica showed an ascorbic acid equivalent of 152.79 ± 7.43 µg mL -1 . The presence of phytoconstituents such as phytol and α-tocopherol from gas chromatography/mass spectrometry analysis confirmed the potential of A. nilotica as an antioxidant. The results were validated using the stress response mechanism in S. cerevisiae wild type and its isogenic deletion strains sod1Δ and tsa1Δ. Acacia nilotica substantially neutralized reactive oxygen species generated by hydrogen peroxide in mutant strains, as evident from spot assay and fluorescence assay using fluorescence microscopy and intensity studies., Conclusion: The results suggested the efficacy of A. nilotica as a potent antioxidant in the S. cerevisiae system for the first time and its use in neutraceuticals/therapeutics. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.) more...