Extracted Trans-Resveratrol from Arachis hypogaea Enhances Expression of Sirtuin Gene and Replicative Life Span in Saccharomyces cerevisiae

Aims: Biotic stress given by Aspergillus niger enhances trans-resveratrol production in Arachis hypogaea plant. This plant extract increases sir2 gene expression and Replicative Life Span in Saccharomyces cerevisiae. Design of Study: Peanut plant was grown in aseptic environment, infected by Aspergillus niger. Plant extract used for quantification of trans-resveratrol by RP-HPLC. Yeast culture was grown in Potato dextrose media along with plant extract. Sir2 gene expression fold calculated by real time pcr. Replicative Life Span of yeast was measured by spectrophotometer. Place and Duration of Study: Allele Life Sciences Pvt. Ltd., Department of Biotechnology between February 2017 to March 2020. Methodology: Biotic stress in Arachis hypogaea plant was induced by wounding the leaves and introducing Aspergillus niger to enhance trans-resveratrol production. Tran-resveratrol was quantified by Reverse Phase High Pressure Liquid Chromatography (RP-HPLC). Two methods conducted to check reverse ageing, first one epigenetic based, when extracted trans-resveratrol from infected Arachis hypogaea plant extract added to Saccharomyces cerevisiae culture, it enhanced expression of Sir2 gene in Saccharomyces cerevisiae measured by qPCR, ABI applied biosystem. Process included RNA isolation, cDNA synthesis and thereafter qPCR. Enhanced expression of sirtuin responsible for gene silencing as sirtuin (Sir2 gene product) is a class of Histone deacetylase transferase enzyme. Second method, Replicative Life Span of Saccharomyces cerevisiae culture increased when Aspergillus niger infected peanut plant extract added to yeast culture which was measured through spectrophotometer at 600nm and showed high absorbance value. Results: Tran-resveratrol was quantified by Reverse Phase High Pressure Liquid Chromatography (RP-HPLC) and yield was 2.24 mg/g. Sir2 gene expression increased by 1.56 fold in yeast grown in infected peanut plant extract. Absorbance of yeast culture grown in infected peanut plant extract was 0.522±0.008 which was higher than control. Conclusion: Sir2 gene expression enhances along with replicative life span in yeast in presence of peanut plant extract.

Almond Skin Extracts and Chlorogenic Acid Delay Replicative Aging by Enhanced Oxidative Stress Response Involving SIR2 and SOD2 in Yeast

Almond (Prunus dulcis (Mill.) D.A.Webb) is one of the largest nut crops in the world. Recently, phenolic compounds, mostly stored in almond skin, have been associated with much of the health-promoting behavior associated with their intake. The almond skin enriched fraction obtained from cold-pressed oil residues of the endemic Moroccan Beldi ecotypes is particularly rich in chlorogenic acid. In this study, both almond skin extract (AE) and chlorogenic acid (CHL) supplements, similar to traditional positive control resveratrol, significantly increased the replicative life-span of yeast compared to the untreated group. Our results showed that AE and CHL significantly reduced the production of reactive oxygen and nitrogen species (ROS/RNS), most likely due to their ability to maintain mitochondrial function during aging, as indicated by the maintenance of normal mitochondrial membrane potential in treated groups. This may be associated with the observed activation of the anti-oxidative stress response in treated yeast, which results in activation at both gene expression and enzymatic activity levels for SOD2 and SIR2, the latter being an upstream inducer of SOD2 expression. Interestingly, the differential gene expression induction of mitochondrial SOD2 gene at the expense of the cytosolic SOD1 gene confirms the key role of mitochondrial function in this regulation. Furthermore, AE and CHL have contributed to the survival of yeast under UV-C-induced oxidative stress, by reducing the development of ROS / RNS, resulting in a significant reduction in cellular oxidative damage as evidenced by decreased membrane lipid peroxidation, protein carbonyl content and 8-oxo-guanine formation in DNA. Together, these results demonstrate the interest of AE and CHL as new regulators in the replicative life-span and control of the oxidative stress response of yeast.

Novel Small Molecule Stimulants of Hematopoietic Stem Cells and Their Mode of Action

Agents that can improve the function and/or numbers of hematopoietic progenitor and stem cells (HSC) are of great importance for the treatment of bone marrow failures of different etiology. However, except for the hematopoietic growth factors, which lead to significant depletion HSC via simultaneous differentiation and some anabolic steroids, the list of capable agents that can improve the function or numbers of HSC is very short. However, such drugs would have a tremendous range of application from ex vivo expansion, to bone marrow regeneration in aplastic anemia (AA) post chemotherapy, HSC transplantation or to improve the function of normal HSC in aging. While performing a multidrug screen for the agents that could simultaneously decrease senescence and overcome proliferative block in pre-senescent cells, we have identified two compounds violuric acid (VA) and 1-naphthoquinone-2-monoxime (N2N1). Both compounds considerably extended the replicative life span (RLS) of normal cells. We applied these drugs to stromal/mesenchymal cells obtained from healthy bone marrows, primary human normal dermal fibroblasts, progeroid primary cells derived from the patients diagnosed with Werner or Bloom syndromes and small panel of cancer cell lines (SKM-1, K562, KG-1, THP-1). Both compounds, in dose dependent manner prolonged the RLS of replicatively pre-aged cells. On an average, 10-15 additional population doublings (PD) were achieved after addition of N2N1 at 1μM. VA treatment has added 8-10 extra population doublings. If compared with untreated controls that can propagate up to 45-50 PD, the treatment with VA or N2N1 adds from 16 to 30% increase in replicative life span. To compare, the effect of rapamycin (1nM) on human fibroblasts showed the RLS increase ranged from 5 to 10%. Treatment with both VA and N2N1 results in restoration of cell cycle progression, decreased activity of SAβG, down-regulation of p16, p21 and γH2A.X and, up-regulation of lamin B1 protein. Treatment with both compounds resulted in maintenance of normal telomere length. In term of HSC these agents in vivo increased the performance of HSC in competitive repopulation assay. Bone marrow cells were isolated from mice (CD45.1) treated with the vehicle or experimental drugs for three weeks. After that, they were mixed with the equal number of competitor bone marrow cells CD45.2 and injected into irradiated CD45.2 host animals. In three weeks, we observed a substantial domination of CD45.1 cells over CD45.2 in experimental groups, while control (vehicle) group exhibited equal representation of both genotypes. In vitro, treatment with VA or N2N1 contributed to prolonged availability of HSC in serial replating CFU assays in methylcellulose and long-term culture initiating cell (LTC-IC) assays. Addition of VA or N2N1 to the short-term cultures (7-14 days) of normal bone marrow cells in a medium containing a cocktail of growth factors (Il6, IL3, FLT3L, TPO, SCF) resulted in maintenance and growth of HSC or progenitors. Gated on lymphocyte sub-population, treated with N2N1 or VA samples revealed ~0.3%±0.02 or 0.2% ±0.02 of CD34+, CD45+ cells correspondingly, while control samples had 0.08% of these cells (the result of three independent experiments). Most importantly, we observed colonies formation, after application of these drugs to the bone marrow isolated from the patients diagnosed with severe AA. Further studies also indicated that these agents do not promote growth of leukemic cell. Analysis of mechanism of action showed that VA and N1N2 function as redox co-factors in oxidations of NAD(P)H. VA transfers electrons non-enzymaticly from NAD(P)H to oxidized glutathione or peroxides. N2N1 is a redox co-factor for the NAD(P)H dehydrogenase (quinone) 1 (NQO1) and together they move electrons from NAD(P)H to cytochrome c or CoQ10. As such, we presented here a comprehensive prove that pharmacologic manipulation of redox balance controlled by glutathione or NQO1 activity via redox catalysts can ameliorate the detrimental consequences of HSC loss during normal aging by interfering with direct ROS mediated signaling or attenuating collateral ROS mediated damages. Figure. Figure. Disclosures Maciejewski: Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Loss of Ubp3 increases silencing, decreases unequal recombination in rDNA, and shortens the replicative life span in Saccharomyces cerevisiae

Ubp3 is a conserved ubiquitin protease that acts as an antisilencing factor in MAT and telomeric regions. Here we show that ubp3∆ mutants also display increased silencing in ribosomal DNA (rDNA). Consistent with this, RNA polymerase II occupancy is lower in cells lacking Ubp3 than in wild-type cells in all heterochromatic regions. Moreover, in a ubp3∆ mutant, unequal recombination in rDNA is highly suppressed. We present genetic evidence that this effect on rDNA recombination, but not silencing, is entirely dependent on the silencing factor Sir2. Further, ubp3∆ sir2∆ mutants age prematurely at the same rate as sir2∆ mutants. Thus our data suggest that recombination negatively influences replicative life span more so than silencing. However, in ubp3∆ mutants, recombination is not a prerequisite for aging, since cells lacking Ubp3 have a shorter life span than isogenic wild-type cells. We discuss the data in view of different models on how silencing and unequal recombination affect replicative life span and the role of Ubp3 in these processes.