Aging

Published by Impact Journals
Online ISSN: 1945-4589
Publications
Article
In addition to its traditional role in the regulation of calcium homeostasis and bone metabolism, vitamin D also exhibits immunomodulatory, anti-proliferative and cancer preventive activities. Molecular mechanisms that confer the chemo-preventive properties to vitamin D are poorly understood. We previously reported that constitutive phosphorylation of histone H2AX on Ser139 (γH2AX) and activation of ATM (Ser1981 phosphorylation), seen in untreated normal or tumor cells predominantly in S phase of the cell cycle, is to a large extent indicative of DNA replication stress occurring as a result of persistent DNA damage caused by endogenous oxidants, by-products of oxidative metabolism. In the present study we observed that exposure of mitogenically stimulated human lymphocytes, pulmonary carcinoma A549 and lymphoblastoid TK6 cells to 1,25-dihydroxyvitamin D3 (1,25-VD) reduced the level of constitutive expression of γH2AX and ATM-S1981P. We also observed that the H2O2-induced rise in the level of γH2AX in lymphocytes was attenuated by 1,25-VD. Whereas in lymphocytes 1,25-VD reduced by 50-70% the level of endogenous oxidants as determined by their ability to oxidize 2,7-dichlorodihydrofluorescein (DCFH) in A549 and TK6 cells the attenuation of DNA damage signaling by 1,25-VD was seen in the absence of detectable reduction in DCFH oxidation. These findings suggest that while the anti-oxidant activity of 1,25-VD may contribute to a reduction in the intensity of DNA replication stress in lymphocytes, other factors play a role in the 1,25-VD effects seen in A549 and TK6 cells. The data are consistent with the recent report on the interaction between DNA damage signaling (ATM activation) and 1,25D receptor (VDR) phosphorylation that lead to enhancement of DNA repair efficiency, and provide further support for the chemo-preventive and anti-aging properties of this vitamin/hormone.
 
Article
In adult mammals, neural stem cells (NSCs) generate new neurons that are important for specific types of learning and memory. Controlling adult NSC number and function is fundamental for preserving the stem cell pool and ensuring proper levels of neurogenesis throughout life. Here we study the importance of the microRNA gene cluster miR-106b~25 (miR-106b, miR-93, and miR-25) in primary cultures of neural stem/progenitor cells (NSPCs) isolated from adult mice. We find that knocking down miR-25 decreases NSPC proliferation, whereas ectopically expressing miR-25 promotes NSPC proliferation. Expressing the entire miR-106b~25 cluster in NSPCs also increases their ability to generate new neurons. Interestingly, miR-25 has a number of potential target mRNAs involved in insulin/insulin-like growth factor-1 (IGF) signaling, a pathway implicated in aging. Furthermore, the regulatory region of miR-106b~25 is bound by FoxO3, a member of the FoxO family of transcription factors that maintains adult stem cells and extends lifespan downstream of insulin/IGF signaling. These results suggest that miR-106b~25 regulates NSPC function and is part of a network involving the insulin/IGF-FoxO pathway, which may have important implications for the homeostasis of the NSC pool during aging.
 
List of miRNAs differentially expressed in the liver of three months old Wrn hel/hel mutant compared to wild type mice with an adjusted P-value < 0.1
Life span of wild type and mutant C. elegans animals. (A) Survival curves of wild type (N2) and wrn-1(gk99) animals grown at 25°C. (B) Survival curves of wild type (N2) and mir-124 (n4255) strains grown at 25°C. (C) Survival curves of wild type (N2) and wrn-1;mir-124 double mutant animals grown at 25°C. All experiments were performed with five different pools of 20 to 30 animals of each genotype. The indicated P-values were obtained using the log-rank test method.  
Reactive oxygen species (ROS) and ATP levels in wild type and mutant C. elegans strains. (A) ROS levels in whole body of 7-day adult old worms (post-larval L4 stage) detected with dichlorofluorescein. Data are expressed as units of fluorescence per micrograms of proteins. (Unpaired Student's t-test; *P = 0.0442 vs. wild type; **P = 0.0008 vs. wild type). (B) ATP levels in whole body of 7-day old adult worms (post-larval L4 stage). (Unpaired Student's t-test; *P = 0.0410 vs. wild type; **P = 0.0137 vs. wild type). Data are expressed as units of fluorescence per micrograms of proteins. Twenty-five worms of each genotype were collected for the ROS or ATP measurements. Experiments were performed with three independent pools of animals.
The aging marker lipofuscin is increased in mutant C. elegans strains. Representative photographs of wild type (N2), wrn1(gk99), mir-124(n4255), and wrn-1;mir-124 double mutant worms at three days into adulthood. Panels on the right represent the lipofuscin autofluorescence alone. All pictures were taken at the same exposure time. Magnification is 10 X. The histogram at the bottom represents the average intensity of lipofuscin autofluorescence in the different C. elegans strains. Ten to fifteen three-days old (three days into adulthood) worms of each strain were photographed and the fluorescence intensity was quantified using Adobe Photoshop. The fold increase in fluorescence intensity compared to wild type animals is indicated. (Unpaired Student's t-test; *P = 0.00002 for wrn1(gk99) vs. wild type; **P = 0.00222 for mir-124(n4255) vs. wild type; and ***P = 0.00078 for wrn-1;mir-124 vs. wild type).
Impact of vitamin C on the life span of wild type and mutant C. elegans strains. (A) Survival curves of vitamin C treated wrn-1(gk99) and of untreated wild-type (N2) and wrn- 1(gk99) C. elegans strains grown at 25°C (vitamin C treated wrn-1(gk99) vs. untreated wrn-1(gk99) worms: P = 1.4 × 10 -7 ; vitamin C treated wrn-1(gk99) vs. untreated wild-type worms: P = 0.0778). (B) Survival curves of vitamin C treated mir- 124(n4255) and of untreated wild-type (N2) and mir- 124(n4255) C. elegans strains grown at 25°C (vitamin C treated mir-124(n4255) vs. untreated mir-124(n4255) worms: P = 3.0 × 10 -9 ; vitamin C treated wrn-1(gk99) vs. untreated wild-type worms: P = 0.359). (C) Survival curves of vitamin C treated wrn-1;mir-124 and of untreated wild-type (N2) and wrn-1;mir- 124 double mutant C. elegans strains grown at 25°C (vitamin C treated wrn-1;mir-124 vs. untreated wrn-1;mir-124 worms: P = 3.1 × 10 -6  
Article
Small non-coding microRNAs are believed to be involved in the mechanism of aging but nothing is known on the impact of microRNAs in the progeroid disorder Werner syndrome (WS). WS is a premature aging disorder caused by mutations in a RecQ-like DNA helicase. Mice lacking the helicase domain of the WRN ortholog exhibit many phenotypic features of WS, including a pro-oxidant status and a shorter mean life span.Caenorhabditis elegans (C. elegans) with a nonfunctional wrn-1 DNA helicase also exhibit a shorter life span. Thus, both models are relevant to study the expression of microRNAs involved in WS. In this study, we show that miR-124 expression is lost in the liver of Wrn helicase mutant mice. Interestingly, the expression of this conserved miR-124 in whole wrn-1 mutant worms is also significantly reduced. The loss of mir-124 in C. elegans increases reactive oxygen species formation and accumulation of the aging marker lipofuscin, reduces whole body ATP levels and results in a reduction in life span. Finally, supplementation of vitamin C normalizes the median life span of wrn-1 and mir-124 mutant worms. These results suggest that biological pathways involving WRN and miR-124 are conserved in the aging process across different species.
 
Relative miR-126-3p expression in plasma from 136 healthy subjects (CTR) and 193 patients (T2DM) divided into age groups. Error bars (a) showing relative miR-126-3p expression (in a.u.) in plasma of young (20-45 years, 
Characterization of young, intermediate-age and senescent HUVECs. Bar chart showing cumulative population doubling (CPD) (a), SA-β-gal activity (%) (b), p16INK4a relative expression reported as fold changes vs. young cells (c) and telomere length (T/S) reported as fold changes vs. young cells (d), in young, intermediate age and senescent HUVECs. *GLM, p<0.05. 
Article
Circulating miR-126-3p levels were determined in 136 healthy subjects (CTRs) aged 20-90 years and 193 patients with type-2 diabetes mellitus (T2DMs) aged 40-80 years, to explore the combined effect of age and glycemic state on miR-126-3p expression. Moreover, intra/extracellular miR-126-3p levels were measured in human endothelial cells (HUVECs) undergoing senescence under normo/hyper-glycemic conditions. Plasma miR-126-3p was significantly higher in the oldest compared with the youngest CTRs (<45 vs. >75 years; relative expression: 0.27±0.29 vs. 0.48±0.39, p=0.047). Age-based comparison between CTRs and T2DM demonstrated significantly different miR-126-3p levels only in the oldest (0.48±0.39 vs. 0.22±0.23, p<0.005). After multiple adjustments, miR-126-3p levels were seen to be lower in patients with poor glycemic control, compared with age-matched CTRs. The age-related increase in plasma miR-126-3p found in CTRs was paralleled by a 5/6-fold increase in intra/extracellular miR-126-3p in in vitro-cultured HUVECs undergoing senescence. Notably, significant down-regulation of SPRED-1 protein, a validated miR-126-3p target, was found in senescent HUVECs. Moreover, miR-126-3p expression was down-regulated in intermediate-age HUVECs grown in high-glucose medium until senescence. Aging/senescence-associated miR-126-3p up-regulation is likely a senescence-associated compensatory mechanism that is blunted when endothelial cells are exposed to high glucose levels, a phenomenon that probably occurs in vivo in T2DM patients.
 
Dynamics of food consumption in male and female 129/Sv mice treated or non-treated with metformin.
Dynamics of drinking water consumption in male and female 129/Sv mice treated or non-reated with metformin.
Effect of antidiabetic drugs on life span and spontaneous carcinogenesis in rodents 
Article
Studies in mammals have led to the suggestion that hyperglycemia and hyperinsulinemia are important factors both in aging and in the development of cancer. It is possible that the life-prolonging effects of calorie restriction are due to decreasing IGF-1 levels. A search of pharmacological modulators of insulin/IGF-1 signaling pathway (which mimetic effects of life span extending mutations or calorie restriction) could be a perspective direction in regulation of longevity. Antidiabetic biguanides are most promising among them. The chronic treatment of inbred 129/Sv mice with metformin (100 mg/kg in drinking water) slightly modified the food consumption but failed to influence the dynamics of body weight, decreased by 13.4% the mean life span of male mice and slightly increased the mean life span of female mice (by 4.4%). The treatment with metformin failed influence spontaneous tumor incidence in male 129/Sv mice, decreased by 3.5 times the incidence of malignant neoplasms in female mice while somewhat stimulated formation of benign vascular tumors in the latter.
 
Article
Insertion of 144-base pair (bp) containing six extra octapeptide repeats between residues 51 and 91 of prion protein (PrP) gene is associated with inherited prion diseases. Most cases linked to this insertion examined by Western blotting showed detectable proteinase K-resistant PrPSc (rPrPSc) resembling PrPSc type 1 and type 2 in sporadic Creutzfeldt-Jakob disease (sCJD), or PrP7-8 in Gerstmann-Sträussler-Scheinker disease. However, cases lacking detectable rPrPSc also have been reported. Which PrP conformer is associated with neuropathological changes in the cases without detectable rPrPSc remains to be determined. Here we report that while all six but one subjects with the 144-bp insertion mutations examined display the pathognomonic PrP patches in the cerebellum, one of them exhibits no detectable typical rPrPSc even in PrPSc-enriched preparations. Instead, a large amount of abnormal PrP is captured from this case by gene 5 protein and sodium phosphotungstate, reagents that have been proved to specifically capture abnormal PrP. All captured abnormal PrP from the cerebellum and other brain regions is virtually sensitive to PK-digestion (termed sPrPSc). The presence of the predominant sPrPSc but absence of rPrPSc in this 144-bp insertion-linked inherited CJD case suggests that mutant sPrPSc is the main component of the PrP deposit patches and sPrPSc is sufficient to cause neurotoxicity and prion disease.
 
Article
Senescence is a cellular program that irreversibly arrests the proliferation of damaged cells and induces the secretion of the inflammatory mediators IL- 6 and IL-8 which are part of a larger senescence associated secretory phenotype (SASP). We screened quiescent and senescent human fibroblasts for differentially expressed microRNAS (miRNAs) and found that miRNAs 146a and 146b (miR-146a/b) were significantly elevated during senescence. We suggest that delayed miR-146a/b induction might be a compensatory response to restrain inflammation. Indeed, ectopic expression of miR-146a/b in primary human fibroblasts suppressed IL-6 and IL-8 secretion and downregulated IRAK1, a crucial component of the IL-1 receptor signal transduction pathway. Cells undergoing senescence without induction of a robust SASP did not express miR-146a/b. Further, IL-1alpha neutralizing antibodies abolished both miR-146a/b expression and IL-6 secretion. Our findings expand the biological contexts in which miRNA-146a/b modulates inflammatory responses. They suggest that IL-1 receptor signaling initiates both miR-146a/b upregulation and cytokine secretion, and that miR-146a/b is expressed in response to rising inflammatory cytokine levels as part of a negative feedback loop that restrains excessive SASP activity.
 
Replicative senescence in human dermal fibroblasts (HDFn). (A) Growth curve of cultured HDFn. (B) SA‐β‐gal staining of HDFn cells at p1 and p16 in culture. (C) HDFn cultured cells at p1 and p16 (p, passage) were subjected to a 4h BrdU‐pulse, then collected, PI stained and analyzed by flow cytometry. BrdU positive cells are indicated as S phase fluorescent population and are assessed by PI staining of DNA content of 2n or 4n (fixed to values of 200 and 400 in the plots). (D) Western blots performed on proteins extracts from HDFn at p1 and p16 showing the analysis of some senescence markers, such as p53 and p16 levels. β‐actin was used as loading control. (E) Real Time RT‐qPCR was employed to analyze the expression levels of miR‐152 and miR‐181a in HDFn cells at a growing number of passages in culture (p1‐p16). Values reported are the average ± SD of three independent experiments.  
miR‐152 and miR‐181a induce cellular senescence in HDFn cells. (A) 96 h after transfection of HDFn cells with scramble control (Ctr), miR‐152 or miR‐181a sequence, cells were subjected to a 4h BrdU‐ pulse, then collected, PI stained and analyzed by flow cytometry as described in Figure 1. (B) Western blot analysis of protein extract of HDFn transfected with miR‐152 and miR‐181a versus scramble control sequence (ctr). MiR‐152and miR‐181a overexpression increase p53 and p16INK4 protein level. β‐actin was used as loading control. (C‐D) SA‐β‐gal staining and quantification by blue cells counting/field (as fold over control). Values reported are average ± SD of three independent stains. *p‐Value <0.01 by Student's t test.  
miR-152 represses ITGA5 expression and controls HDFn cell adhesion. (A) Predicted miR-152 target sites on human ITGA5 3'UTR were identified by TargetScan 6.2 software. (B) Real Time RT-qPCR was employed to analyze the expression level of ITGA5 in HDFn cells at a growing number of passages in culture (p1p16). Values reported are the average ± SD of three independent experiment. (C) Western blot of HDFn cells protein extracts collected at increasing passage number in culture (p1-p16). Itga5 protein level is shown and β-actin was used as loading control. (D) Insertion of ITGA5 3'UTR target sequence in a luciferase reporter vector leads to diminished luciferase activity in presence of miR-152 in HEK293 cells 24h after co-transfection. Histograms show the values resulting as the average ± SD from three independent co-transfections.(E) Real Time RT-qPCR was employed to analyze the expression level of ITGA5 in proliferating HDFn cells transfected with miR-152 versus a scrambled control sequence (Ctr). Values reported are the average ± SD of three independent experiment. (F) Western blot analysis of protein extracts of HDFn transfected with miR-152 versus a scrambled control sequence (Ctr). miR-152 overexpression decreases ITGA5 protein levels; β-actin was used as a loading control. (G) Adhesion assay performed on proliferating HDFn cells transfetcted with miR-152 versus scrambled control sequence. (H) Histogram shows adhesion ability of proliferating HDFn 96h after transfection with control or miR-152. Values reported are the average ± SD of three independent experiment. *p-Value <0.01 by Student's t test.
COL16A1 expression is downregulated in senescent HDFn cells and its 3'‐UTR is direct miR‐ 181a target. (A) Predicted miR‐181a target site on human COL16A1 3'UTR was identified by TargetScan 6.2 software. (B) Real Time RT‐qPCR was employed to analyze the expression level of COL16A1 in HDFn cells at a growing number of passages in culture (p1‐p16). Values reported are the average ± SD of three independent experiment. (C) Western blot of HDFn cells protein extracts collected at increasing passage number in culture (p1‐p16). Col16a1 protein level is shown and β‐actin was used as loading control. (D) Insertion of COL16A1 3'UTR target sequence in a luciferase reporter vector leads to diminished luciferase activity in presence of miR‐181a in HEK293 cells 24h after co‐transfection. Histograms show the values resulting as the average ± SD from three independent co‐transfections. (E) Real Time RT‐qPCR was employed to analyze the expression level of COL16A1 in proliferating HDFn cells transfected with miR‐181a versus a scrambled control sequence (ctr). Values reported are the average ± SD of three independent experiment. (F) Western blot analysis of protein extracts of HDFn transfected with miR‐181a versus a scrambled control sequence (ctr). Itga5 protein levels is shown and β‐actin was used as a loading control.  
Article
Ageing of human skin is associated with phenotypic changes in the cutaneous cells; the major functional markers of ageing occur as consequences of dermal and epidermal cell senescence and of structural and compositional remodeling of normally long-lived dermal extracellular matrix proteins. Understanding the contribution of the dermal cells in skin ageing is a key question, since this tissue is particularly important for skin integrity and its properties can affect the epidermis. Several microRNAs have been shown to be involved in the regulation of pathways involved in cellular senescence and exerted important effects on tissues ageing. In this study, we demonstrate that the expression of miR-152 and miR-181a increased during the human dermal fibroblasts senescence and that their overexpression, is sufficient to induce cellular senescence in early-passage cells. The increase of these miRNAs during cells senescence was accompanied by a decrease in integrin α5 and collagen XVI expression at mRNA and/or protein levels resulting in reduced cellular adhesion and suggesting extracellular matrix remodeling. These findings indicate that changes in miRNAs expression, by modulating the levels of adhesion proteins and extra-cellular matrix components, such as integrin α5 and collagen XVI, could contribute to the compositional remodelling of the dermis and epidermis occurring during skin aging.
 
Article
This model however was not yet complete: It was not obvious why the expression of Class II genes - which are only directly controlled by PQM-1 and not by DAF-16 - should rise when DAF-16 function is lost in a daf-2(-) background. To address this paradox, we performed additional experiments that revealed an active avoidance of DAF-16 and PQM-1 residing in the nucleus together. The translocation of DAF-16 to the nucleus in response to loss of DAF-2 signaling causes PQM-1 to be displaced to the cytoplasm; conversely, DAF-16 becomes more nuclear in wild-type (N2) worms after RNAi knockdown of PQM-1. Together, these observations suggest an elegant mechanism for switching between stress response and growth/development. While stress response is required for survival of an acute insult, it is likely to inhibit development and may be energetically costly to maintain. Through its antagonism with DAF-16, nuclear PQM-1 may help the worm maintain an "unstressed" transcriptional state that is critical to the animal's ability to develop. Loss of PQM-1 suppresses daf-2 longevity and thermotolerance and further slows development. We also observed progressive loss of nuclear PQM-1 during wild-type aging, along with declining expression of its target genes. By day seven of adulthood, the tightly antagonistic coupling between DAF-16 and PQM-1 is lost, with both factors residing in the cytoplasm.
 
Quantitative analysis of IGF1R protein levels. (A) Alignment of the 50 amino acids comprising the epitope recognized by the anti-IGF1R polyclonal antibody used (SC-712, see Experimental Procedures). This region maps to the N-terminus of the protein, which is on the alpha-chain peptide of IGF1R. It excludes the signaling peptide, which is cleaved during protein maturation, and was not used in the immunogen. Human sequence is included due to it being used to generate the antibody. Sequence identity is indicated by dots. (B) Western blot of serial dilutions of mouse brain whole tissue protein extracts. Linear region was between 25 and 3.12 μg. (C) Band intensity in arbitrary units, plotted against protein loaded to verify linearity. 
Levels of IGF1R protein in brain tissue are highly negatively correlated to lifespan, and weakly negatively correlated to body mass. (A) Western blots showing IGF1R and actin bands. (B) Log-transformed graph of IGF1R intensity plotted against maximum lifespan shows strong correlation (r 2 =0.61, p=0.0008). (C) Log-transformed graph of IGF1R intensity plotted against average adult body mass show marginally significant correlation (r 2 =0.31, p=0.037). Error bars: 1 SD. Correlation of brain IGF1R to lifespan was still strong (r 2 = 0.58, p = 0.0009) after multiple regression analysis factoring in the contribution of body mass to lifespan, whereas correlation between body mass and lifespan was non-significant (adjusted r 2 = 0.14, p=0.1). Phylogenetic correction by independent contrasts maintained a significant correlation to lifespan (r 2 = 0.374, p= 0.0261) while the correlation to body mass was rendered non-significant (r 2 = 0.189, p= 0.136). 
Levels of IGF1R protein in lung tissue do not correlate with lifespan or body mass. (A) Western blots showing IGF1R and actin bands. (B) Logtransformed graph of IGF1R intensity plotted against maximum lifespan shows very weak (non-significant) correlation (r 2 =0.05). (C) Log-transformed graph of IGF1R intensity plotted against average adult body mass show very weak (non-significant) correlation (r 2 =0.18, p=0.11). Error bars are s.d. 
Levels of IGF1R protein in heart tissue do not correlate with lifespan or body mass. (A) Western blots showing IGF1R and actin bands. (B) Logtransformed graph of IGF1R intensity plotted against maximum lifespan shows no correlation. (C) Logtransformed graph of IGF1R intensity plotted against average adult body mass shows no correlation. Error bars are s.d. 
Levels of IGF1R protein in kidney tissue do not correlate with lifespan or body mass. (A) Western blots showing IGF1R and actin bands. (B) Log-transformed graph of IGF1R intensity plotted against maximum lifespan shows no correlation. (C) Log-transformed graph of IGF1R intensity plotted against average adult body mass shows no correlation. Error bars are s.d. 
Article
The insulin/insulin-like growth factor signaling (IIS) pathway is a major conserved regulator of aging. Nematode, fruit fly and mouse mutants with reduced IIS signaling exhibit extended lifespan. These mutants are often dwarfs leading to the idea that small body mass correlates with longevity within species. However, when different species are compared, larger animals are typically longer-lived. Hence, the role of IIS in the evolution of life history traits remains unresolved. Here we used comparative approach to test whether IGF1R signaling changes in response to selection on lifespan or body mass and whether specific tissues are involved. The IGF1R levels in the heart, lungs, kidneys, and brains of sixteen rodent species with highly diverse lifespans and body masses were measured via immunoblot after epitope conservation analysis. We report that IGF1R levels display strong negative correlation with maximum lifespan only in brain tissue and no significant correlations with body mass for any organ. The brain-IGF1R and lifespan correlation holds when phylogenetic non-independence of data-points is taken into account. These results suggest that modulation of IGF1R signaling in nervous tissue, but not in the peripheral tissues, is an important factor in the evolution of longevity in mammals.
 
Article
Cardiac failure is a leading cause of age-related death, though its root cause remains unknown. Mounting evidence implicates a decline in mitochondrial function due to increased opening of the mitochondrial permeability transition pore (mPTP). Here we report that the NAD+-dependent deacetylase SIRT3 deacetylates the regulatory component of the mPTP, cyclophilin D (CypD) on lysine 166, adjacent to the binding site of cyclosporine A, a CypD inhibitor. Cardiac myocytes from mice lacking SIRT3 exhibit an age-dependent increase in mitochondrial swelling due to increased mPTP opening, a phenotype that is rescued by cyclosporine A. SIRT3 knockout mice show accelerated signs of aging in the heart including cardiac hypertrophy and fibrosis at 13 months of age. SIRT3 knockout mice are also hypersensitive to heart stress induced by transverse aortic constriction (TAC), as evidenced by cardiac hypertrophy, fibrosis, and increased mortality. Together, these data show for the first time that SIRT3 activity is necessary to prevent mitochondrial dysfunction and cardiac hypertrophy during aging and shed light on new pharmacological approaches to delaying aging and treating diseases in cardiac muscle and possibly other post-mitotic tissues.
 
The osteogenic capacity of old mice is significantly reduced both in vivo and in vitro. Statistically analyzed values show the mean ± SD (n=10). * p < 0.05. (A) Micro‐CT analysis of trabecular bone mass in the tibiae of 4 (young) and 16 month‐old (old) mice. Quantitative analyses were performed via volumetric bone mineral density (BMD) and trabecular bone volume fraction (BV/TV) measurements. (B) HE stainings of histological sections from femur derived from young and old mice for detection of the number of bone trabeculae. (C‐D) Representative images of the CFU‐F assay for determination of proliferation capacity and of the CFU‐Ob assay for osteogenic differentiation ability of BMMSCs obtained from young and old mice and stained with crystal violet and alizarin red, respectively. CFU efficiency was determined by the number of colonies relative to the total number of seeded cells in each plate. (E) Serum levels of TNF‐α and INF‐γ in young and old mice determined via ELISA. Results are expressed as pg/ml.  
BMMSCs from old mice express higher levels of senescence markers and lower osteoblast markers compared to young ones. Statistically analyzed values show the mean ± SD (n=10). * p < 0.05. (A) In vitro staining of the senescence-related marker ß-galactosidase in BMMSCs cultures derived from young and old mice. Quantitative analysis of the total number of positively stained cells. (B-C) Real-time PCR analyses on whole bone tissue extracts (B) and on BMMSCs (C) for the senescence-related genes p16, p21 and p53. Normalization to ß-actin. (D) The western blot showed that the protein level changed as the mRNA. (E) Alizarin red staining of BMMSCs from young and old mice osteogenically induced for 14 d. Cont = Control, OS = osteogenically induced. (F-G) Real-time PCR and western blot analyses on BMMSCs for the osteogenic markers Runx2, ALP, osterix. Normalization to ß-actin.
Overexpression of p53 changed the phenotype of young BMMSCs into old BMMSCs. BMMSCs from young mice were lentivirally transduced to upregulate the expression level of p53 (= pLenti-p53) or were transduced as lentiviral control (= pLenti-Cont). Statistically analyzed values show the mean ± SD (n=10). * p < 0.05. (A) Alizarin red staining of pLenti-p53 and of pLenti-Cont after osteogenic inducing for 14 days. Cont = Control, OS = osteogenically induced. The values show the mean ± SD (n=10). * p < 0.05. (B-C) Real-time PCR and western blot analyses on BMMSCs with lentiviral transduction (pLenti-p53 and pLenti-Cont) and with/without osteogenic induction for the osteogenic markers Runx2, ALP, osterix. Normalization to ß-actin. (D-E) Histological analyses and corresponding statistical analysis of tissue sections from subcutaneous pockets on the backs of 6-week-old NOD/SCID mice with implanted HA/TCP ceramic particles mixed with BMMSCs from young mice with lentiviral transduction of p53 and control.
Schematic diagram of p53/miR‐17/Smurf1 cascade. (A, B) p53 regulates the osteogenic differentiation of BMMSCs through inhibiting transcription of miR‐17‐92 cluster and subsequent modulating Smurf1, a direct target gene of miR‐17, aslo acts as a negative regulator for osteogenic differentiation of mesenchymal stem cells.  
Article
Osteoporosis is an age-related progressive bone disease. Trp53 (p53) is not only a famous senescence marker but also a transcription regulator which played a critical role in osteogenesis. However, how p53 contributes to the bone mass loss in age-related osteoporosis is still unclear. Here, we found that bone mass and osteogenic differentiation capacity of mesenchymal stem cells (MSCs) is significantly reduced with advancing age. Serum levels of TNF-α and INF-γ and senescence-associated β-galactosidase, p16, p21 and p53 are significantly increased in elder mice, but antipodally, osteogenic marker expression of Runx2, ALP and osterix are reduced. Overexpression p53 by lentivirus inhibits osteogenesis in young MSCs in culture and upon implantation in NOD/SCID mice through inhibiting the transcription of miR-17-92 cluster, which is decreased in old mice. In addition, miR-17 mimics could partially rescue the osteogenesis of old MSCs both in vitro an in vivo. More importantly, Smurf1 as a direct target gene of miR-17, plays an important role in the p53/miR-17 cascade acting on osteogenesis. Our findings reveal that p53 inhibits osteogenesis via affecting the function of MSCs through miRNA signaling pathways and provide a new potential target for treatment in future.
 
Article
The protein tyrosine phosphatase nonreceptor 22 gene (PTPN22) is an important negative regulator of signal transduction through the T-cell receptors (TCR). Recently a single-nucleotide polymorphism (SNP) 1858 C/T within this gene was shown to be a risk factor for several autoimmune diseases, such as rheumatoid arthritis (RA), Graves' Disease (GD), systemic lupus erythematosus (SLE), Wegener's granulomatosis (WG) and type 1 diabetes mellitus (T1D). The aim of this study was to analyze a possible association between 1858 C/T SNP and a number of autoimmune diseases, including RA, GD and T1D in Russian population. Patients with T1D, GD, RA and healthy controls were genotyped for the 1858 C/T SNP in PTPN22 gene. We found a significant association between PTPN22 1858 C/T SNP and T1D and GD. 1858T/T genotype was observed more frequently in T1D and GD patients compared to control subjects. No such association was observed for RA. In concordance with a previous data establishing PTPN22 1858 C/T SNP association with several autoimmune diseases, our findings provide further evidence that the PTPN22 gene may play an important role in the susceptibility to some autoimmune diseases.
 
Article
The editorial board of Aging reviews research papers published in 2009, which they believe have or will have a significant impact on aging research. Among many others, the topics include genes that accelerate aging or in contrast promote longevity in model organisms, DNA damage responses and telomeres, molecular mechanisms of life span extension by calorie restriction and pharmacologic interventions into aging. The emerging message in 2009 is that aging is not random but determined by a genetically-regulated longevity network and can be decelerated both genetically and pharmacologically.
 
Article
Antibody and B cell responses to influenza A viruses were measured over a period of 2 months in 30 aged and 15 middle-aged individuals following vaccination with the 2011/12 trivalent inactivated influenza vaccine by micro-neutralization assays, ELISAs, ELISpot assays and cell surface staining with lineage-defining antibodies followed by multicolor flow cytometry. Both cohorts developed comparable antibody responses to the H3N2 virus of the vaccine while responses to the H1N1 virus were compromised in the aged. ELISpot assays of peripheral blood mononuclear cells (PBMCs) gave comparable results for the two cohorts. Analysis by flow cytometry upon staining of CD19+IgD-CD20- PBMCs with antibodies to CD27 and CD38 showed markedly reduced increases of such cells following vaccination in the aged. Additional analysis of cells from a subset of 10 younger and 10 aged individuals indicated that in the aged a portion of IgG producing cells lose expression of CD27 and reduce expression of CD38.
 
Increased IL-21 secretion from aged subjects is not due to age-associated alteration in dendritic cell function. A. Bar graph depicts the levels of IL-12p70 in the supernatant from stimulated aged and young DC. B. Bar graph depicts the percent proliferation of aged and young CD4+ T cells after culture with aged DCs. C. Bar graph depicts the level of IL-21 in the supernatant of aged and young CD4+ T cells after culture with aged DCs. D. Bar graph depicts the level of IFN-in the supernatant of aged and young CD4+ T cells after culture with aged DCs. E. Bar graph depicts the percent proliferation of aged and young CD4+ T cells after culture with young DCs. F. Bar graph depicts the level of IL-21 in the supernatant of aged and young CD4+ T cells after culture with young DCs. G. Bar graph depicts the level of IFN- in the supernatant of aged and young CD4+ T cells after culture with young DCs. Data is mean +/-S.E. of 8 different aged and young subjects.
STAT-4 phosphorylation is altered in CD4+ T cells from aged subjects. A. Histograms depict the phosphorylation of STAT-4 in naïve CD4+ T cells from aged and young at 0, 20, 60and 90 m after stimulation with IL-12. Graph is representative of 12 such experiments. B. Bar graph depicts the mean fluorescence intensity (MFI) of phosphorylation of STAT-4 in aged and young CD4+ T cells before and after activation with IL-12. Data is mean +/-S.E. of 12 different aged and young subjects. C. Bar graph also depicts phospho STAT-4 levels, the same as B using in cell ELISA. D. Western blot represents the level of non-phosphorylated STAT-4 in aged and young CD4+ T cells. Data represents pooled samples from 5 different aged and young subjects. E. Bar graph also depicts total STAT as in D using in cell ELISA. F. Graph depicts the level of IL-21 after treatment with JAK-2 inhibitor in aged CD4+ T cells. G. Graph depicts the level of IFN- after treatment with JAK-2 inhibitor in aged CD4+ T cells. Data is mean +/-S.E. of 6 different aged subjects.
Article
Advancing age leads to significant decline in immune functions. IL-21 is produced primarily by T follicular helper (Tfh) cells and is required for effective immune cell functions. Here we compared the induction of IL-21 in aged and young subjects. Our investigation demonstrates that CD4+T cells from healthy elderly individuals (age ≥ 65) secreted significantly higher levels of IL-21 on priming with aged and young dendritic cells (DC). Though the aged and young DCs secreted comparable levels of IL-12 on stimulation with anti-CD40 antibody and LPS, culture of DCs with aged CD4+ T cells resulted in increased production of IL-21 as compared to that with young CD4+ T cells. Further examination revealed that the response of aged naïve CD4+ T cells to IL-12 was altered, resulting in increased differentiation of aged Th cells towards Tfh cells. Investigation into the signaling mechanism suggested that phosphorylation of STAT-4 in response to IL-12 was sustained for a longer duration in aged CD4+ T cells as compared to CD4+ T cells from young subjects. Additional analysis demonstrated that increased IL-21 secretion correlated with chronic CMV infection in aged subjects. These findings indicate that chronic CMV infection alters the response of aged CD4+ T cells to IL-12 resulting in an increased secretion of IL-21 and that aging affects Tfh cell responses in humans which may contribute to age-associated inflammation and immune dysfunctions.
 
miR-29 and cellular arrest proteins are increased and IGF-1, p85 and B-myb are decreased in the muscles of aged rodents. (A) Total RNA obtained from hind 
miR-29 expression decreases muscle cell proliferation and induces cellular senescence in MPCs. (A) MPCs were transduced with Ad-miR-29 or the control adenovirus (Ad-empty). Proliferation was assessed using a chromogenic substrate and a commercial kit (Millipore) monitoring absorbance at 420 nm (Methods). The bars show the mean ± s.e. (n=6; *p<0.05 vs. Ad-empty). (B) MPCs were transduced with Ad-miR-29 or Ad-empty. The Ki67 expression, a marker of proliferation, was assessed by immunohistology. A positive Ki67 was determined as a bright red spot localized in the nuclei. The bar graph shows cells that were positively stained for Ki67 in miR-29 treated cells and are expressed as a percent of the positive cells in the Ad-empty treated cells. Counts were made in 5 pre-defined, randomly chosen fields; each field had >500 nuclei. Red staining in nuclei was assessed using the Micro-suite Five Biological Software (Bars: mean ± s.e.; n=6/condition; *p<0.05 vs. ad-empty). (C) MPCs were transduced with Ad-miR-29 or with the Ad-empty adenovirus. To block miR-29 expression in MPCs, vectors that express an antisense of Zip-miR29 (pmiRZip29a plus pmiRZip29c) were transfected into MPCs 4 hours before the Ad-miR-29 or Adempty viruses were added. The blue color identifies SA-βgal is present in cells with the "fried egg morphology" that signifies cellular senescence. The bar graph shows the percentage of cells with positive staining of SA-βgal in 5 pre-defined, randomly chosen fields (Bars: mean ± s.e.; n=6/condition; *p<0.05 vs. controls; #p<0.05 vs. Ad-miR-29). (D) MPCs were transduced with Ad-miR-29 or with Ad-empty adenovirus. To block miR-29 expression in MPCs, vectors that express an antisense of miR-29a+c (pmiRZip29a plus pmiRZip29c) were transfected into MPCs 4 hours before the Ad-miR29 or Adempty (Ctrl) viruses were added. miR-29a expression was measured using real-time qPCR; U6 was the internal control. The bar graph shows the amount of miR-29a expressed as a fold change from the level in controls (Bars: mean ± s.e.; ctrl defined as 1 fold; n=3 determinations per condition; *p<0.05 and #p<0.01 vs. Ad-miR29 only). 
miR-29 expression in muscles of mice decreases IGF-1, p85α, B-myb but increases cell cycle arrest proteins. The mouse microRNA mimic, miR-29a (mmumiR-29a), was injected into the right tibialis anterior (TA) muscle of 4 month old normal mice; permeation increased by muscle electroporation. The left TA muscle was used as a control and injected with mouse mimic miR-ctrl (mmu-miR-ctrl) and electroporated. Muscles were harvested at 7 and 30 days after electroporation. (A) RNA isolated from TA muscles and miR-29a expressions were measured using real-time qPCR; U6 was the internal control. The bar graph shows the amount of miR-29a expressed as a fold change from the level in controls (Bars: mean ± s.e.; ctrl defined as 1 fold; n=3 determinations per condition; *p<0.05 and **p<0.01 vs. control). (B) IGF-1 protein level was measured by ELISA in muscle lysates. Results in the bar graph compare the protein amount of IGF-1 in control muscle (ctrl) vs. miR-29 overexpressing muscle. All data were normalized to the muscle total protein concentration (Bars: mean ± s.e.; n=6; *p<0.05 vs. control). (C) Protein levels of P85α, B-myb, P53, P16 INK4A , RB and GAPDH in lysates from muscles at 7 days after electroporation were measured by Western blotting. There were two RB protein bands: the lower is hypophosphoryated RB (pRB; MW 107 kDa) and the upper band is more highly phosphorylated RB protein (ppRB; MW 112kDa). The bar graph shows the density of each protein band expressed as a fold-change from control levels (mimic miR-ctrl was set to 1, indicated by a horizontal line in the graph). All band densities were normalized to the density of GAPDH (Bars: mean ± s.e.; n=6; *p<0.05 vs. ctrl). (D) The proliferation marker (Ki67) was assessed by immuno-histochemistry in TA muscles at 30 days after electroporation of mmu-miR-29 or mmu-miR-ctrl into muscles. The bar graph shows the percentage of positive staining nuclei of Ki67 in 10 predetermined, random fields for each condition (Bars: mean ± s.e.; n=6/condition; *p<0.05 vs. controls). (E) The level of the senescence marker, SA-βgal, was assessed in cross sections of TA muscles at 30 days after electroporation of the mmu-miR-29 or mu-miR-ctrl. The blue color shows the presence of SA-βgal indicating the presence of cellular senescence. The bar graph shows the percentage of positive staining in 10 predetermined, random fields for each condition (Bars: mean ± s.e.; n=6/condition; *p<0.05 vs. controls). 
Wnt-3a induces miR-29 promoter activation. (A) MPCs were transfected with pGL3-miR-29a/b1 (gray bar) or pGL3-miR-29C/b2 (black bar) to assay miR-29 promoter activities. Transfected cells were treated as follows: left to right, treated with TGF-β; co-transfected with NFκB plasmid (pCMV1.p65); transduced with Ad-Wnt-3a; or treated with Wnt3a-conditioned media. The bar graph represents firefly luciferase activity corrected for renilla luciferase activity (FFL/RL) and compared to control (luciferase activity of pGL3 untreated, set to 1 and designated by a horizontal line in the graph). The data represent the means ± s.e.; (n=9, *p<0.05 vs. pGL3 (Ctrl). (B) MPCs were cultured in Wnt-3a conditioned media and cells harvested at the indicated times. Total RNA was isolated and miR-29a expression was measured by qPCR. The bar graph shows miR-29a from Wnt-3a conditional medium expressed as a fold-change vs. miR-29a levels in cells cultured with control media (set to 1). Results are normalized to U6 RNA as an internal control. The data represent the means ± s.e.; (n=3 pairs; *p<0.05 vs. control). 
Article
The mechanisms underlying the development of aging-induced muscle atrophy are unclear. By microRNA array and individual qPCR analyses, we found significant up-regulation of miR-29 in muscles of aged rodents vs. results in young. With aging, p85α, IGF-1 and B-myb muscle levels were lower while the expression of certain cell arrest proteins (p53, p16 and pRB) increased. When miR-29 was expressed in muscle progenitor cells (MPC), their proliferation was impaired while SA-βgal expression increased signifying the development of senescence. Impaired MPC proliferation resulted from interactions between miR-29 and the 3'-UTR of p85a, IGF-1 and B-myb, suppressing the translation of these mediators of myoblast proliferation. In vivo, electroporation of miR-29 into muscles of young mice suppressed the proliferation and increased levels of cellular arrest proteins, recapitulating aging-induced responses in muscle. A potential stimulus of miR-29 expression is Wnt-3a since we found that exogenous Wnt-3a stimulated miR-29 expression 2.7-fold in primary cultures of MPCs. Thus, aging-induced muscle senescence results from activation of miR-29 by Wnt-3a leading to suppressed expression of several signaling proteins (p85α, IGF-1 and B-myb) that act coordinately to impair the proliferation of MPCs contributing to muscle atrophy. The increase in miR-29 provides a potential mechanism for aging-induced sarcopenia.
 
Article
The RNA-binding protein tristetraprolin (TTP) regulates expression of many cancer-associated and proinflammatory factors through binding AU-rich elements (ARE) in the 3'-untranslated region (3'UTR) and facilitating rapid mRNA decay. Here we report on the ability of TTP to act in an anti-proliferative capacity in HPV18-positive HeLa cells by inducing senescence. HeLa cells maintain a dormant p53 pathway and elevated telomerase activity resulting from HPV-mediated transformation, whereas TTP expression counteracted this effect by stabilizing p53 protein and inhibiting hTERT expression. Presence of TTP did not alter E6 and E7 viral mRNA levels indicating that these are not TTP targets. It was found that TTP promoted rapid mRNA decay of the cellular ubiquitin ligase E6-associated protein (E6-AP). RNA-binding studies demonstrated TTP and E6-AP mRNA interaction and deletion of the E6-AP mRNA ARE-containing 3'UTR imparts resistance to TTP-mediated downregulation. Similar results were obtained with high-risk HPV16-positive cells that employ the E6-AP pathway to control p53 and hTERT levels. Furthermore, loss of TTP expression was consistently observed in cervical cancer tissue compared to normal tissue. These findings demonstrate the ability of TTP to act as a tumor suppressor by inhibiting the E6-AP pathway and indicate TTP loss to be a critical event during HPV-mediated carcinogenesis.
 
Age-dependent expression levels of two miRNAs in blood and tissue samples of C57/B6 mice A graphical representation of expression levels of miRNAs using qualitative PCR, represented as 1/ΔCt values as box plots for brain, PBMC and plasma samples. Age groups included are from early to old age [from 2 days (d) to 25 months (m)]. Panel (A) shows composite graphs for expression of miR-34a, while panel (B) shows the levels of expression of miR-196a in brain, PBMCs, and plasma. (n = 3; three different animals were used from each age group selected for the study.)
Age‐dependent expression levels of SIRT1 in blood and tissue samples of C57/B6 mice Western blot analysis of SIRT1 expression in age groups from 2 days (d) to 25 months (m); panels (A) SIRT1 expression in plasma samples normalized with selected Ponceau S stained band, showing constant levels for all samples used, along with histograms presenting average densitometric values, (B) SIRT1 expression in PBMC samples normalized with β‐actin, along with histograms presenting average densitometric values, (C) SIRT1 expression in brain samples normalized with β‐actin, along with histograms presenting average densitometric values, (D) Composite graph presenting SIRT1 expression in plasma, PBMC and brain samples. (*p < 0.01, **p < 0.0001; all histograms represent Mean ± SD; n = 3; three different mice used from each selected age group.)  
Expression of miRNA‐34a and SIRT1 in brain tissue sections from middle and old age C57/B6 mice In situ hybridization detected miR‐34a expression using LNA probes. Panels (A) and (B) show expression of miR‐34a in cortex and hippocampus of 10 and 31 month old mice. Scrambled LNA probes were used as control for this experiment (Image not shown); (C) graphical presentation of mean intensity values of miR‐34a expression in cortex and hippocampus of 10 and 31 month old mice, by immunostaining assay to detect SIRT1 expression. Panels (D‐E) and (F‐G) show expression of SIRT1 in cortex and hippocampus of 10 and 31 month old mice, by DAPI staining to detect nucleus and section integrity (Image not shown); (H) graphical presentation of mean intensity values of SIRT1 expression in cortex and hippocampus of 10 and 31 month old mice. (*p < 0.01, **p < 0.0001; all histograms represent Mean ± SD; n = 3; three different biological samples from each age group.)  
Age-dependent expression levels of Bcl-2 in blood and brain tissue samples of C57/B6 mice Western blot analysis for Bcl-2 levels in age groups from 2 days (d) to 25 months (m); panels (A-B-C) Bcl-2 expression normalized with Ponceau S stained bands in plasma, and with β-actin in brain and PBMC samples, along with histograms presenting average Bcl-2 expression densitometric values; (D) Composite graph presenting Bcl-2 expression in plasma, PBMC and brain samples as average densitometric intensity. All graphs represent Mean ± SD. n = 3; three different mice from each age group.
Age-dependent changes in ratio of acetylated to total p53 in blood and brain samples of C57/B6 mice. Western blot analysis of acetylated p53 proportion in age groups from 2 days to 25 months; Panels A, B, and C show acetylated P53 proportion normalized with Ponceau S in plasma (Panel A), and with β-actin in PBMCs and brain samples, along with histograms presenting the average densitometric values. Panel D presents a composite graph of the percentage of acetylated P53 expression in the total p53 protein pool in plasma, PBMC and brain samples. (*p < 0.01, **p < 0.0001; all histograms represent Mean ± SD; n = 3; three different biological samples from each age group)
Article
MicroRNAs in blood samples have been identified as an important class of biomarkers, which can reflect physiological changes from cancer to brain dysfunction. In this report we identify concordant increases in levels of expression of miR-34a in brain and two components of mouse blood samples, peripheral blood mononuclear cells (PBMCs) and plasma, from 2 day old neonates through young adulthood and mid-life to old age at 25 months. Levels of this microRNA's prime target, silent information regulator 1 (SIRT1), in brain and the two blood-derived specimens decrease with age inversely to miR-34a, starting as early as 4 months old, when appreciable tissue aging has not yet begun. Our results suggest that: 1. Increased miR-34a and the reciprocal decrease of its target, SIRT1, in blood specimens are the accessible biomarkers for age-dependent changes in brain; and 2. these changes are predictors of impending decline in brain function, as early as in young adult mice.
 
Article
WRN protein, defective in Werner syndrome (WS), a human segmental progeria, is a target of serine/threonine kinases involved in sensing DNA damage. DNA-PK phosphorylates WRN in response to DNA double strand breaks (DSBs). However, the main phosphorylation sites and functional importance of the phosphorylation of WRN has remained unclear. Here, we identify Ser-440 and -467 in WRN as major phosphorylation sites mediated by DNA-PK.In vitro, DNA-PK fails to phosphorylate a GST-WRN fragment with S440A and/or S467A substitution. In addition, full length WRN with the mutation expressed in 293T cells was not phosphorylated in response to DSBs produced by bleomycin. Accumulation of the mutant WRN at the site of laser-induced DSBs occurred with the same kinetics as wild type WRN in live HeLa cells. While the wild type WRN relocalized to the nucleoli after 24 hours recovery from etoposide-induced DSBs, the mutant WRN remained mostly in the nucleoplasm. Consistent with this, WS cells expressing the mutants exhibited less DNA repair efficiency and more sensitivity to etoposide, compared to those expressing wild type. Our findings indicate that phosphorylation of Ser-440 and -467 in WRN are important for relocalization of WRN to nucleoli, and that it is required for efficient DSB repair.
 
Infinium 450k probes classification and BOPs definition. (A) The 485577 probes included in the Illumina HumanMethylation450 BeadChip were divided in 4 classes on the basis of their genomic localization. (B) Graphic representation of how probes were grouped in BOPs. Probes mapping in the island and in the surrounding regions of the same CpG island were grouped in 5 functional units: probes in the N-Shelf of the island, probes in the N-Shore of the island, probes in the island, probes in the S-Shore of the island, probes in the S-Shelf of the island. Probes mapping in gene bodies were grouped on the basis of the gene in which they are located.
Characteristics of the BOPs belonging to different probe classes. (A) Numbers of BOPs and CpG probes in Class A, Class B and Class C. For Class A and Class B, subdivision in CpG islands, N-Shores, S-Shores, N-Shelves and S-Shelves is reported. In the lower part of the tables, descriptive statistics for the distribution of number of probes/BOP in the 3 Classes are reported. (B) Descriptive statistics for the distribution of mean bp distance between probes /BOP in the 3 Classes are reported.(C) Density distributions (upper panel) and frequency histograms (lower panels) of the mean bp distance between the probes/BOP.
Proposed analytical pipeline for Infinium 450k data. (A) Workflow for the use of single-probe or regioncentric approaches on Infinium 450k data. (B) Graphical representation of the sliding window MANOVA used to normalize for BOPs lengths. CpG probes are represented as circles. The CpG probes considered in each round of MANOVA are highlighted in yellow. (C) Example of methylation values of CpG probes within a BOP. The BOP includes 22 CpG probes, 5 of which define a "bubble" of differential methylation between Group A and Group B. The p-value derived from MANOVA on this BOP is 2.70*10e-11. We hypothesized to have a shorter BOP including only the 5 CpG probes differentially methylated between Group A and Group B, plus a probe on both the sites whose methylation level is comparable between the two samples. In this case, although the extent of the bubble of differentially methylation is the same of the longer BOP, the p-value derived from MANOVA is lower, equal to 8.64*10e-14. This simple example shows that if we do not normalize for the length of the BOP, short BOPs tend to rank at higher positions than long BOPs.
Number of significant CpG probes per significant BOP. For each dataset, the boxplot reports the-log 10 (q-value) of each significant Class A BOP (MANOVA analysis) against the number of significant CpG probes (q-value < 0.05, ANOVA analysis) included in each BOP. 
The region-centric approach increases the common findings between the 3 datasets. (A) Intersection between the results provided by Hannum et al. and Heyn et al. (left panel) and between the results of the region-centric approach on the two datasets. (B) Intersection between a progressively increasing number of top ranking features (BOPs for the region-centric analysis, CpG probes for the single-probe analysis) in the three datasets.
Article
Aging is characterized by a profound remodeling of the epigenetic architecture in terms of DNA methylation patterns. To date the most effective tool to study genome wide DNA methylation changes is Infinium HumanMethylation450 BeadChip (Infinium 450k). Despite the wealth of tools for Infinium 450k analysis, the identification of the most biologically relevant DNA methylation changes is still challenging. Here we propose an analytical pipeline to select differentially methylated regions (DMRs), tailored on microarray architecture, which is highly effective in highlighting biologically relevant results. The pipeline groups microarray probes on the basis of their localization respect to CpG islands and genic sequences and, depending on probes density, identifies DMRs through a single-probe or a region-centric approach that considers the concomitant variation of multiple adjacent CpG probes. We successfully applied this analytical pipeline on 3 independent Infinium 450k datasets that investigated age-associated changes in blood DNA methylation. We provide a consensus list of genes that systematically vary in DNA methylation levels from 0 to 100 years and that have a potentially relevant role in the aging process.
 
Article
Compounds that delay aging in model organisms may be of significant interest to anti-aging medicine, since these substances potentially provide pharmaceutical approaches to promote healthy lifespan in humans. We here aimed to test whether pharmaceutical concentrations of three fibrates, pharmacologically established serum lipid-lowering drugs and ligands of the nuclear receptor PPARalpha in mammals, are capable of extending lifespan in a nematodal model organism for aging processes, the roundworm Caenorhabditis elegans. Adult C. elegans (wild-type N2 as well as two nhr-49-deficient strains, RB1716 and VC870) were maintained on agar plates and were fed E. coli strain OP50 bacteria. Bezafibrate, clofibrate, and fenofibrate were applied to the agar, respectively, to test whether they may promote longevity by quantifying survival in the presence and absence of the respective compounds. All three fibrates extended C. elegans N2 lifespan when applied at a concentration of 10 micromolar. Bezafibrate additionally extended C. elegans N2 lifespan at concentrations of 1 micromolar and 0.1 micromolar. In strains deficient for nhr-49, a functional orthologue of the mammalian peroxisome proliferator-activated receptor alpha (PPARalpha), all three compounds were incapable of extending lifespan. Taken together, fibrates promote C. elegans longevity in an NHR-49-dependent manner possibly by promoting mitohormesis and suggesting that these compounds may promote lifespan also in mammals.
 
Influence of miR-519 on WI-38 senescence. (A) Fold differences in miR-519 expression in S relative to Y cells, calculated as explained in the legend of Figure 2. (B) Forty-eight h after transfection of either control (Ctrl) siRNA or miR-519a, the levels of miR-519a were measured by RT-qPCR. (C,D) In cells transfected as explained in panel (B), the levels of HuR protein and loading control GAPDH were assessed by Western blot analysis (C), and the levels of HuR mRNA and normalization control 18S rRNA were measured by RT-qPCR (D). (E) WI-38 cell numbers in cells transfected as in (B) were counted every 7 days. (F) SA-β-galactosidase activity in WI-38 cells by week 4 after sequential transfection (every 7 days) of either Ctrl siRNA or miR-519a. (G) Western blot analysis of p27 and loading control GAPDH in Y and S WI-38 cells (top) or in WI-38 cells by week 4 after transfection as explained in (E) (bottom). The data in B,D,E represent the means and S.D. from three independent experiments. 
Influence of miR-519 on the senescent phenotype of HeLa cells. (A) Forty-eight h after transfection of HeLa cells with either control (Ctrl) siRNA or miR-519a, miR-519a levels were measured by RTqPCR. (B) Number of HeLa cells remaining by 72 h after transfection of Ctrl siRNA or miR-519a as explained in (A). (C) β-galactosidase activity in HeLa cells 5 days after transfection with either Ctrl siRNA or miR-519a. (D) Seventy-two hours after transfection as indicated in (A), the levels of the proteins shown were assessed by Western blot analysis. The data in A,B represent the means and S.D. from three independent experiments. 
Article
MicroRNAs (miRNAs) are short non-coding RNAs that regulate diverse biological processes by controlling the pattern of expressed proteins. In mammalian cells, miRNAs partially complement their target sequences leading to mRNA degradation and/or decreased mRNA translation. Here, we have analyzed transcriptome-wide changes in miRNAs in senescent relative to early-passage WI-38 human diploid fibroblasts (HDFs). Among the miRNAs downregulated with senescence were members of the let-7 family, while upregulated miRNAs included miR-1204, miR-663 and miR-519. miR-519 was recently found to reduce tumor growth at least in part by lowering the abundance of the RNA-binding protein HuR. Overexpression of miR-519a in either WI-38 or human cervical carcinoma HeLa cells triggered senescence, as measured by monitoring beta-galactosidase activity and other senescence markers. These data suggest that miR-519 can suppress tumor growth by triggering senescence and that miR-519 elicits these actions by repressing HuR expression.
 
Depletion of p53 or Ku80 increases the rate of spontaneous micronuclei formation in WI38 human fibroblasts. 
53BP1 depletion increases chromosomal instability in WI38 human fibroblasts. WI38 normal human fibroblasts were infected with one of two 53BP1 shRNA constructs (sh53BP1 A or sh53BP1 B) or a scrambled shRNA construct for 24 hours and selected in puromycin for 72 hours. (A) Representative Western blot and densitometric quantitation showing 53BP1 protein levels as a percentage of scrambled control (set as 1 on y-axis). β-actin levels are included as a loading control. (B) Quantitation of micronuclei-positive cells following infection with scrambled or 53BP1 shRNA constructs. Both control, nondamaged cells and cells damaged with 1.5ug/mL neocarzinostatin (NCS) for 2 hours are shown. Data are pooled from three replicates, and at least 400 cells were counted for each condition. Positive cells contain at least one micronucleus. Error bars indicate standard deviation. **P<0.01; ***P<0.001. (C) Representative images depicting micronuclei and 53BP1 foci in control and 53BP1-depleted human fibroblasts. Both non-damaged and damaged (NCS) cells were prepared as in (B) and stained with DAPI and anti-53BP1 (green). Images showing nuclear structure are depicted for both wild-type fibroblasts and fibroblasts infected with one of two 53BP1 shRNA constructs, and examples of micronuclei are marked with white arrows.
Article
Faithful repair of damaged DNA is a crucial process in maintaining cell viability and function. A multitude of factors and pathways guides this process and includes repair proteins and cell cycle checkpoint factors. Differences in the maintenance of genomic processes are one feature that may contribute to species-specific differences in lifespan. We predicted that 53BP1, a key transducer of the DNA damage response and cell cycle checkpoint control, is highly involved in maintaining genomic stability and may function differently in cells from different species. We demonstrate a difference in the levels and recruitment of 53BP1 in mouse and human cells following DNA damage. In addition, we show that unresolved DNA damage persists more in mouse cells than in human cells, as evidenced by increased numbers of micronuclei. The difference in micronuclei seems to be related to the levels of 53BP1 present in cells. Finally, we present evidence that unresolved DNA damage correlates with species lifespan. Taken together, these studies suggest a link between recruitment of 53BP1, resolution of DNA damage, and increased species lifespan.
 
Article
Human TP53 gene is characterised by a polymorphism at codon 72 leading to an Arginine-to-Proline (R/P) substitution. The two resulting p53 isoforms have a different subcellular localisation after stress (more nuclear or more mitochondrial for the P or R isoform, respectively). p53P72 variant is more efficient than p53R72 in inducing the expression of genes involved in nuclear DNA repair. Since p53 is involved also in mitochondrial DNA (mtDNA) maintenance, we wondered whether these p53 isoforms are associated with different accumulation of mtDNA damage. We observed that cells bearing p53R72 accumulate lower amount of mtDNA damage upon rotenone stress with respect to cells bearing p53P72, and that p53R72 co-localises with polymerase gamma more than p53P72. We also analysed the in vivo accumulation of heteroplasmy in a 300 bp fragment of mtDNA D-loop of 425 aged subjects. We observed that subjects with heteroplasmy higher than 5% are significantly less than expected in the p53R72/R72 group. On the whole, these data suggest that the polymorphism of TP53 at codon 72 affects the accumulation of mtDNA mutations, likely through the different ability of the two p53 isoforms to bind to polymerase gamma, and may contribute to in vivo accumulation of mtDNA mutations.
 
Short hairpin RNAs (shRNAs) for Ku70/80 inhibit the proliferation of ALT cells. (A) shRNAs for Ku70/80 reduce the levels of Ku70/80 in CCL75.1 cells. CCL75.1 cells were infected with lentiviruses for the conditional expression of shRNAs targeting Ku70 and Ku80 or GFP and cultured for 3, 7 and10 days in the presence of 1.0 mg/ml Doxycycline (DOX). The levels of Ku70 and Ku80 were analyzed by immunoblotting with antibodies against Ku70 and Ku80. Antibodies against tubulin were used as a loading control. We estimated that 7 to 10 days after induction, Ku70/80 shRNAs-expressing cells display an 80 to 85% decrease in Ku70/80 expression compared to GFP shRNAs control. (B) Growth curves of CCL75.1 expressing shRNAs for Ku70/80 or GFP. After 5 days in Geneticin and hygromycin selection, 1.0ug/ml DOX was added to the media and the growth rate of each CCL75.1 cell line was measured by counting viable cells every 2 days. Cells were seeded at a low density, and the medium was changed every 2 days. Values represent the mean ± the standard deviation of three experiments (n = 3). (C) Detection of SA-β-gal activity. CCL75.1 cells transduced with lentiviruses for the conditional expression of shRNAs targeting K70/80 or GFP were grown for 3, 6, 9, or 12 days after addition of DOX and were stained for SA-βgal activity as previously described [14]. Values are the mean ± the standard deviation of three independent experiments (n = 3) carried out in duplicates in which 500 cells were scored for SA-β galactosidase. Student's t test was used to evaluate differences in means between two groups, and P < 0.05 was considered statistically significant. ALT cells (CCL75.1 and Saos2) were purchased from ATCC.
Depletion of Ku70/80 in CCL75.1 cells induces transient p53 activation. The levels of p21, p53, and p53 phosphorylated at serine 15 were assessed by Western blotting in extracts prepared from CCL75.1 cells collected either before, or 3 to 6 days after addition of DOX. Tubulin serves as a loading control. Western blot analyses were performed with antibodies against p53, p53-phosphoserine15, p21, and tubulin.
Depletion of Ku70/80 in CCL75.1 cells does not affect telomere length nor overhangs signal. (A) Terminal Restriction Fragment (TRF) analysis. CCL75.1 cells transduced with lentiviruses for the conditional expression of shRNAs targeting Ku70 and Ku80 or GFP were harvested at 7 (shKu70/80) and 10 days (shKu70/80 and shGFP) after induction with DOX. Equal amounts of genomic DNA digested with HinfI and RsaI were separated by electrophoresis on a 0.8% agarose gel and analyzed by Southern blotting with a radiolabeled (CCCTAA) 4 probe. The molecular mass standards shown on the right lane were generated by digestion of lambda DNA with the restriction endonuclease HindIII. (B) Telomere 3' overhang signal analysis. CCL75.1 cells transduced with lentiviruses for the conditional expression of shRNAs targeting either Ku70/80 or GFP were harvested at 7 (shKu70/80) and 10 days (shKu70/80 and shGFP) after induction with DOX. Equal amounts of genomic DNA digested with HinfI and RsaI were separated by electrophoresis on a 0.8% agarose gel. The gel was dried under native conditions and then hybridized to a radiolabeled (CCCTAA) 4 probe.
Depletion of Ku70/80 reduces the levels of t-circles in CCL75.1 and Saos2 cells. (A) Genomic DNA 
Article
In normal cells, telomeres shorten each time a cell divides ultimately resulting in cell senescence. In contrast, cancer cells counteract the loss of telomeric DNA either by inducing the expression of telomerase or by activating the alternative lengthening of telomeres (ALT) pathway. ALT cells are characterized by heterogeneous telomeres and the presence of extrachromosomal circular double-stranded DNA molecules containing telomeric repeat sequences. These telomeric circles (t-circles) are though to be generated through a recombination process and utilized as templates for telomere elongation by rolling-circle-replication, although their precise mechanism of formation and role in telomere maintenance and cell proliferation is largely unknown. Here we show that shRNA-mediated knockdown of the Ku70/80 heterodimer, a factor with functions at both pathological and natural DNA ends, inhibits ALT cell growth and results in a significant decrease in the levels of t-circles without affecting overall telomere length. These findings demonstrate that non homology-based processes contribute to the maintenance of t-circles and proliferation of ALT cells.
 
Cell proliferation and heterochromatin marks. (A) Mean ± SEM of population doublings (PD) per 24 h determined for each donor in five to six independent cultures by seeding a defined amount of cells onto a standardised area of substratum and monitoring the time required for growth to confluency and the final cell yield. Results of linear regression are stated as R 2 : Pearson's coefficient for goodness of fit, p: probability for slope = 0, dotted lines: 95% confidence limits of linear regression, and N.S.: linear regression of the data revealed no significant age‐related change in the parameters. (B) Heterochromatin marks HP1β (left) and mH2A (right) determined by immunoblotting in whole cell lysates of dermal fibroblasts from the young (open symbols) and old donor group (closed symbols) at PD < 14. Data were normalised to values obtained in control fibroblasts subjected to replicative senescence. Data points represent means of triplicate determinations in separate cell cultures. Errors were < 30% of the values and are omitted for the sake of clarity. Horizontal lines indicate the mean of the respective age group. Numbers next to data points indicate the chronological age of the respective donor.  
Trajectories of DNA strand break repair. Alkaline DNA unwinding was monitored in cell lysates prepared at the indicated time points subsequent to exposure of the cells to 3.8 Gy (arrows). Values are normalised to the amplitude of the initial IR‐induced increase in unwinding. Data represent mean values ± SEM of five independent cell cultures analysed for each individual donor. Numbers on the right margins of the age groups indicate the chronological ages of the donors of the samples.  
Age‐correlated regulation of DNA‐repair genes.  
Article
Dermal fibroblasts provide a paradigmatic model of cellular adaptation to long-term exogenous stress and ageing processes driven thereby. Here we addressed whether fibroblast ageing analysedex vivo entails genome instability. Dermal fibroblasts from human female donors aged 20-67 years were studied in primary culture at low population doubling. Under these conditions, the incidence of replicative senescence and rates of age-correlated telomere shortening were insignificant. Genome-wide gene expression analysis revealed age-related impairment of mitosis, telomere and chromosome maintenance and induction of genes associated with DNA repair and non-homologous end-joining, most notably XRCC4 and ligase 4. We observed an age-correlated drop in proliferative capacity and age-correlated increases in heterochromatin marks, structural chromosome abnormalities (deletions, translocations and chromatid breaks), DNA strand breaks and histone H2AX-phosphorylation. In a third of the cells from old and middle-aged donors repair of X-ray induced DNA strand breaks was impaired despite up-regulation of DNA repair genes. The distinct phenotype of genome instability, increased heterochromatinisation and (in 30% of the cases futile) up-regulation of DNA repair genes was stably maintained over several cell passages indicating that it represents a feature of geroconversion that is distinct from cellular senescence, as it does not encompass a block of proliferation.
 
From the macroscopic to the microscopic: ICT reconstruction of the young and aged mouse eyelid. (A) Dissecting microscope images of a 5‐month old and (B) 2‐year old mouse eyelid. Meibomian glands (black arrowheads) were identified as whitish tissue underneath transparent conjunctiva and gland dropout was observed in the older mouse eyelids (asterisk). Eyelids were embedded in BMMA and serially sectioned; the red box highlights the areas 3‐D reconstructed through sequential immunostaining, as shown in C‐H (C) A 5month old mouse eyelid 2µm BMMA section stained with CK1 and DAPI before antibody elution with glycine HCl and subsequent immunostaining with (D) CK5, (E) CK6 and (F) Ki67 antibodies. The resulting overlays (G and H) show the meibomian gland parallel to the conjunctiva (CJ) and embedded in the orbicularis muscle (OM). The meibomian gland duct (DU) and orifice (O) are CK5 + /CK6 + , whereas acinar (AC) and skin (S) epithelium are CK5 + /CK6 ‐ . CK5‐based reconstructions of a (I) 5month and (L) 2year old mouse eyelid visualised the hair follicle (red arrowheads) and meibomian gland epithelia (green), confirming the truncation of meibomian glands with age. In 3‐D, CK1 staining at the lid margin of (J) young and (K) old lids revealed a loss of a layer of epidermal cells posterior to meibomian gland orifices in the older lid. This caused an anterior shift of the mucocutaneous junction (white arrows) to the level of the meibomian gland orifices.  
Segmented ICT reconstructions of (A-D) a 5month and (E-H) 2 year old mouse meibomian gland. In older meibomian glands, there is a notable loss of acini (A:E), ductal epithelium (B:F), lipid volume (C:G) and total cell number (D:H).
Quantification of the age‐related changes in the Mouse meibomian gland by ICT.  
Keratinization of the meibomian gland orifice. (A‐C) A 5month and (D‐F) 2year old mouse lid margin cross‐sections. (D) Gland plugging and (D‐F) a loss of CK1 + cells posterior to the orifice causes the mucocutaneous junction to shift anteriorly in the aged mouse eyelid. CK1 immunostaining shows no hyper‐keratinization or obstruction in the atrophic meibomian glands.  
Nascent acini and their absence with aging. (A) A 3-D reconstruction of the 5 month old meibomian gland and an orthoslice taken at the region of interest: the transition between duct and acini (yellow arrowhead). (B) An acinus with a significant CK6 + component (C) also exhibits localized proliferation in the same region, according to Ki67 staining (D) and the resulting overlay (E). (F) The 3-D reconstruction of a 2 year meibomian gland with ductal truncation was evaluated at the same transitional region between duct and multiple acini (G-red arrowheads) and no prominent CK6 (H) or (I) Ki67 staining was evident when overlaying the sequential immunostains (J), suggesting a loss of proliferation in these zones.
Article
Meibomian gland dysfunction (MGD) is frequent with aging and is the primary cause of dry eye disease, the most prevalent ocular complaint. We used a novel 3-D reconstruction technique, immunofluorescent computed tomography (ICT), to characterize meibomian gland keratinization and cell proliferation in a mouse model of age-related meibomian gland dysfunction (ARMGD). To visualize the changes associated with ARMGD, 5-month and 2-year old mouse eyelids were 3-D reconstructed by ICT using antibodies to cytokeratin (CK) 1, 5 and 6 and the proliferation marker Ki67. We quantified total gland, ductal and lipid volume from the reconstructions, observing a dramatic decrease in old glands. In young glands, proliferative ductules suggest a potential site of acinar progenitors that were found to be largely absent in aged, atrophic glands. In the aged mouse, we observed an anterior migration of the mucocutaneous junction (MCJ) and an absence of hyper-keratinization with meibomian gland atrophy. Thus, we propose that changes in the MCJ and glandular atrophy through a loss of meibocyte progenitors are most likely responsible for ARMGD and not ductal hyper-keratinization and gland obstruction.
 
Schematic figures depicting pathways by which leptin exerts anti-diabetic actions: (A) leptin acts on leptin receptors in hypothalamic proopiomelanocortin (POMC) neurons to regulate hepatic insulin sensitivity hence improve glucose levels in the blood of T2DM mice [17]. (B) leptin acts on leptin receptors in hypothalamic POMC and γ-aminobutyric acid (GABA)ergic neurons to regulate liver, interscapular brown adipose tissue and soleus muscle function to reduce glucose levels in the blood of T1DM mice [4]. LHA; lateral hypothalamic area, ARC; hypothalamic arcuate nucleus, DMH; dorsomedial nucleus and VMH; ventromedial hypothalamic nucleus. Anatomical location of LHA, ARC and DMH is shown in blue-colored circle [48]. POMC neurons are depicted in green while GABAergic neurons in blue.
Article
Diabetes afflicts hundreds of millions worldwide. People affected by type 1 diabetes mellitus (T1DM; the insulin-deficient form of diabetes) or type 2 diabetes mellitus (T2DM; the insulin-resistant form of diabetes) have significantly reduced life expectancy compared to normal individuals. This is due in part to the fact that (despite improvements) current anti-diabetic approaches are suboptimal. Indeed, severe morbidities (e.g.: cardiovascular disease, hypertension) are still too often associated with diabetes. Recent preclinical results indicate that different types of hypothalamic neurons are endowed with the ability to mediate the hyperglycemia-lowering action of the adipocyte-derived hormone leptin in an insulin-dependent and insulin-independent fashion. These results may pave the way for better anti-diabetic approaches and therefore positively impact on life expectancy of diabetic subjects.
 
Dynamic changes of miRNAs with age. (A) Survivorship of a population of N2 (wild type) animals, from which individuals were harvested for analysis at Day 1 and Day 12 of adulthood. (B-G) Normalized Ct values describing miRNA abundance in individual worms (each point represents data from a single animal) at two ages are shown for mir-44 (B), mir-51 (C), mir-240 (D), mir-58 (E), mir-60 (F), and mir-235 (G). (H) Graphical representation of the fold change in miRNA abundance between young and old animals. In all graphs with error bars they indicate the standard error of the mean. In individual worm graphs each point is the average from 2-3 technical replicates. 
Dynamic changes of miRNAs with age in long-lived germline ablated animals. (A) Survivorship of a population of N2 (wild type) animals, from which individuals were harvested for analysis at Day 1, Day 6, Day 12 and Day 18 of adulthood. (B-G) miRNA levels in individual worms (columns) at four time points are shown for mir-54 (B), mir-67 (C), mir-354 (D), mir-53 (E), mir-64 (F), and mir-81 (G). (H) Graphical representation of the fold change in miRNA abundance between young and old animals using averaged values. In all graphs with error bars they indicate the standard error of the mean. In individual worm graphs, each point is the average from 2-3 technical replicates. 
mir-71 acts at least partly in parallel to the IIS pathway. (A) The integrated transgene maIs352[Pmir-71::mir71::GFP] partially rescues the lifespan of mir-71 null mutants. (B) The longevity phenotype of daf-2(e1368) is significantly suppressed by the presence of the mir-71(n4115) deletion. (C) daf-16; mir-71 double null mutants show significant enhancement relative to the single mutants. (D) Over-expression of mir-71 extends the lifespan of daf-16 null mutants. 
Article
Non-coding small RNAs of the micro-RNA class (miRNA) are conserved regulators of gene function with a broad impact on biological processes. We screened miRNA levels for age-related changes in individual worms and investigated their influence on the lifespan of the nematode C. elegans. We measured the abundance of 69 miRNAs expressed in individual animals at different ages with over thirty five thousand discrete quantitative nano-fluidic polymerase chain reactions. We found that miRNA abundance was highly variable between individual worms raised under identical conditions and that expression variability generally increased with age. To identify expression differences associated with either reproductive or somatic tissues, we analyzed wild type and mutants that lacked germlines. miRNAs from the mir-35-41 cluster increased in abundance with age in wild type animals, but were nearly absent from mutants lacking a germline, suggesting their age-related increase originates from the germline. Most miRNAs with age-dependent levels did not have a major effect on lifespan, as corresponding deletion mutants exhibited wild-type lifespans. The major exception to this was mir-71, which increased in abundance with age and was required for normal longevity. Our genetic characterization indicates that mir-71 acts at least partly in parallel to insulin/IGF like signals to influence lifespan.
 
Article
Telomere shortening represents a causal factor of cellular senescence. At the same time, several lines of evidence indicate a pivotal role of oxidative DNA damage for the aging process in vivo. A causal connection between the two observations was suggested by experiments showing accelerated telomere shorting under conditions of oxidative stress in cultured cells, but has never been studied in vivo. We therefore have analysed whether an increase in mitochondrial derived oxidative stress in response to heterozygous deletion of superoxide dismutase (Sod2(+/-)) would exacerbate aging phenotypes in telomere dysfunctional (mTerc(-/-)) mice. Heterozygous deletion of Sod2 resulted in reduced SOD2 protein levels and increased oxidative stress in aging telomere dysfunctional mice, but this did not lead to an increase in basal levels of oxidative nuclear DNA damage, an accumulation of nuclear DNA breaks, or an increased rate of telomere shortening in the mice. Moreover, heterozygous deletion of Sod2 did not accelerate the depletion of stem cells and the impairment in organ maintenance in aging mTerc(-/-) mice. In agreement with these observations, Sod2 haploinsufficiency did not lead to a further reduction in lifespan of mTerc(-/-) mice. Together, these results indicate that a decrease in SOD2-dependent antioxidant defence does not exacerbate aging in the context of telomere dysfunction.
 
Article
Pathogenesis of age-related macular degeneration (AMD), the leading cause of blindness in the world, remains poorly understood. This makes it necessary to create animal models for studying AMD pathogenesis and to design new therapeutic approaches. Here we showed that retinopathy in OXYS rats is similar to human AMD according to clinical signs, morphology, and vascular endothelium growth factor (VEGF) and pigment epithelium-derived factor (PEDF) genes expression. Clinical signs of retinopathy OXYS rats manifest by the age 3 months against the background of significantly reduced expression level of VEGF and PEDF genes due to the decline of the amount of retinal pigment epithelium (RPE) cells and alteration of choroidal microcirculation. The disruption in OXYS rats' retina starts at the age of 20 days and appears as reduce the area of RPE cells but does not affect their ultrastructure. Ultrastructural pathological alterations of RPE as well as develop forms of retinopathy are observed in OXYS rats from age 12 months and manifested as excessive accumulation of lipofuscin in RPE regions adjacent to the rod cells, whirling extentions of the basement membrane into the cytoplasm. These data suggest that primary cellular degenerative alterations in the RPE cells secondarily lead to choriocapillaris atrophy and results in complete loss of photoreceptor cells in the OXYS rats' retina by the age of 24 months.
 
Article
Many data pertaining to the accelerated telomere loss in cultured cells derived from Werner syndrome (WS), a representative premature aging syndrome, have been accumulated. However, there have been no definitive data on in vivo telomere shortening in WS patients. In the present study, we measured terminal restriction fragment (TRF) lengths of 10 skin samples collected from extremities of 8 WS patients aged between 30 and 61 years that had been surgically amputated because of skin ulceration, and estimated the annual telomere loss. Whereas the values of TRF length in younger WS patients (in their thirties) were within the normal range, those in older WS patients were markedly shorter relative to non‐WS controls. Regression analyses indicated that the TRF length in WS was significantly shorter than that in controls (p < 0.001). Furthermore, we found that TRF lengths in muscle adjacent to the examined epidermis were also significantly shorter than those of controls (p = 0.047). These data demonstrate for the first time that in vivo telomere loss is accelerated in systemic organs of WS patients, suggesting that abnormal telomere erosion is one of the major causes of early onset of age‐related symptoms and a predisposition to sarcoma and carcinoma in WS.
 
Article
Males, who are bigger and stronger than females, live shorter in most species from flies to mammals including humans. Cellular mass growth is driven in part by mTOR (Target of Rapamycin). When developmental growth is completed, then, instead of growth, mTOR drives aging, manifested by increased cellular functions, such as hyper-secretion by fibroblasts, thus altering homeostasis, leading to age-related diseases and death. We hypothesize that MTOR activity is elevated in male mice compared with females. Noteworthy, 6 months old males were 28 % heavier than females. Also levels of phosphorylated S6 (pS6) and phospho-AKT (p-AKT, Ser 473), markers of the mTOR activity, were higher in male organs tested. Levels of pS6 were highly variable among mice and correlated with body weight and p-AKT. With age, the difference between levels of pS6 between sexes tended to minimize, albeit males still had hyperactive mTOR. Unlike fasting, the intraperitoneal (i.p.) administration of rapamycin eliminated pS6 in all organs of all females measured by immunoblotting and immunohistochemistry without affecting p-AKT and blood insulin. Although i.p. rapamycin dramatically decreased levels of pS6 in males too, it was still detectable by immunoblotting upon longer exposure. Our study demonstrated that both tissue p-AKT and pS6 were higher in young male mice and were associated with increased body weight and insulin. These data can explain bigger body size and faster aging in males. Our data suggest higher efficacy of rapamycin compared to fasting. Higher sensitivity of females to rapamycin may explain more pronounced life extension by rapamycin observed in females compared to males in several studies.
 
Primers used for analysis of chromosome 1
Plots of the distribution of trait linkage probability in the population of F2 rats (bred by reciprocal cross of OXYS and WAG) at age 3-4 months. The linkage of: (panel A) retinopathy; (panel B) cataract with markers on chromosome 1 was analyzed. Dotted vertical line at LOD = 2.0 is the threshold for suggestive statistical significance. The OXYS regions introgressed into WAG rats in the construction of congenic strains are shown as inserts to the left of the linkage map.
The breeding scheme for the construction of congenic strains. Selection is made at microsatellite marker locus M through a series of backcrosses such that the M1 allele of a donor (OXYS) replaces the M2 allele of a recipient (WAG). Rats are genotyped at M by PCR reaction using DNA obtained from tail tips. The genome of the donor strain is shown as an open symbol, and the genome of the recipient strain as a solid symbol. Increasing shades of gray from light to dark represent an increase in the percentage of genetic background of the recipient that occurs with each backcross (BC). The scheme is based on [26].
Cataract and retinopathy incidence in rats of WAG/OXYS-1.1 and WAG/OXYS-1.2 congenic strains at the ages of 1.5-2 and 3-4 months. Data are presented as a distribution of animal eyes with the stages of cataract and retinopathy (colors labeled with 0 through 2 correspond to a stage of cataract or retinopathy).
Article
Age-related macular degeneration (AMD) and cataract are common age-related diseases in humans. Previously we showed that senescence-accelerated OXYS rats develop retinopathy and cataract, which are comparable to human AMD and senile cataract. Here we focused on the identification of quantitative trait loci (QTLs), which affect early-onset cataract and retinopathy in OXYS rats, using F2 hybrids bred by a reciprocal cross (OXYS×WAG and WAG×OXYS). Chromosome 1 showed significant associations between retinopathy and loci in the regions of markers D1Rat30 and D1Rat219 (QTL1) as well as D1Rat219 and D1Rat81 (QTL2); and between early cataract development with the locus in the region of the markers D1Rat219 and D1Rat81 (QTL2). To determine the effects of these QTLs, we generated two congenic strains by transferring chromosome 1 regions from OXYS into WAG background. Both congenic strains (named WAG/OXYS-1.1 and WAG/OXYS-1.2, respectively) display early cataract and retinopathy development. Thus, we confirmed that genes located in the analyzed regions of chromosome 1 are associated with the development of these diseases in OXYS rats.
 
Article
Hutchinson-Gilford Progeria (HGPS) and Werner syndromes are diseases that clinically resemble some aspects of accelerated aging. HGPS is caused by mutations in theLMNA gene resulting in post-translational processing defects that trigger Progeria in children. Werner syndrome, arising from mutations in the WRN helicase gene, causes premature aging in young adults. What are the molecular mechanism(s) underlying these disorders and what aspects of the diseases resemble physiological human aging? Much of what we know stems from the study of patient derived fibroblasts with both mutations resulting in increased DNA damage, primarily at telomeres. However, in vivo patients with Werner's develop arteriosclerosis, among other pathologies. In HGPS patients, including iPS derived cells from HGPS patients, as well as some mouse models for Progeria, vascular smooth muscle (VSM) appears to be among the most severely affected tissues. Defective Lamin processing, associated with DNA damage, is present in VSM from old individuals, indicating processing defects may be a factor in normal aging. Whether persistent DNA damage, particularly at telomeres, is the root cause for these pathologies remains to be established, since not all progeroid Lmna mutations result in DNA damage and genome instability.
 
Effect of rapamycin (0.5 mg/kg per day) on Tau, Tau phospho T181 and APP in the hippocampus of OXYS and Wistar rats (n=6). A: Levels of App by immunoblot. B: Levels of total Tau. C: Levels of Tau phospho T181. D: Representative immunoblots of App, Tau and Tau phosphor T181 in the hippocampus of OXYS and Wistar rat. *-P < 0.05, statistically significant effect of rapamycin; #-P < 0.05 between OXYS and Wistar rats. Data are presented as mean ± SEM. 
Effect of rapamycin (0.5 mg/kg per day) on the levels and phosphorylation of S6 ribosomal protein in the hippocampus of OXYS and Wistar rats (n=6) measured by immunoblot. A: S6. B: pS6. C: Representative immunoblots of S6 and pS6 in the hippocampus of OXYS and Wistar rats. 
Effect of rapamycin (0.5 mg/kg per day) on the levels and phosphorylation of S6 ribosomal protein in the frontal cortex OXYS and Wistar rats (n=4) by immunoblot. A: S6. B: pS6. C: Representative immunoblots of S6 and pS6 in the frontal cortex of OXYS and Wistar rats. 
MRI image of brain 4 month-old OXYS, Wistar rats and OXYS rats after rapamycin supplementation. (A) Hydrocephaly of lateral ventricle of OXYS rats (arrow). (B) Loci of demyelinization of the brain OXYS rats (arrow). 
Article
Cellular and organismal aging are driven in part by the MTOR (mechanistic target of rapamycin) pathway and rapamycin extends life span inC elegans, Drosophila and mice. Herein, we investigated effects of rapamycin on brain aging in OXYS rats. Previously we found, in OXYS rats, an early development of age-associated pathological phenotypes similar to several geriatric disorders in humans, including cerebral dysfunctions. Behavioral alterations as well as learning and memory deficits develop by 3 months. Here we show that rapamycin treatment (0.1 or 0.5 mg/kg as a food mixture daily from the age of 1.5 to 3.5 months) decreased anxiety and improved locomotor and exploratory behavior in OXYS rats. In untreated OXYS rats, MRI revealed an increase of the area of hippocampus, substantial hydrocephalus and 2-fold increased area of the lateral ventricles. Rapamycin treatment prevented these abnormalities, erasing the difference between OXYS and Wister rats (used as control). All untreated OXYS rats showed signs of neurodegeneration, manifested by loci of demyelination. Rapamycin decreased the percentage of animals with demyelination and the number of loci. Levels of Tau and phospho-Tau (T181) were increased in OXYS rats (compared with Wistar). Rapamycin significantly decreased Tau and inhibited its phosphorylation in the hippocampus of OXYS and Wistar rats. Importantly, rapamycin treatment caused a compensatory increase in levels of S6 and correspondingly levels of phospo-S6 in the frontal cortex, indicating that some downstream events were compensatory preserved, explaining the lack of toxicity. We conclude that rapamycin in low chronic doses can suppress brain aging.
 
Article
NaCl induces DNA breaks, thus leading to cellular senescence. Here we showed that Ku86 deficiency accelerated the high NaCl-induced cellular senescence. We find that 1) high NaCl induces rapid cellular senescence in Ku86 deficient(xrs5) cells, 2) Ku86 deficiency shortens lifespan of C. elegans in high NaCl, and 3) cellular senescence is greatly accelerated in renal inner medullas of Ku86 (-/-) mice. Further, although water balance is known to be compromised in old mice, this occurs at much earlier age in Ku86(-/-) mice. When subjected to mild water restriction, 3 month old Ku86(-/-), but not Ku86(+/+),mice rapidly become dehydrated as evidenced by decrease in body weight, increased production of antidiuretic hormone,increased urine osmolality and decreased urine volume. The deficiency in water balance does not occur in Ku86(+/+)mice until they are much older (14 months). We conclude that Ku86 deficiency accelerates high NaCl(-) induced cellular senescence,particularly in the renal medulla where NaCl normally is high.
 
Effect of the exposure to various light regimens on survival and tumorigenesis in rats. (A)-survival, males;
Dynamics of the
                                        coefficient of homeostatic stability (CHS) in female (A) and male (B)
                                        rats maintained at various light regimens.
                                       ^ The difference with the relevant parameter in the group LD is significant, р<0,05;
                                       * The difference with the parameter at the age of 3 months in the same group is significant, р<0,05 (Wilcoxon-Mann-Whytney test).
Age-related dynamics of incidence of irregular estrous cycles in rats maintained
                                            at various light/dark  regimens. Ordinate, number of rats with irregular
                                            estrous cycles (%).
                                        The difference with the relevant parameter in the group LD is significant, р<0.05.
(A) - survival, males; (B)- survival-
                                            females; (C) - total tumor incidence, males; (D) - total
                                            tumor incidence, females; (E) - malignant tumor incidence, males; (F)
                                            - malignant tumorincidence, females.
Article
We evaluated the effect of various light/dark regimens on the survival, life span and tumorigenesis in rats. Two hundred eight male and 203 females LIO rats were subdivided into 4 groups and kept at various light/dark regimens: standard 12:12 light/dark (LD); natural lighting of the North-West of Russia (NL); constant light (LL), and constant darkness (DD) since the age of 25 days until natural death. We found that exposure to NL and LL regimens accelerated development of metabolic syndrome and spontaneous tumorigenesis, shortened life span both in male and females rats as compared to the standard LD regimen. We conclude that circadian disruption induced by light-at-night accelerates aging and promotes tumorigenesis in rats. This observation supports the conclusion of the International Agency Research on Cancer that shift-work that involves circadian disruption is probably carcinogenic to humans.
 
Article
We have recently reported that a novel muscle-specific inositide phosphatase (MIP/MTMR14) plays a critical role in [Ca2+]i homeostasis through dephosphorylation of sn-1-stearoyl-2-arachidonoyl phosphatidylinositol (3,5) bisphosphate (PI(3,5)P2). Loss of function mutations in MIP have been identified in human centronuclear myopathy. We developed a MIP knockout (MIPKO) animal model and found that MIPKO mice were more susceptible to exercise-induced muscle damage, a trademark of muscle functional changes in older subjects. We used wild-type (Wt) mice and MIPKO mice to elucidate the roles of MIP in muscle function during aging. We found MIP mRNA expression, MIP protein levels, and MIP phosphatase activity significantly decreased in old Wt mice. The mature MIPKO mice displayed phenotypes that closely resembled those seen in old Wt mice: i) decreased walking speed, ii) decreased treadmill activity, iii) decreased contractile force, and iv) decreased power generation, classical features of sarcopenia in rodents and humans. Defective Ca2+ homeostasis is also present in mature MIPKO and old Wt mice, suggesting a putative role of MIP in the decline of muscle function during aging. Our studies offer a new avenue for the investigation of MIP roles in skeletal muscle function and as a potential therapeutic target to treat aging sarcopenia.
 
(A) - survival, males;  (B) - total tumor incidence, males; (C)
                                            - fatal tumor incidence, males; (D) - survival, females; (E)
                                            - total tumor incidence, females; (F) - fatal tumor incidence,
                                            females.
(a) - the
                                            difference with the relevant parameter in the group LD is significant,
                                            p<0.05; (b) - the
                                            difference with the relevant parameter in the group LL-1 is significant,
                                            p<0.05.
(a) - the
                                            difference with the relevant parameter in the group LD is significant,
                                            p<0.05;
                                            (b)
                                            - the difference with the relevant parameter in the group LL-1 is
                                            significant, p<0.05.
Article
We evaluated the effect of exposure to constant light started at the age of 1 month and at the age of 14 months on the survival, life span, tumorigenesis and age-related dynamics of antioxidant enzymes activity in various organs in comparison to the rats maintained at the standard (12:12 light/dark) light/dark regimen. We found that exposure to constant light started at the age of 1 month accelerated spontaneous tumorigenesis and shortened life span both in male and female rats as compared to the standard regimen. At the same time, the exposure to constant light started at the age of 14 months failed to influence survival of male and female rats. While delaying tumors in males, constant light accelerated tumors in females. We conclude that circadian disruption induced by light-at-night started at the age of 1 month accelerates aging and promotes tumorigenesis in rats, however failed affect survival when started at the age of 14 months.
 
Chronic exposure to metformin suppresses the proliferative activity of human [A431] cancer cells (A) to enter a form of permanent cell-cycle arrest accompanied by the induction of “self-digesting” autophagy (B).
Chronic exposure to metformin activates the DNA damage sensor kinase ATM in poly-morphic, polyploidy [A431] cancer cells that contained giant nuclei and/or multiple varied size nuclei, including micronuclei (A, B). Chronically-treated cells demonstrated a large, spreading and flattening morphology, typical of senescent cells (C).
Chronic exposure to metformin accelerates the onset of replicative senescence in human [WI-38 and BJ-1] fibroblast cultures confirmed by senescence-associated β-galactosidase (SA-β-gal) staining (A). Chronic exposure to metformin sensitizes [MCF-7] cancer cells to the senescence program activated by the DNA-damaging drug doxorubicin (B). Chronic exposure to metformin transcriptionally activates a senescence-associated secretory phenotype (SASP) or senescence messaging secretome (SMS) involving the production of factors that reinforce the senescence arrest, alter the surrounding microenvironment, and trigger immune surveillance of the senescent cells.
Article
By activating the ataxia telangiectasia mutated (ATM)-mediated DNA Damage Response (DDR), the AMPK agonist metformin might sensitize cells against further damage, thus mimicking the precancerous stimulus that induces an intrinsic barrier against carcinogenesis. Herein, we present the new hypothesis that metformin might function as a tissue sweeper of pre-malignant cells before they gain stem cell/tumor initiating properties. Because enhanced glycolysis (the Warburg effect) plays a causal role in the gain of stem-like properties of tumor-initiating cells by protecting them from the pro-senescent effects of mitochondrial respiration-induced oxidative stress, metformin's ability to disrupt the glycolytic metabotype may generate a cellular phenotype that is metabolically protected against immortalization. The bioenergetic crisis imposed by metformin, which may involve enhanced mitochondrial biogenesis and oxidative stress, can lower the threshold for cellular senescence by pre-activating an ATM-dependent pseudo-DDR. This allows an accelerated onset of cellular senescence in response to additional oncogenic stresses. By pushing cancer cells to use oxidative phosphorylation instead of glycolysis, metformin can rescue cell surface major histocompatibility complex class I (MHC-I) expression that is downregulated by oncogenic transformation, a crucial adaptation of tumor cells to avoid the adaptive immune response by cytotoxic T-lymphocytes (CTLs). Aside from restoration of tumor immunosurveillance at the cell-autonomous level, metformin can activate a senescence-associated secretory phenotype (SASP) to reinforce senescence growth arrest, which might trigger an immune-mediated clearance of the senescent cells in a non-cell-autonomous manner. By diminishing the probability of escape from the senescence anti-tumor barrier, the net effect of metformin should be a significant decrease in the accumulation of dysfunctional, pre-malignant cells in tissues, including those with the ability to initiate tumors. As life-long or late-life removal of senescent cells has been shown to prevent or delay the onset or progression of age-related disorders, the tissue sweeper function of metformin may inhibit the malignant/metastatic progression of pre-malignant/senescent tumor cells and increase the human lifespan.
 
Retinoic acid induces differentiation of embryonic H9c2 myoblasts to cardiomyocytes in low serum supplemented with retinoic acid. Rat embryonic heart-derived myoblasts maintained in DMEM containing 10% FBS (A) were cultured in DMEM containing 1% FBS for seven days resulting in differentiation to skeletal muscle (B). A seven day culture in low serum media supplemented with 10 nM retinoic acid resulted in differentiation into cardiac myocytes (C). RT-PCR quantitation of MLC-2v transcripts (which display absolute cardiac tissue specificity) indicates overexpression of the gene in retinoic acid treated cells compared to cells cultured in low serum without retinoic acid (mean ± standard deviations of triplicates from a representative experiment, total of three independent experiments, (D). Bar = 5 μm; x 20 magnification.
Doxorubicin (Dox) induces a dose-dependent hypertrophic response in rat H9c2 cardiomyocytes. Cells were treated with the indicated concentrations of doxorubicin for two hrs and cultured in fresh media for a further 24 hours prior to quantitation of cell volume (A) and total protein content per cell (B). Phase-contrast images of control untreated H9c2 myocytes (C) compared to cells treated with 1 μM doxorubicin (2 hr treatment followed by 24 hour incubation in fresh media, D). Bar = 5 μm; x 20 magnification.
Doxorubicin modulates the expression of cardiac hypertrophy-associated genes in H9c2 myocytes. QT-PCR analysis of MLC-2v, α-MHC and ANP transcripts in cells treated with the indicated concentrations of doxorubicin for 2 hours followed by a 24 hour treatment in fresh media. Fold change of mRNA expression shown relative to untreated control H9c2 cells; mean ± standard deviations of triplicates from a representative experiment (total of three independent experiments) are indicated.
Trichostatin A (TSA) potentiates doxorubicin-induced hypertrophic responses in H9c2 cardiomyocytes by modulating the expression of hypertrophy-associated genes. QT-PCR analysis of MLC-2v, α-MHC and ANP transcripts in cells treated with 1 μM doxorubicin (Dox) for 2 hours followed by a 24 hour treatment in fresh media in the presence and absence of 1 μM TSA. Fold change of mRNA expression shown relative to untreated control H9c2 cells; mean ± standard deviations of triplicates from a representative experiment (total of three independent experiments) are indicated.
Trichostatin A (TSA) augments doxorubicin-induced accumulation of γH2AX foci in H9c2 cardiomyoctes. Cells pretreated with the indicated concentration of TSA for 24 hours were exposed to 1 μM doxorubicin for 1 hour, followed by a 24 hour treatment in fresh media. Cells were then stained for γH2AX foci, images were acquired with a Zeiss LSM 510 Meta Confocal microscope using 0.5 μm Z-sectioning and foci were quantitated using Metamorph (A). Mean ± standard deviations from two independent experiments (total of five independent experiments) are indicated (*p<0.001). Immunofluorescence visualization of γH2AX foci (B) in untreated H9c2 cells (i), cells treated with 1 μM TSA (ii), cells treated with 1 μM doxorubicin (iii) and cells treated with a combination of TSA and doxorubicin (iv) as described above. Bar = 2 μm; x 63 magnification.
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Top-cited authors
Steve Horvath
  • University of California, Los Angeles
Mikhail Blagosklonny
  • Roswell Park Cancer Institute
Morgan Elyse Levine
  • Yale University
Brian H Chen
  • U.S. Department of Health and Human Services
Themistocles Assimes
  • Stanford University