Continuous multiparametric monitoring of cell metabolism in response to transient overexpression of the sirtuin deacetylase SIRT3

The analysis and visualisation of research data in an environment which is most similar to living conditions belong to the most challenging claims of present scientific research endeavours. To date, the effect of protein function on cell metabolism is most commonly assessed from a series of end point analyses, which finally allows an approximate estimation on how a specific effect takes its course. In the study presented herein, we demonstrate how the combination of transient transfection and a biosensor chip system gives the opportunity to analyse the effect of a specific protein on cell metabolism in living cells through real-time monitoring of metabolically relevant parameters, such as oxygen consumption, acidification rate and cell adhesion. In addition, this method allows online monitoring of the time course of metabolic changes due to changes in expression levels of metabolic regulative proteins from the time of transfection to maximum overexpression. The methodology presented herein was assessed for the transient overexpression of the sirtuin deacetylase SIRT3, a mitochondrial key element in the regulation of energy metabolism, metabolic disease, cancer and ageing

Fragile DNA Repair Mechanism Reduces Ageing in Multicellular Model

DNA damages, as well as mutations, increase with age. It is believed that these result from increased genotoxic stress and decreased capacity for DNA repair. The two causes are not independent, DNA damage can, for example, through mutations, compromise the capacity for DNA repair, which in turn increases the amount of unrepaired DNA damage. Despite this vicious circle, we ask, can cells maintain a high DNA repair capacity for some time or is repair capacity bound to continuously decline with age? We here present a simple mathematical model for ageing in multicellular systems where cells subjected to DNA damage can undergo full repair, go apoptotic, or accumulate mutations thus reducing DNA repair capacity. Our model predicts that at the tissue level repair rate does not continuously decline with age, but instead has a characteristic extended period of high and non-declining DNA repair capacity, followed by a rapid decline. Furthermore, the time of high functionality increases, and consequently slows down the ageing process, if the DNA repair mechanism itself is vulnerable to DNA damages. Although counterintuitive at first glance, a fragile repair mechanism allows for a faster removal of compromised cells, thus freeing the space for healthy peers. This finding might be a first step toward understanding why a mutation in single DNA repair protein (e.g. Wrn or Blm) is not buffered by other repair proteins and therefore, leads to severe ageing disorders

Lithic technological responses to Late Pleistocene glacial cycling at Pinnacle Point Site 5-6, South Africa

There are multiple hypotheses for human responses to glacial cycling in the Late Pleistocene, including changes in population size, interconnectedness, and mobility. Lithic technological analysis informs us of human responses to environmental change because lithic assemblage characteristics are a reflection of raw material transport, reduction, and discard behaviors that depend on hunter-gatherer social and economic decisions. Pinnacle Point Site 5-6 (PP5-6), Western Cape, South Africa is an ideal locality for examining the influence of glacial cycling on early modern human behaviors because it preserves a long sequence spanning marine isotope stages (MIS) 5, 4, and 3 and is associated with robust records of paleoenvironmental change. The analysis presented here addresses the question, what, if any, lithic assemblage traits at PP5-6 represent changing behavioral responses to the MIS 5-4-3 interglacial-glacial cycle? It statistically evaluates changes in 93 traits with no a priori assumptions about which traits may significantly associate with MIS. In contrast to other studies that claim that there is little relationship between broad-scale patterns of climate change and lithic technology, we identified the following characteristics that are associated with MIS 4: increased use of quartz, increased evidence for outcrop sources of quartzite and silcrete, increased evidence for earlier stages of reduction in silcrete, evidence for increased flaking efficiency in all raw material types, and changes in tool types and function for silcrete. Based on these results, we suggest that foragers responded to MIS 4 glacial environmental conditions at PP5-6 with increased population or group sizes, 'place provisioning', longer and/or more intense site occupations, and decreased residential mobility. Several other traits, including silcrete frequency, do not exhibit an association with MIS. Backed pieces, once they appear in the PP5-6 record during MIS 4, persist through MIS 3. Changing paleoenvironments explain some, but not all temporal technological variability at PP5-6.Social Science and Humanities Research Council of Canada; NORAM; American-Scandinavian Foundation; Fundacao para a Ciencia e Tecnologia [SFRH/BPD/73598/2010]; IGERT [DGE 0801634]; Hyde Family Foundations; Institute of Human Origins; National Science Foundation [BCS-9912465, BCS-0130713, BCS-0524087, BCS-1138073]; John Templeton Foundation to the Institute of Human Origins at Arizona State Universit

Deficit of circulating stem – progenitor cells in opiate addiction: a pilot study

A substantial literature describes the capacity of all addictive drugs to slow cell growth and potentiate apoptosis. Flow cytometry was used as a means to compare two lineages of circulating progenitor cells in addicted patients. Buprenorphine treated opiate addicts were compared with medical patients. Peripheral venous blood CD34+ CD45+ double positive cells were counted as haemopoietic stem cells (HSC's), and CD34+ KDR+ (VEGFR2+) cells were taken as endothelial progenitor cells (EPC's). 10 opiate dependent patients with substance use disorder (SUD) and 11 non-addicted (N-SUD) were studied. The ages were (mean + S.D.) 36.2 + 8.6 and 56.4 + 18.6 respectively (P <0.01). HSC's were not different in the SUD (2.38 + 1.09 Vs. 3.40 + 4.56 cells/mcl). EPC's were however significantly lower in the SUD (0.09 + 0.14 Vs. 0.26 + 0.20 cells/mcl; No. > 0.15, OR = 0.09, 95% C.I. 0.01–0.97), a finding of some interest given the substantially older age of the N-SUD group. These laboratory data are thus consistent with clinical data suggesting accelerated ageing in addicted humans and implicate the important stem cell pool in both addiction toxicology and ageing. They carry important policy implications for understanding the fundamental toxicology of addiction, and suggest that the toxicity both of addiction itself and of indefinite agonist maintenance therapies may have been seriously underestimated

Dietary Restriction Depends on Nutrient Composition to Extend Chronological Lifespan in Budding Yeast Saccharomyces cerevisiae

10.1371/journal.pone.0064448PLoS ONE85

Loss of p53 Ser18 and Atm Results in Embryonic Lethality without Cooperation in Tumorigenesis

Phosphorylation at murine Serine 18 (human Serine 15) is a critical regulatory process for the tumor suppressor function of p53. p53Ser18 residue is a substrate for ataxia-telangiectasia mutated (ATM) and ATM-related (ATR) protein kinases. Studies of mice with a germ-line mutation that replaces Ser18 with Ala (p53S18A mice) have demonstrated that loss of phosphorylation of p53Ser18 leads to the development of tumors, including lymphomas, fibrosarcomas, leukemia and leiomyosarcomas. The predominant lymphoma is B-cell lymphoma, which is in contrast to the lymphomas observed in Atm−/− animals. This observation and the fact that multiple kinases phosphorylate p53Ser18 suggest Atm-independent tumor suppressive functions of p53Ser18. Therefore, in order to examine p53Ser18 function in relationship to ATM, we analyzed the lifespan and tumorigenesis of mice with combined mutations in p53Ser18 and Atm. Surprisingly, we observed no cooperation in survival and tumorigenesis in compound p53S18A and Atm−/− animals. However, we observed embryonic lethality in the compound mutant animals. In addition, the homozygous p53Ser18 mutant allele impacted the weight of Atm−/− animals. These studies examine the genetic interaction of p53Ser18 and Atm in vivo. Furthermore, these studies demonstrate a role of p53Ser18 in regulating embryonic survival and motor coordination

Genomic Instability Is Associated with Natural Life Span Variation in Saccharomyces cerevisiae

Increasing genomic instability is associated with aging in eukaryotes, but the connection between genomic instability and natural variation in life span is unknown. We have quantified chronological life span and loss-of-heterozygosity (LOH) in 11 natural isolates of Saccharomyces cerevisiae. We show that genomic instability increases and mitotic asymmetry breaks down during chronological aging. The age-dependent increase of genomic instability generally lags behind the drop of viability and this delay accounts for ∼50% of the observed natural variation of replicative life span in these yeast isolates. We conclude that the abilities of yeast strains to tolerate genomic instability co-vary with their replicative life spans. To the best of our knowledge, this is the first quantitative evidence that demonstrates a link between genomic instability and natural variation in life span

Both telomeric and non-telomeric DNA damage are determinants of mammalian cellular senescence

<p>Abstract</p> <p>Background</p> <p>Cellular senescence is a state reached by normal mammalian cells after a finite number of cell divisions and is characterized by morphological and physiological changes including terminal cell-cycle arrest. The limits on cell division imposed by senescence may play an important role in both organismal aging and in preventing tumorigenesis. Cellular senescence and organismal aging are both accompanied by increased DNA damage, seen as the formation of γ-H2AX foci (γ-foci), which may be found on uncapped telomeres or at non-telomeric sites of DNA damage. However, the relative importance of telomere- and non-telomere-associated DNA damage to inducing senescence has never been demonstrated. Here we present a new approach to determine accurately the chromosomal location of γ-foci and quantify the number of telomeric versus non-telomeric γ-foci associated with senescence in both human and mouse cells. This approach enables researchers to obtain accurate values and to avoid various possible misestimates inherent in earlier methods.</p> <p>Results</p> <p>Using combined immunofluorescence and telomere fluorescence <it>in situ </it>hybridization on metaphase chromosomes, we show that human cellular senescence is not solely determined by telomeric DNA damage. In addition, mouse cellular senescence is not solely determined by non-telomeric DNA damage. By comparing cells from different generations of telomerase-null mice with human cells, we show that cells from late generation telomerase-null mice, which have substantially short telomeres, contain mostly telomeric γ-foci. Most notably, we report that, as human and mouse cells approach senescence, all cells exhibit similar numbers of total γ-foci per cell, irrespective of chromosomal locations.</p> <p>Conclusion</p> <p>Our results suggest that the chromosome location of senescence-related γ-foci is determined by the telomere length rather than species differences <it>per se</it>. In addition, our data indicate that both telomeric and non-telomeric DNA damage responses play equivalent roles in signaling the initiation of cellular senescence and organismal aging. These data have important implications in the study of mechanisms to induce or delay cellular senescence in different species.</p

Association between Urinary Excretion of Cortisol and Markers of Oxidatively Damaged DNA and RNA in Humans

Chronic psychological stress is associated with accelerated aging, but the underlying biological mechanisms are not known. Prolonged elevations of the stress hormone cortisol is suspected to play a critical role. Through its actions, cortisol may potentially induce oxidatively generated damage to cellular constituents such as DNA and RNA, a phenomenon which has been implicated in aging processes. We investigated the relationship between 24 h excretion of urinary cortisol and markers of oxidatively generated DNA and RNA damage, 8-oxo-7,8-dihydro-2′-deoxyguanosine and 8-oxo-7,8-dihydroguanosine, in a sample of 220 elderly men and women (age 65 – 83 years). We found a robust association between the excretion of cortisol and the oxidation markers (R2 = 0.15, P<0.001 for both markers). Individuals in the highest quartile of cortisol excretion had a 57% and 61% higher median excretion of the DNA and RNA oxidation marker, respectively, than individuals in the lowest quartile. The finding adds support to the hypothesis that cortisol-induced damage to DNA/RNA is an explanatory factor in the complex relation between stress, aging and disease

Premature Senescence and Increased TGFβ Signaling in the Absence of Tgif1

Transforming growth factor β (TGFβ) signaling regulates cell cycle progression in several cell types, primarily by inducing a G1 cell cycle arrest. Tgif1 is a transcriptional corepressor that limits TGFβ responsive gene expression. Here we demonstrate that primary mouse embryo fibroblasts (MEFs) lacking Tgif1 proliferate slowly, accumulate increased levels of DNA damage, and senesce prematurely. We also provide evidence that the effects of loss of Tgif1 on proliferation and senescence are not limited to primary cells. The increased DNA damage in Tgif1 null MEFs can be partially reversed by culturing cells at physiological oxygen levels, and growth in normoxic conditions also partially rescues the proliferation defect, suggesting that in the absence of Tgif1 primary MEFs are less able to cope with elevated levels of oxidative stress. Additionally, we show that Tgif1 null MEFs are more sensitive to TGFβ-mediated growth inhibition, and that treatment with a TGFβ receptor kinase inhibitor increases proliferation of Tgif1 null MEFs. Conversely, persistent treatment of wild type cells with low levels of TGFβ slows proliferation and induces senescence, suggesting that TGFβ signaling also contributes to cellular senescence. We suggest that in the absence of Tgif1, a persistent increase in TGFβ responsive transcription and a reduced ability to deal with hyperoxic stress result in premature senescence in primary MEFs