SIRT3 gene expression: a link between inherited mitochondrial DNA variants and oxidative stress

Signaling pathways between mitochondrial and nuclear genomes are activated to preserve cellular homeostasis, especially in the event of stress. Using cybrid cell lines, we investigated whether inherited mitochondrial DNA (mtDNA) variants modulate the expression profiles of mammalian sirtuins (SIRT1-7) under oxidative stress conditions. We found that the expression of the SIRT3 gene was down-regulated in cybrids harboring mtDNA of the J haplogroup, which correlated with mitochondrial function, resulting in a decline of NAD(+)/NADH and ATP levels. Overall, the data reported here highlight a link between SIRT3, mitochondrial DNA variability and mitochondrial functionality, three fundamental components of the cellular stress response.     

Epidemiological, genetic and epigenetic aspects of the research on healthy ageing and longevity

ABSTRACT: Healthy ageing and longevity in humans result from a number of factors, including genetic background, favorable environmental and social factors and chance.In this article we aimed to overview the research on the biological basis of human healthy ageing and longevity, discussing the role of epidemiological, genetic and epigenetic factors in the variation of quality of ageing and lifespan, including the most promising candidate genes investigated so far. Moreover, we reported the methodologies applied for their identification, discussing advantages and disadvantages of the different approaches and possible solutions that can be taken to overcome them. Finally, we illustrated the recent approaches to define healthy ageing and underlined the role that the emerging field of epigenetics is gaining in the search for the determinants of healthy ageing and longevity.     

Phosphoinositide 3-kinase is involved in the tumor-specific activation of human breast cancer cell Na(+)/H(+) exchange, motility, and invasion induced by serum deprivation

Whereas the tumor acidic extracellular pH plays a crucial role in the invasive process, the mechanism(s) behind this acidification, especially in low nutrient conditions, are unclear. The regulation of the Na(+)/H(+) exchanger (NHE) and invasion by serum deprivation were studied in a series of breast epithelial cell lines representing progression from non-tumor to highly metastatic cells. Whereas serum deprivation reduced lactate production in all three cells lines, it inhibited NHE activity in the non-tumor cells and stimulated it in the tumor cells with a larger stimulation in the metastatic cells. The stimulation of NHE in the tumor cell lines was the result of an increased affinity of the internal H(+) regulatory site of the NHE without changes in sodium kinetics or expression. Serum deprivation conferred increased cell motility and invasive ability that were abrogated by specific inhibition of the NHE. Inhibition of phosphoinositide 3-kinase by overexpression of a dominant-negative mutant or wortmannin incubation inhibited NHE activity and invasion in serum replete conditions while potentiating the serum deprivation-dependent activation of the NHE and invasion. These results indicate that the up-regulation of the NHE by a phosphoinositide 3-kinase-dependent mechanism plays an essential role in increased tumor cell invasion induced by serum deprivation.     

CRISPR/Cas9 System as an Agent for Eliminating Polyomavirus JC Infection

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the central nervous system (CNS) caused by reactivation of the human polyomavirus JCV gene expression and its replication in oligodendrocytes, the myelin producing cells in the brain. Once a rare disease seen in patients with lymphotproliferative and myeloproliferative disorders, PML has been seen more frequently in HIV-1 positive/AIDS patients as well as patients undergoing immunomodulatory therapy due for autoimmune disorders including multiple sclerosis, rheumatoid arthritis, and others. As of now there is no cure for PML and in most cases disease progression leads to death within two years. Similar to other polyomaviruses, the JCV genome is small circular double stranded DNA that includes coding sequences for the viral early protein, T-antigen, which is critical for directing viral reactivation and lytic infection. Here, we employ a newly developed gene editing strategy, CRISPR/Cas9, to introduce mutations in the viral genome and, by inactivating the gene encoding T-antigen, inhibit viral replication. We first used bioinformatics screening and identified several potential targets within the JCV T-antigen gene that can serve as sites for the creation of guide RNAs (gRNAs) for positioning the Cas9 nuclease on the designated area of the viral genome for editing. Results from a series of integrated genetic and functional studies showed that transient or conditional expression of Cas9 and gRNAs specifically targets the DNA sequences corresponding to the N-terminal region of T-antigen, and by introducing mutation, interferes with expression and function of of the viral protein, hence suppressing viral replication in permissive cells. Results from SURVEYOR assay revealed no off-target effects of the JCV-specific CRISPR/Cas9 editing apparatus. These observations provide the first evidence for the employment of a gene editing strategy as a promising tool for the elimination of the JCV genome and a potential cure for PML.     

Characterization of a bidirectional promoter shared between two human genes related to aging: SIRT3 and PSMD13

The human SIRT3 gene contains an intronic VNTR enhancer whose variability is correlated with life span. The SIRT3 5' flanking region encompasses the PSMD13 gene encoding the p40.5 regulator subunit of the 26S proteasome. Proteasome is a multicatalytic proteinase whose function declines with aging. SIRT3 and PSMD13 are linked in a head-to-head configuration (788-bp intergenic region). The molecular configuration of two genes that are both related to aging prompted us to search for shared regulatory mechanisms between them. Transfection experiments carried out in HeLa cells by deletion mutants of the PSMD13-SIRT3 intergenic region showed a complex pathway of coregulation acting in both directions. Furthermore, linkage disequilibrium (LD) analyses carried out in a sample of 710 subjects (18-108 years of age) screened for A21631G (marker of PSMD13), and for G477T and VNTR(intron5) (markers of SIRT3), revealed high LD, with significantly different PSMD13-SIRT3 haplotype pools between samples of centenarians and younger people.     

Construction and Application of Efficient Ac-Ds Transposon Tagging Vectors in Rice

Transposons are effective mutagens alternative to T-DNA for the generation of insertional mutants in many plant species including those whose transformation is inefficient. The current strategies of transposon tagging are usually slow and labor-intensive and yield low frequency of tagged lines. We have constructed a series of transposon tagging vectors based on three approaches: (i) Ac TPase controlled by glucocorticoid binding domain/VP16 acidic activation domain/Gal4 DNA-binding domain (GVG) chemical-inducible expression system; (ii) deletion of Ac TPase via Cre- lox site-specific recombination that was initially triggered by Ds excision; and (iii) suppression of early transposition events in transformed rice callus through a dual-functional hygromycin resistance gene in a novel Ds element ( HPT-Ds ). We tested these vectors in transgenic rice and characterized the transposition events. Our results showed that these vectors are useful resources for functional genomics of rice and other crop plants. The vectors are freely available for the community.     

Faster top running speeds are achieved with greater ground forces not more rapid leg movements.

We twice tested the hypothesis that top running speeds are determined by the amount of force applied to the ground rather than how rapidly limbs are repositioned in the air. First, we compared the mechanics of 33 subjects of different sprinting abilities running at their top speeds on a level treadmill. Second, we compared the mechanics of declined (-6 degrees ) and inclined (+9 degrees ) top-speed treadmill running in five subjects. For both tests, we used a treadmill-mounted force plate to measure the time between stance periods of the same foot (swing time, t(sw)) and the force applied to the running surface at top speed. To obtain the force relevant for speed, the force applied normal to the ground was divided by the weight of the body (W(b)) and averaged over the period of foot-ground contact (F(avge)/W(b)). The top speeds of the 33 subjects who completed the level treadmill protocol spanned a 1.8-fold range from 6.2 to 11.1 m/s. Among these subjects, the regression of F(avge)/W(b) on top speed indicated that this force was 1.26 times greater for a runner with a top speed of 11.1 vs. 6.2 m/s. In contrast, the time taken to swing the limb into position for the next step (t(sw)) did not vary (P = 0.18). Declined and inclined top speeds differed by 1.4-fold (9.96+/-0.3 vs. 7.10+/-0.3 m/s, respectively), with the faster declined top speeds being achieved with mass-specific support forces that were 1.3 times greater (2.30+/- 0.06 vs. 1.76+/-0.04 F(avge)/ W(b)) and minimum t(sw) that were similar (+8%). We conclude that human runners reach faster top speeds not by repositioning their limbs more rapidly in the air, but by applying greater support forces to the ground.     

High-speed running performance is largely unaffected by hypoxic reductions in aerobic power.

We tested the importance of aerobic metabolism to human running speed directly by altering inspired oxygen concentrations and comparing the maximal speeds attained at different rates of oxygen uptake. Under both normoxic (20.93% O2) and hypoxic (13.00% O2) conditions, four fit adult men completed 15 all-out sprints lasting from 15 to 180 s as well as progressive, discontinuous treadmill tests to determine maximal oxygen uptake and the metabolic cost of steady-state running. Maximal aerobic power was lower by 30% (1.00 +/- 0.15 vs. 0.77 +/- 0.12 ml O2. kg-1. s-1) and sprinting rates of oxygen uptake by 12-25% under hypoxic vs. normoxic conditions while the metabolic cost of submaximal running was the same. Despite reductions in the aerobic energy available for sprinting under hypoxic conditions, our subjects were able to run just as fast for sprints of up to 60 s and nearly as fast for sprints of up to 120 s. This was possible because rates of anaerobic energy release, estimated from oxygen deficits, increased by as much as 18%, and thus compensated for the reductions in aerobic power. We conclude that maximal metabolic power outputs during sprinting are not limited by rates of anaerobic metabolism and that human speed is largely independent of aerobic power during all-out runs of 60 s or less.