Elevated PP is also an independent predictor of gait speed in older adults, a measure of physical function that in and of itself is a predictor of survival in older adults. Older adults with higher PP had PR-171 significantly slower gait speed compared to older adults with lower PP. These findings raise the intriguing possibility that ageassociated decline in vascular function may be inextricably linked to decline in physical function. In well-functioning older adults, PP/arterial stiffness may not be a predictor of short distance gait speed. Our findings are consistent with this as we noted no association between PP and 4 m gait speed in older adults with mobility limitations from the LIFE-P cohort. We noted an association between long distance gait speed and PP in older adults with mobility limitations. Recent work from the Health Aging and Body Composition Study has noted that arterial stiffness is a predictor of gait speed in older adults with peripheral arterial disease. Previous studies have noted that arterial stiffness and pressure from wave reflections are also predictors of walking distance in patients with PAD. Recently, our group has reported an association between PP and long-distance gait performance in adults with multiple sclerosis. Mobility limitations, as seen with PAD and MS, perpetuate a sedentary lifestyle and physical inactivity is a potent instigator of vascular mal-adaptation. Older adults with mobilitylimitations from LIFE-P with the slowest gait speed had substantially higher pulse pressure than older well-functioning adults from the Health ABC Study, suggesting hastened vascular senescence in the LIFE-P group. Thus our findings support previous conclusions that arterial stiffness may be especially detrimental to older adults with established compromised mobility and significantly impaired vascular function and suggest that long distance gait performance but not short distance gait performance may be influenced by PP in older adults at risk for mobility disability. Several potential mechanisms may explain the association between PP and gait performance. Left ventricular ejection of stroke volume into a stiff aorta coupled with early return of reflected pressure waves of greater magnitude increases cardiac energetic demand, reduces stroke volume, reduces myocardial oxygen supply/consumption and reduces subendocardial perfusion. Pulsatile pressure and flow damages the endothelium which may alter oxygen delivery to and impair oxygen uptake by the working skeletal muscle. Finally, pulsatile load may damage cerebral blood vessels, reduce cerebrovascular reactivity, and contribute to cerebral white matter hyperintensities and cognitive decline. Indeed white matter lesions may be an intermediate factor in the relation of hypertension and lower gait speed in older adults and cognitive function is associated with physical function. Older adults taking beta-blockers had higher PP and a trend toward lower gait speed than older adults not taking these agents. This appears to have been mediated by the secondary effect of beta-blockers on heart rate as heart rate was significantly lower in those taking beta-blockers versus those not taking these agents. Adjusting for heart rate abolished differences in PP and gait speed. Reductions in heart rate with beta-blocker use may alter pressure wave temporal associations, increasing late systolic pressure augmentation and widening PP. Moreover, increased arterial stiffness, as occurs with natural aging, may exacerbate the influence of HR on wave reflections. Thus, therapies that negatively influence pressure from wave reflections and increase PP may have a detrimental effect on physical function in older adults with low already low vascular compliance.

These two conditions, which account for 1–2% of all SAR131675 congenital heart disease in humans, are still difficult to treat. Given the fact that NFATC1 is at the center of valve formation in mammals, we hypothesize that mutations in the gene encoding it would be associated with valve malformations in humans. We have previously shown that a tandem repeat in the intronic region of NFATC1 is associated with ventricular septal defects but with no valvular phenotype. We therefore screened for such mutations in patients with different valve diseases registered at the congenital heart disease genetics program at the American University of Beirut Medical Center. Results showed 2 novel missense single nucleotide polymorphisms in only one patient with tricuspid atresia. Functional analyses of the mutated protein do show a defect in its cellular localization, transcriptional activities and DNA binding patterns suggesting that the mutations are disease causing. Congenital heart diseases are still the leading cause of death in newborns in addition to being the most frequent congenital diseases in humans. The genetic mechanisms underlying such diseases however, are being unraveled slowly in the last decade because of the tremendous work done on understanding the molecular mechanisms governing cardiac development in numerous organisms. These mechanisms include the collaborative interaction between transcription factors and their occupancy of conserved cis regulatory elements on different cardiac-specific promoters. Led to the formulation of hypotheses that mutations in these genes could cause heart malformations in humans. More importantly, the available data on genes such as GATA4, NKX2-5 and TBX5 do point to a dose-dependent genotype-phenotype correlation whereby haploinsufficiency is by itself diseases-causing. Our results go along with what is published in that regard by adding the NFATC1 gene to the list of mutated genes linked to congenital heart disease in humans, particularly valve diseases. We have shown two heterozygous mutations on one allele of the NFATC1 gene in one patient with tricuspid atresia out of 19. The fact that the double mutation is also found in the father who has a normal phenotype argues for incomplete penetrance, a phenomenon seen in other genes encoding transcription factors involved in cardiac and non-cardiac congenital diseases. One such example is the Arg25Cys mutation, which was shown to abrogate the transcriptional activity of the NKX2-5 protein and yet has reduced penetrance depending on the population study groups. In mice, the Holt-Oram syndrome recreated with the heterozygous Tbx5 model is the best example of a dosage dependent phenotype-genotype correlation. In fact, null mice for both Tbx5 alleles showed a very severe cardiac phenotype leading to early embryonic lethality, while mice carrying only one Tbx5 allele display a spectrum of phenotypes recapitulating the ones observed in humans. Unfortunately, in our case the indexedpatient was evaluated for the first time at the age of 16 years at our center when he presented with severe cyanosis and complications of his condition which was not well taken care of at earlier stages and had led to the his death few days after his admission to the hospital. Exon by exon sequencing of different genes encoding transcription factors, including GATA4,5,6, TBX5,20, NKX2-5, PITX2, and NFATC1 was carried out on the whole family and none except NFATC1 showed polymorphisms that could be disease causing. We cannot exclude however, that other not tested gene could also be mutated and carried on the maternally inherited allele, and that the combination of such mutations is responsible for the observed phenotype.

Demonstrated that nucleolar proteins are in continuous exchange with other nuclear and cellular compartments in response to specific cellular conditions. Of importance, the nucleolus is also the target of viruses including HIV-1, hCMV, HSV and KSHV, as part of their replication strategy. Proteomics studies analysing the nucleoli of cells infected with Human respiratory syncytial virus, influenza A virus, avian coronavirus infectious bronchitis virus or adenovirus highlighted how viruses can distinctively disrupt the distribution of nucleolar proteins. Interestingly, both HIV-1 regulatory proteins Tat and Rev localise to the nucleoplasm and nucleolus. Both their sequences encompass a nucleolar localisation signal overlapping with their nuclear localisation signal, which governs their nucleolar localisation. AbMole BioScience Furthermore, Tat and Rev interact with the nucleolar antigen B23, which is essential for their nucleolar localisation. Nevertheless, a recent study described that in contrast to Jurkat T-cells and other transformed cell lines where Tat is associated with the nucleus and nucleolus, in primary T-cells Tat primarily accumulates at the plasma membrane, while trafficking via the nucleus where it functions. While the regulation of their active nuclear import and/or export, as mediated by the karyopherin/importin family have been well described, the mechanisms distributing Tat and Rev between the cytoplasm, nucleoplasm and the nucleolus remains elusive. The CBDs are typically characterized by an eight stranded jellyroll b-sandwich, flanked by helices at the N- and C-termini as well as a small intervening helix situated between strands b6 and b7. Recent methods aimed at comparing patterns of amino acid conservations in sequence and in space have identified four conserved structural elements that are universally present in eukaryotic CBDs: the N-terminal helical bundle, the b2- b3 loop, the phosphate binding cassette and the hinge helix. Previous investigations on the CBD of EPAC1, have established the former three structural elements as crucial determinants underlying auto-inhibition. However, the role of the hinge helix as an auto-inhibitory determinant of the EPAC CBD is currently not fully understood. The last two turns of the EPAC hinge helix partially unfold as a6 rotates towards the a5 helix of the PBC upon cAMP binding. This hinge rotation has been rationalized as a consequence of the cAMP-induced repositioning of the PBC L273 residue, which contacts with F300 in the hinge helix. The repositioning of the conserved L273, and consequently F300, retracts the hinge helix toward the PBC helix upon activation. Recent studies mapping the EPAC allosteric network through chemical shift covariance analysis have revealed that L273 and F300 are part of a larger cluster of allosteric residues, which includes also a hydrophobic spine at the interface between the a4 and a6 helices. Such spine spans residues in the C-terminal end of the hinge helix that unwinds upon cAMP binding. Based on these observations, here we hypothesize that the Cterminal residues of the hinge helix are key determinants of EPAC auto-inhibition and that perturbations that destabilize the helix or induce unwinding shift the apo/inactive vs. apo/active pre-equilibrium toward the latter state, i.e. an active state without cAMP. To test this hypothesis, we designed three successive deletion mutations of the 149–318 EPAC1 construct, which spans the CBD and which from here on forth will be referred to as the Wt-EPAC. Specifically, these mutants are C-terminally truncated at positions 305, 310, and 312 and act as perturbations that destabilize the hinge helix of apoEPAC, mimicking the cAMP-induced unwinding.

Interestingly lacking mitochondrial genes or the entire mtDNA. Remarkably, fusion inhibition was observed under fermentative conditions, when glycolysis provides ATP for mitochondrial biogenesis and growth. The dominant inhibition of fusion in heterogenic crosses demonstrated that the fusion defects of OXPHOS deficient mitochondria cannot be compensated, in trans, by functional mitochondria. Fusion assays with fluorescently labeled outer membranes demonstrated that OXPHOS defects selectively inhibit inner membrane fusion. Electron microscopy revealed that fusion inhibition was associated to the presence of elongated, unfused inner membranes that were connected to boundary membranes. These ultrastructural features are reminiscent of those observed upon inhibition of inner membrane fusion with ionophores or in Mgm1-mutant strains. The selective inhibition of inner membrane fusion in OXPHOS-deficient cells Nilotinib confirms that outer and inner membrane fusions are catalyzed by machineries that can function separately and have different energetic requirements. The requirement of DYm for inner, and not outer membrane fusion, suggests that the observed fusion inhibition is related to the lower DYm in OXPHOS deficient cells. However, given the interdependence of respiration, ATP-synthesis and DYm, we cannot exclude that other parameters also contribute to fusion inhibition. Surprisingly, fusion inhibition was not systematically associated to major alterations in mitochondrial distribution and morphology, implying that such fusion defects escape detection in studies that were solely based on the analysis of mitochondrial morphology. Similarly, cells devoid of subunit e of the ATP-synthase, defective in ATP-synthase oligomerization, showed significant alterations of mitochondrial ultrastructure that were not paralleled by defects in overall morphology or fusion. The fact that major alterations in overall distribution and morphology were restricted to Datp6 and atp6-L247R strains, suggests that this phenotype is associated to the low levels of Atp6 protein rather than to a defect in fusion. In addition, it is interesting to note that among the mutants identified in the screen for altered mitochondrial distribution and morphology, only 9 encoded OXPHOS-related proteins, and of those, 8 were components or assembly factors of ATP-synthase. Further work is required to unravel the exact links between ATP-synthase and mitochondrial ultrastructure, morphology and/ or dynamics. In mammals, the inhibition of fusion by bioenergetic defects and/or loss of DYm is paralleled by fast and quantitative changes in the isoform-pattern of OPA1. We observed that, in yeast, the patterns of Mgm1-isoforms varied somewhat between strains and culture conditions, but that these variations did not correlate with the fusion capacity. We conclude that, in OXPHOS-deficient strains, fusion capacity was not lowered through changes in the isoform pattern of Mgm1. The fact that fusion inhibition by dissipation of DYm was not associated to changes in the isoform pattern of Mgm1 further indicates that, in yeast, a factor other than Mgm1 requires DYm for inner membrane fusion. This points to differences in the properties and regulation of mitochondrial fusion and Mgm1/OPA1 in yeast and in mammals. Current models of mitochondrial biogenesis and maintenance include the hypothesis that defective mitochondria have a lower fusion capacity, that this leads to their exclusion from the network of functional mitochondria and that this facilitates their selective degradation by autophagy. The dominant inhibition of fusion demonstrated in this work provides a mechanism for the exclusion of defective mitochondria and thus for the selective degradation of mitochondria by autophagy. Further work is required to elucidate the complex relationships.

We developed them into cleavage-stage embryos for analyzing the expression of different ligand-receptor pairs known to play autocrine/paracrine functions in animal embryos. We also demonstrated that culturing these abnormally fertilized embryos in serum-free culture media supplemented with GDC-0941 PI3K inhibitor growth factors substantially promoted their development by more than 2-fold. The improvement of sequential culture systems for human IVF during the last decades has allowed extended culture of human early embryos to the blastocyst stage. Blastocyst transfer facilitates the selection of the best embryos with high implantation potential and therefore reduces the number of transferred embryos to avoid multiple pregnancies. However, the current human embryo culture system is still suboptimal and many embryos cannot develop to the blastocyst stage. Our results using normally fertilized day 3 embryos suggest that key autocrine/paracrine growth factors are beneficial to human embryonic development in vitro. These growth factors not only increase the rate of blastocyst formation, but also the quality of blastocysts. Indeed, culturing good-quality day 3 embryos in culture medium supplemented with these growth factors resulted in a 3.3-fold increase in the blastocyst formation rate and a 7.6-fold increase in the proportion of high quality blastocysts as compared to controls. These findings are consistent with the hypothesis that autocrine/paracrine factors secreted by early embryos are diluted during culture and growth factor supplementation is necessary to promote optimal blastocyst formation. Because most of the commercially available, chemically-defined media for human embryo cultures in IVF-ET do not contain growth factors, the present supplementation of widely used culture media with autocrine/paracrine growth factors has practical value in future IVF-ET procedures. Different from previously published reports showing small stimulatory effects of individual growth factors on human embryo development, our combined treatment with several autocrine/ paracrine factors showed a robust stimulation of normally fertilized day 3 embryos likely due to additive effects of different growth factors in the promotion of early embryonic development. Inclusion of IGF-I or GM-CSF increased the proportion of embryos developing to the blastocyst stage by 1.51-fold and 2.53-fold, respectively. In our study, treatment embryos with the growth factor cocktail showed a 3.3-fold increase in the proportion of blastocyst-stage-embryos. The ability of these paracrine/autocrine factors to promote development of early human embryos is consistent with findings showing zygote genome activation in human embryos at 4- to 8-cell stages on day 3 after fertilization when the expression of these growth factors begun to increase. In the present combination treatment protocol, several distinct signaling pathways could be activated by the autocrine/paracrine factors used: EGF, IGF-I and BDNF bind to respective receptor tyrosine kinases to activate downstream phophotidyinositol-3-kinase-Akt signaling, CSF1 and GM-CSF interact with type I cytokine receptors to activate the downstream JAK/STAT pathway, whereas GDNF and artemin interact with glycosylphosphatidyl- inositol-anchored receptors to activate downstream cRET and Src kinase pathways. Although the fresh tri-pronuclear zygotes used here were treated with five growth factors due to reagent availability, thawed normallyfertilized and SCNT embryos were treated with seven growth factors. It is likely that these divergent pathways exert overlapping and redundant actions on early embryo development and not all growth factors are needed for optimal embryo growth.