Long-term calorie restriction in humans is not associated with indices of delayed immunologic aging: A descriptive study.

BACKGROUND: Delayed immunologic aging is purported to be a major mechanism through which calorie restriction (CR) exerts its anti-aging effects in non-human species. However, in non-obese humans, the effect of CR on the immune system has been understudied relative to its effects on the cardiometabolic system. OBJECTIVE: To examine whether CR is associated with delayed immunologic aging in non-obese humans. METHODS: We tested whether long-term CR practitioners (average 10.03 years of CR) evidenced decreased expression of T cell immunosenescence markers and longer immune cell telomeres compared to gender-, race/ethnicity-, age-, and education-matched "healthy" Body Mass Index (BMI) and "overweight"/"obese" BMI groups. RESULTS: Long-term human CR practitioners had lower BMI (p <  0.001) and fasting glucose (p <  0.001), as expected. They showed similar frequencies of pre-senescent cells (CD8+CD28- T cells and CD57 and PD-1 expressing T cells) to the comparison groups. Even after adjusting for covariates, including cytomegalovirus status, we observed shorter peripheral blood mononuclear cell telomeres in the CR group (p = 0.012) and no difference in granulocyte telomeres between groups (p = 0.42). CONCLUSIONS: We observed no clear evidence that CR as it is currently practiced in humans delays immune aging related to telomere length or T cell immunosenescent markers

Flags for future disease: postpartum cardiometabolic risk & prevention by understanding the contribution of breastfeeding and the influences of the COVID-19 pandemic

Cardiovascular disease is a global leading cause of mortality in women. Pregnancy has been called a ‘stress test’ due to pathological indicators such as gestational diabetes (GDM) that can ‘flag’ increased risk for cardiometabolic disease. Of concern is the global rising prevalence of type 2 diabetes (T2DM) and cardiometabolic disease, with pandemics identified to increase population risk for both. There are measures to reduce cardiometabolic disease risk such as exercise and diet. Breastfeeding has also been identified to reduce T2DM risk, however the impact on next pregnancy GDM risk is unknown. It is also unknown how changes in maternity care during the COVID-19 pandemic impact cardiometabolic risk. The aim of my thesis was to investigate cardiometabolic risk for women during the pandemic with a focus on gestational diabetes and breastfeeding. Quantitative and qualitative studies were undertaken for this thesis. An initial novel quantitative pilot study identified that high intensity breastfeeding (HIBF) was associated with a lower glucose level on the OGTT (published). In my follow-up study of 5,374 participants, HIBF was associated with 47% reduced odds of an abnormal fasting glucose in a subsequent pregnancy OGTT (aOR 0.53; 95%CI 0.38-0.75; p<0.01). A mixed methods approach examined potential influences on cardiometabolic health in the first year of the pandemic during the low COVID-19 prevalence period (three publications). Further qualitative methodology was used to explore the disruption to maternity care and identified disrupted social support and vulnerable groups in the high migrant population. This thesis presents novel findings identifying optimal breastfeeding patterns can have direct metabolic benefits in a subsequent. The results of our COVID-19 research provides unique insights into how service and community changes alone impact obstetric outcomes and potential cardiometabolic risk in a high migrant population

Transcription-Independent Heritability of Induced Histone Modifications in the Mouse Preimplantation Embryo

Enzyme-catalyzed, post-translational modifications of core histones have been implicated in the complex changes in gene expression that drive early mammalian development. However, until recently the small number of cells available from the preimplantation embryo itself has prevented quantitative analysis of histone modifications at key regulator genes. The possible involvement of histone modifications in the embryo's response to extracellular signals, or as determinants of cell fate or lineage progression, remains unclear. Here we describe the use of a recently-developed chromatin immunoprecipitation technique (CChIP) to assay histone modification levels at key regulator genes (Pou5f1, Nanog, Cdx2, Hoxb1, Hoxb9) as mouse embryos progress from 8-cell to blastocyst in culture. Only by the blastocyst stage, when the embryonic (Inner Cell Mass) and extra-embryonic (Trophoblast) lineages are compared, do we see the expected association between histone modifications previously linked to active and silent chromatin, and transcriptional state. To explore responses to an environmental signal, we exposed embryos to the histone deacetylase inhibitor, anti-epileptic and known teratogen valproic acid (VPA), during progression from 8-cell to morula stage. Such treatment increased H4 acetylation and H3 lysine 4 methylation at the promoters of Hoxb1 and Hoxb9, but not the promoters of Pou5f1, Nanog,Cdx2 or the housekeeping gene Gapdh. Despite the absence of detectable Hoxb transcription, these VPA-induced changes were heritable, following removal of the inhibitor, at least until the blastocyst stage. The selective hyperacetylation of Hoxb promoters in response to a histone deacetylase inhibitor, suggests that Hox genes have a higher turnover of histone acetates than other genes in the preimplantation embryo. To explain the heritability, through mitosis, of VPA-induced changes in histone modification at Hoxb promoters, we describe how an epigenetic feed-forward loop, based on cross-talk between H3 acetylation and H3K4 methylation, might generate a persistently increased steady-state level of histone acetylation in response to a transient signal

Pol II–Expressed shRNA Knocks Down Sod2 Gene Expression and Causes Phenotypes of the Gene Knockout in Mice

RNA interference (RNAi) has been used increasingly for reverse genetics in invertebrates and mammalian cells, and has the potential to become an alternative to gene knockout technology in mammals. Thus far, only RNA polymerase III (Pol III)–expressed short hairpin RNA (shRNA) has been used to make shRNA-expressing transgenic mice. However, widespread knockdown and induction of phenotypes of gene knockout in postnatal mice have not been demonstrated. Previous studies have shown that Pol II synthesizes micro RNAs (miRNAs)—the endogenous shRNAs that carry out gene silencing function. To achieve efficient gene knockdown in mammals and to generate phenotypes of gene knockout, we designed a construct in which a Pol II (ubiquitin C) promoter drove the expression of an shRNA with a structure that mimics human miRNA miR-30a. Two transgenic lines showed widespread and sustained shRNA expression, and efficient knockdown of the target gene Sod2. These mice were viable but with phenotypes of SOD2 deficiency. Bigenic heterozygous mice generated by crossing these two lines showed nearly undetectable target gene expression and phenotypes consistent with the target gene knockout, including slow growth, fatty liver, dilated cardiomyopathy, and premature death. This approach opens the door of RNAi to a wide array of well-established Pol II transgenic strategies and offers a technically simpler, cheaper, and quicker alternative to gene knockout by homologous recombination for reverse genetics in mice and other mammalian species

Genetic Correction of Huntington's Disease Phenotypes in Induced Pluripotent Stem Cells

SummaryHuntington's disease (HD) is caused by a CAG expansion in the huntingtin gene. Expansion of the polyglutamine tract in the huntingtin protein results in massive cell death in the striatum of HD patients. We report that human induced pluripotent stem cells (iPSCs) derived from HD patient fibroblasts can be corrected by the replacement of the expanded CAG repeat with a normal repeat using homologous recombination, and that the correction persists in iPSC differentiation into DARPP-32-positive neurons in vitro and in vivo. Further, correction of the HD-iPSCs normalized pathogenic HD signaling pathways (cadherin, TGF-β, BDNF, and caspase activation) and reversed disease phenotypes such as susceptibility to cell death and altered mitochondrial bioenergetics in neural stem cells. The ability to make patient-specific, genetically corrected iPSCs from HD patients will provide relevant disease models in identical genetic backgrounds and is a critical step for the eventual use of these cells in cell replacement therapy

Expression of protocadherin gamma in skeletal muscle tissue is associated with age and muscle weakness

Background: The skeletal muscle system plays an important role in the independence of older adults. In this study we examine differences in the skeletal muscle transcriptome between healthy young and older subjects and (pre-)frail older adults. Additionally, we examine the effect of resistance-type exercise training on the muscle transcriptome in healthy older subjects and (pre-)frail older adults. Methods: Baseline transcriptome profiles were measured in muscle biopsies collected from 53 young, 73 healthy older subjects and 61 frail older subjects. Follow-up samples from these frail older subjects (31 samples) and healthy older subjects (41 samples) were collected after 6 months of progressive resistance-type exercise training. Frail older subjects trained twice per week and the healthy older subjects trained three times per week. Results: At baseline genes related to mitochondrial function and energy metabolism were differentially expressed between older and young subjects, as well as between healthy and frail older subjects. 307 genes were differentially expressed after training in both groups. Training affected expression levels of genes related to extracellular matrix, glucose metabolism and vascularization. Expression of genes that were modulated by exercise training were indicative of muscle strength at baseline. Genes that strongly correlated with strength belonged to the protocadherin gamma gene cluster (r=-0.73). Conclusions: Our data suggest significant remaining plasticity of ageing skeletal muscle to adapt to resistance-type exercise training. Some age-related changes in skeletal muscle gene expression appear to be partially reversed by prolonged resistance-type exercise training. The protocadherin gamma gene cluster may be related to muscle denervation and re-innervation in ageing muscle

Supplementary Material: PREMATURE EXPRESSION OF AN AGE-ASSOCIATED MUSCLE SENESCENCE PATHWAY IN HIV INFECTION

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Nitric oxide availability is increased in contracting skeletal muscle from aged mice, but does not differentially decrease muscle superoxide

Reactive oxygen and nitrogen species have been implicated in the loss of skeletal muscle mass and function that occurs during aging. Nitric oxide (NO) and superoxide are generated by skeletal muscle and where these are generated in proximity their chemical reaction to form peroxynitrite can compete with the superoxide dismutation to hydrogen peroxide. Changes in NO availability may therefore theoretically modify superoxide and peroxynitrite activities in tissues, but published data are contradictory regarding aging effects on muscle NO availability. We hypothesised that an age-related increase in NO generation might increase peroxynitrite generation in muscles from old mice, leading to an increased nitration of muscle proteins and decreased superoxide availability. This was examined using fluorescent probes and an isolated fiber preparation to examine NO content and superoxide in the cytosol and mitochondria of muscle fibers from adult and old mice both at rest and following contractile activity. We also examined the 3-nitrotyrosine (3-NT) and peroxiredoxin 5 (Prx5) content of muscles from mice as markers of peroxynitrite activity. Data indicate that a substantial age-related increase in NO levels occurred in muscle fibers during contractile activity and this was associated with an increase in muscle eNOS. Muscle proteins from old mice also showed an increased 3-NT content. Inhibition of NOS indicated that NO decreased superoxide bioavailability in muscle mitochondria, although this effect was not age related. Thus increased NO in muscles of old mice was associated with an increased 3-NT content that may potentially contribute to age-related degenerative changes in skeletal muscle