Dagmara McGuinness

Socio-economic status is associated with epigenetic differences in the pSoBid cohort

BACKGROUND Epigenetic programming and epigenetic mechanisms driven by environmental factors are thought to play an important role in human health and ageing. Global DNA methylation has been postulated as an epigenetic marker for epidemiological studies as it is reflective of changes in gene expression linked to disease. How epigenetic mechanisms are affected by psychological, sociological and biological determinants of health still remains unclear. The aim of this study was to investigate the relationship between socio-economic and lifestyle factors and epigenetic status, as measured by global DNA methylation content, in the pSoBid cohort, which is characterized by an extreme socio-economic and health gradient. METHODS DNA was extracted from peripheral blood leukocytes using the Maxwell® 16 System and Maxwell® 16 Blood DNA Purification kit (Promega, UK). Global DNA methylation was assessed using Methylamp™ Global DNA Methylation Quantification Ultra kit (Epigentek, USA). Associations between global DNA methylation and socio-economic and lifestyle factors were investigated in linear regression models. RESULTS Global DNA hypomethylation was observed in the most socio-economically deprived subjects. Job status demonstrated a similar relationship, with manual workers having 24% lower DNA methylation content than non-manual. Additionally, associations were found between global DNA methylation content and biomarkers of cardiovascular disease (CVD) and inflammation, including fibrinogen and interleukin-6 (IL-6), after adjustment for socio-economic factors. CONCLUSIONS This study has indicated an association between epigenetic status and socio-economic status (SES). This relationship has direct implications for population health and is reflected in further associations between global DNA methylation content and emerging biomarkers of CVD.

Accelerated biological ageing in HIV-infected individuals in South Africa: a case–control study

Objectives:Little is known about the impact of HIV infection on biological ageing in sub-Saharan Africa. The study aimed to assess biological ageing in South African HIV-infected adults and HIV-seronegative individuals using two validated biomarkers, telomere length and CDKN2A expression (a mediator of cellular senescence). Design:A case–control study. Methods:Two hundred and thirty-six HIV-infected adults aged at least 30 years and 250 age and sex frequency matched HIV-seronegative individuals were recruited from clinics in township communities in Cape Town. Biological ageing was evaluated by measurement of telomere length and CDKN2A expression in peripheral blood leukocytes. Results:The median ages of the HIV-infected and HIV-seronegative participants were 39 and 40 years, respectively. Among HIV-infected participants, 87.1% were receiving antiretroviral therapy (ART), their median CD4+ cell count was 468 cells/&mgr;l and 84.3% had undetectable viral load. Both biomarkers were validated against chronological age in HIV-seronegative individuals. Telomere length was significantly shorter in HIV-infected individuals than in HIV-seronegative individuals (mean relative T/S ratio ±SE:0.91 ± 0.007 vs. 1.07 ± 0.008, P < 0.0001). CD2NKA expression was higher in HIV-infected participants than in HIV-seronegative individuals (mean expression: 0.45 ± 0.02 vs. 0.36 ± 0.03, P = 0.003). Socioeconomic factors were not associated with biological ageing in HIV-infected participants. However, in participants on ART with undetectable viral load, biomarker levels indicated greater biological ageing in those with lower current CD4+ cell counts. Conclusion:Telomere length and CDKN2A expression were both consistent with increased biological ageing in HIV-infected individuals. Prospective studies of the impact of HIV on biological ageing in sub-Saharan Africa are warranted.

Chip-Based Dynamic Real-Time Quantification of Drug-Induced Cytotoxicity in Human Tumor Cells

Cell cytotoxicity tests are among the most common bioassays using flow cytometry and fluorescence imaging analysis. The permeability of plasma membranes to charged fluorescent probes serves, in these assays, as a marker distinguishing live from dead cells. Since it is generally assumed that probes, such as propidium iodide (PI) or 7-amino-actinomycin D (7-AAD), are themselves cytotoxic, they are currently generally used only as the end-point markers of assays for live versus dead cells. In the current study, we provide novel insights into potential applications of these classical plasma membrane integrity markers in the dynamic tracking of drug-induced cytotoxicity. We show that treatment of a number of different human tumor cell lines in cultures for up to 72 h with the PI, 7-AAD, SYTOX Green (SY-G), SYTOX Red (SY-R), TO-PRO, and YO-PRO had no effect on cell viability assessed by the integrity of plasma membrane, cell cycle progression, and rate of proliferation. We subsequently explore the potential of dynamic labeling with these markers in real-time analysis, by comparing results from both conventional cytometry and microfluidic chips. Considering the simplicity of the staining protocols and their low cost combined with the potential for real-time data collection, we show how that real-time fluorescent imaging and Lab-on-a-Chip platforms have the potential to be used for automated drug screening routines.

Biological implications of polymeric microdevices for live cell assays.

Lab-on-a-chip technologies have the potential to deliver significant technological advances in modern biomedicine, through the ability to provide appropriate low-cost microenvironments for screening cells. However, to date, few studies have investigated the suitability of poly(dimethylsiloxane) (PDMS) for live cell culture. Here, we describe an inexpensive method for production of reusable, optical-grade PDMS microculture chips which provide a static and self-contained microwell system analogous to conventional polystyrene multiwell plates. We use these structures to probe the effects of PDMS upon live cell culture bioassays, using time-lapse fluorescence imaging to explore the toxicity of the substrate. We use three model systems to explore the efficacy of the microstructured devices: (i) live cell culture, (ii) adenoviral gene delivery to mammalian cells, and (iii) gravity enforced formation of multicellular tumor spheroids (MCTS). Results show that PDMS is nontoxic to cells, as their viability and growth characteristic in PDMS-based platforms is comparable to that of their polystyrene counterparts.

Pre-transplant CDKN2A expression in kidney biopsies predicts renal function and is a future component of donor scoring criteria.

CDKN2A is a proven and validated biomarker of ageing which acts as an off switch for cell proliferation. We have demonstrated previously that CDKN2A is the most robust and the strongest pre-transplant predictor of post- transplant serum creatinine when compared to “Gold Standard” clinical factors, such as cold ischaemic time and donor chronological age. This report shows that CDKN2A is better than telomere length, the most celebrated biomarker of ageing, as a predictor of post-transplant renal function. It also shows that CDKN2A is as strong a determinant of post-transplant organ function when compared to extended criteria (ECD) kidneys. A multivariate analysis model was able to predict up to 27.1% of eGFR at one year post-transplant (p = 0.008). Significantly, CDKN2A was also able to strongly predict delayed graft function. A pre-transplant donor risk classification system based on CDKN2A and ECD criteria is shown to be feasible and commendable for implementation in the near future.

Sirtuins, Bioageing, and Cancer

The Sirtuins are a family of orthologues of yeast Sir2 found in a wide range of organisms from bacteria to man. They display a high degree of conservation between species, in both sequence and function, indicative of their key biochemical roles. Sirtuins are heavily implicated in cell cycle, cell division, transcription regulation, and metabolism, which places the various family members at critical junctures in cellular metabolism. Typically, Sirtuins have been implicated in the preservation of genomic stability and in the prolongation of lifespan though many of their target interactions remain unknown. Sirtuins play key roles in tumourigenesis, as some have tumour-suppressor functions and others influence tumours through their control of the metabolic state of the cell. Their links to ageing have also highlighted involvement in various age-related and degenerative diseases. Here, we discuss the current understanding of the role of Sirtuins in age-related diseases while taking a closer look at their roles and functions in maintaining genomic stability and their influence on telomerase and telomere function.

Identification of Molecular Markers of Delayed Graft Function Based on the Regulation of Biological Ageing

Introduction Delayed graft function is a prevalent clinical problem in renal transplantation for which there is no objective system to predict occurrence in advance. It can result in a significant increase in the necessity for hospitalisation post-transplant and is a significant risk factor for other post-transplant complications. Methodology The importance of microRNAs (miRNAs), a specific subclass of small RNA, have been clearly demonstrated to influence many pathways in health and disease. To investigate the influence of miRNAs on renal allograft performance post-transplant, the expression of a panel of miRNAs in pre-transplant renal biopsies was measured using qPCR. Expression was then related to clinical parameters and outcomes in two independent renal transplant cohorts. Results Here we demonstrate, in two independent cohorts of pre-implantation human renal allograft biopsies, that a novel pre-transplant renal performance scoring system (GRPSS), can determine the occurrence of DGF with a high sensitivity (>90%) and specificity (>60%) for donor allografts pre-transplant, using just three senescence associated microRNAs combined with donor age and type of organ donation. Conclusion These results demonstrate a relationship between pre-transplant microRNA expression levels, cellular biological ageing pathways and clinical outcomes for renal transplantation. They provide for a simple, rapid quantitative molecular pre-transplant assay to determine post-transplant allograft function and scope for future intervention. Furthermore, these results demonstrate the involvement of senescence pathways in ischaemic injury during the organ transplantation process and an indication of accelerated bio-ageing as a consequence of both warm and cold ischaemia.

Identification of androgen receptor phosphorylation in the primate ovary in vivo

The androgen receptor (AR) is a member of the nuclear receptor superfamily, and is important for both male and female reproductive health. The receptor is a target for a number of post-translational modifications including phosphorylation, which has been intensively studied in vitro. However, little is known about the phosphorylation status of the receptor in target tissues in vivo. The common marmoset is a useful model for studying human reproductive functions, and comparison of the AR primary sequence from this primate shows high conservation of serines known to be phosphorylated in the human receptor and corresponding flanking amino acids. We have used a panel of phosphospecific antibodies to study AR phosphorylation in the marmoset ovary throughout the follicular phase and after treatment with GNRH antagonist or testosterone propionate. In normal follicular phase ovaries, total AR (both phosphorylated and non-phosphorylated forms) immunopositive staining was observed in several cell types including granulosa cells of developing follicles, theca cells and endothelial cells lining blood vessels. Receptor phosphorylation at serines 81, 308, and 650 was detected primarily in the granulosa cells of developing follicles, surface epithelium, and vessel endothelial cells. Testosterone treatment lead to a modest increase in AR staining in all stages of follicle studied, while GNRH antagonist had no effect. Neither treatment significantly altered the pattern of phosphorylation compared to the control group. These results demonstrate that phosphorylation of the AR occurs, at a subset of serine residues, in a reproductive target tissue in vivo, which appears refractory to hormonal manipulations.

The role of epigenetics in renal ageing

An ability to separate natural ageing processes from processes specific to morbidities is required to understand the heterogeneity of age-related organ dysfunction. Mechanistic insight into how epigenetic factors regulate ageing throughout the life course, linked to a decline in renal function with ageing, is already proving to be of value in the analyses of clinical and epidemiological cohorts. Noncoding RNAs provide epigenetic regulatory circuits within the kidney, which reciprocally interact with DNA methylation processes, histone modification and chromatin. These interactions have been demonstrated to reflect the biological age and function of renal allografts. Epigenetic factors control gene expression and activity in response to environmental perturbations. They also have roles in highly conserved signalling pathways that modulate ageing, including the mTOR and insulin/insulin-like growth factor signalling pathways, and regulation of sirtuin activity. Nutrition, the gut microbiota, inflammation and environmental factors, including psychosocial and lifestyle stresses, provide potential mechanistic links between the epigenetic landscape of ageing and renal dysfunction. Approaches to modify the renal epigenome via nutritional intervention, targeting the methylome or targeting chromatin seem eminently feasible, although caution is merited owing to the potential for intergenerational and transgenerational effects.

Accelerated ageing and renal dysfunction links lower socioeconomic status and dietary phosphate intake

Background We have sought to explore the impact of dietary Pi intake on human age related health in the pSoBid cohort (n=666) to explain the disparity between health and deprivation status in this cohort. As hyperphosphataemia is a driver of accelerated ageing in rodent models of progeria we tested whether variation in Pi levels in man associate with measures of biological ageing and health. Results We observed significant relationships between serum Pi levels and markers of biological age (telomere length (p=0.040) and DNA methylation content (p=0.028), gender and chronological age (p=0.032). When analyses were adjusted for socio-economic status and nutritional factors, associations were observed between accelerated biological ageing (telomere length, genomic methylation content) and dietary derived Pi levels among the most deprived males, directly related to the frequency of red meat consumption. Conclusions Accelerated ageing is associated with high serum Pi levels and frequency of red meat consumption. Our data provide evidence for a mechanistic link between high intake of Pi and age-related morbidities tied to socio-economic status.