Disentangling the effect of dietary restriction on mitochondrial function using recombinant inbred mice

Dietary restriction (DR) extends lifespan and healthspan in many species, but precisely how it elicits its beneficial effects is unclear. We investigated the impact of DR on mitochondrial function within liver and skeletal muscle of female ILSXISS mice that exhibit strain-specific variation in lifespan under 40% DR. Strains TejJ89 (lifespan increased under DR), TejJ48 (lifespan unaffected by DR) and TejJ114 (lifespan decreased under DR) were studied following 10 months of 40% DR (13 months of age). Oxygen consumption rates (OCR) within isolated liver mitochondria were unaffected by DR in TejJ89 and TejJ48, but decreased by DR in TejJ114. DR had no effect on hepatic protein levels of PGC-1a, TFAM, and OXPHOS complexes IV. Mitonuclear protein imbalance (nDNA:mtDNA ratio) was unaffected by DR, but HSP90 protein levels were reduced in TejJ114 under DR. Surprisingly hepatic mitochondrial hydrogen peroxide (H2O2) production was elevated by DR in TejJ89, with total superoxide dismutase activity and protein carbonyls increased by DR in both TejJ89 and TejJ114. In skeletal muscle, DR had no effect on mitochondrial OCR, OXPHOS complexes or mitonuclear protein imbalance, but H2O2 production was decreased in TejJ114 and nuclear PGC-1a increased in TejJ89 under DR. Our findings indicate that hepatic mitochondrial dysfunction associated with reduced lifespan of TejJ114 mice under 40% DR, but similar dysfunction was not apparent in skeletal muscle mitochondria. We highlight tissue-specific differences in the mitochondrial response in ILSXISS mice to DR, and underline the importance and challenges of exploiting genetic heterogeneity to help understand mechanisms of ageing

Rescue of the MERTK phagocytic defect in a human iPSC disease model using translational read-through inducing drugs.

Inherited retinal dystrophies are an important cause of blindness, for which currently there are no effective treatments. In order to study this heterogeneous group of diseases, adequate disease models are required in order to better understand pathology and to test potential therapies. Induced pluripotent stem cells offer a new way to recapitulate patient specific diseases in vitro, providing an almost limitless amount of material to study. We used fibroblast-derived induced pluripotent stem cells to generate retinal pigment epithelium (RPE) from an individual suffering from retinitis pigmentosa associated with biallelic variants in MERTK. MERTK has an essential role in phagocytosis, one of the major functions of the RPE. The MERTK deficiency in this individual results from a nonsense variant and so the MERTK-RPE cells were subsequently treated with two translational readthrough inducing drugs (G418 & PTC124) to investigate potential restoration of expression of the affected gene and production of a full-length protein. The data show that PTC124 was able to reinstate phagocytosis of labeled photoreceptor outer segments at a reduced, but significant level. These findings represent a confirmation of the usefulness of iPSC derived disease specific models in investigating the pathogenesis and screening potential treatments for these rare blinding disorders

Exercise alters the molecular pathways of insulin signaling and lipid handling in maternal tissues of obese pregnant mice.

Obesity during gestation adversely affects maternal and infant health both during pregnancy and for long afterwards. However, recent work suggests that a period of maternal exercise during pregnancy can improve metabolic health of the obese mother and her offspring. This study aimed to identify the physiological and molecular impact of exercise on the obese mother during pregnancy that may lead to improved metabolic outcomes. To achieve this, a 20-min treadmill exercise intervention was performed 5 days a week in diet-induced obese female mice from 1 week before and up to day 17 of pregnancy. Biometric, biochemical and molecular analyses of maternal tissues and/or plasma were performed on day 19 of pregnancy. We found exercise prevented some of the adverse changes in insulin signaling and lipid metabolic pathways seen in the liver, skeletal muscle and white adipose tissue of sedentary-obese pregnant dams (p110β, p110α, AKT, SREBP). Exercise also induced changes in the insulin and lipid signaling pathways in obese dams that were different from those observed in control and sedentary-obese dams. The changes induced by obesity and exercise were tissue-specific and related to alterations in tissue lipid, protein and glycogen content and plasma insulin, leptin and triglyceride concentrations. We conclude that the beneficial effects of exercise on metabolic outcomes in obese mothers may be related to specific molecular signatures in metabolically active maternal tissues during pregnancy. These findings highlight potential metabolic targets for therapeutic intervention and the importance of lifestyle in reducing the burden of the current obesity epidemic on healthcare systems

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform

Investigation of PTC124-mediated translational readthrough in a retinal organoid model of AIPL1-associated Leber congenital amaurosis

Leber congenital amaurosis type 4 (LCA4), caused by AIPL1 mutations, is characterized by severe sight impairment in infancy and rapidly progressing degeneration of photoreceptor cells. We generated retinal organoids using induced pluripotent stem cells (iPSCs) from renal epithelial cells obtained from four children with AIPL1 nonsense mutations. iPSC-derived photoreceptors exhibited the molecular hallmarks of LCA4, including undetectable AIPL1 and rod cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE6) compared with control or CRISPR-corrected organoids. Increased levels of cGMP were detected. The translational readthrough-inducing drug (TRID) PTC124 was investigated as a potential therapeutic agent. LCA4 retinal organoids exhibited low levels of rescue of full-length AIPL1. However, this was insufficient to fully restore PDE6 in photoreceptors and reduce cGMP. LCA4 retinal organoids are a valuable platform for in vitro investigation of novel therapeutic agents

Tortoise or hare? Supporting the chronotype preference of employees with fluctuating chronic illness symptoms

Objective: Our aim is to understand how to facilitate the job retention of employees with chronic illness. We focus on multiple sclerosis (MS) as a criterion chronic illness. Design: An opportunity sample of 20 individuals of working age (13 female; 7 male) were recruited who had been in paid employment for over 28 months with a concurrent diagnosis of MS. Participants took part in one of three focus groups with a topic guide comprising eight keywords: work, coping, performance, support, future, expectations, and sharing symptoms. Data were analysed using dialogical analysis. Main outcome measures: As a qualitative study, no outcome measure was used. However, the specific focus of interest was to search for differential patterns of ‘timespace’ – chronotope - that people with chronic illness utilize to manage their condition in the workplace. Results: Participants oriented to two distinct chronotope types: unsustainable epic (characterized by condensed time) and temporary idyll (characterized by condensed space). Perceived managerial discretion was identified as possibly influencing participants’ chronotope preference. Conclusion: Identifying chronotope preference has practical implications for health psychologists and related professionals who provide and advise on support to facilitate people with chronic illness to thrive in the workplace

I had no other choice but to catch it too : the roles of family history and experiences with diabetes in illness representations.

BACKGROUND: A family history of diabetes and family members\u27 experiences with diabetes may influence individuals\u27 beliefs and expectations about their own diabetes. No qualitative studies have explored the relationship between family history and experiences and individuals\u27 diabetes illness representations. METHODS: Secondary data analysis of 89 exploratory, semi-structured interviews with adults with type 1 or type 2 diabetes seeking care in an urban health system. Participants had a recent diabetes-related ED visit/hospitalization or hemoglobin A1c \u3e 7.5%. Interviews were conducted until thematic saturation was achieved. Demographic data were collected via self-report and electronic medical record review. Interviews were audio-recorded, transcribed, and coded using a conventional content analysis approach. References to family history and family members\u27 experiences with diabetes were analyzed using selected domains of Leventhal\u27s Common Sense Model of Self-Regulation. RESULTS: Participants cited both genetic and behavioral family history as a major cause of their diabetes. Stories of relatives\u27 diabetes complications and death figured prominently in their discussion of consequences; however, participants felt controllability over diabetes through diet, physical activity, and other self-care behaviors. CONCLUSIONS: Findings supported an important role of family diabetes history and experience in development of diabetes illness representations. Further research is needed to expand our understanding of the relationships between these perceptions, self-management behaviors, and outcomes. Family practice providers, diabetes educators and other team members should consider expanding assessment of current family structure and support to also include an exploration of family history with diabetes, including which family members had diabetes, their self-care behaviors, and their outcomes, and how this history fits into the patient\u27s illness representations

Seeing the way: visual sociology and the distance runner's perspective

Employing visual and autoethnographic data from a two‐year research project on distance runners, this article seeks to examine the activity of seeing in relation to the activity of distance running. One of its methodological aims is to develop the linkage between visual and autoethnographic data in combining an observation‐based narrative and sociological analysis with photographs. This combination aims to convey to the reader not only some of the specific subcultural knowledge and particular ways of seeing, but also something of the runner's embodied feelings and experience of momentum en route. Via the combination of narrative and photographs we seek a more effective way of communicating just how distance runners see and experience their training terrain. The importance of subjecting mundane everyday practices to detailed sociological analysis has been highlighted by many sociologists, including those of an ethnomethodological perspective. Indeed, without the competence of social actors in accomplishing these mundane, routine understandings and practices, it is argued, there would in fact be no social order

Protective Effects of Human iPS-Derived Retinal Pigment Epithelium Cell Transplantation in the Retinal Dystrophic Rat

Transformation of somatic cells with a set of embryonic transcription factors produces cells with the pluripotent properties of embryonic stem cells (ESCs). These induced pluripotent stem (iPS) cells have the potential to differentiate into any cell type, making them a potential source from which to produce cells as a therapeutic platform for the treatment of a wide range of diseases. In many forms of human retinal disease, including age-related macular degeneration (AMD), the underlying pathogenesis resides within the support cells of the retina, the retinal pigment epithelium (RPE). As a monolayer of cells critical to photoreceptor function and survival, the RPE is an ideally accessible target for cellular therapy. Here we report the differentiation of human iPS cells into RPE. We found that differentiated iPS-RPE cells were morphologically similar to, and expressed numerous markers of developing and mature RPE cells. iPS-RPE are capable of phagocytosing photoreceptor material, in vitro and in vivo following transplantation into the Royal College of Surgeons (RCS) dystrophic rat. Our results demonstrate that iPS cells can be differentiated into functional iPS-RPE and that transplantation of these cells can facilitate the short-term maintenance of photoreceptors through phagocytosis of photoreceptor outer segments. Long-term visual function is maintained in this model of retinal disease even though the xenografted cells are eventually lost, suggesting a secondary protective host cellular response. These findings have identified an alternative source of replacement tissue for use in human retinal cellular therapies, and provide a new in vitro cellular model system in which to study RPE diseases affecting human patients