The sequence of the Saccharomyces cerevisiae RAD52 gene contains five potential translation start sites and protein-blot analysis typically detects multiple Rad52 species with different electrophoretic mobilities. Here we define the gene products encoded by RAD52 . We show that the multiple Rad52 protein species are due to promiscuous choice of start codons as well as post-translational modification. Specifically, Rad52 is phosphorylated both in a cell cycle-independent and in a cell cycle-dependent manner. Furthermore, phosphorylation is dependent on the presence of the Rad52 C terminus, but not dependent on its interaction with Rad51. We also show that the Rad52 protein can be translated from the last three start sites and expression from any one of them is sufficient for spontaneous recombination and the repair of gamma-ray-induced double-strand breaks.
Introduction & Objective: Early detection of type 2 diabetes mellitus (T2DM) increases the quality of life and the life expectancy of individuals with T2DM and reduces societal health care costs. The aim of the study is to evaluate the efficiency of targeted and free screening using at-home self-sampling A1c tests for early detection of T2DM. Methods: In October 2022 to June 2023, the Danish Diabetes Association mailed free capillary at-home self-sampling A1c tests to 8,000 randomly selected individuals aged 50 to 75 years, who had not had their A1c measured within the past two years. The Clinical Laboratory Information Register, including all laboratory measurements covering hospitals and GPs, was used to identify the population. Results: 3,040 individuals, 38%, returned a blood sample. This is in line with screening studies for other diseases in Denmark. The participation rate was 3.8 %-points higher for women than for men and approximately U-shaped across age for both sexes with the highest participation rate among the 50-year-olds. The age-adjusted share of participants with A1c of 42-47 mmol/mol was 11%, equivalent to the estimated share of individuals with unknown prediabetes in the Danish population aged 50-75 years. The age-adjusted share of the participants with A1c ≥48 mmol/mol was 1.2% for women and 2.5% for men. It is slightly lower than the estimated shares of individuals with unknown T2DM, 1.6% for women and 2.7% for men. Further, individuals between 50-59 years of age constituted 51% of the participants with A1c ≥48 mmol/mol compared to 40% of the population, when diagnosed by the Danish Health Care System (DHCS). Similarly, the median A1c was 52 mmol/mol among the participants with A1c ≥48 mmol/mol compared to a median A1c of 60 mmol/mol in the population, when diagnosed with T2DM by the DHCS. Conclusion: The pilot screening study for at-home self-sampling prove highly efficient in detecting individuals with A1c ≥48 mmol/mol earlier compared to individuals in the same age group diagnosed by the DHCS. Disclosure N. Cayuelas Mateu: Research Support; Novo Nordisk A/S, Bayer Inc., Boehringer-Ingelheim, Sanofi, AstraZeneca, Abbott, Abbott Diagnostics. P. Rossing: Other Relationship; AstraZeneca, Bayer Inc., Boehringer-Ingelheim, Gilead Sciences, Inc., Novo Nordisk, Eli Lilly and Company, Novartis AG, Abbott Diagnostics. K.P. Neergaard: None. T. Thybo: Research Support; Novo Nordisk A/S, Bayer Inc., Boehringer-Ingelheim, Sanofi, AstraZeneca, Abbott, Abbott Diagnostics.
Background: Lifestyle interventions have been shown to delay or prevent the onset of type 2 diabetes among high risk adults. A better understanding of the variability in physiological responses would support the matching of individuals with the best type of intervention in future prevention programmes, in order to optimize risk reduction. The purpose of this study was to determine if phenotypic characteristics at baseline or following a 12 weeks lifestyle intervention could explain the inter-individual variability in change in glucose tolerance in individuals with high risk for type 2 diabetes. Methods: In total, 285 subjects with normal glucose tolerance (NGT, FINDRISC score > 12), impaired fasting glucose (IFG) and impaired glucose tolerance (IGT) were recruited for a 12 weeks lifestyle intervention. Glucose tolerance, insulin sensitivity, anthropometric characteristics and aerobic fitness were measured. Variability of responses was examined by grouping participants by baseline glycemic status, by cluster analysis based on the change in glucose tolerance and by Principal Component Analysis (PCA). Results: In agreement with other studies, the mean response to the 12 weeks intervention was positive for the majority of parameters. Overall, 89% improved BMI, 80% waist circumference, and 81% body fat while only 64% improved fasting plasma glucose and 60% 2 h glucose. The impact of the intervention by glycaemic group did not show any phenotypic differences in response between NGT, IFG, and IGT. A hierarchical cluster analysis of change in glucose tolerance identified four sub-groups of "responders" (high and moderate) and "non-responders" (no response or deteriorated) but there were few differences in baseline clincal and physiological parameters or in response to the intervention to explain the overall variance. A further PCA analysis of 19 clinical and physiological univariables could explain less than half (48%) of total variability. Conclusion: We found that phenotypic characteristics from standard clinical and physiological parameters were not sufficient to account for the inter-individual variability in glucose tolerance following a 12 weeks lifestyle intervention in inidivuals at high risk for type 2 diabetes. Further work is required to identify biomarkers that complement phenotypic traits and better predict the response to glucose tolerance.
With the global escalation of type 2 diabetes and evidence consistently showing that its onset can be prevented or delayed by changing lifestyle behaviours, there is an urgent need to translate practical, affordable and acceptable interventions from the research setting into the real world. One such approach to lifestyle interventions might be the introduction of a programme in which the individual is provided with choice and facilitated to 'self-select' an exercise programme. Previous research has shown that this is likely to be less resource intensive, an essential requirement for success outside the controlled research environment, while at the same time promoting positive responses relating to adherence, competence and self-efficacy, essential attributes for long-term success. Through a two-group parallel-randomised controlled trial, this study aims to assess the clinical and psychological impact of the DEXLIFE 'self-selected' lifestyle modification programme in adults at risk of developing type 2 diabetes. A total of 360 subjects at risk of developing type 2 diabetes are randomly assigned in a 1:3 ratio to a control (n = 90) or intervention arm (n = 270). Randomization is stratified by age, sex and body mass index. The control arm receives general information on lifestyle and diabetes risk. The intervention group participate in a 12 week 'self-selected' supervised exercise training programme accompanied with dietary advice to improve food choices. Participants are given access to Dublin City University Sport (an on-campus gym) and asked to perform four exercise classes per week. Dublin City University Sport offers over 50 classes per week, many of which are medically supervised. If weight loss is indicated, reduction in total calorie intake by 600 kcal/day is advised. Common to all food plans is <10 % saturated fat intake, as well as a dietary fibre intake of >15 g/1000 kcal. Insulin sensitivity is the primary outcome measure. Secondary outcome measures include glucose function, fitness, body composition, anthropometrics, heart rate variability, lipid profiles, blood pressure, physical activity levels, dietary intake and quality of life. "Self-selected" lifestyle intervention has not previously been evaluated in type 2 diabetes prevention and if shown to be successful could be implemented in practice immediately. Current Controlled Trials: ISRCTN66987085 .
Natural products are an important source of drugs and other commercially interesting compounds, however their isolation and production is often difficult. Metabolic engineering, mainly in bacteria and yeast, has sought to circumvent some of the associated problems but also this approach is impeded by technical limitations. Here we describe a novel strategy for production of diverse natural products, comprising the expression of an unprecedented large number of biosynthetic genes in a heterologous host.As an example, genes from different sources, representing enzymes of a seven step flavonoid pathway, were individually cloned into yeast expression cassettes, which were then randomly combined on Yeast Artificial Chromosomes and used, in a single transformation of yeast, to create a variety of flavonoid producing pathways. Randomly picked clones were analysed, and approximately half of them showed production of the flavanone naringenin, and a third of them produced the flavonol kaempferol in various amounts. This reflected the assembly of 5-7 step multi-species pathways converting the yeast metabolites phenylalanine and/or tyrosine into flavonoids, normally only produced by plants. Other flavonoids were also produced that were either direct intermediates or derivatives thereof. Feeding natural and unnatural, halogenated precursors to these recombinant clones demonstrated the potential to further diversify the type of molecules that can be produced with this technology.The technology has many potential uses but is particularly suited for generating high numbers of structurally diverse compounds, some of which may not be amenable to chemical synthesis, thus greatly facilitating access to a huge chemical space in the search for new commercially interesting compounds.
Mitochondria are not only the major site for generation of reactive oxygen species, but also one of the main targets of oxidative damage. One of the major products of DNA oxidation, 8-oxodeoxyguanosine (8-oxodG), accumulates in mitochondrial DNA (mtDNA) at levels three times higher than in nuclear DNA. The main pathway for the repair of 8-oxodG is the base excision repair pathway initiated by oxoguanine DNA glycosylase (OGG1). We previously demonstrated that mammalian mitochondria from mice efficiently remove 8-oxodG from their genomes and isolated a protein from rat liver mitochondria with 8-oxoguanine (8-oxodG) DNA glycosylase/apurinic DNA lyase activity. In the present study, we demonstrated that the mitochondrial 8-oxodG DNA glycosylase/apurinic DNA lyase activity is the mitochondrial isoform of OGG1. Using mouse liver mitochondria isolated from ogg1(-/-) mice, we showed that the OGG1 gene encodes for the mitochondrial 8-oxodG glycosylase because these extracts have no incision activity toward an oligonucleotide containing a single 8-oxodG DNA base lesion. Consistent with an important role for the OGG1 protein in the removal of 8-oxodG from the mitochondrial genome, we found that mtDNA isolated from liver from OGG1-null mutant animals contained 20-fold more 8-oxodG than mtDNA from wild-type animals.
Introduction & Objective: No publicly accessible and highly updated database on diabetes treatment and outcomes has been available before in Denmark. The aim is to provide data to help improve the health of people living with diabetes. Methods: Diabetestal, developed by the Danish Diabetes Association, is a quarterly updated quality database of all people living with diabetes in Denmark. Diabetestal links individual health records from several registers that are internationally recognized for their high validity and quality, e.g. the Patient Register, The Clinical Laboratory Information Register and The Prescription Registry. Data includes lab measurements, drug purchases, accessories, diabetes-related complications, glycemic control separately for demography, diabetes duration and residence etc. Results: Diabetestal integrates clinical practice guidelines through an open, intuitive, chart-based, customizable user experience, enabling the analysis of geographical inequalities in treatment across time, e.g. therapeutic inertia in Danmark has improved while regional inequalities in treatment persist, as depict in the graph. Conclusion: Diabetestal may help health professionals highlight trends or problems and enable them to make informed decisions and thereby improve adherence to quality guidelines and help improve outcomes for people living with diabetes. www.diabetestal.nu Disclosure N. Cayuelas Mateu: Research Support; Novo Nordisk A/S, Bayer Inc., Boehringer-Ingelheim, Sanofi, AstraZeneca, Abbott, Abbott Diagnostics. T. Thybo: Research Support; Novo Nordisk A/S, Bayer Inc., Boehringer-Ingelheim, Sanofi, AstraZeneca, Abbott, Abbott Diagnostics.
The repair pathways involved in the removal of thymine glycol (TG) from DNA by human cell extracts have been examined. Closed circular DNA constructs containing a single TG at a defined site were used as substrates to determine the patch size generated afterin vitro repair by cell extracts. Restriction analysis of the repair incorporation in the vicinity of the lesion indicated that the majority of TG was repaired through the base excision repair (BER) pathways. Repair incorporation 5′ to the lesion, characteristic for the nucleotide excision repair pathway, was not found. More than 80% of the TG repair was accomplished by the single-nucleotide repair mechanism, and the remaining TGs were removed by the long patch BER pathway. We also analyzed the role of the xeroderma pigmentosum, complementation group G (XPG) protein in the excision step of BER. Cell extracts deficient in XPG protein had an average 25% reduction in TG incision. These data show that BER is the primary pathway for repair of TG in DNA and that XPG protein may be involved in repair of TG as an accessory factor.
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