Molecular Characterisation of the Interaction of Microbes with the Insulin Pathway

Full version unavailable due to 3rd party copyright restrictions.Exposure to microorganisms is considered an environmental factor which can contribute to diabetes mellitus via cytotoxicity or autoimmune responses against pancreatic cells. Firstly, the effects on rat insulinoma pancreatic β-cell line of secondary metabolites pyrrolnitrin (Burkholderia spp), phenazine compounds (Pseudomonas aeruginosa and Burkholderia spp) were investigated. Both compounds separately showed significant cytotoxicity after 24 h and at concentrations of 10 & 100 ng/ml potentiated insulin gene transcription, Ca2+ content and glucose-stimulated insulin secretion (GSIS). Furthermore, the outward membrane current was inhibited by phenazine (100 ng/ml) or pyrrolnitrin (10 or 100 ng/ml). Secondly, the capacity of 45 microbial species to bind insulin was screened in order to assess how common insulin binding was amongst microorganisms Burkholderia multivorans, B. cenocepacia and Aeromonas salmonicida bound insulin. A genomic library of B. multivorans was constructed in λ Zap Express and screened successfully for insulin binding recombinants. Recombinant phagemids p1 & p2 were excised, p1, encoded an insulin binding protein (IBP1 30 kDa) with homology to the iron complex siderophore receptor. For p2, two IBPs were detected at 20 & 30 kDa (IBP2 & IBP3), representing an intracellular and outer membrane peptide transporter. Comparison of IBP1 and human insulin receptor (HIR) produced 6 linear epitopes, and for IBP2 & IBP3 produced 3 epitopes. Thirdly, glutamic acid decarboxylase GAD65 is a major pancreatic autoantigen contributing to autoimmune diabetes. To assess the likelihood that microorganisms possess epitopes that mimic regions on GAD, 45 microbial species were tested for homology. This was facilitated by purifying recombinant GAD protein which was used to produce GAD antiserum. Four E. coli cross-reacting proteins were identified using mass spectrometry, outer-membrane protein A, formate dehydrogenase, superoxide dismutase and DNA starvation protein. Epitopes occurred at the C-terminal region of GAD65 (residues 419–565), a region previously reported to be targeted by autoantibodies. This study suggests that pyrrolnitrin and phenazine are cytotoxic to pancreatic β-cells and B. multivorans IBPs linear epitopes may be diabetogenic, particularly in patients with cystic fibrosis related diabetes (CFRD) who suffer a long term infections with Pseudomonas and Burkholderia species. Furthermore, microbial GAD epitopes could potentially induce an autoimmune response leading to diabetes

Insulin acutely improves mitochondrial function of rat and human skeletal muscle by increasing coupling efficiency of oxidative phosphorylation.

Insulin is essential for the regulation of fuel metabolism and triggers the uptake of glucose by skeletal muscle. The imported glucose is either stored or broken down, as insulin stimulates glycogenesis and ATP synthesis. The mechanism by which ATP production is increased is incompletely understood at present and, generally, relatively little functional information is available on the effect of insulin on mitochondrial function. In this paper we have exploited extracellular flux technology to investigate insulin effects on the bioenergetics of rat (L6) and human skeletal muscle myoblasts and myotubes. We demonstrate that a 20-min insulin exposure significantly increases (i) the cell respiratory control ratio, (ii) the coupling efficiency of oxidative phosphorylation, and (iii) the glucose sensitivity of anaerobic glycolysis. The improvement of mitochondrial function is explained by an insulin-induced immediate decrease of mitochondrial proton leak. Palmitate exposure annuls the beneficial mitochondrial effects of insulin. Our data improve the mechanistic understanding of insulin-stimulated ATP synthesis, and reveal a hitherto undisclosed insulin sensitivity of cellular bioenergetics that suggests a novel way of detecting insulin responsiveness of cells

Development and use of Bacteroides 16S rRNA polymerase chain reaction assay for source tracking dog faecal pollution in bathing waters

Faecal pollution on bathing beaches poses a potential threat to human health and as a result may also negatively affect the local economy. In instances where the source of such pollution is not obvious, it may be necessary to track such sources using a host-specific genetic markers technique. Bacteroides species are potential indicators for source tracking of faecal pollution in bathing waters. This study designed specific primer sets to amplify sections of the 16S rRNA gene unique to Bacteroides from domestic dogs and used quantitative PCR (qPCR) to quantify such genetic markers in environmental samples. The sensitivity and specificity of the primer sets was determined; they were specific in silico against known dog Bacteroides sequences and in vitro against Bacteroides sequences originating from human and livestock faeces. Dog faecal Bacteroides contamination was then detected in sea water during the bathing season at a local beach where dogs are banned during the summer months, in spite of the fact that these waters had met EU directive standards based on the culture-based enumeration of faecal indicator bacteria. Quantitative PCR was used to determine the limit of detection (LOD) of the dog Bacteroides genetic markers in these water samples. The copy number of dog Bacteroides genetic markers in the water was low and the LOD of those markers was 4 copies per reaction. The use of these dog primers has the potential to supply important additional information when source tracking faecal pollution at bathing beaches and maintaining water quality

Green revolution in Sub-Saharan Africa: Implications of imposed innovation for the wellbeing of rural smallholders

Green Revolution policies are again being pursued to drive agricultural growth and reduce poverty in Sub-Saharan Africa. However conditions have changed since the well-documented successes of the 1960s and 1970s benefited smallholders in southern Asia and beyond. We argue that under contemporary constraints the mechanisms for achieving improvements in the lives of smallholder farmers through such policies are unclear and that both policy rationale and means of governing agricultural innovation are crucial for pro-poor impacts. To critically analyze Rwanda’s Green Revolution policies and impacts from a local perspective, a mixed methods, multidimensional wellbeing approach is applied in rural areas in mountainous western Rwanda. Here Malthusian policy framing has been used to justify imposed rather than ‘‘induced innovation”. The policies involve a substantial transformation for rural farmers from a traditional polyculture system supporting subsistence and local trade to the adoption of modern seed varieties, inputs, and credit in order to specialize in marketable crops and achieve increased production and income. Although policies have been deemed successful in raising yields and conventionally measured poverty rates have fallen over the same period, such trends were found to be quite incongruous with local experiences. Disaggregated results reveal that only a relatively wealthy minority were able to adhere to the enforced modernization and policies appear to be exacerbating landlessness and inequality for poorer rural inhabitants. Negative impacts were evident for the majority of households as subsistence practices were disrupted, poverty exacerbated, local systems of knowledge, trade, and labor were impaired, and land tenure security and autonomy were curtailed. In order to mitigate the effects we recommend that inventive pro-poor forms of tenure and cooperation (none of which preclude improvements to input availability, market linkages, and infrastructure) may provide positive outcomes for rural people, and importantly in Rwanda, for those who have become landless in recent years. We conclude that policies promoting a Green Revolution in Sub-Saharan Africa should not all be considered to be pro-poor or even to be of a similar type, but rather should be the subject of rigorous impact assessment. Such assessment should be based not only on consistent, objective indicators but pay attention to localized impacts on land tenure, agricultural practices, and the wellbeing of socially differentiated people

Statistics versus livelihoods: questioning Rwanda’s pathway out of poverty

Recent statistics indicate that poverty in Rwanda decreased impressively between 2006 and 2014. This seems to confirm Rwanda’s developmental progress. This article however argues for a more cautious interpretation of household survey data. The authors contrast macro-level statistical analysis with in-depth field research on livelihood conditions. Macro-economic numbers provide interesting information, however differentiated evidence is required to understand how poverty ‘works’ in everyday life. On the basis of the Rwandan case study, the authors conclude that because of the high political stakes of data collection and analysis, and given that relations of power influence the production of knowledge on poverty, cross-checking is crucial

Local anesthetics induce autophagy in young permanent tooth pulp cells

Pulp cells are essential for tooth development, and dentin repair and regeneration. In addition these cells have been identified as an important stem cell source. Local anesthetics are widely used in dental clinics, as well as the other clinical disciplines and have been suggested to interfere with human permanent tooth development and induce tooth agenesis through unknown mechanisms. Using pig model and human young permanent tooth pulp cells, our research has identified that the local anesthetics commonly used in clinics can affect cell proliferation. Molecular pathway profiling suggested that LC3II is one of the earliest molecules induced by the agents and p62 is the only common downstream target identified for all the drugs tested. The effect of the drugs could be partially recovered by V-ATPase inhibitor only if early intervention is performed. Our results provide novel evidence that local anesthetics could affect tooth cell growth that potentially can have impacts on tooth development