Novel genes in the liver of diabetic Psammomys obesus

Type 2 diabetes mellitus is a metabolic disease characterised by defects in insulin secretion and insulin action and disturbances in carbohydrate, fat and protein metabolism. Hepatic insulin resistance contributes to hyperglycemia and also leads to disturbances in fat metabolism in type 2 diabetes. Psammomys obesus is a unique poly genie animal model of type 2 diabetes and obesity, ideally suited for studies examining physiological and genetic aspects of these diseases. To identify metabolic abnormalities potentially contributing to the obesity and diabetes phenotype in P. obesus, indirect calorimetry was used to characterise whole body energy expenditure and substrate utilisation. Lean-NGT, obese-IGT and obese-diabetic animals were examined in fed and fasted states and following 14 days of dietary energy restriction. Energy expenditure and fat oxidation were elevated in the obese-IGT and obese-diabetic groups in proportion to body weight. Glucose oxidation was not different between groups. Obese-diabetic P. obesus displayed elevated nocturnal blood glucose levels and fat oxidation. Following 14 days of dietary energy restriction, body weight was reduced and plasma insulin and blood glucose levels were normalised in all groups. Glucose oxidation was reduced and fat oxidation was increased. After 24 hours of fasting, plasma insulin and blood glucose levels were normalised in all groups. Energy expenditure and glucose oxidation were greatly reduced and fat oxidation was increased. Following either dietary energy restriction or fasting, energy expenditure, glucose oxidation and fat oxidation were not different between groups of P. obesus. Energy expenditure and whole body substrate utilisation in P. obesus was similar to that seen in humans. P. obesus responded normally to short term fasting and dietary energy restriction. Elevated nocturnal fat oxidation rates and plasma glucose levels in obese-diabetic P. obesus may be an important factor in the pathogenesis of obesity and type 2 diabetes in these animals. These studies have further validated P. obesus as an ideal animal model of type 2 diabetes and obesity. It was hypothesised that many genes in the liver of P. obesus involved in glucose and fat metabolism would be differentially expressed between lean-NGT and obese-diabetic animals. These genes may represent significant factors in the pathophysiology of type 2 diabetes. Two gene discovery experiments were conducted using suppression subtractive hybridisation (SSH) to enrich a cDNA library for differentially expressed genes. Experiment 1 used cDNA dot blots to screen 576 clones with cDNA derived from lean-NGT and obese-diabetic animals. 6 clones were identified as overexpressed in lean-NGT animals and 6 were overexpressed in obese-diabetic animals. These 12 clones were sequenced and SYBR-Green PCR was used to confirm differential gene expression. 4 genes were overexpressed (≥1.5 fold) in lean-NGT animals and 4 genes were overexpressed (≥1.5 fold) in obese-diabetic animals. To explore the physiological role of these genes, hepatic gene expression was examined in several physiological conditions. One gene, encoding thyroxine binding globulin (TBG), was confirmed as overexpressed in lean-NGT P. obesus with ad libitum access to food, relative to both obese-IGT and obese-diabetic animals. TBG expression decreased with fasting in all animals. Fasting TBG expression remained greater in lean-NGT animals than obese-IGT and obese-diabetic animals. TBG expression was not significantly affected by dietary energy restriction. TBG is involved in thyroid metabolism and is potentially involved in the regulation of energy expenditure. Fasting increased hepatic site 1 protease (SIP) expression in lean-NGT animals but was not significantly affected in obese-IGT and obese-diabetic animals. SIP expression was not significantly affected by dietary energy restriction. SIP is involved in the proteolytic processing of steroid response element binding proteins (SREBP). SREBPs are insulin responsive and are known to be involved in lipid metabolism. Gene expression studies found TBG and SIP were associated with obesity and diabetes. Future research will determine whether TBG and SIP are important in the pathogenesis of these diseases. Experiment 2 used SSH and cDNA microarray to screen 8064 clones. 223 clones were identified as overexpressed in lean-NGT P. obesus and 274 clones were overexpressed in obese-diabetic P. obesus (p ≤0.05). The 9 most significantly differentially expressed clones identified from the microarray screen were sequenced (p ≤0.01). 7 novel genes were identified as well as; sulfotransferase related protein and albumin. These 2 genes have not previously been associated with either type 2 diabetes or obesity. It is unclear why hepatic expression of these genes may differ between lean-NGT and obese-diabetic groups of P. obesus. Subsequent studies will explore the potential role of these novel and known genes in the pathophysiology of type 2 diabetes

Efficient empirical determination of maximum permissible error in coordinate metrology

Maximum permissible errors (MPEs) are an important measurement system specification and form the basis of periodic verification of a measurement system's performance. However, there is no standard methodology for determining MPEs, so when they are not provided, or not suitable for the measurement procedure performed, it is unclear how to generate an appropriate value with which to verify the system. Whilst a simple approach might be to take many measurements of a calibrated artefact and then use the maximum observed error as the MPE, this method requires a large number of repeat measurements for high confidence in the calculated MPE. Here, we present a statistical method of MPE determination, capable of providing MPEs with high confidence and minimum data collection. The method is presented with 1000 synthetic experiments and is shown to determine an overestimated MPE within 10 % of an analytically true value in 99.2 % of experiments, while underestimating the MPE with respect to the analytically true value in 0.8 % of experiments (overestimating the value, on average, by 1.24 %). The method is then applied to a real test case (probing form error for a commercial fringe projection system), where the efficiently determined MPE is overestimated by 0.3 % with respect to an MPE determined using an arbitrarily chosen large number of measurements

Syntaxin 5 Is Required for Copper Homeostasis in Drosophila and Mammals

Copper is essential for aerobic life, but many aspects of its cellular uptake and distribution remain to be fully elucidated. A genome-wide screen for copper homeostasis genes in Drosophila melanogaster identified the SNARE gene Syntaxin 5 (Syx5) as playing an important role in copper regulation; flies heterozygous for a null mutation in Syx5 display increased tolerance to high dietary copper. The phenotype is shown here to be due to a decrease in copper accumulation, a mechanism also observed in both Drosophila and human cell lines. Studies in adult Drosophila tissue suggest that very low levels of Syx5 result in neuronal defects and lethality, and increased levels also generate neuronal defects. In contrast, mild suppression generates a phenotype typical of copper-deficiency in viable, fertile flies and is exacerbated by co-suppression of the copper uptake gene Ctr1A. Reduced copper uptake appears to be due to reduced levels at the plasma membrane of the copper uptake transporter, Ctr1. Thus Syx5 plays an essential role in copper homeostasis and is a candidate gene for copper-related disease in humans

Manganese causes neurotoxic iron accumulation via translational repression of Amyloid Precursor Protein (APP) and H-Ferritin

For more than 150 years, it is known that occupational overexposure of manganese (Mn) causes movement disorders resembling Parkinson's disease (PD) and PD‐like syndromes. However, the mechanisms of Mn toxicity are still poorly understood. Here, we demonstrate that Mn dose‐ and time‐dependently blocks the protein translation of amyloid precursor protein (APP) and heavy‐chain Ferritin (H‐Ferritin), both iron homeostatic proteins with neuroprotective features. APP and H‐Ferritin are post‐transcriptionally regulated by iron responsive proteins, which bind to homologous iron responsive elements (IREs) located in the 5′‐untranslated regions (5′‐UTRs) within their mRNA transcripts. Using reporter assays, we demonstrate that Mn exposure repressed the 5′‐UTR‐activity of APP and H‐Ferritin, presumably via increased iron responsive proteins‐iron responsive elements binding, ultimately blocking their protein translation. Using two specific Fe2+‐specific probes (RhoNox‐1 and IP‐1) and ion chromatography inductively coupled plasma mass spectrometry (IC‐ICP‐MS), we show that loss of the protective axis of APP and H‐Ferritin resulted in unchecked accumulation of redox‐active ferrous iron (Fe2+) fueling neurotoxic oxidative stress. Enforced APP expression partially attenuated Mn‐induced generation of cellular and lipid reactive oxygen species and neurotoxicity. Lastly, we could validate the Mn‐mediated suppression of APP and H‐Ferritin in two rodent in vivo models (C57BL6/N mice and RjHan:SD rats) mimicking acute and chronic Mn exposure. Together, these results suggest that Mn‐induced neurotoxicity is partly attributable to the translational inhibition of APP and H‐Ferritin resulting in impaired iron metabolism and exacerbated neurotoxic oxidative stress

Palaeoproteomics resolves sloth relationships

The living tree sloths Choloepus and Bradypus are the only remaining members of Folivora, a major xenarthran radiation that occupied a wide range of habitats in many parts of the western hemisphere during the Cenozoic, including both continents and the West Indies. Ancient DNA evidence has played only a minor role in folivoran systematics, as most sloths lived in places not conducive to genomic preservation. Here we utilize collagen sequence information, both separately and in combination with published mitochondrial DNA evidence, to assess the relationships of tree sloths and their extinct relatives. Results from phylogenetic analysis of these datasets differ substantially from morphology-based concepts: Choloepus groups with Mylodontidae, not Megalonychidae; Bradypus and Megalonyx pair together as megatherioids, while monophyletic Antillean sloths may be sister to all other folivorans. Divergence estimates are consistent with fossil evidence for mid-Cenozoic presence of sloths in the West Indies and an early Miocene radiation in South America

High performing hospitals: a qualitative systematic review of associated factors and practical strategies for improvement.

BACKGROUND: High performing hospitals attain excellence across multiple measures of performance and multiple departments. Studying high performing hospitals can be valuable if factors associated with high performance can be identified and applied. Factors leading to high performance are complex and an exclusive quantitative approach may fail to identify richly descriptive or relevant contextual factors. The objective of this study was to undertake a systematic review of qualitative literature to identify methods used to identify high performing hospitals, the factors associated with high performers, and practical strategies for improvement. METHODS: Methods used to collect and summarise the evidence contributing to this review followed the 'enhancing transparency in reporting the synthesis of qualitative research' protocol. Peer reviewed studies were identified through Medline, Embase and Cinahl (Jan 2000-Feb 2014) using specified key words, subject terms, and medical subject headings. Eligible studies required the use of a quantitative method to identify high performing hospitals, and qualitative methods or tools to identify factors associated with high performing hospitals or hospital departments. Title, abstract, and full text screening was undertaken by four reviewers, and inter-rater reliability statistics were calculated for each review phase. Risk of bias was assessed. Following data extraction, thematic syntheses identified contextual factors important for explaining success. Practical strategies for achieving high performance were then mapped against the identified themes. RESULTS: A total of 19 studies from a possible 11,428 were included in the review. A range of process, output, outcome and other indicators were used to identify high performing hospitals. Seven themes representing factors associated with high performance (and 25 sub-themes) emerged from the thematic syntheses: positive organisational culture, senior management support, effective performance monitoring, building and maintaining a proficient workforce, effective leaders across the organisation, expertise-driven practice, and interdisciplinary teamwork. Fifty six practical strategies for achieving high performance were catalogued. CONCLUSIONS: This review provides insights into methods used to identify high performing hospitals, and yields ideas about the factors important for success. It highlights the need to advance approaches for understanding what constitutes high performance and how to harness factors associated with high performance

Identification and characterisation of copper homeostasis genes

© 2010 Dr. Adam SouthonCopper is essential for life, yet also potentially toxic in excess. Copper homeostasis is therefore regulated at the cellular, tissue and organismal levels. Studies with eukaryotic model systems, primarily yeast and mammals, have identified conserved mechanisms for copper uptake, distribution, sequestration and efflux. Nevertheless, there is much we do not know about how copper levels are sensed and regulated. Additional proteins involved in copper homeostasis under both ‘normal’ and ‘diseased’ conditions remain to be identified. The emergence of Drosophila melanogaster as a bona fide model system for the study of copper homeostasis has coincided with research aiming to identify and characterise novel copper regulatory genes in this organism. Whereas others have focused on in vivo studies, the studies reported in this thesis are focussed on cultured D. melanogaster S2 cells. The first aim of this project was to use cDNA microarrays to identify genes transcriptionally regulated by copper levels in S2 cells. The second and third aims were to characterise the function and localisation of novel copper homeostasis genes in vitro and in vivo respectively. Initial characterisation of D. melanogaster S2 cells found these cells express orthologues of key mammalian copper regulatory genes. Copper uptake primarily occurs via Ctr1A and Ctr1B, the orthologues of human Ctr1. Copper efflux occurs via DmATP7, the sole D. melanogaster orthologue of the mammalian P-type ATPases, ATP7A and ATP7B. S2 cells are highly copper tolerant and primarily rely on metallothionein-mediated copper sequestration and copper efflux to maintain homeostasis. Whereas ATP7A and ATP7B undergo copper-induced trafficking between the trans-Golgi network and plasma membrane of mammalian cells, this does not appear to occur with endogenous DmATP7 in cultured D. melanogaster cells. Interestingly, when expressed in mammalian cells DmATP7 does undergo copper-induced trafficking to the plasma membrane and can facilitate copper efflux, demonstrating functional conservation of localisation and trafficking motifs in these P-type ATPases. Malvolio, the orthologue of Divalent Metal ion Transporter 1, also contributes to copper uptake in S2 cells and D. melanogaster, with impaired function associated with sensitivity to copper limitation. D. melanogaster therefore utilises this general metal transporter in addition to the copper-specific Ctr1 pathway. cDNA microarrays were used to identify genes transcriptionally regulated by copper in S2 cells, with RNA interference used to determine whether candidate genes could affect copper homeostasis. Several components of the COPI vesicle trafficking pathway, including ADP-ribosylation factor 1 (Arf1), were found to affect copper levels in S2 cells. Arf1 was found to have a conserved role in regulating copper uptake in cultured mammalian and D. melanogaster cells, and this is likely to be mediated via the localisation of Ctr1 at the plasma membrane. Taken together these studies demonstrate the value of D. melanogaster S2 cells for the study of copper homeostasis, thereby complementing the D. melanogaster model system. These novel findings should stimulate additional research in both D. melanogaster and mammalian systems and facilitate a greater understanding of copper homeostasis

Identification and characterisation of copper homeostasis genes

© 2010 Dr. Adam SouthonCopper is essential for life, yet also potentially toxic in excess. Copper homeostasis is therefore regulated at the cellular, tissue and organismal levels. Studies with eukaryotic model systems, primarily yeast and mammals, have identified conserved mechanisms for copper uptake, distribution, sequestration and efflux. Nevertheless, there is much we do not know about how copper levels are sensed and regulated. Additional proteins involved in copper homeostasis under both ‘normal’ and ‘diseased’ conditions remain to be identified. The emergence of Drosophila melanogaster as a bona fide model system for the study of copper homeostasis has coincided with research aiming to identify and characterise novel copper regulatory genes in this organism. Whereas others have focused on in vivo studies, the studies reported in this thesis are focussed on cultured D. melanogaster S2 cells. The first aim of this project was to use cDNA microarrays to identify genes transcriptionally regulated by copper levels in S2 cells. The second and third aims were to characterise the function and localisation of novel copper homeostasis genes in vitro and in vivo respectively. Initial characterisation of D. melanogaster S2 cells found these cells express orthologues of key mammalian copper regulatory genes. Copper uptake primarily occurs via Ctr1A and Ctr1B, the orthologues of human Ctr1. Copper efflux occurs via DmATP7, the sole D. melanogaster orthologue of the mammalian P-type ATPases, ATP7A and ATP7B. S2 cells are highly copper tolerant and primarily rely on metallothionein-mediated copper sequestration and copper efflux to maintain homeostasis. Whereas ATP7A and ATP7B undergo copper-induced trafficking between the trans-Golgi network and plasma membrane of mammalian cells, this does not appear to occur with endogenous DmATP7 in cultured D. melanogaster cells. Interestingly, when expressed in mammalian cells DmATP7 does undergo copper-induced trafficking to the plasma membrane and can facilitate copper efflux, demonstrating functional conservation of localisation and trafficking motifs in these P-type ATPases. Malvolio, the orthologue of Divalent Metal ion Transporter 1, also contributes to copper uptake in S2 cells and D. melanogaster, with impaired function associated with sensitivity to copper limitation. D. melanogaster therefore utilises this general metal transporter in addition to the copper-specific Ctr1 pathway. cDNA microarrays were used to identify genes transcriptionally regulated by copper in S2 cells, with RNA interference used to determine whether candidate genes could affect copper homeostasis. Several components of the COPI vesicle trafficking pathway, including ADP-ribosylation factor 1 (Arf1), were found to affect copper levels in S2 cells. Arf1 was found to have a conserved role in regulating copper uptake in cultured mammalian and D. melanogaster cells, and this is likely to be mediated via the localisation of Ctr1 at the plasma membrane. Taken together these studies demonstrate the value of D. melanogaster S2 cells for the study of copper homeostasis, thereby complementing the D. melanogaster model system. These novel findings should stimulate additional research in both D. melanogaster and mammalian systems and facilitate a greater understanding of copper homeostasis