Analysis of Beta-Cell Gene Expression Reveals Inflammatory Signaling and Evidence of Dedifferentiation following Human Islet Isolation and Culture

The stresses encountered during islet isolation and culture may have deleterious effects on beta-cell physiology. However, the biological response of human islet cells to isolation remains poorly characterized. A better understanding of the network of signaling pathways induced by islet isolation and culturing may lead to strategies aimed at improving islet graft survival and function. Laser capture microdissection (LCM) was used to extract beta-cell RNA from 1) intact pancreatic islets, 2) freshly isolated islets, 3) islets cultured for 3 days, and changes in gene expression were examined by microarray analysis. We identified a strong inflammatory response induced by islet isolation that continues during in-vitro culture manifested by upregulation of several cytokines and cytokine-receptors. The most highly upregulated gene, interleukin-8 (IL-8), was induced by 3.6-fold following islet isolation and 56-fold after 3 days in culture. Immunofluorescence studies showed that the majority of IL-8 was produced by beta-cells themselves. We also observed that several pancreas-specific transcription factors were down-regulated in cultured islets. Concordantly, several pancreatic progenitor cell-specific transcription factors like SOX4, SOX9, and ID2 were upregulated in cultured islets, suggesting progressive transformation of mature beta-cell phenotype toward an immature endocrine cell phenotype. Our findings suggest islet isolation and culture induces an inflammatory response and loss of the mature endocrine cell phenotype. A better understanding of the signals required to maintain a mature beta-cell phenotype may help improve the efficacy of islet transplantation

Data Analytics for Uncovering Fraudulent Behaviour in Elite Sports

Sports officials around the world are facing societal challenges due to the unfair nature of fraudulent practices performed by unscrupulous athletes. Recently, sample swapping has been raised as a potential practice where some athletes exchange their doped sample with a clean one to evade a positive test. The current detection method for such cases includes laboratory testing like DNA analysis. However, these methods are costly and time-consuming, which goes beyond the budgetary limits of anti-doping organisations. Therefore, there is a need to explore alternative methods to improve decision-making. We presented a data analytical methodology that supports anti-doping decision-makers on the task of athlete disambiguation. Our proposed model helps identify the swapped sample, which outperforms the current state-of-the-art method and different baseline models. The evaluation on real-world sample swapping cases shows promising results that help advance the research on the application of data analytics in the context of anti-doping analysis

A Genome-Wide RNAi Screen Identifies Regulators of Cholesterol-Modified Hedgehog Secretion in Drosophila

Hedgehog (Hh) proteins are secreted molecules that function as organizers in animal development. In addition to being palmitoylated, Hh is the only metazoan protein known to possess a covalently-linked cholesterol moiety. The absence of either modification severely disrupts the organization of numerous tissues during development. It is currently not known how lipid-modified Hh is secreted and released from producing cells. We have performed a genome-wide RNAi screen in Drosophila melanogaster cells to identify regulators of Hh secretion. We found that cholesterol-modified Hh secretion is strongly dependent on coat protein complex I (COPI) but not COPII vesicles, suggesting that cholesterol modification alters the movement of Hh through the early secretory pathway. We provide evidence that both proteolysis and cholesterol modification are necessary for the efficient trafficking of Hh through the ER and Golgi. Finally, we identified several putative regulators of protein secretion and demonstrate a role for some of these genes in Hh and Wingless (Wg) morphogen secretion in vivo. These data open new perspectives for studying how morphogen secretion is regulated, as well as provide insight into regulation of lipid-modified protein secretion

Characterization and prevention of cell death in isolated islets of langerhans

A major limitation to the success of islet cell transplantation as a therapy for type I diabetes is the cell loss induced by the islet isolation procedure. The aim of this thesis was to elucidate signal transduction events and related intracellular activities that are implicated in islet cell survival/death following islet isolation in order to develop therapeutic interventions to promote islet survival.The isolation of pancreatic islets imposes considerable stress on these cells, resulting in significant levels of cell death following isolation which was associated with activation of the stress-activated c-jun NH2-terminal kinase (JNK). However, within 24 hours in culture, JNK activation was greatly reduced concomitant with an increase in AKT activation. Inhibition of phosphatidylinositol 3-kinase (PI3K)/AKT signalling resulted in sustained JNK phosphorylation, while activators of AKT suppressed JNK phosphorylation, indicating that the rise in AKT activity during islet culture suppresses JNK. One of the stimulus of the PI3K/AKT pathway was found to be insulin secreted by the islets themselves, acting in an autocrine manner. The result of this autocrine activation of the prosurvival AKT pathway, and subsequent suppression of JNK, was a decrease in the appearance of apoptotic cells in islets after 72 hours in culture. Caspase inhibition alone was unable to prevent cell death in isolated islets. In addition, the amount of mitochondrial depolarization occurring in isolated islets was unaffected by caspase inhibition, leading to the notion that the commitment to islet cell death could be occurring at the level of, or upstream of, mitochondrial dysfunction. Indeed, inhibition of BAX translocation to the mitochondria, a critical event mediating mitochondrial permeabilization, prevented islet cell death. Inhibition of JNK also prevented mitochondrial permeabilization and cell death.The current results demonstrate that insulin can act as an autocrine survival signal in isolated human islets. These findings also reveal the interdependence of necrosis and apoptosis in isolated islets, suggesting therapeutic strategies which target early events in cell death signalling in order to prevent multiple forms of islet cell death

Improving stroke patients' care: a patient held record is not enough.

BACKGROUND: Stroke patients' care in hospital tends to be poorly organised, with poor communication and a lack of information being frequent sources of complaint. The purpose of this study was to evaluate whether a patient-held record (PHR) would result in greater patient satisfaction and better care planning for stroke patients. METHODS: A time series control (6 months) - intervention (8 months) - control (6 months) was used among London teaching hospital general medical and geriatric medicine inpatient wards. All stroke patients admitted to the wards during the intervention phase received a PHR and were instructed in its use. Demographic, stroke severity, social factors and outcomes were collected from all stroke patients during all phases of the study. RESULTS: Of 252 stroke patients aged 46 to 98 years entered into the study, by six months after admission 118 (46.8%) had died. PHR and control group patients were well matched in terms of socio-demographic characteristics and pre-stroke ability. At six months after admission, 119 (97%) patients responded to the questionnaire. Just over half (56%, 13) of intervention group patients recalled receiving a PHR. Of those patients, 59% reported reading the PHR, 27% had lost their PHR, and two-thirds said they had difficulties encouraging staff to write in the PHR. Half felt that possession of the PHR was more trouble than it was worth. PHR group patients were more satisfied with the recovery they had made (79% vs. 59%, p=0.04), but felt less able to talk to staff about their problems (61% vs. 82%, p=0.02). PHR group patients reported receiving fewer explanations about their condition (18% vs. 33%, p=0.12) and treatment (26% vs. 45%, p=0.07), and were more afraid of asking doctors questions (21% vs. 4%, p=0.01) than controls. PHR group patients were no better prepared for hospital discharge than control group patients, and both groups were ill-informed about services and benefits that might have helped after discharge from hospital. CONCLUSIONS: Stroke patients received poor information and explanations regardless of whether they received a PHR. A PHR did not appear to improve patient satisfaction or discharge planning, and may have reduced opportunities for communication and explanation

The role of kinases in the Hedgehog signalling pathway

The Hedgehog (Hh) signalling pathway has a crucial role in several developmental processes and is aberrantly activated in a variety of cancers. In Drosophila, many of the canonical Hh pathway components are phosphorylated, yet the precise role of these phosphorylation events in the regulation of Hh signal transduction is unclear. Furthermore, the Hh pathway receives input from several kinases that have well-described roles in other cellular functions, some of which have both positive and negative effects on Hh signalling. Several recent studies have characterized the role of specific phosphorylation events in the Hh pathway, and have begun to shed light on how phosphorylation of Hh signalling components affects their subcellular location, stability and activity to mediate the transcriptional response to the Hh gradient