A Multi-Parameter, High-Content, High-Throughput Screening Platform to Identify Natural Compounds that Modulate Insulin and Pdx1 Expression

Diabetes is a devastating disease that is ultimately caused by the malfunction or loss of insulin-producing pancreatic beta-cells. Drugs capable of inducing the development of new beta-cells or improving the function or survival of existing beta-cells could conceivably cure this disease. We report a novel high-throughput screening platform that exploits multi-parameter high-content analysis to determine the effect of compounds on beta-cell survival, as well as the promoter activity of two key beta-cell genes, insulin and pdx1. Dispersed human pancreatic islets and MIN6 beta-cells were infected with a dual reporter lentivirus containing both eGFP driven by the insulin promoter and mRFP driven by the pdx1 promoter. B-score statistical transformation was used to correct systemic row and column biases. Using this approach and 5 replicate screens, we identified 7 extracts that reproducibly changed insulin and/or pdx1 promoter activity from a library of 1319 marine invertebrate extracts. The ability of compounds purified from these extracts to significantly modulate insulin mRNA levels was confirmed with real-time PCR. Insulin secretion was analyzed by RIA. Follow-up studies focused on two lead compounds, one that stimulates insulin gene expression and one that inhibits insulin gene expression. Thus, we demonstrate that multi-parameter, high-content screening can identify novel regulators of beta-cell gene expression, such as bivittoside D. This work represents an important step towards the development of drugs to increase insulin expression in diabetes and during in vitro differentiation of beta-cell replacements

In Vivo Conditional Pax4 Overexpression in Mature Islet β-Cells Prevents Stress-Induced Hyperglycemia in Mice

OBJECTIVE To establish the role of the transcription factor Pax4 in pancreatic islet expansion and survival in response to physiological stress and its impact on glucose metabolism, we generated transgenic mice conditionally and selectively overexpressing Pax4 or a diabetes-linked mutant variant (Pax4R129 W) in β-cells. RESEARCH DESIGN AND METHODS Glucose homeostasis and β-cell death and proliferation were assessed in Pax4- or Pax4R129 W-overexpressing transgenic animals challenged with or without streptozotocin. Isolated transgenic islets were also exposed to cytokines, and apoptosis was evaluated by DNA fragmentation or cytochrome C release. The expression profiles of proliferation and apoptotic genes and β-cell markers were studied by immunohistochemistry and quantitative RT-PCR. RESULTS Pax4 but not Pax4R129 W protected animals against streptozotocin-induced hyperglycemia and isolated islets from cytokine-mediated β-cell apoptosis. Cytochrome C release was abrogated in Pax4 islets treated with cytokines. Interleukin-1β transcript levels were suppressed in Pax4 islets, whereas they were increased along with NOS2 in Pax4R129 W islets. Bcl-2, Cdk4, and c-myc expression levels were increased in Pax4 islets while MafA, insulin, and GLUT2 transcript levels were suppressed in both animal models. Long-term Pax4 expression promoted proliferation of a Pdx1-positive cell subpopulation while impeding insulin secretion. Suppression of Pax4 rescued this defect with a concomitant increase in pancreatic insulin content. CONCLUSIONS Pax4 protects adult islets from stress-induced apoptosis by suppressing selective nuclear factor-κB target genes while increasing Bcl-2 levels. Furthermore, it promotes dedifferentiation and proliferation of β-cells through MafA repression, with a concomitant increase in Cdk4 and c-myc expression

Cyclin-dependent kinase 5 mediates pleiotrophin-induced endothelial cell migration

Pleiotrophin (PTN) stimulates endothelial cell migration through binding to receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) and ανβ3 integrin. Screening for proteins that interact with RPTPβ/ζ and potentially regulate PTN signaling, through mass spectrometry analysis, identified cyclin-dependent kinase 5 (CDK5) activator p35 among the proteins displaying high sequence coverage. Interaction of p35 with the serine/threonine kinase CDK5 leads to CDK5 activation, known to be implicated in cell migration. Protein immunoprecipitation and proximity ligation assays verified p35-RPTPβ/ζ interaction and revealed the molecular association of CDK5 and RPTPβ/ζ. In endothelial cells, PTN activates CDK5 in an RPTPβ/ζ- and phosphoinositide 3-kinase (PI3K)-dependent manner. On the other hand, c-Src, ανβ3 and ERK1/2 do not mediate the PTN-induced CDK5 activation. Pharmacological and genetic inhibition of CDK5 abolished PTN-induced endothelial cell migration, suggesting that CDK5 mediates PTN stimulatory effect. A new pyrrolo[2,3-α]carbazole derivative previously identified as a CDK1 inhibitor, was found to suppress CDK5 activity and eliminate PTN stimulatory effect on cell migration, warranting its further evaluation as a new CDK5 inhibitor. Collectively, our data reveal that CDK5 is activated by PTN, in an RPTPβ/ζ-dependent manner, regulates PTN-induced cell migration and is an attractive target for the inhibition of PTN pro-angiogenic properties

Beta Cell Hubs Dictate Pancreatic Islet Responses to Glucose

N.R.J. was supported by a Diabetes UK RW and JM Collins Studentship (12/0004601). J.B. was supported by a European Foundation for the Study of Diabetes (EFSD) Albert Renold Young Scientist Fellowship and a Studienstiftung des deutschen Volkes PhD Studentship. D.T. was supported by an Advanced Grant from the European Research Commission (268795). G.A.R. was supported by Wellcome Trust Senior Investigator (WT098424AIA) and Royal Society Wolfson Research Merit Awards, and by MRC Programme (MR/J0003042/1), Biological and Biotechnology Research Council (BB/J015873/1), and Diabetes UK Project (11/0004210) grants. G.A.R. and M.W. acknowledge COST Action TD1304 Zinc-Net. D.J.H. was supported by Diabetes UK R.D. Lawrence (12/0004431), EFSD/Novo Nordisk Rising Star and Birmingham Fellowships, a Wellcome Trust Institutional Support Award, and an MRC Project Grant (MR/N00275X/1) with G.A.R. D.J.H and G.A.R. were supported by Imperial Confidence in Concept (ICiC) Grants. J.F. was supported by an MRC Programme grant (MR/L02036X/1). L.P. provided human islets through collaboration with the Diabetes Research Institute, IRCCS San Raffaele Scientific Institute (Milan), within the European islet distribution program for basic research supported by JDRF (1-RSC-2014-90-I-X). P.M. and M.B. were supported by the Innovative Medicine Initiative Joint Undertaking under grant agreement no. 155005 (IMIDIA), resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies in kind contribution, and by the Italian Ministry of University and Research (PRIN 2010-2012). D.B. and E.B. provided human islets through the European Consortium for Islet Transplantation sponsored by JDRF (1-RSC-2014-100-I-X)

Ograniczanie drgań dwumasowego układu sterowania z dodatkowymi sprzężeniami zwrotnymi

In the paper an analysis of control structures for the electrical drive system with elastic joint was carried out. The synthesis of the control structure with PI controller supported by different additional feedbacks was presented. The pole placement method was applied. The analytical equations which allow for calculating the control structure parameters were given. In order to damp the torsional vibration effectively the application of the feedback from one selected state variable is necessary. In the literature a big number of possible feedbacks was reported. In the paper it was shown that all systems with one additional feedback can be divided into three different groups, according to their dynamical characteristics. Simulation results were confirmed experimentally in the laboratory set up.W układach napędowych, elementy mechaniczne przenoszenia momentu posiadają skończoną sztywność. Podlegają one w czasie pracy odkształceniom sprężystym i plastycznym. W większości przypadków, w napędzie elektrycznym pomija się elastyczność połączenia wału z silnikiem. Istnieje jednak grupa napędów, w której nieuwzględnienie skończonej sztywności wału może prowadzić do powstania drgań mechanicznych, a w konsekwencji do znaczących oscylacji zmiennych elektromagnetycznych. Może to spowodować pogorszenie przebiegu procesu technologicznego, skrócenie żywotności napędu itp. Przykładową grupą są napędy walcarek, taśmociągów, napędy robotów przemysłowych [1]-[4]. Aby poprawić właściwości dynamiczne takich napędów i ograniczyć oscylacje prędkości i momentu silnika, powszechnie stosuje się zmodyfikowane struktury sterowania. Wykorzystują one dodatkowe sprzężenia zwrotne od wybranych zmiennych stanu. Tłumienie oscylacji prędkości obciążenia możliwe jest poprzez wprowadzenie dodatkowego sprzężenia zwrotnego od: momentu skrętnego, różnicy prędkości silnika i obciążenia, prędkości obciążenia lub ich pochodnych wprowadzonych do węzła momentu elektromagnetycznego lub prędkości. W referacie przedstawiono analizę układu składającego się silnika napędowego połączonego z maszyną obciążającą za pośrednictwem elastycznego sprzęgła. Przeprowadzono syntezę sterowania układu dwumasowego z regulatorem prędkości PI z różnymi dodatkowymi sprzężeniami zwrotnymi (rys. 1). Przedstawiono wzory umożliwiające dobór nastaw regulatorów i uzyskanie dowolnego współczynnika tłumienia układu regulacji (Tab.1). Wskazano na ograniczenia w kształtowaniu charakterystyk dynamicznych w zależności od stopni swobody układu regulacji. Referacie pokazano, że mimo dużej liczby możliwych sprzężeń zwrotnych przedstawianych w literaturze, wszystkie struktury sterowania z jednym dodatkowym sprzężeniem zwrotnym mogą być podzielone na trzy grupy o identycznych właściwościach dynamicznych (rys. 1). Badania symulacyjne zawarte w pracy zostały zweryfikowane na stanowisku laboratoryjnym którego schemat przedstawiono na rys. 3. Na rys. 4 przedstawiono przebiegi zmiennych układu bez dodatkowych sprzężeń zwrotnych. Podobnie jak w badaniach symulacyjnych w prędkości obciążenia występują duże słabo tłumione oscylacje. Kolejno przebadano układy z dodatkowymi sprzężeniami zwrotnymi. Zgodnie z wzorami przedstawionymi w Tab. 1 nastawy układu regulacji dobrano w sposób zapewniający uzyskanie współczynnika tłumienia drgań &xir = 0.7. Otrzymane wyniki potwierdzały przeprowadzone badania symulacyjne. Układy sterowania z jednym dodatkowym sprzężeniem zwrotnym efektywnie tłumiły drgania skrętne układu (rys. 5-7)

Robust control of the PMSM drive with changeable inertia using TSK-type fuzzy controller

In the work the application of the fuzzy TSK-type controller for the speed control of the nonlinear, nonstationary PMSM drive is considered. The parameters of the fuzzy controller are selected using genetic algorithm. The designed controller is robust against variation of inertia of the drive. The comparative study concerning proposed and classical control structure is considered. The theoretical consideration are verify by simulation tests. The obtained results show the superior performance of the proposed system