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

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

The issue of the functioning land and buildings registration from the conducting perspective

Praca urzędów jest powszechnie znana, jednak panują o niej opinie, mniej lub bardziej pochlebne i nie ma znaczenia czy przedmiotem omówienia są urzędy gmin czy starostwa powiatowe. Na tychże jednostkach oraz na powiatowych ośrodkach dokumentacji geodezyjnej i kartograficznej, od czasu prowadzenia reformy administracyjnej kraju w 1999 roku, spoczął obowiązek prowadzenia ewidencji gruntów i budynków. Stąd w niniejszym artykule zostały poruszone wybrane aspekty pracy urzędu, ze szczególnym uwzględnieniem działań w ewidencji gruntów i budynków przez organ ją prowadzący. Opisany również został problem składanych wniosków o dokonanie aktualizacji ewidencji od strony ich kompletności, braków formalnych, czy zasadnych dokumentów stanowiących podstawę wprowadzania tych zmian. Ponadto język pism urzędowych oraz aktów prawnych, w oparciu o które organy administracyjne działają, jawi się niejasnym komunikatorem ze stroną. Kolejną kwestią wartą uwagi jest komunikacja urzędu z interesantem, która odbywa się głównie w formie tradycyjnej, czyli papierowej korespondencji pocztowej. Poruszane zagadnienia posiadają odniesienie do zapisów kodeksu postępowania administracyjnego, który stanowi fundament pracy urzędów, ze szczególnym zwróceniem uwagi na te prowadzące ewidencję gruntów i budynków. Te z kolei działają w oparciu o prawo geodezyjne i rozporządzenia w sprawie ewidencji gruntów i budynków.Office work is generally known, however prevailing opinions are more or less flattering and it does not matter if the subject of discussion are the county offices or district municipalities. Both these units and the county centres of surveying and cartographic documentation, were established to keep land and buildings registration, since implementing the administrative country reform in 1999. Thus in this article the selected aspects of office work have been addressed, with particular emphasis on investments held in the land and building register, by the body conducting it. Also the problem is described from the side of applications completeness to update the registry, the formal deficiencies, outdated address information and the documents constituting reasonable basis for these changes. Moreover, the official documents language, on which administrative authorities work, appears to be unclear communicator for persons, not knowing expertise law language. The further issue worth attention is the communication between office and the person and opportunities for improving it, because it goes mainly with traditional way via paper mail. Discussed issues refer to the provisions of the Administrative Procedure Code, that is the work office base with particular emphasis on those leading the land and buildings registration. These offices work is based on surveying and cartographic law and order on the land and buildings registration

Administrative update of soil classification

Gleboznawcza klasyfikacja gruntów ściśle wiąże się z prowadzeniem ewidencji gruntów i budynków przez starostów, zgodnie z zapisami prawa geodezyjnego i kartograficznego. Rejestr w postaci operatu gleboznawczej klasyfikacji gruntów powstał, był prowadzony i aktualizowany w oparciu o rozporządzenie z 1956 roku w sprawie gleboznawczej klasyfikacji gruntów aż do 2012 roku. Nowe rozporządzenie zastąpiło wiekowy akt wprowadzając zmiany w dotychczas utrwalonym porządku. Spowodowało to wiele komplikacji dla organów administracyjnych oraz osób wykonujących czynności techniczne. W artykule poruszone zostały kwestie związane z funkcjonowaniem operatu gleboznawczej klasyfikacji gruntów, jego prowadzeniem i aktualizacją w oparciu o uchylony już akt od strony administracyjno-prawnej, jak i w oparciu o rozporządzenie z 12 września 2012 r. Kwestie te dotyczą pojęcia operatu gleboznawczej klasyfikacji gruntów jako rejestru osobnego w stosunku do operatu ewidencji gruntów, czy celu funkcjonowania i zakresu zadań związanych z prowadzeniem operatu gleboznawczej klasyfikacji gruntów. Wiele problemów powoduje niejasne określenie pożądanych kompetencji osoby, która zostanie powołana do wykonania czynności klasyfikacyjnych w terenie i w zakresie robót kameralnych. Poruszony aspekt klasyfikacji gruntów jest tematem bardzo aktualnym w zakresie gospodarowania nieruchomościami. Dynamiczne zmiany w polskim prawodawstwie wymuszają konieczność uaktualniania starszych przepisów. Znowelizowane akty sprzyjają usprawnieniu działań, a z drugiej jednak strony nie odpowiadają na wszystkie nurtujące pytania, na które osoba administracji musi znać odpowiedź.Soil classification is closely linked to conducting of land administration by leading authority, with respect to land surveying act. Register in form of soil classification frame was conducted and updated on the base of regulation from 1956 till 2012. New executive act has replaced old one and entering changes with the previously established order. This caused a lot of complications for the administrative authorities and persons performing technical activities. In this article the problems related to the functioning of the soil classification were discussed, its running and updating based on the already repealed act from the administrative and legal side and moreover in accordance with the executive act from 12th September 2012. These topics are related to concept of soil classification as a separate register with respect to land register or the aim of functioning and the scope of the tasks associated with conducting soil classification register. Many problems are coming with unclear description of proper person who is appointed to perform outdoor and indoor classification operations. Described topic of soil classification is a very up to date problem in the field of property management. Very dynamic changes in Polish legislation force the need updating older legal provisions. The amendment acts conductive to improving operations, but on the other side it do not answer all the key pillar questions, on which the person who work in administrative office have to know the answer

The US Medical Liability System: Evidence for Legislative Reform

BACKGROUND Despite state and federal efforts to implement medical malpractice reform, there is limited evidence on which to base policy decisions. The National Practitioner Data Bank (NPDB) offers an opportunity to evaluate the effects of previous malpractice tort reforms on malpractice payments and premiums. METHODS For every state and the District of Columbia, we calculated the number of malpractice payments, total amount paid, and average payment from NPDB data reported from 1999 through 2001. We analyzed 44,913 claims using logistic regression to study associations between payments, physician premiums, and 10 state statutory tort reforms. RESULTS Wide variations exist in malpractice payments among states. The reforms most associated with lower payments and premiums were total and noneconomic damage caps. Mean payments were 26% lower in states with total damage caps ($196,495.34 vs$265,554.50, P = .001). Mean payments were 22% less in states with noneconomic damage caps ($219,225.98 vs$279,849.86, P = .010). Total damage caps were associated with lower mean annual premiums, especially for obstetricians ($22,371.57 vs$42,728.68, P <.001). Hard noneconomic damage caps were associated with premium reductions for obstetricians (30,283.75 vs 45,740.88; P = .039). CONCLUSIONS Significant reductions in malpractice payments could be realized if total or noneconomic damage caps were operating nationally. Hard noneconomic damage and total damage caps could yield lower premiums. If tied to a comprehensive plan for reform, the money saved could be diverted to implement alternative approaches to patient compensation or be used to achieve other systems reform benefiting patients, employers, physicians, and hospitals