Neue Erkenntnisse zum Diabetes – protektiven Locus IDDM4 der SH Ratte: Charakterisierung einer neuen kongenen BB.6S f Rattenlinie.

Beim Typ-1 Diabetes handelt es sich um eine multifaktorielle Autoimmunerkrankung, die auf dem Zusammenspiel genetischer, immunologischer und umweltbedingter Faktoren beruht. Hilfreich bei der Identifizierung krankheitsassoziierter Genloci sind diverse Tiermodelle, bei denen durch Kreuzungsstudien kongene Linien mit spontanen Rekombinationen im Bereich diabetes-suszeptibler oder diabetes-resistenter Genloci entstehen. Ein „Produkt“ solcher Kreuzungen ist die homozygote BB.6S m Rattenlinie, die obwohl Träger der diabetogenen Iddm1 und Iddm2 Genloci, nur eine T1D-Inzidenz von 10 % aufwies. Ursache für den diabetes-protektive Effekt war der alleinige chromosomale Austausch des diabetogenen Iddm4 Bereiches auf Chromosomen 6 der BB/OK Ratte durch den einer männlichen, diabetes-resistenten SH Ratte. Innerhalb dieser Arbeit sollten daher folgende Fragestellungen geklärt werden. 1. Ist die Iddm4 vermittelte Reduktion der Diabetesinzidenz auf die Expression eines oder mehrere potentieller diabetes-protektiver Gene zurückzuführen? 2. Wird das T1D Risiko durch den in diesem Bereich liegenden Imprintinglokus Dlk1-Dio3 beeinflusst? Zur Beantwortung dieser Fragen wurde eine zweite kongene Rattenlinie generiert. Die BB.6S f Linie, deren Iddm4 Genlocus von weiblichen SH Ratten abstammte, zeichnete sich wie die BB.6S m durch eine massive Reduktion der T1D Inzidenz aus. Nur 13 % der BB.6S f Ratten entwickelten einen T1D. Bei der Analyse der Expression von Strukturgenen konnten keine einheitlichen Muster zwischen BB.6S m und BB.6S f Ratten im Bezug zu BB/OK Ratten detektiert werden. Erst die Analyse des Expressionsmusters von mikroRNAs, deren Gene im Iddm4 Bereich und hier insbesondere im Dlk1-Dio3 Gencluster lokalisiert sind, ergaben sich Hinweise auf die mikroRNAs mir-337, mir-345 und mir-299 als potenzielle Kandidaten für einen T1D Schutz. Im zweiten Teil der Arbeit sollte geklärt werden, ob die T1D Inzidenz durch genomische Prägung der Gene des Dlk1-Dio3 Imprintingclusters beeinflusst wird: So sind auf dem väterlich vererbten Allel unter anderem die Gene Dlk1, Rtl1 und Dio3 aktiviert, während bei mütterlicher Abstammung Gtl2 und mikroRNAs exprimiert werden. Zur Klärung dieser Fragestellung wurden heterozygote F1 Hybride aus den Kreuzungen (BB/OK x BB.6S m) und (BB.6S m x BB/OK) generiert. Die phänotypischen Daten belegen, dass die heterozygoten Tiere in der Regel eine höhere T1D Inzidenz aufwiesen, die zusätzlich in Abhängigkeit vom Geschlecht der Nachkommen und dem Vererbungsmuster zwischen 21 und 74 % variierte. Generell waren weibliche Nachkommen besser vor einem T1D geschützt als männlichen Nachkommen. Außerdem zeigte sich, dass die T1D Inzidenz niedriger war, wenn das SHR Allels väterlicherseits vererbt wurde. Diese Sachverhalte deuten einerseits auf eine Interaktion des Iddm4 Bereichs von Chromosom 6 mit dem biallelischen X-Chromosom hin. Eine weitere Analyse der Dlk1 Expression belegt, dass es beim Fehlen des paternalen SHR Allels überraschenderweise zu einer biallelischen Expression des Dlk1 Genes kommt. Dieser Effekt ist assoziiert mit dem Verlust der Iddm4 vermittelten T1D Protektion. Die Daten der vorliegenden Arbeit lassen zusammenfassend drei Aussagen zu. I. Es konnte unter Einsatz der beiden kongenen BB.6S Rattenlinien kein Gen identifiziert werden, das den durch den Iddm4 Bereich der SH Ratte vermittelten Diabetesschutz bewirkt. II. Die Diabetesinzidenz wird offensichtlich durch Interaktion zwischen Chromosom 6 und dem biallelischen X-Chromosom beeinflusst. III. Hier konnte erstmals tierexperimentell bestätigt werden, dass die Manifestation eines T1D durch Imprinting geprägt wird.Type I Diabetes forms a multifactorial autoimmune disease based on the interaction of genetic, immunologic and environmental factors. To identify gene loci associated with the disease, animal models are helpful in which hybridization studies lead to congene lines carrying spontaneous recombinations in areas of gene loci suspected to be impelling diabetes or to be diabetes –resistant. One of such hybridization “products” is the homozygous BB.6S rat line which, even though carrying the diabetogenic Iddm1 und Iddm2 gene loci, presented a T1D incidence of 10 %. The only cause of the diabetes-protective effect was the chromosomal transposition of the diabetogenic Iddm4 area on chromosome 6 of the BB/OK rat by one of a male, diabetes-resistant SH rat. In this work, the following questions were to be clarified: 1. Is Iddm4 – induced reduction of the incidence of diabetes caused by the expression of one or several potentially diabetes-protective genes? 2. Is the T1D risk affected by the imprinting locus Dlk1-Dio3 in this gene area? To answer this question, a second congene rat line was generated. Likewise the BB.6S line, the BB.6S f line, whose Iddm4 gene locus stems from female SH rats, showed m a massive reduction of the T1D incidence. Only 13 % of the BB.6S f rats developed a T1D. Analyzing the expression of genetic patterns, no uniform pattern between BB.6S m and BB.6S f rats could be detected compared to BB/OK rats. Only when analysing the micro RNA expression pattern with its genes localized in the Iddm4 area, particularly in the Dlk1-Dio3 gene cluster, we found indications that microRNA mir-337, mir-345 und mir-299 are prospective candidates constituting a T1D protection. In a second part of this work it should be cleared if T1D incidence is influenced by genomic shaping of the genes of the Dlk1-Dio3 imprinting clusters. On paternally bequeathed alleles among others the genes Dlk1, Rtl1 and Dio3 are activated, whereas in maternal line of descent Gtl2 und microRNA are exprimed. To further investigate this question, heterozygous F1 hybrids were generated from the hybrids (BB/OK x BB.6S m) and (BB.6S m x BB/OK). Phenotypic data underline that the heterozygous animals showed a higher T1D incidence as a rule, varying between 21 und 74 % depending on the gender of the sibship and the inheritance pattern. Generally, female descendants were better protected from T1D than male descendants. Furthermore, T1D incidence was lower when the SHR allel was left paternally. On the one hand, these facts indicate an interaction of the Iddm4 area of chromosome 6 with the biallelic X-chromosome. Pursuing analyses of the Dlk1 expression prove that a missing paternal SHR allel surprisingly leads to a biallelic expression of the Dlk1 gene. This effect is associated with the loss of Iddm4 mediated T1D protection. In summary, the data of the here presented work allow three conclusions: I By using the two congene BB.6S rat lines no gene could be identified causing the diabetes protection induced by the Iddm4 area in SH rats. II The incidence of diabetes is apparently influenced by the interaction of chromosome 6 and the biallelic X–chromosome. III Here, for the first time in animal experiments, it could be proved that the manifestation of a T1D is characterized by imprinting

The expression of myogenic gene markers during the embryo‐larval‐transition in Pikeperch (Sander lucioperca)

Abstract Pikeperch (Sander lucioperca) has become a species of interest in aquaculture. It is a popular and economically valuable food fish and can produce high numbers of offspring. However, during early development, there are transition phases when high mortality rates concur with growth changes, vital organ transformations and a limited energy budget. Up to now, no study focused on the developmental adaption of muscle tissue in pikeperch, regardless of muscle tissue influencing essential traits such as locomotion and thus the competence to hunt prey and avoid predators. In the present study, therefore, the developmental myogenesis of pikeperch was analysed using specimens from early embryonic to larval development. Myogenic and developmental genes were utilized to gain insights into transcriptomic regulation during these stages by applying a nanofluidic qPCR approach. Result, three phases of myogenic gene expression, during somitogenesis, during the late embryonic development and during the larval development were detected. Increased myostatin expression showed an interim arrest of muscle formation between embryonic and larval myogenesis. Expression patterns of satellite cell gene markers indicated an accumulation of stem cells before myogenesis interruption. The here gained data will help to broaden the knowledge on percid myogenesis and can support pikeperch rearing in aquaculture

An Alternative Promoter in Intron1 of the Renin Gene is Regulated by Glucose Starvation via Serum Response Factor

Background/Aims: Renin is known as a secretory glycoprotein that ultimately leads to angiotensin II generation. In this way renin exerts pro-inflammatory effects and promotes cardiac injury. Additional transcripts have been identified encoding for a cytosolic renin isoform that - in contrast to secretory renin - exhibits cardioprotective effects under ischemic conditions. The promoter of these transcripts is unknown. Methods: Using qRT-PCR and dual-luciferase reporter assay we examined the expression and promotor activity of cytosolic renin as well as the regulation by glucose starvation in H9c2 cardiomyoblasts. Results: We identified a promoter in intron1 of the rat renin gene with two glucose starvation-sensitive regions. One region contains a binding motif for serum response factor (SRF). Under glucose depletion expression of SRF increased prior to cytosolic renin. SRF knock down selectively decreased cytosolic renin expression and attenuated the increase of cytosolic renin expression under glucose depletion. Conclusions: Transcripts encoding for secretory and cytosolic renin are differentially expressed. The low basal expression of cytosolic renin as well as its induction under ischemia-related conditions represents an efficient system regulated in accordance with its previously identified unfavorable effects under control situations but protective effects seen after myocardial infarction or glucose depletion

Overexpression of Transcripts Coding for Renin-b but Not for Renin-a Reduce Oxidative Stress and Increase Cardiomyoblast Survival under Starvation Conditions

A stimulated renin-angiotensin system is known to promote oxidative stress, apoptosis, necrosis and fibrosis. Renin transcripts (renin-b; renin-c) encoding a cytosolic renin isoform have been discovered that may in contrast to the commonly known secretory renin (renin-a) exert protective effects Here, we analyzed the effect of renin-a and renin-b overexpression in H9c2 cardiomyoblasts on apoptosis and necrosis as well as on potential mechanisms involved in cell death processes. To mimic ischemic conditions, cells were exposed to glucose starvation, anoxia or combined oxygen–glucose deprivation (OGD) for 24 h. Under OGD, control cells exhibited markedly increased necrotic and apoptotic cell death accompanied by enhanced ROS accumulation, loss of mitochondrial membrane potential and decreased ATP levels. The effects of OGD on necrosis were exaggerated in renin-a cells, but markedly diminished in renin-b cells. However, with respect to apoptosis, the effects of OGD were almost completely abolished in renin-b cells but interestingly also moderately diminished in renin-a cells. Under glucose depletion we found opposing responses between renin-a and renin-b cells; while the rate of necrosis and apoptosis was aggravated in renin-a cells, it was attenuated in renin-b cells. Based on our results, strategies targeting the regulation of cytosolic renin-b as well as the identification of pathways involved in the protective effects of renin-b may be helpful to improve the treatment of ischemia-relevant diseases

An Alternative Promoter in Intron1 of the Renin Gene is Regulated by Glucose Starvation via Serum Response Factor

Background/Aims: Renin is known as a secretory glycoprotein that ultimately leads to angiotensin II generation. In this way renin exerts pro-inflammatory effects and promotes cardiac injury. Additional transcripts have been identified encoding for a cytosolic renin isoform that - in contrast to secretory renin - exhibits cardioprotective effects under ischemic conditions. The promoter of these transcripts is unknown. Methods: Using qRT-PCR and dual-luciferase reporter assay we examined the expression and promotor activity of cytosolic renin as well as the regulation by glucose starvation in H9c2 cardiomyoblasts. Results: We identified a promoter in intron1 of the rat renin gene with two glucose starvation-sensitive regions. One region contains a binding motif for serum response factor (SRF). Under glucose depletion expression of SRF increased prior to cytosolic renin. SRF knock down selectively decreased cytosolic renin expression and attenuated the increase of cytosolic renin expression under glucose depletion. Conclusions: Transcripts encoding for secretory and cytosolic renin are differentially expressed. The low basal expression of cytosolic renin as well as its induction under ischemia-related conditions represents an efficient system regulated in accordance with its previously identified unfavorable effects under control situations but protective effects seen after myocardial infarction or glucose depletion

X2Rail‐3 -- Deliverable 8.3 -- Protection profile for Adaptable Communication System (ACS) components

This document is a deliverable of the X2RAIL-3 European Project (https://projects.shift2rail.org/s2r_ip2_n.aspx?p=X2RAIL-3).This document provides the first version of the Protection Profile (PP) for an AdaptableCommunication System (ACS) component, the ACS gateway (ACS GW).The scope of the ACS is derived from X2Rail-1, UIC Future Railway MobileCommunication System (FRMCS) and Mission Critical Service group from 3GPP.The technical security requirements for the main network component, the ACS GW, arederived from IEC 62443-4-2 considering security level 3 (SL 3) and X2Rail Shared SecurityServices.Due to the limitation of FRMCS use cases to mission-critical Automatic Train Protection (ATP)and business communication of Wireless internet on-train for passengers, this protectionprofile might not be comprehensive. A future update of the PP is likely due to future mobilecommunication technology research activities, potentially growing FRMCS use cases, andupdates of the specifications given above

X2Rail‐3 -- Deliverable 8.3 -- Protection profile for Adaptable Communication System (ACS) components

This document is a deliverable of the X2RAIL-3 European Project (https://projects.shift2rail.org/s2r_ip2_n.aspx?p=X2RAIL-3).This document provides the first version of the Protection Profile (PP) for an AdaptableCommunication System (ACS) component, the ACS gateway (ACS GW).The scope of the ACS is derived from X2Rail-1, UIC Future Railway MobileCommunication System (FRMCS) and Mission Critical Service group from 3GPP.The technical security requirements for the main network component, the ACS GW, arederived from IEC 62443-4-2 considering security level 3 (SL 3) and X2Rail Shared SecurityServices.Due to the limitation of FRMCS use cases to mission-critical Automatic Train Protection (ATP)and business communication of Wireless internet on-train for passengers, this protectionprofile might not be comprehensive. A future update of the PP is likely due to future mobilecommunication technology research activities, potentially growing FRMCS use cases, andupdates of the specifications given above

Industrie 4.0 - Mitarbeiter einbinden: Fallbeispiele aus der betrieblichen Praxis

Im gemeinsamen Erfahrungs- und Meinungsaustausch der Arbeitsgruppe „Industrie 4.0 - Mitarbeiter einbinden“, in der sich zwölf parallele Forschungsverbundprojekte aus der Ausschreibung „Industrie 4.0 auf den betrieblichen Hallenboden“ engagierten, entstand die gleichnamige Broschüre „Industrie 4.0 - Mitarbeiter einbinden“. Im gemeinschaftlichen Werk, das sich primär an industrielle Anwender richtet, die gegenwärtig oder zukünftig vor ähnlichen Herausforderungen stehen, werden Ansätze, Methoden, Wege und Vorgehensweisen vorgestellt, wie Unternehmen ihre Mitarbeiter bei Industrie 4.0-Technologien eingebunden haben