On noise and single-cell expression dynamics in toxin-driven bacterial competition

Complex microbial communities are composed of a multitude of bacterial strains that interact with each other in many different ways. Stability of such systems is crucial for their long-term survival, especially in fluctuating environments. It is still largely unknown what factors influence bacterial interaction dynamics and how they affect bacterial competition. But, the interaction of strains can be driven by the production of toxins or public goods. Therefore, it is crucial to get further insight into the gene expression dynamics of these compounds in order to understand the development of such complex ecosystems. Factors affecting bacterial competition such as the timing of release of interacting components and the amount being released into the environment have to be studied in order to determine their influence on competition outcome. Additionally, it is unknown how noise in gene expression dynamics of interacting compounds and the resulting release distributions influence bacterial competition. In this study, the plasmid encoded toxin producing ColicinE2 system of the well-known organism Escherichia coli was used as a model system. Bacterial interactions involving this strain are driven by the production and release of a toxin called colicin which kills closely related competitors. Therefore, in this combined experimental and theoretical study, toxin expression dynamics were investigated and how they determine the timing and amount of toxin being released. Additionally, mechanisms of noise control of both, toxin production and release in the ColicinE2 expression system were analyzed. Finally, the influence of stochasticity in single-cell expression dynamics and toxin production on bacterial competition outcome between a colicin producing strain and a toxin sensitive strain were investigated. Using this analysis, I was able to show that both toxin expression dynamics and noise in the ColicinE2 system are mainly controlled by globally acting regulatory proteins such as LexA and CsrA. Regarding CsrA, factors affecting the availability of free CsrA play an important role. Furthermore, I was able to identify single-stranded DNA produced by replication of the toxin producing plasmid as a new, previously unknown regulatory component influencing CsrA abundance in the cell. In addition, I could show that the metabolism of the bacterial cell influences the timing of toxin release, which is in turn correlated to the actual amount of released toxin. Finally, I could show how these toxin expression dynamics affect competition outcome for colicin driven bacterial interaction and could determine the importance of high toxin amounts as well as heterogeneity in toxin release timing for the competitive success of the colicin producing population.Komplexe mikrobielle Gemeinschaften bestehen aus vielen verschiedenen Bakterienstämmen die eine Vielzahl an Interaktionsmöglichkeiten miteinander besitzen. Vor allem in Umgebungen die vielen Schwankungen ausgesetzt sind, ist die Stabilität eines solchen Ökosystems ein wichtiges Überlebenskriterium. Es ist jedoch noch kaum bekannt welche Faktoren die dynamischen Prozesse der bakteriellen Interaktion beeinflussen und wie sich die dadurch veränderten Prozesse auf den bakteriellen Wettbewerb auswirken. Die Interaktion von verschiedenen Bakterienstämmen kann z.B. durch die Produktion und Abgabe von allgemein nutzbaren Substanzen (z.B. Proteine,...) erfolgen. Daher ist es wichtig die Produktionsdynamiken solcher Substanzen in einzelnen Zellen (mikroskopische Interaktionsebene) zu untersuchen um ihren Einfluss auf die Zusammensetzung komplexer Ökosysteme (makroskopische Interaktionsebene) verstehen zu können. Dabei ist eine quantitative Analyse spezifischer Interaktionsparameter von besonderem Interesse, wie z.B. ihre Produktionsmenge und ihr Abgabezeitpunkt, um zu verstehen wie sich Änderungen dieser Parameter auf das Wettbewerbsergebnis zwischen den Interaktionspartnern auswirken. Ein weiterer wichtiger Faktor, der diese Parameter und damit den bakteriellen Wettbewerb beeinflussen kann ist stochastisches Rauschen. In dieser Arbeit wird das plasmidkodierte ColicinE2 System von Escherichia coli als Modellsystem genutzt um oben genannte Aspekte zu studieren. Ein wichtiger Faktor der Interaktionen bei denen ein solcher Stamm beteiligt ist, ist die Produktion und Abgabe eines Toxins (Colicin genannt), das nahe verwandte Bakterien tötet. Daher wird in dieser Arbeit in einer Kombination aus experimenteller und theoretischer Analyse untersucht welchen Einfluss Einzelzellparameter wie der Zeitpunkt der Toxinabgabe und die Menge des abgegebenen Toxins auf den makroskopischen bakteriellen Wettbewerb (Populationsebene) haben. Des Weiteren wird analysiert welche regulatorische Mechanismen des ColicinE2 Systems das Rauschen von Toxinproduktionsmenge und Abgabezeitpunkt des Toxins kontrollieren. Abschließend wird derWettbewerb zwischen einem toxinproduzierenden C-Stamm und einem toxinsensitiven S-Stamm untersucht und wie sich die zuvor untersuchten Expressionsdynamiken der einzelnen Zellen und Stochastizität der Genexpression auf den Wettbewerb zwischen dem C-Stamm und dem S-Stamm auswirken. Anhand dieser Untersuchungen konnte ich zeigen, dass die Toxinexpressionsdynamik und deren Rauschen im ColicinE2 System hauptsächlich durch globale Regulatoren wie die Proteine LexA oder CsrA kontrolliert werden. Im Bezug auf CsrA sind vor allem die Verfügbarkeit von freiem CsrA und welche Regulationskomponenten diese Verfügbarkeit steuern wichtig. Dabei konnte ich einzelsträngige DNA, die bei der Replikation des Colicinplasmids entsteht, als neuen Regulationsfaktor für freies CsrA identifizieren. Außerdem konnte ich zeigen, dass sich der Metabolismus der Bakterienzelle auf die Dynamiken der Toxinproduktion auswirkt und der Abgabezeitpunkt des Toxins mit der abgegebenen Colicinmenge korreliert. Des Weiteren konnte ich zeigen, dass sich die Toxinexpressionsdynamiken auf das Resultat des bakteriellen Wettbewerbs auswirken und dass sowohl die abgegebene Toxinmenge als auch eine zeitlich heterogene Toxinabgabe wichtig für den Wettbewerbserfolg der colicinproduzierenden Population sind

Lasten mitokondriaalisten kardiomyopatioiden molekyylitausta

Mitochondrial diseases comprise a heterogeneous group of disorders. Disease can manifest with various symptoms and can affect almost every tissue or organ of the body. However, organs with a high-energy demand such as skeletal muscle, brain and heart are affected most often. Most patients with a mitochondrial disease have a dysfunctional respiratory chain (RC) that impairs sufficient ATP production, which is needed to maintain basic bodily functions. Mitochondria are under control of both the nuclear and mitochondrial genomes, which makes the identification of pathogenic disease mutations for an individual patient even more challenging. Mitochondrial DNA (mtDNA) mutations are a frequent cause of mitochondrial disease. Presently, more than 250 pathogenic mtDNA point mutations have been described and published. Seizures, myoclonus, myopathy, cardiomyopathy, retinopathy, and sensorineural hearing loss are common clinical features. The aim of this PhD study was to identify causes of mitochondrial disease in adult and child patients in Finland, with special emphasis on infantile disorders. This study utilized material from 114 patients, adults and children. For 69 patients mitochondrial DNA sequencing was performed. 101 patients were screened for nuclear candidate genes (TK2, AARS2, POLG2, DGUOK). Samples from 33 patients were analyzed for respiratory chain deficiencies by blue native electrophoresis. In addition, we used the novel exome sequencing method for one patient to identify the underlying genetic defect. With a combination of these methods 17 out of the 114 patients in this study obtained a molecular diagnosis. Although pathogenic mtDNA point mutations account for a substantial amount of mitochondrial diseases such as MELAS, Leigh syndrome and MERRF, most mitochondrial disorders arise due to nuclear defects in either genes encoding one of the respiratory chain complex subunits, directly affecting the RC, or in a gene which encodes one of the many mitochondrial proteins that are important for mitochondrial function, such as the maintenance of mitochondrial DNA. We have identified eight cases with mitochondrial depletion syndrome (MDS) with two mutations identified within the TK2 gene (c.739 C > T, p.R172W and c.898 C > T, p.R225W), of which p.R172W is novel. Furthermore, this study shows that the novel p.R172W mutation has a Finnish founder. During the development of this thesis it has become apparent that the heart can be the primary organ affected by mitochondrial dysfunction. Mitochondrial cardiomyopathy (CMP) in children is especially devastating as early onset mitochondrial CMP has a poor prognosis with a very high mortality rate. We have utilized the recent development of new analytical techniques for DNA sequencing to study selected patients with early onset fatal CMP. Exome analysis is a breakthrough in the era of molecular genetics and has made it possible to selectively sequence the protein coding regions of the human genome. We sequenced the exome of one patient with infantile onset hypertrophic cardiomyopathy. We identified a homozygous missense mutation c.1777C>T, p.R592W in the AARS2 gene, encoding a putative mitochondrial alanyl-tRNA synthetase (MT-ALARS). A child from another family, who died perinatally of hypertrophic cardiomyopathy, had the same mutation, which was compound heterozygous with another missense mutation c.467T>G, p.L155R. This thesis work has resulted in identifying 9 molecular causes for mitochondrial disease for 16 Finnish patients and one Estonian patient of which four were previously unpublished and one has a novel phenotype. As well as providing molecular diagnosis for patients with mitochondrial disease this study also investigated the basic molecular mechanisms of the individual mutations and enhanced the clinical description of the diseases, making it easier for clinicians in the future to pinpoint the genetic cause for their patient s disease.Mitokondriotaudit ovat monimuotoinen ryhmä sairauksia, joissa oireita voi esiintyä lähes kaikissa elimistön kudoksissa ja elimissä. Vaikeimmat oireet ilmenevät usein kudoksissa, jotka käyttävät eniten energiaa, kuten lihaksissa, aivoissa ja sydämessä. Mitokondriotaudeista kärsivillä potilailla solun hengitysketju on viallinen ja ATP:n eli energian tuotto on häiriintynyt. Sekä tuman että mitokondrion geenit säätelevät mitokondrioiden normaalia toimintaa, joten tautia aiheuttavien geenivirheiden eli mutaatioiden tunnistaminen on hankalaa. Monen mitokondriotaudin taustalla on mutaatio mitokondrion DNA:ssa (mtDNA), ja tautia aiheuttavia mtDNA:n pistemutaatioita on tällä hetkellä tunnistettu ja julkaistu jo yli 250. Näiden tautien yleisimmät kliiniset oireet ovat kouristukset, myoklonus, lihasheikkous, sydänlihasheikkous, retinopatia ja sensorineuraalinen kuulovamma. Tämän väitöskirjan tavoitteena oli tunnistaa mitokondriotautien taustalla olevia geenivirheitä sekä aikuis- että lapsipotilaissa, erityisesti varhaislapsuudessa ilmenevissä taudeissa. Tutkittavana oli 114 potilasta. Näistä 69:n potilaan mtDNA sekvensoitiin ja 101:n potilaan tuman DNA:sta tutkittiin mitokondriotauteja aiheuttavat kandidaattigeenit (TK2, AARS2, POLG2, DGUOK), lisäksi 33:n potilaan näytteistä analysoitiin hengitysketjun toiminta. Yhden potilaan kaikki proteiineja koodaavat tuman genomin alueet tutkittiin uuden eksomisekvensointi-menetelmän avulla. Näiden tutkimusten avulla 114:sta potilaasta 17 sai molekyylitason diagnoosin. Vaikka lukuisia mtDNA:n virheitä on löydetty ja julkaistu, useimmat mitokondriotaudit johtuvat muutoksista tuman koodaamissa geeneissä. Tuman geenivirhe voi vaikuttaa joko hengitysketjun proteiineihin ja näin ollen suoraan hengitysketjun toimintaan, tai muiden mitokondrion normaaliin toimintaan vaikuttavien proteiinien, kuten mtDNA:n ylläpidosta huolehtivien proteiinien toimintaan. Tässä työssä tunnistimme kahdeksan mitokondriaalista depleetiosyndroomaa (MDS), jossa mtDNA:n määrä vähenee. Näistä kahdessa tapauksessa mutaatio löytyi TK2-geenistä (c.739 C > T, p.R172W ja c.898 C > T, p.R225W). Näistä p.R172W on aiemmin tunnistamaton muutos, jonka osoitimme olevan suomalainen valtamutaatio. Tämän tutkimuksen teon aikana sydänoireet nousivat entistä vahvemmin esiin mitokondriopotilaiden primaarisena ongelmana. Erityisesti lasten mitokondriaalinen sydänlihasheikkous on vaikea tauti, jossa on huono ennuste ja korkea kuolleisuus. Eksomi-analyysi on uusi menetelmä, jolla voidaan sekvensoida kaikki proteiineja koodaavat alueet potilaan tuman genomista. Tässä tutkimuksessa eksomisekvensoinnilla analysoitiin yksi näyte lapsipotilaasta, jonka hypertrofinen sydänlihasheikkous johti kuolemaan varhaislapsuudessa. Tunnistimme potilaalta mitokondriaalista alanyyli-tRNA-syntetaasia (MT-ALARS) koodittavassa AARS2-geenissä homotsygoottisen missense-mutaation, c1777C > T, p.R592W. Sama mutaatio löytyi myös toisen varhaislapsuudessa sydänlihasheikkouteen menehtyneen lapsen näytteestä. Tällä potilaalla muutos oli heterotsygoottisessa muodossa ja esiintyi yhdessä c.467T > G, p.L155R muutoksen kanssa. Tämä väitöskirjatutkimus johti yhdeksän molekyylitason muutoksen löytymiseen 16 suomalaisen ja yhden virolaisen mitokondriopotilaan perimästä. Neljä näistä muutoksista oli ennen tuntemattomia ja yhden potilaan taudinkuva oli ennen kuvaamaton. Molekyylidiagnostiikan lisäksi tutkimus paransi tautien kliinistä kuvausta, edesauttaen kliinikoiden työtä tulevaisuudessa

Dynamics of ColicinE2 production and release determine the competitive success of a toxin-producing bacterial population

The release of toxins is one mechanism used by bacterial species to establish dominance over competitors, but how the dynamics of toxin expression determine the competitive success of a toxin-producing population is largely unknown. Here, we investigate how the expression dynamics of ColicinE2 - a toxic bacteriocin - affect competition between toxin-producing and toxin-sensitive strains of Escherichia coli. We demonstrate that, in addition to genetic modifications in the toxin expression system, alterations of the growth medium can be used to modulate the timing of toxin production and the amount of toxin released. Thus cells that release the toxin at later times can accumulate more colicin. In experiments, we found that delaying toxin release does not significantly alter competition outcome. However, our theoretical analysis allowed us to assess the relative contributions of release time and toxin level to the competitive success of the producer strain, that might counteract each other in experiments. The results reveal that the importance of delaying toxin release lies in increasing the toxin amount. This is a more effective strategy for the toxin-producing strain than prompt discharge of the colicin. In summary, our study shows how the toxin release dynamics influence the competitive success of the toxin-producing bacterial population

CsrA and its regulators control the time-point of ColicinE2 release in Escherichia coli

The bacterial SOS response is a cellular reaction to DNA damage, that, among other actions, triggers the expression of colicin - toxic bacteriocins in Escherichia coli that are released to kill close relatives competing for resources. However, it is largely unknown, how the complex network regulating toxin expression controls the time-point of toxin release to prevent premature release of inefficient protein concentrations. Here, we study how different regulatory mechanisms affect production and release of the bacteriocin ColicinE2 in Escherichia coli. Combining experimental and theoretical approaches, we demonstrate that the global carbon storage regulator CsrA controls the duration of the delay between toxin production and release and emphasize the importance of CsrA sequestering elements for the timing of ColicinE2 release. In particular, we show that ssDNA originating from rolling-circle replication of the toxin-producing plasmid represents a yet unknown additional CsrA sequestering element, which is essential in the ColicinE2-producing strain to enable toxin release by reducing the amount of free CsrA molecules in the bacterial cell. Taken together, our findings show that CsrA times ColicinE2 release and reveal a dual function for CsrA as an ssDNA and mRNA-binding protein, introducing ssDNA as an important post-transcriptional gene regulatory element

Effects of stochasticity and division of labor in toxin production on two-strain bacterial competition in Escherichia coli

In phenotypically heterogeneous microbial populations, the decision to adopt one or another phenotype is often stochastically regulated. However, how this stochasticity affects interactions between competing microbes in mixed communities is difficult to assess. One example of such an interaction system is the competition of an Escherichia coli strain C, which performs division of labor between reproducers and self-sacrificing toxin producers, with a toxin-sensitive strain S. The decision between reproduction or toxin production within a single C cell is inherently stochastic. Here, combining experimental and theoretical approaches, we demonstrate that this stochasticity in the initial phase of colony formation is the crucial determinant for the competition outcome. In the initial phase (t < 12h), stochasticity influences the formation of viable C clusters at the colony edge. In the subsequent phase, the effective fitness differences (set primarily by the degree of division of labor in the C strain population) dictate the deterministic population dynamics and consequently competition outcome. In particular, we observe that competitive success of the C strain is only found if (i) a C edge cluster has formed at the end of the initial competition phase and (ii) the beneficial and detrimental effects of toxin production are balanced, which is the case at intermediate toxin producer fractions. Our findings highlight the importance of stochastic processes during the initial phase of colony formation, which might be highly relevant for other microbial community interactions in which the random choice between phenotypes can have long-lasting consequences for community fate

Organisational Health Services Research in Germany:A Scoping Review of Conference Abstracts

Background Health Services Research (HSR) is a growing field in Germany, in which Organisational Health Services Research (OHSR) has emerged as a subfield. The aim of this scoping review was to provide an overview of the field of OHSR within HSR in Germany and to map systematically original contributions by describing the organisational setting, the research design, the research objectives and the theoretical underpinning.Methods A scoping review examined published abstracts from the 19th German Conference on Health Services Research 2020. Abstracts were included if (1) health care organisations, subunits or organisational processes were mentioned as research objects, and (2) if at least one out of five research perspectives from a recent German definition of OHSR was addressed. After intensive pilot screenings within a group of nine researchers, all abstracts were screened independently in three review teams with three researchers each, and data from included abstracts were extracted using content analysis based on a self-developed detailed coding scheme.Results Out of n=468 identified abstracts in German (84%) or English (16%) language, n=138 (29.5%) abstracts were included. The majority of included abstracts addressed acute care in hospitals (34.8%), reported results from observational studies (59.4%) and collected primary data (69.6%). There was a slightly higher use of quantitative (32.6%) than qualitative (24.6%) research methods with a considerable number of studies using more than one method (31.9%). An explicit reference to theory was made in 7.2% and 17.4% used the term ‘organisation’ or related terms explicitly in their abstract.Discussion This review provides a systematic but preliminary overview of the scope to which HSR in Germany addresses OHSR. The organisational perspective is considered extensively in HSR abstracts, but mostly implicitly. The research is reported largely free of theory which can reduce their explanatory power. Therefore, a research agenda, more awareness as well as education and better conceptualisation of OHSR topics within German HSR are needed

Digital Online Patient Informed Consent for Anesthesia before Elective Surgery-Recent Practice in Europe

BACKGROUND: Digitalization in the health system is a topic that is rapidly gaining popularity, and not only because of the current pandemic. As in many areas of daily life, digitalization is becoming increasingly important in the medical field amid the exponential rise in the use of computers and smartphones. This opens up new possibilities for optimizing patient education in the context of anesthesia. The main aim of this study was to assess the implementation of remote consent in Europe.METHODS: An online survey entitled "Digital online Patient Informed Consent for Anesthesia before Elective Surgery. Recent practice in Europe," with a total of 27 questions, was sent by the European Society of Anesthesiology and Intensive Care (ESAIC) to their members in 47 European countries. To assess the effect of the economy on digitalization and legal status with regard to anesthesia consent, data were stratified based on gross domestic product per capita (GDPPC).RESULTS: In total, 23.1% and 37.2% of the 930 participants indicated that it was possible to obtain consent online or via telephone, respectively. This observation was more often reported in countries with high GDPPC levels than in countries with low GDPPC levels. Furthermore, 27.3% of the responses for simple anesthesia, 18.7% of the responses for complex anesthesia, and 32.2% of the responses for repeated anesthesia indicated that remote consent was in accordance with the law, and this was especially prevalent in countries with high GDPPC. Concerning the timing of consent, patients were informed at least one day before in 67.1% of cases for simple procedures and in 85.2% of cases for complex procedures.CONCLUSION: Even European countries with high GDPPC use remote informed consent only in a minority of cases, and most of the time for repeated anesthetic procedures. This might reflect the inconsistent legal situation and inhomogeneous medical technical structures across Europe.</p

Atrial fibrillation is poorly tolerated by patients with hypertrophic concentric cardiomyopathy caused by mitochondrial tRNALeu (UUR) mutations

Peer reviewe

Psychosocial stress induction in vivo vs. in virtuo and the influence of a health app on the acute stress reaction in youths: a study protocol for a randomized controlled trial

Background Acute and everyday stress is substantial for the development of mental and physical diseases, therefore it is crucial to get a better understanding of its pathogenesis. Different methods (e.g., Ambulatory Assessment) and stress reactivity paradigms (e.g., Trier Social Stress Test / TSST) in laboratory settings are often used to investigate basic mechanisms of this process. Due to the technological progress of the last years and especially due to children and adolescents growing up with it, the application of these developments in clinical research is reasonable. The aim of this project is to successfully transfer the TSST for children and adolescents into the virtual world, which will be compared to a real TSST situation. Physiological and psychological stress reactions will be analyzed in order to assess similarities and differences. Moreover, it will be investigated whether a Heart Coherence Exercise (HCE) has a stronger influence on coping with acute stress compared to Natural Relaxation (NR). Methods This single-center experimental study will examine acute and everyday stress and coping processes in eighty-four healthy children and adolescents between the ages of 11 and 17. For everyday stress, different parameters (e.g., hormonal profiles and mood ratings) as well as a history of stressful life events and utilized coping methods will be recorded and a relaxation exercise will be practiced on a smartphone over 2 days. Regarding the acute stress reaction, the participants will be confronted either with the virtual or the real version of the TSST, followed by the trained relaxation exercise (HCE vs. NR). Physiological (e.g., cortisol and heart rate) and psychological stress markers (e.g., mood and gaze behavior) will be recorded continuously. Discussion Studies are sparse using a virtual version of the TSST in children and adolescents. A successful virtual TSST would constitute an economical variant, which would also make it easier to administer it in clinical or population-based samples. Effective ambulatory relaxation exercises would be a useful addition to clinical treatment approaches

Levels of the Autophagy-Related 5 Protein Affect Progression and Metastasis of Pancreatic Tumors in Mice

[Background and Aims]: Cells in pancreatic ductal adenocarcinoma (PDAC) undergo autophagy, but its effects vary with tumor stage and genetic factors. We investigated the consequences of varying levels of the autophagy related 5 (Atg5) protein on pancreatic tumor formation and progression. [Methods]: We generated mice that express oncogenic Kras in primary pancreatic cancer cells and have homozygous disruption of Atg5 (A5;Kras) or heterozygous disruption of Atg5 (A5+/–;Kras), and compared them with mice with only oncogenic Kras (controls). Pancreata were analyzed by histology and immunohistochemistry. Primary tumor cells were isolated and used to perform transcriptome, metabolome, intracellular calcium, extracellular cathepsin activity, and cell migration and invasion analyses. The cells were injected into wild-type littermates, and orthotopic tumor growth and metastasis were monitored. Atg5 was knocked down in pancreatic cancer cell lines using small hairpin RNAs; cell migration and invasion were measured, and cells were injected into wild-type littermates. PDAC samples were obtained from independent cohorts of patients and protein levels were measured on immunoblot and immunohistochemistry; we tested the correlation of protein levels with metastasis and patient survival times. [Results]: A5+/–;Kras mice, with reduced Atg5 levels, developed more tumors and metastases, than control mice, whereas A5;Kras mice did not develop any tumors. Cultured A5+/–;Kras primary tumor cells were resistant to induction and inhibition of autophagy, had altered mitochondrial morphology, compromised mitochondrial function, changes in intracellular Ca2+ oscillations, and increased activity of extracellular cathepsin L and D. The tumors that formed in A5+/–;Kras mice contained greater numbers of type 2 macrophages than control mice, and primary A5+/–;Kras tumor cells had up-regulated expression of cytokines that regulate macrophage chemoattraction and differentiation into M2 macrophage. Knockdown of Atg5 in pancreatic cancer cell lines increased their migratory and invasive capabilities, and formation of metastases following injection into mice. In human PDAC samples, lower levels of ATG5 associated with tumor metastasis and shorter survival time. [Conclusions]: In mice that express oncogenic Kras in pancreatic cells, heterozygous disruption of Atg5 and reduced protein levels promotes tumor development, whereas homozygous disruption of Atg5 blocks tumorigenesis. Therapeutic strategies to alter autophagy in PDAC should consider the effects of ATG5 levels to avoid the expansion of resistant and highly aggressive cells.This study was supported in part by the Mildred-Scheel-Professur der Deutschen Krebshilfe 111464, DFG AL 1174/6-1 to H.A., DFG DI 2299/1-1 to K.N.D., DFG SFB1321 (S01) to K.S. and W.W., and the German Federal Ministry of Education and Research to the German Center for Diabetes Research (DZD e.V.) to J.A