Analyses of the impact of DNA methylation and methylation-modifying enzymes in different cell systems

Im Rahmen der vorliegenden Arbeit wurde die Bedeutung der DNA-Methylierung und methylierungsmodulierender Enzyme in verschiedenen Geweben untersucht. Ein charakteristisches Merkmal der Alzheimer-Krankheit sind reduzierte Somatostatin- und Somatostatin-Rezeptor-Level im Gehirn, die mit dem Ausbruch der sporadischen Form der Krankheit in Verbindung gebracht werden. Um zu klären, ob die Reduktion des Peptidhormons Somatostatin (SST) und des im Kortex von Alzheimer-Patienten am stärksten reduzierten Rezeptor-Subtyps 4 (SSTR4) auf eine Hypermethylierung der entsprechenden Promotorregion zurückzuführen sein könnte, wurden diese Regionen mittels Bisulfit-Sequenzierung in Zellen von Alzheimer-Patienten und nicht-dementen Normalpersonen analysiert. Die ähnlichen Methylierungslevel dieser Promotorregionen in verschiedenen Gehirnregionen von Patienten und nicht-dementen Normalpersonen unterstützen jedoch nicht die Hypothese eines Zusammenhangs von DNA-Hypermethylierung und den reduzierten SST- und SSTR4-Leveln einer Alzheimer-Erkrankung. Es fand sich jedoch ein Trend zu erhöhter Methylierung des SST-Promotors mit erhöhtem Alter. In einem weiteren Teil dieser Arbeit wurden drei unabhängige transgene Linien eines Mausmodells zur ubiquitären Überexpression der somatischen Form der DNA Methyltransferase 1, Dnmt1s, untersucht. Anhand dieses Modells sollten ursprünglich die Konsequenzen einer solchen Überexpression analysiert werden. Es stellte sich heraus, dass transgene Tiere zwar eine bis zu 229-fach erhöhte Dnmt1-mRNA-Expression aufwiesen, jedoch keine erhöhten Dnmt1-Proteinlevel besaßen. Reportergenversuche weisen auf eine reduzierte Translationseffizienz des Transgenkonstrukts hin, die jedoch nicht allein das Ausbleiben erhöhter Proteinlevel im Mausmodell erklären kann. Es bleibt offen, ob die statischen Proteinlevel auf spezifische Eigenschaften des Transgens oder auf eine stringente Kontrolle der Dnmt1-Level in vivo zurückzuführen sind. Im Hauptteil der vorliegenden Arbeit wurde der Einfluss moderat veränderter TET-Dioxygenase-Level auf HEK293-Zellen untersucht. Die Überexpression von TET1 führte zu erhöhten globalen 5hmC-Leveln und verursachte eine leichte Hypomethylierung von Promotoren, Genen und CpG-islands. Im Gegensatz dazu führte der simultane Knockdown von TET1, TET2 und TET3 zu verringerten globalen 5hmC-Leveln und einer leichten Hypermethylierung der genannten Regionen. Auch wenn die Überexpressions- bzw. Knockdown-Experimente entgegengesetzte Methylierungsveränderungen hervorriefen, gab es nur eine kleine Anzahl reziproker Veränderungen. Die Methylierungsveränderungen beider Experimente geschahen mit unterschiedlicher Präferenz in Abhängigkeit vom endogenen Methylierungsgrad und der Stärke der Genexpression. Sowohl nach der TET1-Überexpression als auch nach dem TET-Knockdown wurden geringfügig hoch- und herunterregulierte Gene identifiziert. Die Expressionsveränderungen konnten jedoch nicht mit Methylierungsveränderungen der Genpromotoren in Verbindung gebracht werden und sind womöglich auf nicht-katalytische Funktionen der TET-Enzyme zurückzuführen. Die Kartierung der 5hmC-DNA-Modifikation im genomweiten Maßstab zeigte, dass die TET1-Überexpression die genomweite 5mC-Oxidation ohne einen erkennbaren Verteilungsschwerpunkt induzierte. Detailliertere Analysen offenbarten den Trend einer unterschiedlichen Suszeptibilität für eine Oxidation durch TET1, der abhängig vom endogenen 5hmC-Vorkommen war und in allen untersuchten genomischen Elementen und Regionen vorlag. Zusammengenommen weisen die in dieser Arbeit durchgeführten Untersuchungen auf eine ausgedehnte Rolle der TET-Familie bei der Regulation der DNA-Methylierungslevel hin.This thesis focused on elucidating the relevance of DNA methylation and methylation-modulating enzymes in different tissues. Reduced levels of somatostatin (SST) and its receptors represent pathological characteristics of Alzheimer’s disease (AD) brains, and deregulated somatostatin signaling has been proposed to play a major role in the development of late-onset sporadic AD. To investigate whether the reduced levels of somatostatin and its most affected receptor subtype 4 (SSTR4) could be caused by DNA hypermethylation of the respective promoter regions, these regions were analyzed in cells from the neocortex of AD patients and non-demented controls using bisulfite sequencing. Tissue samples from two areas of the neocortex were analyzed, each separated into cortical gray and infracortical white matter. However, the similar methylation levels observed in the SST and SSTR4 promoter regions in all samples from AD patients and non-demented controls are not supportive of a causal role of DNA hypermethylation with respect to altered somatostatin signaling in AD. But there was a trend toward increased SST promoter methylation with increasing age. Consequences of ubiquitous overexpression of the somatic form of the DNA methyltransferase 1, Dnmt1s, were supposed to be studied in a transgenic mouse model. Three independent lines of this model were analyzed. Transgenic mice ubiquitously overexpressed Dnmt1 mRNA up to 229-fold, but were lacking increased protein levels. Although reporter gene assays indicated a reduced translation efficiency of the transgene construct, this reduction was not strong enough to solely account for the lack of protein increase in the mouse model. It remains unclear whether the static protein levels are due to intrinsic features of the transgene or a stringent control of Dnmt1 levels in vivo. The main part of this thesis shows that altering TET dioxygenase levels within physiological range can affect DNA methylation levels of HEK293 cells. TET1 overexpression led to increased global 5hmC levels and mild hypomethylation of promoters, gene bodies and CpG islands. Conversely, the simultaneous knockdown of TET1, TET2 and TET3 led to decreased global 5hmC levels and was accompanied by mild hypermethylation of above-mentioned regions. Although the overexpression and knockdown studies caused opposite methylation changes, most changes were non-reciprocal and occurred with different preference depending on endogenous methylation and gene expression levels. Both, TET1 overexpression and TET knockdown, led to slight transcriptional up- and downregulation. However, the expression changes could not be linked to methylation changes of the corresponding gene promoters and were probably caused by non-catalytic functions of the TET enzymes. 5hmC profiling performed on a genome-wide scale showed that TET1 overexpression induced 5mC oxidation without a distribution bias. Detailed analyses revealed a trend of different susceptibility to TET1-induced oxidation that was linked to endogenous 5hmC content and found in all genomic elements and regions. Taken together, the experiments conducted point to a widespread role of the TET family in regulating DNA methylation levels

Sidekick compilation with xDSL

Traditionally, compiler researchers either conduct experiments within an existing production compiler or develop their own prototype compiler; both options come with trade-offs. On one hand, prototyping in a production compiler can be cumbersome, as they are often optimized for program compilation speed at the expense of software simplicity and development speed. On the other hand, the transition from a prototype compiler to production requires significant engineering work. To bridge this gap, we introduce the concept of sidekick compiler frameworks, an approach that uses multiple frameworks that interoperate with each other by leveraging textual interchange formats and declarative descriptions of abstractions. Each such compiler framework is specialized for specific use cases, such as performance or prototyping. Abstractions are by design shared across frameworks, simplifying the transition from prototyping to production. We demonstrate this idea with xDSL, a sidekick for MLIR focused on prototyping and teaching. xDSL interoperates with MLIR through a shared textual IR and the exchange of IRs through an IR Definition Language. The benefits of sidekick compiler frameworks are evaluated by showing on three use cases how xDSL impacts their development: teaching, DSL compilation, and rewrite system prototyping. We also investigate the trade-offs that xDSL offers, and demonstrate how we simplify the transition between frameworks using the IRDL dialect. With sidekick compilation, we envision a future in which engineers minimize the cost of development by choosing a framework built for their immediate needs, and later transitioning to production with minimal overhead

Loss of long-term potentiation at hippocampal output synapses in experimental temporal lobe epilepsy

Patients suffering from temporal lobe epilepsy (TLE) show severe problems in hippocampus dependent memory consolidation. Memory consolidation strongly depends on an intact dialog between the hippocampus and neocortical structures. Deficits in hippocampal signal transmission are known to provoke disturbances in memory formation. In the present study, we investigate changes of synaptic plasticity at hippocampal output structures in an experimental animal model of TLE. In pilocarpine-treated rats, we found suppressed long-term potentiation (LTP) in hippocampal and parahippocampal regions such as the subiculum and the entorhinal cortex (EC). Subsequently we focused on the subiculum, serving as the major relay station between the hippocampus proper and downstream structures. In control animals, subicular pyramidal cells express different forms of LTP depending on their intrinsic firing pattern. In line with our extracellular recordings, we could show that LTP could only be induced in a minority of subicular pyramidal neurons. We demonstrate that a well-characterized cAMP-dependent signaling pathway involved in presynaptic forms of LTP is perturbed in pilocarpine-treated animals. Our findings suggest that in TLE, disturbances of synaptic plasticity may influence the information flow between the hippocampus and the neocortex

The association of intra-therapeutic heterogeneity of somatostatin receptor expression with morphological treatment response in patients undergoing PRRT with [177Lu]-DOTATATE

AIM: Purpose of this study was to evaluate the association of the spatial heterogeneity (asphericity, ASP) in intra-therapeutic SPECT/ CT imaging of somatostatin receptor (SSR) positive metastatic gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN) for morphological treatment response to peptide receptor radionuclide therapy (PRRT). Secondly, we correlated ASP derived form a pre-therapeutic OctreoScan (ASP[In]) and an intra-therapeutic [177Lu]-SPECT/CT (ASP[Lu]). MATERIALS AND METHODS: Data from first therapy cycle [177Lu-DOTA0-Tyr3]octreotate ([177Lu]-DOTATATE)-PRRT was retrospectively analyzed in 33 patients (m = 20; w = 13; median age, 72 [46-88] years). The evaluation of response to PRRT was performed according to RECIST 1.1 in responding lesions [RL (SD, PR, CR), n = 104] and non-responding lesions [NRL (PD), n = 27]. The association of SSR tumor heterogeneity with morphological response was evaluated by Kruskal-Wallis test and receiver operating characteristic curve (ROC). The optimal threshold for separation (RL vs. NRL) was calculated using the Youden-index. Relationship between pre- and intra-therapeutic ASP was determined with Spearman's rank correlation coefficient (ρ) and Bland-Altman plots. RESULTS: A total of 131 lesions (liver: n = 59, lymph nodes: n = 48, bone: n = 19, pancreas: n = 5) were analyzed. Lesions with higher ASP values showed a significantly poorer response to PRRT (PD, median: 11.3, IQR: 8.5-15.5; SD, median: 3.4, IQR: 2.1-4.5; PR, median 1.7, IQR: 0.9-2.8; CR, median: 0.5, IQR: 0.0-1.3); Kruskal-Wallis, p5.45% (sensitivity 96% and specificity 82%). The correlation coefficient of pre- and intra-therapeutic ASP revealed ρ = 0.72 (p <0.01). The mean absolute difference between ASP[In] and ASP[Lu] was -0.04 (95% Limits of Agreement, -6.1-6.0). CONCLUSION: Pre- and intra-therapeutic ASP shows a strong correlation and might be an useful tool for therapy monitoring

Minimally Invasive Vacuum-Assisted Closure Therapy With Instillation (Mini-VAC-Instill) for Pleural Empyema

Enthusiasm for minimally invasive thoracic surgery is increasing. Thoracoscopy plays a significant therapeutic role in the fibrinopurulent stage (stage II) of empyema, in which loculated fluid cannot often be adequately drained by chest tube alone. For some debilitated and septic patients, further procedures such as open-window thoracostomy (OWT) with daily wound care or vacuum-assisted closure (VAC) therapy are necessary. In the present article, we propose a new option of minimally invasive VAC therapy including a topical solution of the empyema without open-window thoracostomy (Mini-VAC-instill). Three patients who underwent surgery using this technique are also presented. The discussion is focused on the advantages and disadvantages of the approach

Stroma AReactive Invasion Front Areas (SARIFA) proves prognostic relevance in gastric carcinoma and is based on a tumor–adipocyte interaction indicating an altered immune response

Background Recently, we presented Stroma AReactive Invasion Front Areas (SARIFA) as a new histomorphologic negative prognostic biomarker in gastric cancer. It is defined as direct contact between tumor cells and fat cells. The aim of this study was to further elucidate the underlying genomic, transcriptional, and immunological mechanisms of the SARIFA phenomenon. Methods To address these questions, SARIFA was classified on H&E-stained tissue sections of three cohorts: an external cohort (n = 489, prognostic validation), the TCGA-STAD cohort (n = 194, genomic and transcriptomic analysis), and a local cohort (n = 60, digital spatial profiling (whole transcriptome) and double RNA in situ hybridization/immunostaining of cytokines). Results SARIFA status proved to be an independent negative prognostic factor for overall survival in an external cohort of gastric carcinomas. In TCGA-STAD cohort, SARIFA is not driven by distinct genomic alterations, whereas the gene expression analyses showed an upregulation of FABP4 in SARIFA-positive tumors. In addition, the transcriptional regulations of white adipocyte differentiation, triglyceride metabolism, and catabolism were upregulated in pathway analyses. In the DSP analysis of SARIFA-positive tumors, FABP4 and the transcriptional regulation of white adipocyte differentiation were upregulated in macrophages. Additionally, a significantly lower expression of the cytokines IL6 and TNFα was observed at the invasion front. Conclusions SARIFA proves to be a strong negative prognostic biomarker in advanced gastric cancer, implicating an interaction of tumor cells with tumor-promoting adipocytes with crucial changes in tumor cell metabolism. SARIFA is not driven by tumor genetics but is very likely driven by an altered immune response as a causative mechanism

Symmetric hyperbolic systems in algebras of generalized functions and distributional limits

We study existence, uniqueness, and distributional aspects of generalized solutions to the Cauchy problem for first-order symmetric (or Hermitian) hyperbolic systems of partial differential equations with Colombeau generalized functions as coefficients and data. The proofs of solvability are based on refined energy estimates on lens-shaped regions with spacelike boundaries. We obtain several variants and also partial extensions of previous results and provide aspects accompanying related recent work by C. Garetto and M. Oberguggenberger

Commensal microbiota stimulate systemic neutrophil migration through induction of Serum amyloid A: Microbiota regulate systemic neutrophil function

Neutrophils serve critical roles in inflammatory responses to infection and injury, and mechanisms governing their activity represent attractive targets for controlling inflammation. The commensal microbiota is known to regulate the activity of neutrophils and other leucocytes in the intestine, but the systemic impact of the microbiota on neutrophils remains unknown. Here we utilized in vivo imaging in gnotobiotic zebrafish to reveal diverse effects of microbiota colonization on systemic neutrophil development and function. The presence of a microbiota resulted in increased neutrophil number and myeloperoxidase expression, and altered neutrophil localization and migratory behaviours. These effects of the microbiota on neutrophil homeostasis were accompanied by an increased recruitment of neutrophils to injury. Genetic analysis identified the microbiota-induced acute phase protein serum amyloid A (Saa) as a host factor mediating microbial stimulation of tissue-specific neutrophil migratory behaviours. In vitro studies revealed that zebrafish cells respond to Saa exposure by activating NF-κB, and that Saa-dependent neutrophil migration requires NF-κB-dependent gene expression. These results implicate the commensal microbiota as an important environmental factor regulating diverse aspects of systemic neutrophil development and function, and reveal a critical role for a Saa-NF-κB signalling axis in mediating neutrophil migratory responses

Complex pleural empyema can be safely treated with vacuum-assisted closure

<p>Abstract</p> <p>Objective</p> <p>For patients with postoperative pleural empyema, open window thoracostomy (OWT) is often necessary to prevent sepsis. Vacuum-assisted closure (VAC) is a well-known therapeutic option in wound treatment. The efficacy and safety of intrathoracal VAC therapy, especially in patients with pleural empyema with bronchial stump insufficiency or remain lung, has not yet been investigated.</p> <p>Methods</p> <p>Between October 2009 and July 2010, eight consecutive patients (mean age of 66.1 years) with multimorbidity received an OWT with VAC for the treatment of postoperative or recurrent pleural empyema. Two of them had a bronchial stump insufficiency (BPF).</p> <p>Results</p> <p>VAC therapy ensured local control of the empyema and control of sepsis. The continuous suction up to 125 mm Hg cleaned the wound and thoracic cavity and supported the rapid healing. Additionally, installation of a stable vacuum was possible in the two patients with BPF. The smaller bronchus stump fistula closed spontaneously due to the VAC therapy, but the larger remained open.</p> <p>The direct contact of the VAC sponge did not create any air leak or bleeding from the lung or the mediastinal structures. The VAC therapy allowed a better re-expansion of remaining lung.</p> <p>One patient died in the late postoperative period (day 47 p.o.) of multiorgan failure. In three cases, VAC therapy was continued in an outpatient service, and in four patients, the OWT was treated with conventional wound care. After a mean time of three months, the chest wall was closed in five of seven cases. However, two patients rejected the closure of the OWT. After a follow-up at 7.7 months, neither recurrent pleural empyema nor BPF was observed.</p> <p>Conclusion</p> <p>VAC therapy was effective and safe in the treatment of complicated pleural empyema. The presence of smaller bronchial stump fistula and of residual lung tissue are not a contraindication for VAC therapy.</p