Optimization of a signal transduction model of the mitochondrial apoptosis by training to multi-cell data.

Research in systems biology is a cycle of proposing and validating novel hypotheses through wet lab experiments and computational modelling. Hypotheses based on experimental data are tested in computational models and resulting model predictions propose further biochemical experiments which are once again basis for an improved and more broadly applicable model. Despite best experimental efforts and advances in biotechnology, essential parameters for deterministic models such as biochemical reaction kinetics are often not available in sufficient quality, and adapting these parameters through training of the model to control experiments remains the only alternative. Exemplified by our recently developed model of mitochondria1 apoptosis (I), this thesis presents a structural approach to broaden a computational model by training against single-cell data sets (2). Principal component analysis was used to identify sensitive and adaptable parameters in the model. Parameter optimization by classical Nelder-Mead fitting, a Monte-Carlo approach and a brute-force Screening were then iteratively applied to reduce ambiguity in candidate parameter values. Our approach leads to parameter sets enabling the model to correctly predict apoptotic kinetics of several cancer cell lines. 1. M. Rehm, H.J. Huber, H. Dussmann, and J.H.M. Prehn, The EMBO Journal, 2006, 4338-4349. 2. C.L. O\u27Connor, S. Anguissola, H.J. Huber, H. Dussmann, J.H. Prehn, and M. Rehm, Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2008, 1903- 191

Effektivität des Fremdstuhltransfers bei rezidivierender oder therapierefraktärer Clostridium difficile-Colitis

Diese Arbeit zeigt an Hand einer kleinen Fallserie mit sieben Patienten die Ansprechrate, unerwünschte Nebenwirkungen, sowie mögliche Probleme beim Ablauf der FMT bei rezidivierender und therapierefraktärer CDI, welche im Rahmen eines individuellen Heilversuchs am UKT durchgeführt wurden, auf. Als Therapieerfolg wurde die Symptomfreiheit bis > acht Wochen nach Transfer gewertet. Zur standardisierten Effektivitätskontrolle wurde eine Datenerhebung mittels Fragebögen durchgeführt. Der FEE zur Ermittlung der Ekelempfindlichkeit wurde einmalig vor Therapiebeginn durchgeführt, der SF-12 zum Gesundheitszustand an den Tagen 0, 28 und 56 und der IBDQ-D zur Erfassung der Lebensqualität bei chronisch entzündlichen Darmerkrankungen an den Tagen 0, 14, 28, 46 und 52. Zudem führten die Patienten ein Patiententagebuch, um das Ergebnis, mögliche Nebenwirkungen und Veränderungen in der Lebensqualität der Patienten nach der FMT zu objektivieren. Es wurde die Hypothese aufgestellt, dass die Lebensqualität nach erfolgreichem FMT steigt. Die Durchführung erfolgte nach Aufbereitung einer Stuhlspende von nahestehenden, zuvor auf Infektionskrankheiten, pathogene Keime und Parasiten untersuchten Personen, mit 250 ml 0,9%iger NaCl-Lösung. Appliziert wurde die Suspension in einem Fall via PEJ. In diesem Kasus blieb ein Therapieerfolg initial aus und erst eine weitere FMT in einem externen Krankenhaus führte zur Heilung. In allen anderen Fällen erfolgte die Gabe koloskopisch. Bei fünf der Patienten kam es direkt zu einem positiven Ansprechen auf den FMT, in einem Fall wurde eine zweite Übertragung benötigt. So ergibt dies einen Therapieerfolg von 71,4% nach der ersten FMT und 85,7% respektive 100% nach der zweiten Übertragung. Schwerwiegende Nebenwirkungen waren nicht zu verzeichnen. Der FEE weist auf eine erhöhte Ekelempfindlichkeit bei allen Befragten hin. Eine mögliche Erklärung diesbezüglich könnte sein, dass hygienebezogener Ekel unterbewusst zur Einhaltung intensiverer Hygienemaßnahmen führt, wodurch eine Keimreduktion des Umfeldes erzielt werden kann, um so einer erneuten Infektion vorzubeugen oder Mitmenschen vor einer Ansteckung zu schützen. Für den IBDQ ergibt sich in der Zusammenschau im Vergleich vor Therapiebeginn bis acht Wochen nach der FMT eine Steigerung der erzielten Punktzahl, welche nach Hlavaty et al. als ein Therapieansprechen und eine Remission der Erkrankung zu werten ist. Im SF-12 liegt in der KSK ebenfalls eine Steigerung vor. Der Median liegt 56 Tage nach erfolgreicher Therapie jedoch unterhalb der Gesamtnormstichprobe von 1994, sowie unterhalb der Normstichprobe für Patienten mit chronischen Magen-Darm-Erkrankungen. In der PSK kommt es zu einem Anstieg über den Median der Gesamtnormstichprobe und über der Normstichprobe für Patienten mit chronischen Magen-Darm-Erkrankungen. Auf Grund der geringen Fallzahl kann jedoch statistisch keine quantitative Aussage getroffen werden und die eingangs gestellte Hypothese kann somit nicht belegt werden. In einem Sonderfall wurden bei einem Patienten, dessen Pouchitis mit einer herkömmlichen Therapie nicht einzudämmen war und der sich in einem klinisch schlechten Zustand befand, drei Behandlungsversuche mittels FMT durchgeführt. Durch die FMT sollte die veränderte Darmflora günstig beeinflusst werden um so den inflammatorischen Prozessen entgegenzu-wirken. Dies ließ sich durch eine Pouchoskopie jedoch nicht objektivieren, sodass keine weitere Übertragung durchgeführt wurde (Schmid et al., 2017). Mittels der durch den SF-12 und den IBDQ-Fragebogen erhobenen Daten konnte ebenfalls keine dauerhafte Verbesserung der Symptomatik aufgezeigt werden. Ein Forschungsausblick zeigt weitere Möglichkeiten und Grenzen des Fremdstuhltransfers auf, wie der FMT in Kapselform, bei CED und Adipositas, der Transfer von gereinigten Bakterienkulturen und sterilem Fäkalfiltrat, sowie die Therapie mit monoklonalen Antikörpern bei CDI und die Prävention durch Impfung. Bei rCDI stellt der FMT, wie bereits in anderen Studien gezeigt werden konnte und daher zwischenzeitlich Einzug in die US-Leitlinien erhalten hat, eine sinnvolle Alternative zur Antibiotikatherapie bei rCDI dar (McDonald et al., 2018). Es kann als ein für den Patienten risikoarmes, für medizinisches Personal einfach durchzuführendes und für die Klinik ökonomisch sinnvolles Verfahren angesehen werden. Die bisher publizierten Ergebnisse konnten in einer kleinen Fallserie am UKT reproduziert werden

Small-scale experimental habitat fragmentation reduces colonization rates in species-rich grasslands

Habitat fragmentation is one of the most important threats to biodiversity. Decreasing patch size may lead to a reduction in the size of populations and to an increased extinction risk of remnant populations. Furthermore, colonization rates may be reduced in isolated patches. To investigate the effects of isolation and patch size on extinction and colonization rates of plant species, calcareous grasslands at three sites in the Swiss Jura Mountains were experimentally fragmented into patches of 0.25, 2.25, and 20.25m2 by frequent mowing of the surrounding area from 1993 to 1999. Species richness in the fragment plots and adjacent control plots of the same sizes was recorded during these 7years. In agreement with the theory of island biogeography, colonization rate was reduced by 30% in fragments versus non-isolated controls, and extinction increased in small versus large plots. Habitat specialists, in contrast to generalists, were less likely to invade fragments. In the last 4years of the experiment, extinction rates tended to be higher in fragment than in control plots at two of the three sites. Despite reduced colonization rates and a tendency of increased extinction rates in fragments, fragmented plots had only marginally fewer species than control plots after 7years. Hence, rates were a more sensitive measure for community change than changes in species richness per se. From a conservation point of view, the detected reduced colonization rates are particularly problematic in small fragments, which are more likely to suffer from high extinction rates in the long ru

Effects of hepatocyte nuclear factor-1A and -4A on pancreatic stone protein/regenerating protein and C-reactive protein gene expression: implications for maturity-onset diabetes of the young

BACKGROUND: There is a significant clinical overlap between patients with hepatocyte nuclear factor (HNF)-1A and HNF4A maturity-onset diabetes of the young (MODY), two forms of monogenic diabetes. HNF1A and HNF4A are transcription factors that control common and partly overlapping sets of target genes. We have previously shown that elevated serum pancreatic stone protein / regenerating protein A (PSP/reg1A) levels can be detected in subjects with HNF1A-MODY. In this study, we investigated whether PSP/reg is differentially regulated by HNF1A and HNF4A. METHODS: Quantitative real-time PCR (qPCR) and Western blotting were used to validate gene and protein expression in cellular models of HNF1A- and HNF4A-MODY. Serum PSP/reg1A levels and high-sensitivity C-reactive protein (hsCRP) were measured by ELISA in 31 HNF1A- and 9 HNF4A-MODY subjects. The two groups were matched for age, body mass index, diabetes duration, blood pressure, lipid profile and aspirin and statin use. RESULTS: Inducible repression of HNF1A and HNF4A function in INS-1 cells suggested that PSP/reg induction required HNF4A, but not HNF1A. In contrast, crp gene expression was significantly reduced by repression of HNF1A, but not HNF4A function. PSP/reg levels were significantly lower in HNF4A subjects when compared to HNF1A subjects [9.25 (7.85-12.85) ng/ml vs. 12.5 (10.61-17.87) ng/ml, U-test P = 0.025]. hsCRP levels were significantly lower in HNF1A-MODY [0.22 (0.17-0.35) mg/L] compared to HNF4A-MODY group [0.81 (0.38-1.41) mg/L, U-test P = 0.002], Parallel measurements of serum PSP/reg1A and hsCRP levels were able to discriminate HNF1A- and HNF4A-MODY subjects. CONCLUSION: Our study demonstrates that two distinct target genes, PSP/reg and crp, are differentially regulated by HNF1A and HNF4A, and provides clinical proof-of-concept that serum PSP/reg1A and hsCRP levels may distinguish HNF1A-MODY from HNF4A-MODY subjects

The autophagic marker p62 highlights Alzheimer type astrocytes in metabolic/hepatic encephalopathy

Metabolic/hepatic encephalopathy is neuropathologically characterized by the presence of Alzheimer type II astrocytes (AA II) with large and clear nuclear morphology. To date, there is no good immunohistochemical marker to better identify these cells. Here, we assessed cases of hepatic encephalopathy of different etiologies by immunohistochemistry using an anti-p62 antibody. We observed peripheral or diffuse nuclear staining of variable intensity in AA II in all cases but not in normal controls or reactive astrocytes. We conclude that p62 is a useful immunohistochemical marker for the identification of AA II and may be helpful for the neuropathological diagnosis of metabolic/hepatic encephalopathy in difficult or equivocal cases

MicroRNA-224 is Readily Detectable in Urine of Individuals with Diabetes Mellitus and is a Potential Indicator of Beta-Cell Demise.

MicroRNA (miRNA) are a class of non-coding, 19-25 nucleotide RNA critical for network-level regulation of gene expression. miRNA serve as paracrine signaling molecules. Using an unbiased array approach, we previously identified elevated levels of miR-224 and miR-103 to be associated with a monogenic form of diabetes; HNF1A-MODY. miR-224 is a novel miRNA in the field of diabetes. We sought to explore the role of miR-224 as a potential biomarker in diabetes, and whether such diabetes-associated-miRNA can also be detected in the urine of patients. Absolute levels of miR-224 and miR-103 were determined in the urine of n = 144 individuals including carriers of a HNF1A mutation, participants with type 1 diabetes mellitus (T1DM), type 2 diabetes mellitus (T2DM) and normal controls. Expression levels were correlated with clinical and biochemical parameters. miR-224 was significantly elevated in the urine of carriers of a HNF1A mutation and participants with T1DM. miR-103 was highly expressed in urine across all diabetes cohorts when compared to controls. For both miR-224 and-103, we found a significant correlation between serum and urine levels (p \u3c 0.01). We demonstrate that miRNA can be readily detected in the urine independent of clinical indices of renal dysfunction. We surmise that the differential expression levels of miR-224 in both HNF1A-MODY mutation carriers and T1DM may be an attempt to compensate for beta-cell demise

Systems analysis of cancer cell heterogeneity in caspase-dependent apoptosis subsequent to mitochondrial outer membrane permeabilization.

Deregulation of apoptosis is a hallmark of carcinogenesis. We here combine live cell imaging and systems modeling to investigate caspase-dependent apoptosis execution subsequent to mitochondrial outer membrane permeabilization (MOMP) in several cancer cell lines. We demonstrate that, although most cell lines that underwent MOMP also showed robust and fast activation of executioner caspases and apoptosis, the colorectal cancer cell lines LoVo and HCT-116 Smac(-/-), similar to X-linked inhibitor of apoptosis protein (XIAP)-overexpressing HeLa (HeLa XIAP(Adv)) cells, only showed delayed and often no caspase activation, suggesting apoptosis impairment subsequent to MOMP. Employing APOPTO-CELL, a recently established model of apoptosis subsequent to MOMP, this impairment could be understood by studying the systemic interaction of five proteins that are present in the apoptosis pathway subsequent to MOMP. Using APOPTO-CELL as a tool to study detailed molecular mechanisms during apoptosis execution in individual cell lines, we demonstrate that caspase-9 was the most important regulator in DLD-1, HCT-116, and HeLa cells and identified additional cell line-specific co-regulators. Developing and applying a computational workflow for parameter screening, systems modeling identified that apoptosis execution kinetics are more robust against changes in reaction kinetics in HCT-116 and HeLa than in DLD-1 cells. Our systems modeling study is the first to draw attention to the variability in cell specific protein levels and reaction rates and to the emergent effects of such variability on the efficiency of apoptosis execution and on apoptosis impairment subsequent to MOMP

Activation of executioner caspases is a predictor of progression-free survival in glioblastoma patients: a systems medicine approach.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. GBM cells are highly resistant to apoptosis induced by antitumor drugs and radiotherapy resulting in cancer progression. We assessed whether a systems medicine approach, analysing the ability of tumor cells to execute apoptosis could be utilized to predict the response of GBM patients to treatment. Concentrations of the key proapoptotic proteins procaspase-3, procaspase-9, Smac and Apaf-1 and the antiapopotic protein XIAP were determined in a panel of GBM cell lines and GBM patient tumor resections. These values were used as input for APOPTO-CELL, a systems biological based mathematical model built to predict cellular susceptibility to undergo caspase activation. The modeling was capable of accurately distinguishing between GBM cells that die or survive in response to treatment with temozolomide in 10 of the 11 lines analysed. Importantly the results obtained using GBM patient samples show that APOPTO-CELL was capable of stratifying patients according to their progression-free survival times and predicted the ability of tumor cells to support caspase activation in 16 of the 21 GBM patients analysed. Calculating the susceptibility to apoptosis execution may be a potent tool in predicting GBM patient therapy responsiveness and may allow for the use of APOPTO-CELL in a clinical setting

Diagnostic Workup for Patients with Solid Renal Masses: A Cost-Effectiveness Analysis

Simple Summary There are several benign and malignant types of solid renal masses. For diagnostic and characterization of these masses, a few imaging methods such as magnetic resonance imaging (MRI), computed tomography (CT) or (contrast-enhanced) ultrasound (CEUS) are established in the clinical routine. The aim of our study was to assess the most economical approach for detecting and characterizing these masses. As a result, contrast-enhanced ultrasound turned out to be a cost-effective diagnostic method. Therefore, if available, this method should be considered in the routine. Alternatively, MRI also offers excellent diagnostic accuracy, but it is associated with higher costs. This result may lead to a change in the diagnostic workup of solid renal masses in clinical routine, as contrast-enhanced ultrasound should be considered as an appropriate method for the first analysis compared to CT and MRI. Background: For patients with solid renal masses, a precise differentiation between malignant and benign tumors is crucial for forward treatment management. Even though MRI and CT are often deemed as the gold standard in the diagnosis of solid renal masses, CEUS may also offer very high sensitivity in detection. The aim of this study therefore was to evaluate the effectiveness of CEUS from an economical point of view. Methods: A decision-making model based on a Markov model assessed expenses and utilities (in QALYs) associated with CEUS, MRI and CT. The utilized parameters were acquired from published research. Further, a Monte Carlo simulation-based deterministic sensitivity analysis of utilized variables with 30,000 repetitions was executed. The willingness-to-pay (WTP) is at USD 100,000/QALY. Results: In the baseline, CT caused overall expenses of USD 10,285.58 and an efficacy of 11.95 QALYs, whereas MRI caused overall expenses of USD 7407.70 and an efficacy of 12.25. Further, CEUS caused overall expenses of USD 5539.78, with an efficacy of 12.44. Consequently, CT and MRI were dominated by CEUS, and CEUS remained cost-effective in the sensitivity analyses. Conclusions: CEUS should be considered as a cost-effective imaging strategy for the initial diagnostic workup and assessment of solid renal masses compared to CT and MRI