Entwicklung der Transkriptomsequenzierung und Anwendung zur Analyse des Transkriptoms von Corynebacterium glutamicum

Pfeifer-Sancar K. Entwicklung der Transkriptomsequenzierung und Anwendung zur Analyse des Transkriptoms von Corynebacterium glutamicum. Bielefeld: Universität Bielefeld; 2014

Is risk-stratified breast cancer screening economically efficient in Germany?

Objectives Risk stratification has so far been evaluated under the assumption that women fully adhere to screening recommendations. However, the participation in German cancer screening programs remains low at 54%. The question arises whether risk-stratified screening is economically efficient under the assumption that adherence is not perfect. Method We have adapted a micro-simulation Markov model to the German context. Annual, biennial, and triennial routine screening are compared with five risk-adapted strategies using thresholds of relative risk to stratify screening frequencies. We used three outcome variables (mortality reduction, quality-adjusted life years, and false-positive results) under the assumption of full adherence vs. an adherence rate of 54%. Strategies are evaluated using efficiency frontiers and probabilistic sensitivity analysis (PSA). Results The reduced adherence rate affects both performance and cost; incremental cost-effectiveness ratios remain constant. The results of PSA show that risk-stratified screening strategies are more efficient than biennial routine screening under certain conditions. At any willingness-to-pay (WTP), there is a risk-stratified alternative with a higher likelihood of being the best choice. However, without explicit decision criteria and WTP, risk-stratified screening is not more efficient than biennial routine screening. Potential improvements in the adherence rates have significant health gains and budgetary implications. Conclusion If the participation rate for mammographic screening is as low as in Germany, stratified screening is not clearly more efficient than routine screening but dependent on the WTP. A more promising design for future stratified strategies is the combination of risk stratification mechanisms with interventions to improve the low adherence in selected high-risk groups

Metabolic changes during pregnancy in glucose-intolerant NZO mice: A polygenic model with prediabetic metabolism

Gestational diabetes mellitus (GDM) is a complex metabolic disease involving genetic and environmental factors. Recent studies have underlined its heterogeneity, so it is reasonable to divide patients into subpopulations depending on whether an insulin secretion or sensitivity defect is predominant. Since testing for GDM is usually performed in the second trimester, misinterpretation of prediabetes as gestational diabetes may occur. As with type 2 diabetes (T2DM), rodent models are needed for both GDM and prediabetes, but few do exist. Here, we compared the metabolic changes in pregnant normal NMRI mice with those in New Zealand obese (NZO) mice. Male animals of this strain are an established model of T2DM, whereas female mice of this strain are protected from hyperglycemia and β-cell death. We demonstrate that female NZO mice exhibited impaired glucose tolerance, preconceptional hyperinsulinemia, and hyperglucagonemia without any signs of manifest diabetes. The NZO model showed, compared with the NMRI control strain, a reduced proliferative response of the Langerhans islets during pregnancy (3.7 ± 0.4 vs. 7.2 ± 0.8% Ki-67-positive nuclei, p = .004). However, oral glucose tolerance tests revealed improved stimulation of insulin secretion in both strains. But this adaption was not sufficient to prevent impaired glucose tolerance in NZO mice compared with the NMRI control (p = .0002). Interestingly, glucose-stimulated insulin secretion was blunted in isolated primary NZO islets in perifusion experiments. In summary, the NZO mouse reflects important characteristics of human GDM and prediabetes in pregnancy and serves as a model for subpopulations with early alterations in glucose metabolism and primary insulin secretion defect

Comprehensive discovery and characterization of small RNAs in Corynebacterium glutamicum ATCC 13032

Mentz A, Neshat A, Pfeifer-Sancar K, Pühler A, Rückert C, Kalinowski J. Comprehensive discovery and characterization of small RNAs in Corynebacterium glutamicum ATCC 13032. BMC Genomics. 2013;14(1): 714.BACKGROUND: Recent discoveries on bacterial transcriptomes gave evidence that small RNAs (sRNAs) have important regulatory roles in prokaryotic cells. Modern high-throughput sequencing approaches (RNA-Seq) enable the most detailed view on transcriptomes offering an unmatched comprehensiveness and single-base resolution. Whole transcriptome data obtained by RNA-Seq can be used to detect and characterize all transcript species, including small RNAs. Here, we describe an RNA-Seq approach for comprehensive detection and characterization of small RNAs from Corynebacterium glutamicum, an actinobacterium of high industrial relevance and model organism for medically important Corynebacterianeae, such as C. diphtheriae and Mycobacterium tuberculosis. RESULTS: In our RNA-Seq approach, total RNA from C. glutamicum ATCC 13032 was prepared from cultures grown in minimal medium at exponential growth or challenged by physical (heat shock, cold shock) or by chemical stresses (diamide, H2O2, NaCl) at this time point. Total RNA samples were pooled and sequencing libraries were prepared from the isolated small RNA fraction. High throughput short read sequencing and mapping yielded over 800 sRNA genes. By determining their 5[prime]- and 3[prime]-ends and inspection of their locations, these potential sRNA genes were classified into UTRs of mRNAs (316), cis-antisense sRNAs (543), and trans-encoded sRNAs (262). For 77 of trans-encoded sRNAs significant sequence and secondary structure conservation was found by a computational approach using a whole genome alignment with the closely related species C. efficiens YS-314 and C. diphtheriae NCTC 13129. Three selected trans-encoded sRNAs were characterized by Northern blot analysis and stress-specific transcript patterns were found. CONCLUSIONS: The study showed comparable numbers of sRNAs known from genome-wide surveys in other bacteria. In detail, our results give deep insight into the comprehensive equipment of sRNAs in C. glutamicum and provide a sound basis for further studies concerning the functions of these sRNAs

In wenigen Schritten zur Zweitveröffentlichung : Workflows für Publikationsservices

Zweitveröffentlichungen von bereits veröffentlichten Publikationen haben eine freie Nutzbarmachung zum Ziel und nutzen den "Grünen Weg" im Open Access. Der Zweitveröffentlichungsworkflow umfasst mehrere Schritte, deren Reihenfolge zum Teil flexibel ist. Für die genaue Umsetzung der einzelnen Schritte gibt es stets verschiedene Optionen, die je nach den Rahmenbedingungen der jeweiligen Einrichtung (z. B. technische Infrastruktur, Personalressourcen) festgelegt werden. Dabei sollten bei der Planung bzw. Weiterentwicklung des Workflows möglichst die Automatisierungspotenziale einzelner Prozesse genutzt werden. Basierend auf einem Workflow-Vergleich der Zweitveröffentlichungsservices der beteiligten Einrichtungen empfiehlt die Unterarbeitsgruppe Workflows (Fokusgruppe Zweitveröffentlichungen) des open-access.network folgende neun Schritte: (1) Auswahl von Informationsquellen zur Identifikation potentieller Zweitveröffentlichungen (2) Rechtliche Prüfung, ob eine Zweitveröffentlichung zulässig ist; (3) Einholen der Publikationsgenehmigung von den Autor*innen; (4) Prüfung, welche Manuskriptversion veröffentlicht werden darf; (5) Bearbeitung der Publikationsdatei; (6) Dublettenprüfung, Eingabe der Metadaten und Ablage der Datei; (7) Dokumentation der Rechtsgrundlage für die Veröffentlichung; (8) Öffentlichkeitsarbeit für die Zweitveröffentlichung; (9) Monitoring und Dokumentation sowie Werbung für den eigenen Service

In wenigen Schritten zur Zweitveröffentlichung. Workflows für Publikationsservices

Zweitveröffentlichungen von bereits veröffentlichten Publikationen haben eine freie Nutzbarmachung zum Ziel und nutzen den "Grünen Weg" im Open Access. Der Zweitveröffentlichungsworkflow umfasst mehrere Schritte, deren Reihenfolge zum Teil flexibel ist. Für die genaue Umsetzung der einzelnen Schritte gibt es stets verschiedene Optionen, die je nach den Rahmenbedingungen der jeweiligen Einrichtung (z. B. technische Infrastruktur, Personalressourcen) festgelegt werden. Dabei sollten bei der Planung bzw. Weiterentwicklung des Workflows möglichst die Automatisierungspotenziale einzelner Prozesse genutzt werden. Basierend auf einem Workflow-Vergleich der Zweitveröffentlichungsservices der beteiligten Einrichtungen empfiehlt die Unterarbeitsgruppe Workflows (Fokusgruppe Zweitveröffentlichungen) des open-access.network folgende neun Schritte: (1) Auswahl von Informationsquellen zur Identifikation potentieller Zweitveröffentlichungen (2) Rechtliche Prüfung, ob eine Zweitveröffentlichung zulässig ist; (3) Einholen der Publikationsgenehmigung von den Autor*innen; (4) Prüfung, welche Manuskriptversion veröffentlicht werden darf; (5) Bearbeitung der Publikationsdatei; (6) Dublettenprüfung, Eingabe der Metadaten und Ablage der Datei; (7) Dokumentation der Rechtsgrundlage für die Veröffentlichung; (8) Öffentlichkeitsarbeit für die Zweitveröffentlichung; (9) Monitoring und Dokumentation sowie Werbung für den eigenen Service

Tenomodulin knockout mice exhibit worse late healing outcomes with augmented trauma-induced heterotopic ossification of Achilles tendon

Heterotopic ossification (HO) represents a common problem after tendon injury with no effective treatment yet being developed. Tenomodulin (Tnmd), the best-known mature marker for tendon lineage cells, has important effects in tendon tissue aging and function. We have reported that loss of Tnmd leads to inferior early tendon repair characterized by fibrovascular scaring and therefore hypothesized that its lack will persistently cause deficient repair during later stages. Tnmd knockout (Tnmd−/−) and wild-type (WT) animals were subjected to complete Achilles tendon surgical transection followed by end-to-end suture. Lineage tracing revealed a reduction in tendon-lineage cells marked by ScleraxisGFP, but an increase in alpha smooth muscle actin myofibroblasts in Tnmd−/− tendon scars. At the proliferative stage, more pro-inflammatory M1 macrophages and larger collagen II cartilaginous template were detected in this group. At the remodeling stage, histological scoring revealed lower repair quality in the injured Tnmd−/− tendons, which was coupled with higher HO quantified by micro-CT. Tendon biomechanical properties were compromised in both groups upon injury, however we identified an abnormal stiffening of non-injured Tnmd−/− tendons, which possessed higher static and dynamic E-moduli. Pathologically thicker and abnormally shaped collagen fibrils were observed by TEM in Tnmd−/− tendons and this, together with augmented HO, resulted in diminished running capacity of Tnmd−/− mice. These novel findings demonstrate that Tnmd plays a protecting role against trauma-induced endochondral HO and can inspire the generation of novel therapeutics to accelerate repair

Genomic CDKN2A/2B deletions in adult Ph+ ALL are adverse despite allogeneic stem cell transplantation

We investigated the role of copy number alterations to refine risk stratification in adult Philadelphia chromosome positive (Ph)+ ALL treated with tyrosine kinase inhibitors (TKI) and allogeneic stem cell transplantation (aSCT). 97 Ph+ ALL patients (median age 41 years, range 18-64 years) within the prospective multicenter GMALL studies 06/99 (n=8) and 07/2003 (n=89) were analysed. All patients received TKI and aSCT in first complete remission (CR1). Copy number analysis was performed with SNP arrays and validated by multiplex ligation-dependent probe amplification (MLPA). The frequencies of recurrently deleted genes were: IKZF1, 76%, CDKN2A/2B, 45%, PAX5, 43%, BTG1, 18%, EBF1, 13%, ETV6, 5%, RB, 14%. In univariate analyses, the presence of CDKN2A/2B deletions had a negative impact on all endpoints: overall survival (p=0.023), disease free survival (p=0.012) and remission duration (p=0.036). The negative predictive value of CDKN2A/2B deletions was retained in multivariable analysis along with other factors such as timing of TKI therapy, intensity of conditioning, achieving remission after induction phase I and BTG1 deletions. We therefore conclude that acquired genomic CDKN2A/2B deletions identify a subgroup of Ph+ ALL patients, who have an inferior prognosis despite aSCT in CR1. Their poor outcome was attributable primarily to a high relapse rate after aSCT

Novel insights into the mechanisms mediating the local antihypertrophic effects of cardiac atrial natriuretic peptide: role of cGMP-dependent protein kinase and RGS2

Cardiac atrial natriuretic peptide (ANP) locally counteracts cardiac hypertrophy via the guanylyl cyclase-A (GC-A) receptor and cGMP production, but the downstream signalling pathways are unknown. Here, we examined the influence of ANP on β-adrenergic versus Angiotensin II (Ang II)-dependent (Gs vs. Gαq mediated) modulation of Ca2+i-handling in cardiomyocytes and of hypertrophy in intact hearts. L-type Ca2+ currents and Ca2+i transients in adult isolated murine ventricular myocytes were studied by voltage-clamp recordings and fluorescence microscopy. ANP suppressed Ang II-stimulated Ca2+ currents and transients, but had no effect on isoproterenol stimulation. Ang II suppression by ANP was abolished in cardiomyocytes of mice deficient in GC-A, in cyclic GMP-dependent protein kinase I (PKG I) or in the regulator of G protein signalling (RGS) 2, a target of PKG I. Cardiac hypertrophy in response to exogenous Ang II was significantly exacerbated in mice with conditional, cardiomyocyte-restricted GC-A deletion (CM GC-A KO). This was concomitant to increased activation of the Ca2+/calmodulin-dependent prohypertrophic signal transducer CaMKII. In contrast, β-adrenoreceptor-induced hypertrophy was not enhanced in CM GC-A KO mice. Lastly, while the stimulatory effects of Ang II on Ca2+-handling were absent in myocytes of mice deficient in TRPC3/TRPC6, the effects of isoproterenol were unchanged. Our data demonstrate a direct myocardial role for ANP/GC-A/cGMP to antagonize the Ca2+i-dependent hypertrophic growth response to Ang II, but not to β-adrenergic stimulation. The selectivity of this interaction is determined by PKG I and RGS2-dependent modulation of Ang II/AT1 signalling. Furthermore, they strengthen published observations in neonatal cardiomyocytes showing that TRPC3/TRPC6 channels are essential for Ang II, but not for β-adrenergic Ca2+i-stimulation in adult myocytes