Abiotic stress - a challenge for the master enzyme plasma membrane H+ -ATPase : a case study for maize with special consideration of salt stress and Mg deficiency

The plasma membrane (PM) H+-ATPase is the powerhouse of the plant. The enzyme establishes an electrochemical proton gradient across the PM. This gradient is responsible for cell-wall acidification which is a requisite for cell-extension growth and it energizes transport processes of symporters and antiporters at the PM. Abiotic stresses often have a negative impact on the functions of enzymes which can disturb major physiological processes and interfere with plant development. Therefore, the effect of abiotic stress on the PM H+-ATPase´s two main functions were investigated in this thesis. The effect of Mg deficiency on apoplastic acidification was studied in developing maize leaves and the effect of salt stress on energization of transport processes was examined in developing maize kernels. Magnesium (Mg) deficiency is often observed in agricultural crops with an insufficient Mg fertilization regime, finally resulting in yield depression. One goal was to identify the process that primarily limits maize (Zea mays L.) growth and yield under insufficient Mg supply. A sufficient (500 µM) and a low (25 µM) Mg concentration were used in time-course hydroponic experiments to investigate parameters which are considered to be important for the characterization of the key processes of growth, namely cell division and cell-extension growth. It was shown that cell division was not susceptible to Mg deficiency, since neither DNA replication nor sugar or protein availability limited this growth process. However, PM H+-ATPase activity was inhibited by a lack of the enzyme´s co-substrate Mg under Mg deficiency. The inhibition of the PM H+-ATPase reduced apoplastic acidification causing a reduced cell-extension growth. This primarily limited maize growth under Mg deficiency. Salt stress affects yield formation of maize at various physiological levels resulting in an overall decrease of grain yield. In this thesis it was investigated how salt stress affects kernel development at and shortly after pollination. Maize kernels grown under control and salt stress were harvested 0 and 2 d after pollination (DAP) and at kernel maturity. Kernel development was not inhibited 0 DAP, but it was inhibited 2 DAP under salt stress. On this day, kernel PM H+-ATPase activity was reduced which caused a lower pH gradient across the plasma membranes of endosperm and embryo tissue. The lower pH gradient supposedly resulted in a decreased energization of transporters responsible for hexose import into the cytoplasm of the kernels. A lack of hexoses reduced the energy status of the cells which impaired cell division. The impaired cell division probably caused the observed growth reduction of kernels 2 DAP, which resulted in an observed lower kernel number and grain yield at maize maturity under salt stress. It is concluded that the impairment of PM H+-ATPase by Mg deficiency and salt stress in maize is a major reason for growth and yield reduction under these environmental constraints. However, while the impairment of the cell-wall acidification is the reason for a reduced extension growth under Mg deficiency in leaves, the decrease in pH gradient at the PM caused kernel abortion under salt stress.Die Plasmamembran (PM) H+-ATPase wird aufgrund ihrer herausragenden Stellung im pflanzlichen Stoffwechsel als Kraftwerk oder als Masterenzym der Pflanze bezeichnet. Sie baut an der Plasmamembran pflanzlicher Zellen einen elektrochemischen Protonengradienten auf, der zweierlei Funktionen hat: 1. Die Ansäuerung der pflanzlichen Zellwand, welches die Voraussetzung für die Turgor-getriebene Zellstreckung ist. 2. Den Antrieb von Transportprozessen durch die Plasmamembran. Abiotischer Stress hat oft einen negativen Einfluss auf diese Funktionen. Eine Störung einer dieser beiden Funktionen hat oft negative Einflüsse auf wichtige physiologische Prozesse, wodurch wiederum die normale Entwicklung der Pflanze gestört werden kann. Daher wurde in dieser Arbeit untersucht, welche Auswirkungen abiotischer Stress auf die beiden Funktionen des pH-Gradienten hat. Der Einfluss von Magnesiummangel auf die Ansäuerung des Apoplasten wurde in noch wachsenden Maisblättern untersucht, während in sich entwickelnden Maiskörnern der Einfluss von Salzstress auf den Antrieb von Transportprozessen in der PM untersucht wurde. In landwirtschaftlichen Kulturen wird häufig Magnesiummangel aufgrund einer unzureichenden Magnesiumdüngung beobachtet, welcher zu einem reduzierten Ertrag führen kann. Ein Ziel dieser Arbeit war es daher, den Prozess zu identifizieren, der unter Magnesiummangel primär das Wachstum und den Ertrag von Mais (Zea mays L.) reduziert. Mit einer niedrigen (25 µM) und einer hohen Magnesiumkonzentration (500 µM) wurden daher Nährlösungsversuche durchgeführt, um Parameter für die physiologischen Wachstumsprozesse wie Zellteilung und Zellstreckung zu erfassen. Es konnte gezeigt werden, dass die Zellteilung weder durch die DNA-Replikation noch durch die Verfügbarkeit von Zuckern oder Proteinen negativ durch Magnesiummangel beeinflusst wurde. Jedoch war unter Magnesiummangel die Aktivität der PM H+-ATPase durch einen Mangel an dem für das Enzym notwendigen Co-Substrats Magnesium reduziert. Durch die Hemmung der Aktivität der PM H+-ATPase unter Magnesiummangel konnte die Ansäuerung des Apoplasten nicht aufrechterhalten werden, sodass hierdurch das Zellstreckungswachstum eingeschränkt wurde. Dies war der primäre Grund für das reduzierte Wachstum von Mais unter Magnesiummangel. Salzstress behindert die Ertragsbildung von Mais auf verschiedenen physiologischen Ebenen, was schließlich zu einem verminderten Ertrag führt. In dieser Arbeit wurde daher untersucht, welche Auswirkungen Salzstress auf die Entwicklung von jungen Maiskörnern während oder kurz nach der Bestäubung hat. Hierzu wurde Mais unter Kontrollbedingungen und unter Salzstress angebaut und die Körner am Tag der Bestäubung, zwei Tage später und zur Kornreife geerntet. Das Wachstum der Körner war zum Zeitpunkt der Bestäubung noch nicht durch den Salzstress reduziert, jedoch zwei Tage später schon. An diesem Tag wurde eine Reduktion der Aktivität der PM H+-ATPase gemessen, die einen reduzierten pH-Gradienten an den Plasmamembranen von Endosperm und Embryo zur Folge hatte. Diese Reduktion verursachte wahrscheinlich einen reduzierten Transport von Hexosen in das Cytoplasma der Körner. Der Mangel an Hexosen im Cytoplasma rief mutmaßlich einen verringerten Energiestatus der Zelle hervor, der in einer verringerten Zellteilung resultierte. Dies war vermutlich die Ursache für die beobachtete Wachstumsreduktion der Körner zwei Tage nach der Bestäubung. Dies mündete in eine verminderte Anzahl von Körnern und in einen verringerten Ertrag bei der Kornreife. Es bleibt festzuhalten, dass die Reduzierung der Aktivität PM H+-ATPase durch Magnesiummangel und Salzstress ein Hauptgrund für vermindertes Wachstum und Ertragsdepression war. Die reduzierte Ansäuerung des Apoplasten war der Grund für das reduzierte Streckungswachstum der Blätter unter Magnesiummangel, während der reduzierte pH-Gradient an der Plasmamembran unter Salzstress das Absterben der Körner verursachte

A Novel Predictor Tool of Biochemical Recurrence after Radical Prostatectomy Based on a Five-MicroRNA Tissue Signature

Within five to ten years after radical prostatectomy (RP), approximately 15-34% of prostate cancer (PCa) patients experience biochemical recurrence (BCR), which is defined as recurrence of serum levels of prostate-specific antigen >0.2 µg/L, indicating probable cancer recurrence. Models using clinicopathological variables for predicting this risk for patients lack accuracy. There is hope that new molecular biomarkers, like microRNAs (miRNAs), could be potential candidates to improve risk prediction. Therefore, we evaluated the BCR prognostic capability of 20 miRNAs, which were selected by a systematic literature review. MiRNA expressions were measured in formalin-fixed, paraffin-embedded (FFPE) tissue RP samples of 206 PCa patients by RT-qPCR. Univariate and multivariate Cox regression analyses were performed, to assess the independent prognostic potential of miRNAs. Internal validation was performed, using bootstrapping and the split-sample method. Five miRNAs (miR-30c-5p/31-5p/141-3p/148a-3p/miR-221-3p) were finally validated as independent prognostic biomarkers. Their prognostic ability and accuracy were evaluated using C-statistics of the obtained prognostic indices in the Cox regression, time-dependent receiver-operating characteristics, and decision curve analyses. Models of miRNAs, combined with relevant clinicopathological factors, were built. The five-miRNA-panel outperformed clinically established BCR scoring systems, while their combination significantly improved predictive power, based on clinicopathological factors alone. We conclude that this miRNA-based-predictor panel will be worth to be including in future studies

Circular RNAs in Clear Cell Renal Cell Carcinoma: Their Microarray-Based Identification, Analytical Validation, and Potential Use in a Clinico-Genomic Model to Improve Prognostic Accuracy

Circular RNAs (circRNAs) may act as novel cancer biomarkers. However, a genome-wide evaluation of circRNAs in clear cell renal cell carcinoma (ccRCC) has yet to be conducted. Therefore, the objective of this study was to identify and validate circRNAs in ccRCC tissue with a focus to evaluate their potential as prognostic biomarkers. A genome-wide identification of circRNAs in total RNA extracted from ccRCC tissue samples was performed using microarray analysis. Three relevant differentially expressed circRNAs were selected (circEGLN3, circNOX4, and circRHOBTB3), their circular nature was experimentally confirmed, and their expression-along with that of their linear counterparts-was measured in 99 malignant and 85 adjacent normal tissue samples using specifically established RT-qPCR assays. The capacity of circRNAs to discriminate between malignant and adjacent normal tissue samples and their prognostic potential (with the endpoints cancer-specific, recurrence-free, and overall survival) after surgery were estimated by C-statistics, Kaplan-Meier method, univariate and multivariate Cox regression analysis, decision curve analysis, and Akaike and Bayesian information criteria. CircEGLN3 discriminated malignant from normal tissue with 97% accuracy. We generated a prognostic for the three endpoints by multivariate Cox regression analysis that included circEGLN3, circRHOBT3 and linRHOBTB3. The predictive outcome accuracy of the clinical models based on clinicopathological factors was improved in combination with this circRNA-based signature. Bootstrapping as well as Akaike and Bayesian information criteria confirmed the statistical significance and robustness of the combined models. Limitations of this study include its retrospective nature and the lack of external validation. The study demonstrated the promising potential of circRNAs as diagnostic and particularly prognostic biomarkers in ccRCC patients

Renal AA-amyloidosis in intravenous drug users - a role for HIV-infection?

Background: Chronic renal disease is a serious complication of long-term intravenous drug use (IVDU). Recent reports have postulated a changing pattern of underlying nephropathy over the last decades. Methods: Retrospective investigation including all patients with prior or present IVDU that underwent renal biopsy because of chronic kidney disease between 01.04.2002 and 31.03.2012 in the city of Frankfurt/Main, Germany. Results: Twenty four patients with IVDU underwent renal biopsy because of progressive chronic kidney disease or proteinuria. Renal AA-amyloidosis was the predominant cause of renal failure in 50% of patients. Membranoproliferative glomerulonephritis (GN) was the second most common cause found in 21%. Patients with AA-amyloidosis were more likely to be HIV infected (67 vs.17%; p=0.036) and tended to have a higher rate of repeated systemic infections (92 vs. 50%; p=0.069). Patients with AA-amyloidosis presented with progressive renal disease and nephrotic-range proteinuria but most patients had no peripheral edema or systemic hypertension. Development of proteinuria preceded the decline of GFR for approximately 1--2 years. Conclusions: AA-amyloidosis was the predominant cause of progressive renal disease in the last 10 years in patients with IVDU. The highest rate of AA-amyloidosis observed was seen in HIV infected patients with IVDU. We speculate that chronic HIV-infection as well as the associated immunosuppression might promote development of AA-amyloidosis by increasing frequency and duration of infections acquired by IVDU

Funktionelle Charakterisierung von humanen Methioninsulfoxidreduktasen vom Typ B

Im Laufe dieser Arbeit wurde gezeigt, dass drei humane Methioninsulfoxidreduktasen vom Typ B (MSRB) existieren, die ausschließlich Met-R-O als Substrat verwenden, wovon für zwei Enzyme (hMSRB1 und hMSRB2) jeweils ähnliche Umsatzraten bestimmt wurden. Das katalytische Zentrum der humanen MSRB-Enzyme enthält ein aktives Cystein, welches im Gegensatz zu den Methioninsulfoxidreduktasen anderer Organismen nicht durch intramolekulare Disulfidbrücken regeneriert wird. Zur Untersuchung der Lebensspanne von Drosophila Fruchtfliegen wurde aus den humanen Methioninsulfoxidreduktasen vom Typ B und Typ A ein Fusionsprotein hergestellt, welches sowohl Met-R-O als auch Met-S-O als Substrat umsetzt. Die regulative Rolle von Methioninsulfoxidreduktasen konnte an zwei verschiedenen Kaliumkanälen verdeutlicht werden. Die durch Methioninoxidation hervorgerufene, variable Inaktivierungszeitspanne des Drosophila ShC/B-Kanals konnte sowohl durch humane Methioninsulfoxidreduktasen vom Typ A als auch vom Typ B beschleunigt werden. Zusätzlich konnte für die Bindung von humanen EAG Kanälen und dem Calciumbindeprotein Calmodulin gezeigt werden, dass die Oxidation der zwei C-terminalen Methionine in Calmodulin (M145 bzw. M146) zu einer verringerten Bindungaffinität von Calmodulin zum Kanal führt, welche durch die Methioninsulfoxidreduktasen wieder hergestellt werden konnte

Detection of Simultaneous Group Effects in MicroRNA Expression and Related Target Gene Sets

Expression levels of mRNAs are among other factors regulated by microRNAs. A particular microRNA can bind specifically to several target mRNAs and lead to their degradation. Expression levels of both, mRNAs and microRNAs, can be obtained by microarray experiments. In order to increase the power of detecting microRNAs that are differentially expressed between two different groups of samples, we incorporate expression levels of their related target gene sets. Group effects are determined individually for each microRNA, and by enrichment tests and global tests for target gene sets. The resulting lists of p-values from individual and set-wise testing are combined by means of meta analysis. We propose a new approach to connect microRNA-wise and gene set-wise information by means of p-value combination as often used in meta-analysis. In this context, we evaluate the usefulness of different approaches of gene set tests. In a simulation study we reveal that our combination approach is more powerful than microRNA-wise testing alone. Furthermore, we show that combining microRNA-wise results with ‘competitive’ gene set tests maintains a pre-specified false discovery rate. In contrast, a combination with ‘self-contained’ gene set tests can harm the false discovery rate, particularly when gene sets are not disjunct

Health indices for the evaluation and monitoring of health in children and adolescents in prevention and health promotion: a scoping review

BACKGROUND Health indicators are used in different settings to monitor health outcomes. Child and adolescent health is arguably one of the most important areas for the application of indices and indicators in prevention and health promotion. Although single health indicators may be better suited to display the complexity of the health status and its determinants, a selected set of indicators will still offer a complex picture. Therefore, it is argued that a group of indicators combined into an index may offer a pragmatic tool that is easier to use in order to inform stakeholders. METHODS A scoping review was conducted to identify and describe health indices that monitor and evaluate health of children and adolescents and to appraise the quality and value of the identified indices that may guide the further applications of these indices in particular settings. The three bibliographic databases MEDLINE, EMBASE and PsycINFO were searched and a double screening of titles and abstracts as well as double screening of full texts was performed. Indices contained in these studies were analysed in terms of focus and composition and evaluated in terms of quality criteria. RESULTS The scoping review identified 36 eligible studies with 18 health indices in six thematic categories. Of the identified indices, seven indices focus on anthropometrical variables, three indices focus on special aspects of newborns and five indices focus on oral health. One index assesses \textquotedblhealthy lifestyle\textquotedbl and one \textquotedblfunctional ability\textquotedbl whereas one index a combination of different aspects. Most indices are calculated by using primary health data. CONCLUSIONS Alone or in combination with single sets of indicators, indices in six major thematic domains may be used as pragmatic tools for monitoring children's and adolescents´ health and the evaluation of interventions in health promotion and prevention settings

The translational potential of microRNAs as biofluid markers of urological tumours

MicroRNAs (miRNAs) are secreted by cells in vesicles, bound in a ribonucleoprotein complex or as free molecules. These miRNA secretion pathways are dysregulated in cancer, making miRNAs attractive candidate molecules for liquid biopsies. A number of studies have investigated the regulation of miRNA secretion into blood and urine and suggested that miRNAs are noninvasive diagnostic, prognostic and surveillance markers in urological carcinomas, and research in this area has increased over the past 5 years. However, methodological and analytical pitfalls exist and require addressing to enable future translation of the laboratory findings regarding miRNAs as biomarkers into clinical practice in bladder cancer, kidney cancer, prostate cancer and testicular cancer