Bedeutung neuronaler microRNAs während der myofibroblastischen Transdifferenzierung hepatischer Sternzellen

Chronische Lebererkrankungen, unabhängig von ihrer Ätiologie, münden in der Leberfibrose. Die Leberfibrose ist gekennzeichnet durch einen unkontrollierten Vernarbungsprozess, der mit einer deutlichen Akkumulation von extrazellulärer Matrix im interstitiellen Raum verbunden ist. Hepatische Sternzellen (HSC) stellen dabei den zentralen Zelltyp in der Deposition extrazellulärer Matrix dar, indem sie nach Aktivierung eine myofibroblastische Transdifferenzierung durchlaufen. Neben den Eigenschaften als Myofibroblast wurden in der Vergangenheit auch neuronale Marker für diese Zellen beschrieben. MicroRNAs spielen eine Rolle in diversen Zellprozessen, wie Entwicklung, Differenzierung und Apoptose. Diese kleinen, einzelsträngigen RNA-Moleküle haben die Funktion, Gene posttranskriptionell zu regulieren. Für die primär in neuronalen Geweben vorkommenden microRNAs miR-9, miR-125b und miR-128 ist eine Beteiligung in der neuronalen Differenzierung bekannt. Innerhalb der vorliegenden Arbeit wurde die Bedeutung neuronaler miRNAs während der myofibroblastischen Transdifferenzierung von HSC untersucht. Dafür wurde zunächst die Isolierung primärer HSC etabliert, da dieses Zellsystem die in vivo ablaufenden Vorgänge während der Differenzierung auch in vitro widerspiegelt. Durch Real-Time-PCR-Quantifizierung konnte nachgewiesen werden, dass die Expression der neuronalen microRNAs miR-9, miR-125b und miR-128 während der in vitro-Transdifferenzierung primärer HSC hochreguliert wurde. Um vertiefend eine Bedeutung dieser microRNAs in HSC zu untersuchen, wurden verschiedene Ansätze zur Bestimmung der Zieltranskripte angegangen. Durch in silico-Analysen mit Hilfe von Datenbanken zeigten sich für miR-9, miR-125b und miR-128 Übereinstimmungen beim Vergleich ihrer potenziellen Ziel-mRNAs. Dabei konnten für das Transkript Lin28 in dessen 3`-untranslatierten Region (UTR) für miR-9, miR-125b, und miR-128 mögliche Bindungsstellen gefunden werden. Deshalb wurden die potenziellen Bindungssequenzen der einzelnen miRNAs sowohl als Wildtyp als auch als Mutante an das 3`-Ende eines Luziferase-Reporters flankierend kloniert und nach Co-Transfektion mit der miRNA in Reporterassays eingesetzt. Diese zeigten eine Interaktion der neuronalen miRNAs mit der 3`-UTR der Lin28 mRNA. Während der myofibroblastischen Differenzierung nahm die Expression von Lin28 stark ab. Dieser inverse Exressionsverlauf von Lin28 zu den neuronalen miRNAs spricht ebenfalls für einen direkten Einfluss von miR-9, miR-125b und miR-128 auf die Lin28 Expression. Zusätzlich zu Lin28 zeigte die Datenbankrecherche Klf4, einen weiteren Pluripotenzfaktor, als potenzielles Zieltranskript für miR-128. Die spezifische Interaktion von miR-128 mit der 3`-UTR von Klf4 konnte ebenfalls erfolgreich über einen Reporterassay dargestellt werden. In zwei weiteren Ansätzen zur Identifizierung möglicher Zieltranskripte in vitro wurde sowohl ein Hybridisierungs-Microarray als auch erstmals die Methode der Ago2-Immunopräzipitation eingesetzt. In den miR-128 behandelten HSC zeigten sich vor allem in der Microarray-Analyse viele Mitglieder der CC- und CXC-Chemokinfamilie als neue, nicht in den Datenbanken vorkommende Ziel-mRNAs für miR-128. Mit Hilfe der Ago2-Immunopräzipitation konnte eine direkte Interaktion der microRNA mit ihrem Zieltranskript im RNA induced silencing complex (RISC) in HSC analysiert werden. Die im RISC von den neuronalen miRNAs gebundenen Transkripte wurden primär durch Klonierung der cDNA mit direkter Sequenzierung und des Weiteren in einer umfangreichen Analyse durch Next Generation Sequencing identifiziert. In diesen Ansätzen wurden für miR-9, miR-125b, aber vor allem für die miR-128 erneut die Mitglieder der Chemokinfamilie als Bindungspartner der neuronalen miRNAs gefunden. Das in der Leberfibrose prädominante Chemokin Ccl2, das bereits durch die Expressionsprofilanalyse nach Überexpression von miR-128 durch eine verringerte Expression aufgefallen war, wurde auf seine direkte Interaktion mit miR-128 durch Reporterassays überprüft. Dabei konnte Ccl2 als Ziel-mRNA für diese neuronale microRNA bestätigt werden. In der myofibroblastischen Transdifferenzierung von HSC ist Ccl2 gegenläufig zur miR-128 Induktion reprimiert. Mit Hilfe des Next Generation Sequencing wurde in Übereinstimmung zu den in silico-Daten zusätzlich Notch1 als Zieltranskript für die neuronalen microRNAs identifiziert. In jüngster Vergangenheit wurde Notch1 als ein wesentlicher Faktor, der die myofibroblastische Transdifferenzierung von HSC hemmt, diskutiert. In der vorgestellten Arbeit wurde für die drei neuronalen microRNAs eine spezifische Interaktion mit der 3`-UTR von Notch1 dargelegt. Mit der Hochregulierung von miR-9, miR-125b und miR-128 während der Transdifferenzierung von HSC konnte umgekehrt eine Herunterregulierung von Notch1 auf Transkript- und Proteinebene veranschaulicht werden. Daher hat die hier gezeigte Interaktionsachse von neuronalen miRNAs und Notch1 eine besonders hohe Bedeutung für den Initiierungsmechanismus der myofibroblastischen Transdifferenzierung

A Classification System for Teachers’ Motivational Behaviours Recommended in Self-Determination Theory Interventions

Teachers’ behaviour is a key factor that influences students’ motivation. Many theoretical models have tried to explain this influence, with one of the most thoroughly researched being self-determination theory (SDT). We used a Delphi method to create a classification of teacher behaviours consistent with SDT. This is useful because SDT-based interventions have been widely used to improve educational outcomes. However, these interventions contain many components. Reliably classifying and labelling those components is essential for implementation, reproducibility, and evidence synthesis. We used an international expert panel (N = 34) to develop this classification system. We started by identifying behaviours from existing literature, then refined labels, descriptions, and examples using the Delphi panel’s input. Next, the panel of experts iteratively rated the relevance of each behaviour to SDT, the psychological need that each behaviour influenced, and its likely effect on motivation. To create a mutually exclusive and collectively exhaustive list of behaviours, experts nominated overlapping behaviours that were redundant, and suggested new ones missing from the classification. After three rounds, the expert panel agreed upon 57 teacher motivational behaviours that were consistent with SDT. For most behaviours (77%), experts reached consensus on both the most relevant psychological need and influence on motivation. Our classification system provides a comprehensive list of teacher motivational behaviours and consistent terminology in how those behaviours are labelled. Researchers and practitioners designing interventions could use these behaviours to design interventions, to reproduce interventions, to assess whether these behaviours moderate intervention effects, and could focus new research on areas where experts disagree

Measuring exercise in eating disorder patients : a Delphi study to aggregate clinical and research knowledge

Background: Exercise is a prominent feature of most eating disorders, and has been shown to have a number of detrimental effects on treatment outcome. There is some disagreement in the literature regarding the construct of compulsive exercise, and assessment and treatment varies significantly. This study therefore aimed to aggregate expert clinicians’ and researchers’ views on how to define and measure compulsive exercise in eating disorder patients. The expert panel was also asked about questionnaire design, and possible problems when measuring compulsive exercise. Method: This study used the Delphi method to establish consensus amongst an expert panel. Three successive rounds of questionnaires were distributed to the panel over a period of six months. The first round consisted of four open-ended questions regarding the definition and measurement of compulsive exercise in eating disorder patients. For Round 2, 70 statements were derived from the answers, and panelists were asked to rate each item on a Likert-based scale. An 85% consensus level was chosen. In Round 3, 44 statements were re-rated by the panel. Results: Seventeen of 24 participants completed all three rounds of the study. Consensus was achieved for 63% of the items, while 18.5% reached near consensus, and 18.5% did not reach consensus after Round 3. The panel agreed on a number of important aspects of compulsive exercise. Several suggestions regarding the format of a questionnaire assessing this behavior were also endorsed. The panel further identified common difficulties when assessing compulsive exercise in eating disorder patients, notably a lack of consensus still apparent in the literature. Conclusion: The current findings constitute a further step towards a unified definition of compulsive exercise, and contribute important suggestions to the measurement of this behavior

The intersection of gender, social class, and cultural context: a meta-analysis

Expectancy value theory is often evoked by educational psychologists to explain gender differences in Science, Technology, Engineering, and Mathematics (STEM) variables. Yet gender does not operate in isolation. Nor are gender effects likely to be context free. In the current meta-analysis, we explore gender differences in STEM-related expectancy for success, and the task values of intrinsic, utility, attainment, and cost. We find that gender differences were generally small in size. Invoking the concept of intersectionality, we find that heterogeneity in gender effect sizes are large and gender differences are moderated, primarily, by socioeconomic status, ethnic diversity, and somewhat by national gender equalit

Evaluating remote facilitation intensity for multi-national translation of nurse-initiated stroke protocols (QASC Australasia): a protocol for a cluster randomised controlled trial

Economic evaluation; Process evaluation; StrokeAvaluació econòmica; Avaluació de processos; IctusEvaluación económica; Evaluación de procesos; IctusBackground Facilitated implementation of nurse-initiated protocols to manage fever, hyperglycaemia (sugar) and swallowing difficulties (FeSS Protocols) in 19 Australian stroke units resulted in reduced death and dependency for stroke patients. However, a significant gap remains in translating this evidence-based care bundle protocol into standard practice in Australia and New Zealand. Facilitation is a key component for increasing implementation. However, its contribution to evidence translation initiatives requires further investigation. We aim to evaluate two levels of intensity of external remote facilitation as part of a multifaceted intervention to improve FeSS Protocol uptake and quality of care for patients with stroke in Australian and New Zealand acute care hospitals. Methods A three-arm cluster randomised controlled trial with a process evaluation and economic evaluation. Australian and New Zealand hospitals with a stroke unit or service will be recruited and randomised in blocks of five to one of the three study arms—high- or low-intensity external remote facilitation or a no facilitation control group—in a 2:2:1 ratio. The multicomponent implementation strategy will incorporate implementation science frameworks (Theoretical Domains Framework, Capability, Opportunity, Motivation – Behaviour Model and the Consolidated Framework for Implementation Research) and include an online education package, audit and feedback reports, local clinical champions, barrier and enabler assessments, action plans, reminders and external remote facilitation. The primary outcome is implementation effectiveness using a composite measure comprising six monitoring and treatment elements of the FeSS Protocols. Secondary outcome measures are as follows: composite outcome of adherence to each of the combined monitoring and treatment elements for (i) fever (n=5); (ii) hyperglycaemia (n=6); and (iii) swallowing protocols (n=7); adherence to the individual elements that make up each of these protocols; comparison for composite outcomes between (i) metropolitan and rural/remote hospitals; and (ii) stroke units and stroke services. A process evaluation will examine contextual factors influencing intervention uptake. An economic evaluation will describe cost differences relative to each intervention and study outcomes. Discussion We will generate new evidence on the most effective facilitation intensity to support implementation of nurse-initiated stroke protocols nationwide, reducing geographical barriers for those in rural and remote areas.This study is funded by a National Health and Medical Research Council Investigator Grant (Grant ID: APP1196352) awarded to SM. The funding body has no role in the design of the study and collection, analysis and interpretation of data and in writing the manuscript

Simultaneous fitting of real-time PCR data with efficiency of amplification modeled as Gaussian function of target fluorescence

<p>Abstract</p> <p>Background</p> <p>In real-time PCR, it is necessary to consider the efficiency of amplification (EA) of amplicons in order to determine initial target levels properly. EAs can be deduced from standard curves, but these involve extra effort and cost and may yield invalid EAs. Alternatively, EA can be extracted from individual fluorescence curves. Unfortunately, this is not reliable enough.</p> <p>Results</p> <p>Here we introduce simultaneous non-linear fitting to determine – without standard curves – an optimal common EA for all samples of a group. In order to adjust EA as a function of target fluorescence, and still to describe fluorescence as a function of cycle number, we use an iterative algorithm that increases fluorescence cycle by cycle and thus simulates the PCR process. A Gauss peak function is used to model the decrease of EA with increasing amplicon accumulation. Our approach was validated experimentally with hydrolysis probe or SYBR green detection with dilution series of 5 different targets. It performed distinctly better in terms of accuracy than standard curve, DART-PCR, and LinRegPCR approaches. Based on reliable EAs, it was possible to detect that for some amplicons, extraordinary fluorescence (EA > 2.00) was generated with locked nucleic acid hydrolysis probes, but not with SYBR green.</p> <p>Conclusion</p> <p>In comparison to previously reported approaches that are based on the separate analysis of each curve and on modelling EA as a function of cycle number, our approach yields more accurate and precise estimates of relative initial target levels.</p

Paths to the light and dark sides of human nature : A meta-analysis of the prosocial benefits of autonomy and the antisocial costs of control

Self-determination theory (SDT) posits that experiences of autonomy lead people to be more prosocial, whereas experiences of control lead to antisocial actions. In this meta-analysis, we tested the links between autonomy and prosociality and control and antisociality, across 139 reports (167 studies) with 1,189 effect sizes (N = 75,546 participants). We used two-stage structural equation modeling including both correlational and longitudinal study designs. We found support for the hypothesized direct links between autonomy and prosociality and between control and antisociality, with cross-paths between these constructs being weaker. In line with SDT’s claims that the salutary effects of autonomy are universal, results also showed that the hypothesized links were consistent across cultures, genders, and age categories. We also reviewed emerging experimental research on the effect of autonomy-priming interventions on prosociality. To conclude, we discuss the theoretical and practical implications of these findings and lay out an agenda for future research. (PsycInfo Database Record (c) 2022 APA, all rights reserved