Optogenetische Stimulation des nativen Herzmuskels durch Adeno-assoziierte Virustransduktion von Channelrhodopsin-2

Die Optogenetik ist eine innovative Methode, die die transgene Expression von lichtsensitiven Proteinen verwendet zur elektrophysiologischen oder biochemischen Stimulation von Zellen durch Beleuchtung. Das Beleuchten mit blauem Licht der Wellenlänge von λ = 470 nm löst in lichtsensitiven Kardiomyozyten, die den Kationenkanal Channelrhodopsin-2 (ChR2) exprimieren, eine Depolarisation mit anschließendem Aktionspotential aus (Boyden et al., 2005; Nagel et al., 2003). Dies ermöglicht eine optogenetische Stimulation von Herzen, was zuvor in vivo im transgenen Zebrafisch-Embryo und in der adulten Maus gezeigt wurde, wobei in diesen transgenen Tiermodellen alle Kardiomyozyten das ChR2 Protein exprimierten (Arrenberg et al., 2010; Bruegmann et al., 2010). Der nächste Schritt zu einer medizinischen Therapie als optogenetischer Herzschrittmacher oder Defibrillator, ist daher der Gentransfer von Channelrhodopsin-2 in ein natives, nicht transgenes Herz. Die vorliegende Arbeit untersuchte zwei Applikationswege für Adeno-assoziierte Virusvektoren, um eine stabile Expression von ChR2 im Mausherz zu erreichen: 1. Die lokale Injektion direkt in das Myokard und 2. die systemische Injektion über die Blutbahn durch Punktion einer Vene. Beide Methoden resultierten in Genexpression von ChR2 im nativen Herzen und erfolgreicher optischer Stimulierbarkeit.Die lokale Injektion von AAV2/1-CAG-hChR2-mCherry ermöglichte im Bereich der Injektionsstelle die optische Stimulation des Myokards. Die Stimulierbarkeit des Herzens dauerte jedoch nur drei Wochen an. Darüber hinaus wurde neben dem Verlust der optischen Stimulierbarkeit auch ein Verlust von ventrikulären Myokardgewebe festgestellt. Durch histologische Analysen wurde im Bereich der Injektionsstelle eine Infiltration von Leukozyten nachgewiesen und fibrotisches Narbengewebe gefunden. Die systemische Injektion von AAV2/9-CAG-hChR2-mCherry ermöglichte die optische Stimulation des Herzens über beide Ventrikel und über das rechte Atrium. Durch die Fluoreszenz des mit ChR2 fusionierten mCherry Proteins wurde die Expression von ChR2-mCherry in allen Bereichen des Herzens in 58 % der Herzmuskelzellen nachgewiesen. Die optische Stimulierbarkeit wurde bereits 4 Wochen nach der Injektion von 2 x 1011 gc AAV2/9-CAG-hChR2-mCherry festgestellt und hielt nachweislich bis zu 16 Monate an. In histologischen Analysen dieser Herzen wurden keine Anzeichen für negative Immunreaktionen oder Narbengewebe gefunden. Durch in vivo und ex vivo Stimulationsanalysen wurden die unteren Grenzwerte der Lichtintensität ermittelt, die für eine optische Stimulation notwendig waren. Weiterhin war eine zehn Minuten andauernde Stimulation durch Lichtimpulse ohne Aussetzer des Herzschlags möglich. Der künstlich beschleunigte Herzrhythmus kehrte nach der Lichtstimulation sofort zum normalen Sinusrhythmus zurück. Die optische Stimulation hatte somit keinen negativen Einfluss auf die kardiale Elektrophysiologie. Die Ergebnisse der vorliegenden Arbeit bestätigen, dass die optische Herzstimulation nach Gentransfer von ChR2 ein großes Potential für eine therapeutische Anwendung als optogenetischer Herzschrittmacher hat

Global standards of Constitutional law : epistemology and methodology

Just as it led the philosophy of science to gravitate around scientific practice, the abandonment of all foundationalist aspirations has already begun making political philosophy into an attentive observer of the new ways in which constitutional law is practiced. Yet paradoxically, lawyers and legal scholars are not those who understand this the most clearly. Beyond analyzing the jurisprudence that has emerged from the expansion of constitutional justice, and taking into account the development of international and regional law, the ongoing globalization of constitutional law requires comparing the constitutional laws of individual nations. Following Waldron, the product of this new legal science can be considered as ius gentium. This legal science is not as well established as one might like to think. But it can be developed on the grounds of the practice that consists in ascertaining standards. As abstract types of best “practices” (and especially norms) of constitutional law from around the world, these are only a source of law in a substantive, not a formal, sense. They thus belong to what I should like to call a “second order legal positivity.” In this article I will undertake, both at a methodological and an epistemological level, the development of a model for ascertaining global standards of constitutional law

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The World Urban Database and Access Portal Tools (WUDAPT) is a community initiative to collect worldwide data on urban form (i.e., morphology, materials) and function (i.e., use and metabolism). This is achieved through crowdsourcing, which we define here as the collection of data by a bounded crowd, composed of students. In this process, training data for the classification of urban structures into Local Climate Zones (LCZ) are obtained, which are, like most volunteered geographic information initiatives, of unknown quality. In this study, we investigated the quality of 94 crowdsourced training datasets for ten cities, generated by 119 students from six universities. The results showed large discrepancies and the resulting LCZ maps were mostly of poor to moderate quality. This was due to general difficulties in the human interpretation of the (urban) landscape and in the understanding of the LCZ scheme. However, the quality of the LCZ maps improved with the number of training data revisions. As evidence for the wisdom of the crowd, improvements of up to 20% in overall accuracy were found when multiple training datasets were used together to create a single LCZ map. This improvement was greatest for small training datasets, saturating at about ten to fifteen sets

The power of monitoring: optimizing survey designs to detect occupancy changes in a rare amphibian population

Biodiversity conservation requires reliable species assessments and rigorously designed surveys. However, determining the survey effort required to reliably detect population change can be challenging for rare, cryptic and elusive species. We used a tropical bromeliad-dwelling frog as a model system to explore a cost-effective sampling design that optimizes the chances of detecting a population decline. Relatively few sampling visits were needed to estimate occupancy and detectability with good precision, and to detect a 30% change in occupancy with 80% power. Detectability was influenced by observer expertise, which therefore also had an effect on the sampling design – less experienced observers require more sampling visits to detect the species. Even when the sampling design provides precise parameter estimates, only moderate to large changes in occupancy will be detected with reliable power. Detecting a population change of 15% or less requires a large number of sites to be surveyed, which might be unachievable for range-restricted species occurring at relatively few sites. Unless there is high initial occupancy, rare and cryptic species will be particularly challenging when it comes to detecting small population changes. This may be a particular issue for long-term monitoring of amphibians which often display low detectability and wide natural fluctuations

Impact of COVID-19 on cardiovascular testing in the United States versus the rest of the world

Objectives: This study sought to quantify and compare the decline in volumes of cardiovascular procedures between the United States and non-US institutions during the early phase of the coronavirus disease-2019 (COVID-19) pandemic. Background: The COVID-19 pandemic has disrupted the care of many non-COVID-19 illnesses. Reductions in diagnostic cardiovascular testing around the world have led to concerns over the implications of reduced testing for cardiovascular disease (CVD) morbidity and mortality. Methods: Data were submitted to the INCAPS-COVID (International Atomic Energy Agency Non-Invasive Cardiology Protocols Study of COVID-19), a multinational registry comprising 909 institutions in 108 countries (including 155 facilities in 40 U.S. states), assessing the impact of the COVID-19 pandemic on volumes of diagnostic cardiovascular procedures. Data were obtained for April 2020 and compared with volumes of baseline procedures from March 2019. We compared laboratory characteristics, practices, and procedure volumes between U.S. and non-U.S. facilities and between U.S. geographic regions and identified factors associated with volume reduction in the United States. Results: Reductions in the volumes of procedures in the United States were similar to those in non-U.S. facilities (68% vs. 63%, respectively; p = 0.237), although U.S. facilities reported greater reductions in invasive coronary angiography (69% vs. 53%, respectively; p < 0.001). Significantly more U.S. facilities reported increased use of telehealth and patient screening measures than non-U.S. facilities, such as temperature checks, symptom screenings, and COVID-19 testing. Reductions in volumes of procedures differed between U.S. regions, with larger declines observed in the Northeast (76%) and Midwest (74%) than in the South (62%) and West (44%). Prevalence of COVID-19, staff redeployments, outpatient centers, and urban centers were associated with greater reductions in volume in U.S. facilities in a multivariable analysis. Conclusions: We observed marked reductions in U.S. cardiovascular testing in the early phase of the pandemic and significant variability between U.S. regions. The association between reductions of volumes and COVID-19 prevalence in the United States highlighted the need for proactive efforts to maintain access to cardiovascular testing in areas most affected by outbreaks of COVID-19 infection

Judging the Quality of Legal Research: A Qualified Response to the Demand for Greater Methodological Rigour

Capstone programs are increasing recognised as high-impact value-laden learning experiences for students, especially in aiding the transition of final year students to professional practice. Although these learning programs have a long history in many disciplines, they are a very recent inclusion in law curriculum in Australia. This paper reports on a project designed to renew the final year law curriculum in Australia by developing a principled approach to designing capstone programs. This framework is based on six key principles, which are transition, integration and closure, diversity, engagement, assessment, and evaluation. The project also identified two themes underpinning capstone experiences which sit behind the six principles - integration and closure, and transition. The principled approach advocated by the authors’ bridges the gap between curriculum theorising and practice. This paper discusses the theme of transition and how the resources provided in the project Toolkit assist unit coordinators to design effective final year capstone programs. The toolkit outlines the six principles, providing commentary on each, and also offers suggestions for subject models along with examples of actual capstone programs, all of which have been designed to facilitate smooth transitions for final year students as they move from undergraduate to emerging professional