Optogenetic regulation of endogenous proteins

Techniques of protein regulation, such as conditional gene expression, RNA interference, knock-in and knock-out, lack sufficient spatiotemporal accuracy, while optogenetic tools suffer from non-physiological response due to overexpression artifacts. Here we present a near-infrared light-activatable optogenetic system, which combines the specificity and orthogonality of intrabodies with the spatiotemporal precision of optogenetics. We engineer optically-controlled intrabodies to regulate genomically expressed protein targets and validate the possibility to further multiplex protein regulation via dual-wavelength optogenetic control. We apply this system to regulate cytoskeletal and enzymatic functions of two non-tagged endogenous proteins, actin and RAS GTPase, involved in complex functional networks sensitive to perturbations. The optogenetically-enhanced intrabodies allow fast and reversible regulation of both proteins, as well as simultaneous monitoring of RAS signaling with visible-light biosensors, enabling all-optical approach. Growing number of intrabodies should make their incorporation into optogenetic tools the versatile technology to regulate endogenous targets. Optogenetic approaches to control protein-protein interactions usually require overexpression of the target proteins. Here the authors integrate intrabodies into near-infrared- and blue-light activatable optogenetic tools to control endogenous proteins in mammalian cells.Peer reviewe

Variable pulmonary manifestations in Chitayat syndrome: Six additional affected individuals

Hand hyperphalangism leading to shortened index fingers with ulnar deviation, hallux valgus, mild facial dysmorphism and respiratory compromise requiring assisted ventilation are the key features of Chitayat syndrome. This condition results from the recurrent heterozygous missense variant NM_006494.2:c.266A>G; p.(Tyr89Cys) in ERF on chromosome 19q13.2, encoding the ETS2 repressor factor (ERF) protein. The pathomechanism of Chitayat syndrome is unknown. To date, seven individuals with Chitayat syndrome and the recurrent pathogenic ERF variant have been reported in the literature. Here, we describe six additional individuals, among them only one presenting with a history of assisted ventilation, and the remaining presenting with variable pulmonary phenotypes, including one individual without any obvious pulmonary manifestations. Our findings widen the phenotype spectrum caused by the recurrent pathogenic variant in ERF, underline Chitayat syndrome as a cause of isolated skeletal malformations and therefore contribute to the improvement of diagnostic strategies in individuals with hand hyperphalangism

State-of-the-art analytical methods of viral infections in human lung organoids

Human-based organ models can provide strong predictive value to investigate the tropism, virulence, and replication kinetics of viral pathogens. Currently, such models have received widespread attention in the study of SARS-CoV-2 causing the COVID-19 pandemic. Applicable to a large set of organoid models and viruses, we provide a step-by-step work instruction for the infection of human alveolar-like organoids with SARS-CoV-2 in this protocol collection. We also prepared a detailed description on state-of-the-art methodologies to assess the infection impact and the analysis of relevant host factors in organoids. This protocol collection consists of five different sets of protocols. Set 1 describes the protein extraction from human alveolar-like organoids and the determination of protein expression of angiotensin-converting enzyme 2 (ACE2), transmembrane serine protease 2 (TMPRSS2) and FURIN as exemplary host factors of SARS-CoV-2. Set 2 provides detailed guidance on the extraction of RNA from human alveolar-like organoids and the subsequent qPCR to quantify the expression level of ACE2, TMPRSS2, and FURIN as host factors of SARS-CoV-2 on the mRNA level. Protocol set 3 contains an in-depth explanation on how to infect human alveolar-like organoids with SARS-CoV-2 and how to quantify the viral replication by plaque assay and viral E gene-based RT-qPCR. Set 4 provides a step-by-step protocol for the isolation of single cells from infected human alveolar-like organoids for further processing in single-cell RNA sequencing or flow cytometry. Set 5 presents a detailed protocol on how to perform the fixation of human alveolar-like organoids and guides through all steps of immunohistochemistry and in situ hybridization to visualize SARS-CoV-2 and its host factors. The infection and all subsequent analytical methods have been successfully validated by biological replications with human alveolar-like organoids based on material from different donors

Eleven strategies for making reproducible research and open science training the norm at research institutions

Reproducible research and open science practices have the potential to accelerate scientific progress by allowing others to reuse research outputs, and by promoting rigorous research that is more likely to yield trustworthy results. However, these practices are uncommon in many fields, so there is a clear need for training that helps and encourages researchers to integrate reproducible research and open science practices into their daily work. Here, we outline eleven strategies for making training in these practices the norm at research institutions. The strategies, which emerged from a virtual brainstorming event organized in collaboration with the German Reproducibility Network, are concentrated in three areas: (i) adapting research assessment criteria and program requirements; (ii) training; (iii) building communities. We provide a brief overview of each strategy, offer tips for implementation, and provide links to resources. We also highlight the importance of allocating resources and monitoring impact. Our goal is to encourage researchers - in their roles as scientists, supervisors, mentors, instructors, and members of curriculum, hiring or evaluation committees - to think creatively about the many ways they can promote reproducible research and open science practices in their institutions

Effects of basal and acute cortisol on cognitive flexibility in an emotional task switching paradigm in men

Item does not contain fulltextThe stress hormone cortisol is assumed to influence cognitive functions. While cortisol-induced alterations of declarative memory in particular are well-investigated, considerably less is known about its influence on executive functions. Moreover, most research has been focused on slow effects, and rapid non-genomic effects have not been studied. The present study sought to investigate the impact of acute cortisol administration as well as basal cortisol levels on cognitive flexibility, a core executive function, within the non-genomic time frame. Thirty-eight healthy male participants were randomly assigned to intravenously receive either cortisol or a placebo before performing a task switching paradigm with happy and angry faces as stimuli. Cortisol levels were measured at six points during the experiment. Additionally, before the experiment, basal cortisol measures for the cortisol awakening response were collected on three consecutive weekdays immediately following awakening and 30, 45, and 60 min after. First and foremost, results showed a pronounced impact of acute and basal cortisol on reaction time switch costs, particularly for angry faces. In the placebo group, low basal cortisol was associated with minimal switch costs, whereas high basal cortisol was related to maximal switch costs. In contrast, after cortisol injection, basal cortisol levels showed no impact. These results show that cognitive flexibility-enhancing effects of acute cortisol administration are only seen in men with high basal cortisol levels. This result supports the context dependency of cortisol administration and shows the relevance of taking basal cortisol levels into account.8 p

Mobilität der Zukunft - muss produziert werden. Positionspapier

Motorisierte Mobilität wird zunehmend anders: multimodaler, flexibler, spontaner und situativer. Eines bleibt jedoch gleich, für die Umsetzung werden weiterhin Fahrzeuge benötigt. Wie diese aussehen, ob sie elektrisch oder durch fossile Brennstoffe angetrieben werden, ob sie automatisiert oder sogar autonom fahren und wann welcher Schritt erreicht wird, ist heute nicht mit Bestimmtheit vorhersagbar. Auch was die Nutzung der Mobilität betrifft, gibt es verschiedene Konzepte, die in Abhängigkeit von Generationen und geographischen Regionen zu unterschiedlichsten Szenarien führen. Dennoch: Es geht um Fahrzeuge, und diese müssen auch zukünftig in Deutschland produziert werden. Technologieführerschaft eingeschlossen. Automobilproduktion wird dabei verstärkt von vielfältigen Abhängigkeiten gekennzeichnet sein, die neben den dargestellten Unbestimmtheiten, noch weitere Herausforderungen meistern muss: Fachkräftemangel, Digitalisierung, Diversifizierung, Nachhaltigkeit, globale Wertschöpfungsketten und Ambivalenzen zwischen Massenfertigung und Losgröße 1 wirken sich massiv auf deren konkurrenzfähige Realisierung aus. Bestehendes Know-how stellt derzeit eine ausgezeichnete Basis für eine zukünftige Produktion im Hochlohnland Deutschland dar. Jedoch wird akuter Handlungsbedarf für die Sicherung und den Ausbau des Produktionsstandortes deutlich. Das neue Positionspapier stellt zentrale Treiber dar: Digitalisierung, Markt, Mensch, Antriebstechnologien. Abgeleitet von sich daraus ergebenden Herausforderungen, stellt die Fraunhofer-Allianz autoMOBILproduktion Optionen und einen konkreten Lösungsraum vor