Mechanistic Insight into RET Kinase Inhibitors Targeting the DFG-out Conformation in RETrearranged Cancer

RET Fusions-Gene können in 1-2% aller Lungen-Adenokarzinom Patienten nachgewiesen werden. Diese genetischen Veränderungen stellen mittels Tyrosin-Kinase Inhibitoren potentiell therapierbare molekulare Zielstrukturen dar (Pao and Hutchinson 2012), jedoch haben klinische Studien im Jahr 2018 für diese Lungenkarzinome bisher noch keine ausreichend erfolgreichen Therapieansätze zeigen können (Drilon et al. 2018). Um systematisch das therapeutische Profil von AD80 und einer Vielzahl weiterer Tyrosinkinase Inhibitoren gegen RET-Fusion getriebene Zellmodelle auswerten zu können, habe ich zunächst Ba/F3 Zellen viral mit KIF5B-RET sowie CCDC6-RET transduziert. Das Wachstum von Ba/F3 Zellen in vitro ist im Normalzustand abhängig von IL-3. Sobald sie mit einem starken Onkogen jedoch transduziert werden, sind sie unabhängig von IL-3 und proliferieren nur noch abhängig von der Aktivität des entsprechenden Onkogens. Durch dieses Modelsystem konnte ich eine große Anzahl verschiedener Inhibitoren gegen RET testen und ihre Potenz untereinander vergleichen. Die selbst etablierten Ba/F3 Zell-Linien zusammen mit der RET-Fusion getriebenen Lungen-Adenokarzinom Zelllinie LC-2/AD bildeten den Ausgangspunkt für mein Projekts. Zusätzlich habe ich ein endogen RET-mutiertes Zell-Model mittels der Genom-Editierungstechnik CRISPR/Cas9 etabliert. Dafür habe ich einen Vektor mit Cas9 mRNA sowie zwei Promotoren für die Expression von spezifischen „single-guided RNAs“ (sgRNA) kloniert und murine Fibroblast Zellen (NIH-3T3) transfiziert. Mittels sgRNAs gegen die jeweiligen spezifischen Introns von RET und KIF5B konnte ich somit in selektionierten NIH-3T3 Zellen KIF5B-RET Translokationen generieren. Indem ich diese neu etablierten Zelllinien zusammen mit einer größeren Anzahl humaner Lungenkrebs Zell-Linien gegen potentielle RET-Inhibitoren getestet habe, konnte ich die klinische Erfahrung in vitro bestätigen, dass die zurzeit gängigen Therapieansätze mit Tyrosinkinase- Inhibitoren wie z.B. Cabozantinib, Alectinib oder Vandetanib wahrscheinlich eine nicht ausreichend hohe therapeutische Potenz besitzen, um eine effektive Wirkung auf RET getriebene Tumore zu entwickeln. Weiterhin deuteten die Daten darauf hin, dass andere Tyrosin-Kinase Inhibitoren, wie z.B. AD80 und Ponatinib, im Vergleich dazu 100 bis 1000-fach potenter sind und spezifisch die RET-Kinase inhibieren. Als nächstes habe ich die Unterschiede in den Zell-Viabilitäts-Assays mit den Veränderungen auf der Protein-Ebene zu verglichen. Die folgenden Western Blot und Phosphoproteom Analysen haben eine entsprechende Reduktion in phospho-RET und den weiteren nachgeschalteten Signal-Molekülen gezeigt. Zusätzlich haben die in vivo Ergebnisse unserer CCDC6-RET PDX-Mausmodelle unsere in vitro Daten mit gutem Tumoransprechen unter Therapie mit AD80 bestätigt. Parallel zu meiner Arbeit für das Projekt haben wir mit anderen Arbeitsgruppen zusammengearbeitet, um ein tieferes Verständnis über die funktionellen Mechanismen hinter der hohen Aktivität von AD80 gegen RET zu erhalten. Mittels computerbasierter Modelle haben wir ableiten können, dass AD80 mit hoher Wahrscheinlichkeit als Typ II Inhibitor die RET-Kinase in der inaktiven „DFG-out“ Konformation bindet, was mit einer erhöhten Kinase- Thermostabilität im Vergleich zu Typ I Inhibitoren als Bindungspartner einhergeht. Dies wiederum ist ein Surrogat-Parameter für eine engere Kinase-Bindung durch die Inhibitoren und könnte eine mögliche Erklärung für die hohe Cytotoxitität in unseren Experimenten sein. Im Folgenden hat sich das Projekt mehr auf die Rolle von Resistenz-Mechanismen in RET mutierten Zelllinien konzentriert. Mittels zielgerichteter Mutagenese (site-directed mutagenesis) habe ich an der sogenannten „Gatekeeper Position“ mutierte Ba/F3 KIFRETV804M und CCDC6-RETV804M etablieren können, um die Wirksamkeit der Inhibitoren dagegen zu testen. Wieder zeigten AD80 und Ponatinib den stärksten inhibitorischen Effekt gegen RET mit nur einer geringen Reduktion der Cytotoxitität in den Zell-Viabilitäts-Assays und RET-Dephosphorylierung im Vergleich zu RETwt. Mittels Sättigungsmutagenese (saturated mutagenesis screening) habe ich versucht, Resistenzmutationen zu finden, die neben der bekannten „Gatekeeper Position“ pV804M zur Resistenz gegen AD80 führen könnten. Es zeigte sich in der Sequenzierung von resistenten Ba/F3CCDC6-RET Zellen die missense Mutation pI788N (c.2363T>A) im Bereich der RET-Kinase Domäne als potentielle sekundäre Resistenzmutation unter Therapie mit AD80. Diesen Resistenz-Effekt durch die neue Mutation konnte ich dann in den folgenden zellulären Modellen mittels Zell-Viabilitäts-Assays und Western Blots bestätigen. Zusätzlich haben Ergebnisse einer sekundär gegen AD80 resistent gewordenen TPC-1 Schilddrüsenkarzinom-Zelllinie sowie RNASequenzierungen von LC-2/AD Zelllinien unter Therapie gegen AD80 ergeben, dass MAPKReaktivierung potentiell eine Rolle als Resistenz-Mechanismus in RET-getriebenen Tumoren besitzen könnte. Um formell die Rolle von MAPK-Reaktivierung in Bezug auf Resistenz-Effekte zu testen, wurden LC-2/AD Zellen von mir lentiviral mit KRASG12V transduziert, was zu einer Überexprimierung von KRAS und zu einer folgenden Resistenz gegen die Behandlung mit AD80 geführt hat. Für Ponatinib gibt es bereits klinische Phase 3 Studien an Patienten mit Chronisch Myeloischer Leukämie, die jedoch trotz gutem initialen Tumor Ansprechen aufgrund von erhöhten Therapie assoziierten Komplikationen beendet werden musste (Lipton et al. 2016). Der RET-Kinase Inhibitor AD80 wäre daher ein geeigneter Kandidat für zukünftige klinische Studien. Bedenkt man den hohen Bedarf an neuen, wirksamen und verträglichen Therapieansätzen im Rahmen der individualisierten Lungenkarzinom-Therapie, bietet unsere Studie eine Vielzahl neuer mechanistischer Einsichten in die aktuelle anti-RET Therapie und trägt zur deren zukünftigen Entwicklung bei

Gestaltung der Rahmenbedingungen von gelingenden kollegialen Austausch-prozessen : eine Untersuchung in Teams der Offenen Kinder- und Jugendarbeit in Leipzig

Die vorliegende Arbeit versucht anhand von teilnehmenden Beobachtungen kollegialer Beratungssituationen, Rahmenbedingungen herauszuarbeiten, die für das Gelingen des Austauschs von Bedeutung sind. Die Gestaltung dieser Rahmenbedingungen wird exemplarisch im sozialarbeiterischen Feld der Offene Kinder- und Jugendarbeit untersucht. Das Ergebnis soll jedoch allgemein für Beratungs- und Anleitungskontexte nutzbar sein

Cross-border effects of R&D tax incentives

Existing evidence shows that R&D tax incentives boost countries’ private sector R&D. Given the importance ofmultinational enterprises (MNEs) for private sector innovation, it is unclear, however, whether firms engagein genuinely new R&D or whether R&D is reallocated across borders. Drawing on data on unconsolidated R&Dactivity of MNEs in Europe, we provide evidence that responses are dominated by cross-border relocations:More generous tax incentives in one country increase MNEs’ R&D investments in affiliates located there, whilelowering R&D investments in affiliates of the same MNE group located in other countries. Globally, firmshardly raise their R&D activities when tax incentives become more generous

Exam preparatory course for the 2nd part of the German medical examination in obstetrics and gynecology – a potential tool for the recruitment of new residents during the occupational decision process before the practical year?

Background: The “Second Stage of the Physician Exam” at the end of the 5th year of medical school in Germany is the final step before the “Practical Year.” An exam preparatory class can cover the complete content of Obstetrics and Gynecology (OB/GYN) in two days. We raise the question of whether such training might promote students’ interest in the given specialty during occupational decision making and whether it could even be used by hospitals as a recruitment tool. This investigation is even more important in the context of fierce competition among young professionals at clinics and in different specialties. Methods: We conducted a multimodal course evaluation for four exam preparatory courses (each of which lasted two days and involved 8.5 h of teaching), including pre- and post-course tests with 20 multiple-choice questions to quantify the level of skill gain. Additionally, a standardized evaluation of course satisfaction was performed, followed by a post-exam questionnaire that dealt with studying activities and individual professional objectives. Results: Overall, n = 197 students took part in four identical courses. Among them, n = 121 completed the pre−/post-course tests, n = 170 completed the evaluation, and n = 110 completed the post-exam questionnaire. An average improvement from 13.9 to 17.2 correct answers was observed (max. 20; pre−/post-difference 95%-CI: [2.77; 3.86], t-test: p < 0.0001). By trend, the students noted that course participation positively influenced their later choice of specialty training (m = 3.63; scale 1 = “strongly disagree,” 5 = “strongly agree”). Conclusions: In addition to self-studying, condensed classroom training is effective and reasonable and might also increase the attractivity of OB/GYN among students and have a positive effect on recruitment

Long Cycle‐Life Ca Batteries with Poly(anthraquinonylsulfide) Cathodes and Ca−Sn Alloy Anodes

Calcium (Ca) batteries are attractive post-lithium battery technologies, due to their potential to provide high-voltage and high-energy systems in a sustainable manner. We investigated herein 1,5-poly(anthraquinonylsulfide) (PAQS) for Ca-ion storage with calcium tetrakis(hexafluoroisopropyloxy)borate Ca[B(hfip)$_4$]$_2$ [hfip=OCH(CF$_3$)$_2$] electrolytes. It is demonstrated that PAQS could be synthesized in a cost-effective approach and be processed environmentally friendly into the electrodes. The PAQS cathodes could provide 94 mAh g$^{−1}$ capacity at 2.2 V vs. Ca at 0.5C (1C=225 mAh g$^{−1}$). However, cycling of the cells was severely hindered due to the fast degradation of the metal anode. Replacing the Ca metal anode with a calcium-tin (Ca−Sn) alloy anode, the PAQS cathodes exhibited long cycling performance (45 mAh g$^{−1}$ at 0.5C after 1000 cycles) and superior rate capability (52 mAh g$^{−1}$ at 5C). This is mainly ascribed to the flexible structure of PAQS and good compatibility of the alloy anodes with the electrolyte solutions, which allow reversible quinone carbonyl redox chemistry in the Ca battery systems. The promising properties of PAQS indicate that further exploration of the organic cathode materials could be a feasible direction towards green Ca batteries

ChatGPT’s performance in German OB/GYN exams – paving the way for AI-enhanced medical education and clinical practice

BackgroundChat Generative Pre-Trained Transformer (ChatGPT) is an artificial learning and large language model tool developed by OpenAI in 2022. It utilizes deep learning algorithms to process natural language and generate responses, which renders it suitable for conversational interfaces. ChatGPT’s potential to transform medical education and clinical practice is currently being explored, but its capabilities and limitations in this domain remain incompletely investigated. The present study aimed to assess ChatGPT’s performance in medical knowledge competency for problem assessment in obstetrics and gynecology (OB/GYN).MethodsTwo datasets were established for analysis: questions (1) from OB/GYN course exams at a German university hospital and (2) from the German medical state licensing exams. In order to assess ChatGPT’s performance, questions were entered into the chat interface, and responses were documented. A quantitative analysis compared ChatGPT’s accuracy with that of medical students for different levels of difficulty and types of questions. Additionally, a qualitative analysis assessed the quality of ChatGPT’s responses regarding ease of understanding, conciseness, accuracy, completeness, and relevance. Non-obvious insights generated by ChatGPT were evaluated, and a density index of insights was established in order to quantify the tool’s ability to provide students with relevant and concise medical knowledge.ResultsChatGPT demonstrated consistent and comparable performance across both datasets. It provided correct responses at a rate comparable with that of medical students, thereby indicating its ability to handle a diverse spectrum of questions ranging from general knowledge to complex clinical case presentations. The tool’s accuracy was partly affected by question difficulty in the medical state exam dataset. Our qualitative assessment revealed that ChatGPT provided mostly accurate, complete, and relevant answers. ChatGPT additionally provided many non-obvious insights, especially in correctly answered questions, which indicates its potential for enhancing autonomous medical learning.ConclusionChatGPT has promise as a supplementary tool in medical education and clinical practice. Its ability to provide accurate and insightful responses showcases its adaptability to complex clinical scenarios. As AI technologies continue to evolve, ChatGPT and similar tools may contribute to more efficient and personalized learning experiences and assistance for health care providers

Virtual Axle Detector Based on Analysis of Bridge Acceleration Measurements by Fully Convolutional Network

In the practical application of the Bridge Weigh-In-Motion (BWIM) methods, the position of the wheels or axles during the passage of a vehicle is a prerequisite in most cases. To avoid the use of conventional axle detectors and bridge type-specific methods, we propose a novel method for axle detection using accelerometers placed arbitrarily on a bridge. In order to develop a model that is as simple and comprehensible as possible, the axle detection task is implemented as a binary classification problem instead of a regression problem. The model is implemented as a Fully Convolutional Network to process signals in the form of Continuous Wavelet Transforms. This allows passages of any length to be processed in a single step with maximum efficiency while utilising multiple scales in a single evaluation. This allows our method to use acceleration signals from any location on the bridge structure and act as Virtual Axle Detectors (VADs) without being limited to specific structural types of bridges. To test the proposed method, we analysed 3787 train passages recorded on a steel trough railway bridge of a long-distance traffic line. Results of the measurement data show that our model detects 95% of the axles, which means that 128,599 out of 134,800 previously unseen axles were correctly detected. In total, 90% of the axles were detected with a maximum spatial error of 20 cm, at a maximum velocity of vmax=56.3m/s. The analysis shows that our developed model can use accelerometers as VADs even under real operating conditions

Psychological distress, fear and coping strategies during the second and third waves of the COVID-19 pandemic in Southern Germany

Background: The COVID-19 pandemic has imposed enormous psychological discomfort and fear across the globe, including Germany. Objectives: To assess the levels of COVID-19 associated psychological distress and fear amongst Southern German population, and to identify their coping strategies. Methods: A cross-sectional survey using an online questionnaire was conducted in healthcare and community settings in the region of Ulm, Southern Germany. Assessment inventories were the Kessler Psychological Distress Scale (K-10), the Brief Resilient Coping Scale (BRCS), and the Fear of COVID-19 Scale (FCV-19S), which were valid and reliable tools. Results: A total of 474 Individuals participated in the study. The mean age was 33.6 years, and 327 (69%) were females. Most participants (n = 381, 80.4%) had high levels of psychological distress, whereas only 5.1% had high levels of fear, and two-thirds of participants showed higher levels of coping. Moderate to very high levels of psychological distress were associated with being female, living alone, distress due to employment changes, experiencing financial impact, having multiple co-morbidities, being a smoker, increased alcohol use over the previous 6 months, contact with COVID-19 cases and healthcare providers for COVID-19-related stress. Individuals who wer

Long‐term validation of virtual sensing of a railway bridge with ballasted superstructure

Railway bridges have a long lifespan, which is challenged by the constant development of vehicles leading to increased loads that they were not originally designed for. To ensure the longest possible use of existing structures, a sensor‐based structural health monitoring system can make a significant contribution. However, due to economic reasons and the inaccessibility of many points of interest, sensors cannot be installed everywhere. Therefore, in most cases, only a few sensors are available at a few points of interest, and methods that aim to reconstruct structural responses at unmeasured points from these measurements are referred to as virtual sensing. In this paper, we have analyzed 19,075 passages recorded on a steel trough bridge with a ballast superstructure and a span of 16.4 m, together with weather data. Our findings show that the influence of train type and speed has a significantly higher impact on the results than environmental factors. The investigation revealed that the model‐based analysis produced similar results to the data‐driven analysis concerning acceleration signals. However, when analyzing strain signals, the two approaches yielded distinctly different results