IDH3 mediates apoptosis of alveolar epithelial cells type 2 due to mitochondrial Ca2+ uptake during hypocapnia

In adult respiratory distress syndrome (ARDS) pulmonary perfusion failure increases physiologic dead-space (VD/VT) correlating with mortality. High VD/VT results in alveolar hypocapnia, which has been demonstrated to cause edema formation, atelectasis, and surfactant depletion, evoked, at least in part, by apoptosis of alveolar epithelial cells (AEC). However, the mechanism underlying the hypocapnia-induced AEC apoptosis is unknown. Here, using fluorescent live-cell imaging of cultured AEC type 2 we could show that in terms of CO2 sensing the tricarboxylic acid cycle enzyme isocitrate dehydrogenase (IDH) 3 seems to be an important player because hypocapnia resulted independently from pH in an elevation of IDH3 activity and subsequently in an increase of NADH, the substrate of the respiratory chain. As a consequence, the mitochondrial transmembrane potential (ΔΨ) rose causing a Ca2+ shift from cytosol into mitochondria, whereas the IDH3 knockdown inhibited these responses. Furthermore, the hypocapnia-induced mitochondrial Ca2+ uptake resulted in reactive oxygen species (ROS) production, and both the mitochondrial Ca2+ uptake and ROS production induced apoptosis. Accordingly, we provide evidence that in AEC type 2 hypocapnia induces elevation of IDH3 activity leading to apoptosis. This finding might give new insight into the pathogenesis of ARDS and may help to develop novel strategies to reduce tissue injury in ARDS

ATM Limits Incorrect End Utilization during Non-Homologous End Joining of Multiple Chromosome Breaks

Chromosome rearrangements can form when incorrect ends are matched during end joining (EJ) repair of multiple chromosomal double-strand breaks (DSBs). We tested whether the ATM kinase limits chromosome rearrangements via suppressing incorrect end utilization during EJ repair of multiple DSBs. For this, we developed a system for monitoring EJ of two tandem DSBs that can be repaired using correct ends (Proximal-EJ) or incorrect ends (Distal-EJ, which causes loss of the DNA between the DSBs). In this system, two DSBs are induced in a chromosomal reporter by the meganuclease I-SceI. These DSBs are processed into non-cohesive ends by the exonuclease Trex2, which leads to the formation of I-SceI–resistant EJ products during both Proximal-EJ and Distal-EJ. Using this method, we find that genetic or chemical disruption of ATM causes a substantial increase in Distal-EJ, but not Proximal-EJ. We also find that the increase in Distal-EJ caused by ATM disruption is dependent on classical non-homologous end joining (c-NHEJ) factors, specifically DNA-PKcs, Xrcc4, and XLF. We present evidence that Nbs1-deficiency also causes elevated Distal-EJ, but not Proximal-EJ, to a similar degree as ATM-deficiency. In addition, to evaluate the roles of these factors on end processing, we examined Distal-EJ repair junctions. We found that ATM and Xrcc4 limit the length of deletions, whereas Nbs1 and DNA-PKcs promote short deletions. Thus, the regulation of end processing appears distinct from that of end utilization. In summary, we suggest that ATM is important to limit incorrect end utilization during c-NHEJ

Ku Regulates the Non-Homologous End Joining Pathway Choice of DNA Double-Strand Break Repair in Human Somatic Cells

The repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity and viability for all organisms. Mammals have evolved at least two genetically discrete ways to mediate DNA DSB repair: homologous recombination (HR) and non-homologous end joining (NHEJ). In mammalian cells, most DSBs are preferentially repaired by NHEJ. Recent work has demonstrated that NHEJ consists of at least two sub-pathways—the main Ku heterodimer-dependent or “classic” NHEJ (C-NHEJ) pathway and an “alternative” NHEJ (A-NHEJ) pathway, which usually generates microhomology-mediated signatures at repair junctions. In our study, recombinant adeno-associated virus knockout vectors were utilized to construct a series of isogenic human somatic cell lines deficient in the core C-NHEJ factors (Ku, DNA-PKcs, XLF, and LIGIV), and the resulting cell lines were characterized for their ability to carry out DNA DSB repair. The absence of DNA-PKcs, XLF, or LIGIV resulted in cell lines that were profoundly impaired in DNA DSB repair activity. Unexpectedly, Ku86-null cells showed wild-type levels of DNA DSB repair activity that was dominated by microhomology joining events indicative of A-NHEJ. Importantly, A-NHEJ DNA DSB repair activity could also be efficiently de-repressed in LIGIV-null and DNA-PKcs-null cells by subsequently reducing the level of Ku70. These studies demonstrate that in human cells C-NHEJ is the major DNA DSB repair pathway and they show that Ku is the critical C-NHEJ factor that regulates DNA NHEJ DSB pathway choice

Entwicklung und Validierung eines EPA-basierten Weiterbildungscurriculums im Bereich der Anästhesiologie: Eine Delphi-Studie

Background: Postgraduate training curricula should not be based on time-spans or predefined numbers of performed procedures. One approach to link competencies to clinical tasks is the concept of Entrustable Professional Activities (EPA). The goal of this study was the definition, ranking and validation of EPAs for anaesthesiology postgraduate training and the creation of an anaesthesiologic core curriculum. Methods: Anaesthesiologists of different levels of training participated in the study (single-center, cross-sectional) . First, an expert group defined a preliminary list of EPAs. Then a first Delphi round (n= 47 participants) was applied to identify daily anaesthesiology tasks with the goal to define EPAs. From the first Delphi round a new set of EPAs was defined, using the template and mapping method. Through an alignment process, conducted by the expert group, the preliminary EPAs and the new set of EPAs from the first Delphi round were summarised into a new list of EPAs. This list was presented to the study participants in a second Delphi round (n=80 participants), with the goal to validate and rank each EPA and to define the year of entrustment. For this purpose, participants were asked in the second Delphi round if each EPA should be included into an anaesthesiology core curriculum and in which year of training entrustment should take place. Furthermore, they were asked to rank each EPA on a numeric scale, defining its importance. From this numeric scale, the content validity index (CVI) for each EPA was calculated.Consensus of the results from the second Delphi round was calculated, using the one-way random effects model to calculate Intra-Class-Correlations (ICC). Percentages of agreement among the whole set of EPAs of this study and a previously published set of EPAs were computed.Results: A core-curriculum comprising of 39 EPAs was developed. The EPAs were subdivided into superior/high and inferior/low scoring EPAs, reflecting their importance and were mapped to the year of entrustment. The results reached high consensus among the different participating anaesthesiologist groups (overall agreement was 0.96 for the CVI of each EPA and 0.83 for the year in which the EPAs should be entrusted). Agreement with the previously defined set of EPAs was 73.3%.Conclusion: This study provides a further step in transforming postgraduate anaesthesiology training into a more contemporary approach. Other studies are necessary to complete and amend the presented core curriculum of EPA based postgraduate anaesthesiology training.Hintergrund: Postgraduelle Weiterbildungscurricula sollten sich weder an Zeiten, noch an vorgegebenen Richtzahlen von durchgeführten Prozeduren orientieren. Ein vielversprechender Ansatz, um kompetenzbasierte Curricula in den klinischen Alltag zu integrieren, ist das Konzept der Entrustable Professional Activities (EPA). Methode: Die Teilnehmer dieser monozentrischen Querschnittsstudie waren Anästhesiologen verschiedener Ausbildungsgrade. Im ersten Schritt wurde durch eine Expertengruppe eine vorläufige Liste von EPAs definiert. Im Anschluss wurde die erste Delphi-Runde (N=47 Teilnehmer) durchgeführt, um alltägliche anästhesiologische Tätigkeiten zu identifizieren. Aus den Ergebnissen der ersten Delphi-Runde wurde durch die Mapping-Methode und durch eine Template-Analyse eine weitere Liste an EPAs definiert. Diese Liste wurde den Studienteilnehmern in einer zweiten Delphi-Runde (N=80 Teilnehmer) mit dem Ziel präsentiert, jede EPA zu validieren, eine Reihung vorzunehmen und das Jahr, in welchem diese anvertraut werden sollte, zu definieren. Dazu wurden die Teilnehmer in der zweiten Runde zu jeder EPA befragt, ob diese Teil eines anästhesiologischen Kerncurriculums sein sollte und in welchem Jahr der Weiterbildung diese anvertraubar sein sollte. Desweiteren wurden die Teilnehmer gebeten, jede EPA auf einer numerischen Skala hinsichtlich ihrer Relevanz zu bewerten. Aus den Ergebnissen der numerischen Skala wurde der Content Validity Index (CVI) berechnet. Die Übereinstimmung der Ergebnisse in der zweiten Delphi-Runde wurde untersucht, indem das Paneldatenmodell angewendet wurde, um die Intraklassen-Korrelation (ICC) zu berechnen. Weiterhin wurde die prozentuale Übereinstimmung zwischen den EPAs dieser Studie und bereits publizierten EPAs berechnet. Ergebnisse: Ein Kerncurriculum bestehend aus 39 EPAs wurde entwickelt. Die EPAs wurden entsprechend ihrer Relevanz in eine hoch und niedrig bewertete Gruppe unterteilt. Ferner wurde das Weiterbildungsjahr festgelegt, in welchem die EPAs anvertraut werden sollten. Die Ergebnisse zeigten eine hohe Übereinstimmung in den unterschiedlichen Gruppen von Anästhesisten (der CVI als Maß für die Zustimmung betrug 0.96 für die EPAs und 0.83 für das Ausbildungsjahr, in welchem die EPA anvertraut werden sollte). Die Übereinstimmung mit den publizierten EPAs betrug 73.3%. Schlussfolgerung: Diese Studie ist ein weiterer Schritt, um die postgraduelle anästhesiologische Weiterbildung zeitgemäß zu strukturieren. Weitere Untersuchungen sind notwendig, um dieses EPA basierte anästhesiologische Kerncurriculum zu vervollständigen und anzupassen

Ku is a 5'-dRP/AP lyase that excises nucleotide damage near broken ends

Mammalian cells require non-homologous end joining (NHEJ) for the efficient repair of chromosomal DNA double-strand breaks. A key feature of biological sources of strand breaks is associated nucleotide damage, including base loss (abasic or apurinic/apyrimidinic (AP) sites). At single-strand breaks, 5'-terminal abasic sites are excised by the 5'-deoxyribose-5-phosphate (5'-dRP) lyase activity of DNA polymerase beta (pol beta): here we show, in vitro and in cells, that accurate and efficient repair by NHEJ of double-strand breaks with such damage similarly requires 5'-dRP/AP lyase activity. Classically defined NHEJ is moreover uniquely effective at coupling this end-cleaning step to joining in cells, helping to distinguish this pathway from otherwise robust alternative NHEJ pathways. The NHEJ factor Ku can be identified as an effective 5'-dRP/AP lyase. In a similar manner to other lyases, Ku nicks DNA 3' of an abasic site by a mechanism involving a Schiff-base covalent intermediate with the abasic site. We show by using cell extracts that Ku is essential for the efficient removal of AP sites near double-strand breaks and, consistent with this result, that joining of such breaks is specifically decreased in cells complemented with a lyase-attenuated Ku mutant. Ku had previously been presumed only to recognize ends and recruit other factors that process ends; our data support an unexpected direct role for Ku in end-processing steps as well