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Resistance to CD19-directed immunotherapies in lymphoblastic leukemia has been attributed, among other factors, to several aberrant CD19 pre-mRNA splicing events, including recently reported excision of a cryptic intron embedded within CD19 exon 2. While “exitrons” are known to exist in hundreds of human transcripts, we discovered, using reporter assays and direct long-read RNA sequencing (dRNA-seq), that the CD19 exitron is an artifact of reverse transcription. Extending our analysis to publicly available datasets, we identified dozens of questionable exitrons, dubbed “falsitrons,” that appear only in cDNA-seq, but never in dRNA-seq. Our results highlight the importance of dRNA-seq for transcript isoform validation.
Background: The Objective Structured Clinical Examination (OSCE) is increasingly used at medical schools to assess practical competencies. To compare the outcomes of students at different medical schools, we introduced standardized OSCE stations with identical checklists.
Methods: We investigated examiner bias at standardized OSCE stations for knee- and shoulder-joint examinations, which were implemented into the surgical OSCE at five different medical schools. The checklists for the assessment consisted of part A for knowledge and performance of the skill and part B for communication and interaction with the patient. At each medical faculty, one reference examiner also scored independently to the local examiner. The scores from both examiners were compared and analysed for inter-rater reliability and correlation with the level of clinical experience. Possible gender bias was also evaluated.
Results: In part A of the checklist, local examiners graded students higher compared to the reference examiner; in part B of the checklist, there was no trend to the findings. The inter-rater reliability was weak, and the scoring correlated only weakly with the examiner’s level of experience. Female examiners rated generally higher, but male examiners scored significantly higher if the examinee was female.
Conclusions: These findings of examiner effects, even in standardized situations, may influence outcome even when students perform equally well. Examiners need to be made aware of these biases prior to examining.
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) offers potential cure to acute myeloid leukemia (AML) patients. However, infections with commensal bacteria are an important cause for non-relapse mortality (NRM). We have previously described the impact of multidrug-resistant organism (MDRO) colonization on the survival of allo-HSCT patients. In the aforementioned publication, according to consensus, we there did not consider the opportunistic gram-negative bacterium Stenotrophomonas maltophilia (S. maltophilia) to be an MDRO. Since rate of S. maltophilia colonization is increasing, and it is not known whether this poses a risk for allo-HSCT patients, we here analyzed here its effect on the previously described and now extended patient cohort. We report on 291 AML patients undergoing allo-HSCT. Twenty of 291 patients (6.9%) were colonized with S. maltophilia. Colonized patients did not differ from non-colonized patients with respect to their age, remission status before allo-HSCT, donor type and HSCT-comorbidity index. S. maltophilia colonized patients had a worse overall survival (OS) from 6 months up to 60 months (85% vs. 88.1% and 24.7% vs. 59.7%; p = 0.007) due to a higher NRM after allo-HSCT (6 months: 15% vs. 4.8% and 60 months: 40.1% vs. 16.2% p = 0.003). The main cause of mortality in colonized patients was infection (46.2% of all deaths) and in non-colonized patients relapse (58.8% of all deaths). 5/20 colonized patients developed an invasive infection with S. maltophilia. The worse OS after allo-HSCT due to higher infection related mortality might implicate the screening of allo-HSCT patients for S. maltophilia and a closer observation of colonized patients as outpatients.
The teaching of professional roles in medical education is an interdisciplinary concern. However, surgeons require specific standards of professionalism for certain context-based situations. In addition to communication, studies require collaboration, leadership, error-/conflict-management, patient-safety and decision-making as essential competencies for surgeons. Standards for corresponding competencies are defined in special chapters of the German National Competency-based Learning Objectives for Undergraduate Medical Education (NKLM; chapter 8, 10). The current study asks whether these chapters are adequately taught in surgical curricula. Eight German faculties contributed to analysing mapping data considering surgical courses of undergraduate programs. All faculties used the MERlin mapping platform and agreed on procedures for data collection and processing. Sub-competency and objective coverage, as well as the achievement of the competency level were mapped. Overall counts of explicit citations were used for analysis. Collaboration within the medical team is a strongly represented topic. In contrast, interprofessional cooperation, particularly in healthcare sector issues is less represented. Patient safety and dealing with errors and complications is most emphasized for the Manager/Leader, while time management, career planning and leadership are not addressed. Overall, the involvement of surgery in teaching the competencies of the Collaborator and Manager/Leader is currently low. However, there are indications of a curricular development towards explicit teaching of these roles in surgery. Moreover, implicitly taught roles are numerous, which indicates a beginning awareness of professional roles.
HLA-DRB1 and HLA-DQB1 genetic diversity modulates response to lithium in bipolar affective disorders
(2021)
Bipolar affective disorder (BD) is a severe psychiatric illness, for which lithium (Li) is the gold standard for acute and maintenance therapies. The therapeutic response to Li in BD is heterogeneous and reliable biomarkers allowing patients stratification are still needed. A GWAS performed by the International Consortium on Lithium Genetics (ConLiGen) has recently identified genetic markers associated with treatment responses to Li in the human leukocyte antigens (HLA) region. To better understand the molecular mechanisms underlying this association, we have genetically imputed the classical alleles of the HLA region in the European patients of the ConLiGen cohort. We found our best signal for amino-acid variants belonging to the HLA-DRB1*11:01 classical allele, associated with a better response to Li (p < 1 × 10−3; FDR < 0.09 in the recessive model). Alanine or Leucine at position 74 of the HLA-DRB1 heavy chain was associated with a good response while Arginine or Glutamic acid with a poor response. As these variants have been implicated in common inflammatory/autoimmune processes, our findings strongly suggest that HLA-mediated low inflammatory background may contribute to the efficient response to Li in BD patients, while an inflammatory status overriding Li anti-inflammatory properties would favor a weak response.
Bei Cryptochromen handelt es sich um Blaulichtrezeptoren der Cryptochrom-Photolyase-Proteinfamilie (CPF). Mitglieder dieser Proteinfamilie sind in allen Domänen des Lebens zu finden und haben eine essentielle Rolle in der Reparatur der DNA sowie der lichtgesteuerten Regulation der Expression. Cryptochrome sind in der Regel keine DNA-reparierenden Proteine. Sie sind regulativ an der Steuerung der inneren Uhr und des Zellzyklus der Organismen beteiligt. In der Kieselalge Phaeodactylum tricornutum konnten bisher sechs phylogenetisch unterschiedliche Mitglieder der CPF identifiziert werden. Bei CryP handelt es sich um das einzige pflanzenähnliche Cryptochrom der photoautotrophen Diatomee. Für das Protein CryP konnte bereits ein blaulichtinduzierter Photozyklus durch die Absorption der Chromophore 5-Methenlytetrahydrofolat (MTHF) und Flavinadenindinukleotid (FAD) gezeigt werden. Außerdem ist eine regulative Wirkung des Proteins auf die Lichtsammelkomplexe (Lhc) der Diatomee bekannt. Für eine weitere Charakterisierung des CryPs wurde in dieser Arbeit zunächst das Absorptionsverhalten unter verschiedenen Wellenlängen beobachtet, um so einen Einblick in eine mögliche Aktivierung und Deaktivierung des Proteins durch Licht unterschiedlicher Wellenlängen zu erlangen. Es zeigte sich hierbei eine mit pflanzlichen Cryptochromen vergleichbare Anreicherung verschiedener Redoxzustände des FADs in Abhängigkeit von der Wellenlänge.
Für eine Aufklärung der Wirkungsweise des CryP-Proteins wurden verschiedene Hypothesen untersucht: Die phylogenetische Nähe und ein ähnliches Absorptionsverhalten des CryPs zu Cryptochromen mit Reparaturfähigkeit für einzelsträngige DNA (Cry-DASH) führte zu einer Untersuchung des Proteins als möglicher Transkriptionsfaktor. Hierfür konnte eine Kernlokalisation des Proteins nachgewiesen werden, was Rückschlüsse auf eine potentielle Regulation der Expression mittels DNA-Bindung zulässt. Außerdem wurde gezeigt, dass CryP DNA-Bindefähigkeit besitzt. Die bisher nachgewiesenen Bindungen waren jedoch unspezifischer Art. Dies konnte auch für die Promotersequenz eines der durch CryP regulierten Gene lhcf1 festgestellt werden. Auf Grund der unspezifischen DNA-Bindung wurde eine zweite Hypothese für CryP untersucht: CryP wirkt regulativ auf die Expression verschiedener Gene durch Protein-Protein-Interaktionen und ist Teil einer Reaktionskaskade zur Signalweiterleitung in P. tricornutum.
Durch die Untersuchung der zweiten Hypothese konnten drei Interaktionspartner für CryP identifiziert und eine Interaktion verifiziert werden. Hierbei handelt es sich um das Protein AAA mit einer bisher unbekannten Funktion und das Protein BolA, welches Teil der zuerst in Escherichia coli identifizierten BolA-like-Proteinfamilie ist. Außerdem konnte eine Interaktion mit dem Cold-Shock-Domänen-Protein CSDP gezeigt werden. Bei den Proteinen BolA und CSDP handelt es sich um potentiell regulierende Faktoren der Transkription und Translation, was Teil einer Reaktionskaskade sein kann. Die aus anderen Organismen bekannten Funktionen des BolA-Proteins überschneiden sich mit den in CryP-Knockdown-Mutanten beobachteten Effekten. Sie zeigen eine erhöhte Sensitivität für Stresssituationen wie abweichende Nährstoffkonzentration, Osmolaritäten und Temperaturen. Diese Beobachtungen stellen einen Zusammenhang der durch einen CryP-Knockdown beobachteten Effekte und der CryP-BolA-Interaktion her. Durch Homologien zu Cold-Shock-Proteinen aus Chlamydomonas reinhardtii gibt die CryP-Interaktion mit dem Protein CSDP Hinweise auf einen potentiellen Mechanismus zur Regulation der Lhc-Proteine, für welche zuvor ein CryP-abhängiger Effekt beschrieben war.
Über die Protein-Protein-Interaktionen hinaus wurde die Phosphorylierung des CryPs als Möglichkeit der Signalweiterleitung untersucht. Es konnte eine reversible Phosphorylierung des heterolog aus E. coli isolierten CryPs gezeigt werden. Diese zeigt Ähnlichkeiten zu bekannten Phosphorylierungen pflanzlicher Cryptochrome und gibt Hinweise auf einen Mechanismus der Signalweiterleitung.
Durch die Untersuchung der CryP-regulierten Transkription mit P. tricornutum CryP-Knockdown-Mutanten durch Next-Generation-Sequencing (NGS) konnte die Hypothese der regulativen Proteinkaskade und der Signalweiterleitung weiter bestätigt werden. Die Auswirkungen des CryPs auf die Transkription erwiesen sich als nicht auf einen Teilbereich des Metabolismus begrenzt, sondern sind in einem großen Teil der funktionellen Gengruppen in P. tricornutum zu sehen. Außerdem konnten drei Klassen CryP-regulierter Gene festgestellt werden. Kategorie 1: die ausschließlich unter Blaulicht regulierten Gene; Kategorie 2: die sowohl unter Blaulicht als auch im Dunkeln regulierten Gene und Kategorie 3: die ausschließlich im Dunkeln regulierten Gene. Ein im Dunkeln und unter Blaulicht jeweils unterschiedlicher regulativer Effekt deutet auf eine Doppelfunktion des CryPs hin. Möglicherweise hat das Cryptochrom unterschiedliche lichtabhängige und lichtunabhängige Funktionen.
Durch die Analyse der CryP-regulierten Genexpression konnte außerdem ein Zusammenhang zwischen CryP und weiteren Photorezeptoren gezeigt werden. Der CryP-Proteingehalt in der Zelle hat einen regulativen Einfluss auf das CPF1-Protein, eine Photolyase mit dualer Funktion aus der gleichen Proteinfamilie. Zusätzlich konnte auch ein Einfluss auf die Lichtsensitivität der Genexpression des Rotlichtrezeptors Phytochrom (DPH) durch CryP gezeigt werden. Vergleichbar mit höheren Pflanzen scheint ein regulatives Netzwerk der Photorezeptoren auch in der Diatomee P. tricornutum vorhanden zu sein.
Bipolar disorder (BD) is a genetically complex mental illness characterized by severe oscillations of mood and behavior. Genome-wide association studies (GWAS) have identified several risk loci that together account for a small portion of the heritability. To identify additional risk loci, we performed a two-stage meta-analysis of >9 million genetic variants in 9,784 bipolar disorder patients and 30,471 controls, the largest GWAS of BD to date. In this study, to increase power we used ~2,000 lithium-treated cases with a long-term diagnosis of BD from the Consortium on Lithium Genetics, excess controls, and analytic methods optimized for markers on the Xchromosome. In addition to four known loci, results revealed genome-wide significant associations at two novel loci: an intergenic region on 9p21.3 (rs12553324, p = 5.87×10-9; odds ratio = 1.12) and markers within ERBB2 (rs2517959, p = 4.53×10-9; odds ratio = 1.13). No significant X-chromosome associations were detected and X-linked markers explained very little BD heritability. The results add to a growing list of common autosomal variants involved in BD and illustrate the power of comparing well-characterized cases to an excess of controls in GWAS.
Measurements of charged-particle production in pp, p−Pb, and Pb−Pb collisions in the toward, away, and transverse regions with the ALICE detector are discussed. These regions are defined event-by-event relative to the azimuthal direction of the charged trigger particle, which is the reconstructed particle with the largest transverse momentum (ptrigT) in the range 8<ptrigT<15 GeV/c. The toward and away regions contain the primary and recoil jets, respectively; both regions are accompanied by the underlying event (UE). In contrast, the transverse region perpendicular to the direction of the trigger particle is dominated by the so-called UE dynamics, and includes also contributions from initial- and final-state radiation. The relative transverse activity classifier, RT=NTch/⟨NTch⟩, is used to group events according to their UE activity, where NTch is the charged-particle multiplicity per event in the transverse region and ⟨NTch⟩ is the mean value over the whole analysed sample. The energy dependence of the RT distributions in pp collisions at s√=2.76, 5.02, 7, and 13 TeV is reported, exploring the Koba-Nielsen-Olesen (KNO) scaling properties of the multiplicity distributions. The first measurements of charged-particle pT spectra as a function of RT in the three azimuthal regions in pp, p−Pb, and Pb−Pb collisions at sNN−−−√=5.02 TeV are also reported. Data are compared with predictions obtained from the event generators PYTHIA 8 and EPOS LHC. This set of measurements is expected to contribute to the understanding of the origin of collective-like effects in small collision systems (pp and p−Pb).
Measurements of charged-particle production in pp, p−Pb, and Pb−Pb collisions in the toward, away, and transverse regions with the ALICE detector are discussed. These regions are defined event-by-event relative to the azimuthal direction of the charged trigger particle, which is the reconstructed particle with the largest transverse momentum (ptrigT) in the range 8<ptrigT<15 GeV/c. The toward and away regions contain the primary and recoil jets, respectively; both regions are accompanied by the underlying event (UE). In contrast, the transverse region perpendicular to the direction of the trigger particle is dominated by the so-called UE dynamics, and includes also contributions from initial- and final-state radiation. The relative transverse activity classifier, RT=NTch/⟨NTch⟩, is used to group events according to their UE activity, where NTch is the charged-particle multiplicity per event in the transverse region and ⟨NTch⟩ is the mean value over the whole analysed sample. The energy dependence of the RT distributions in pp collisions at s√=2.76, 5.02, 7, and 13 TeV is reported, exploring the Koba-Nielsen-Olesen (KNO) scaling properties of the multiplicity distributions. The first measurements of charged-particle pT spectra as a function of RT in the three azimuthal regions in pp, p−Pb, and Pb−Pb collisions at sNN−−−√=5.02 TeV are also reported. Data are compared with predictions obtained from the event generators PYTHIA 8 and EPOS LHC. This set of measurements is expected to contribute to the understanding of the origin of collective-like effects in small collision systems (pp and p−Pb).
Results on the transverse spherocity dependence of light-flavor particle production (π, K, p, ϕ, K∗0, K0S, Λ, Ξ) at midrapidity in high-multiplicity pp collisions at s√=13 TeV were obtained with the ALICE apparatus. The transverse spherocity estimator (SpT=1O) categorizes events by their azimuthal topology. Utilizing narrow selections on SpT=1O, it is possible to contrast particle production in collisions dominated by many soft initial interactions with that observed in collisions dominated by one or more hard scatterings. Results are reported for two multiplicity estimators covering different pseudorapidity regions. The SpT=1O estimator is found to effectively constrain the hardness of the events when the midrapidity (|η|<0.8) estimator is used. The production rates of strange particles are found to be slightly higher for soft isotropic topologies, and severely suppressed in hard jet-like topologies. These effects are more pronounced for hadrons with larger mass and strangeness content, and observed when the topological selection is done within a narrow multiplicity interval. This demonstrates that an important aspect of the universal scaling of strangeness enhancement with final-state multiplicity is that high-multiplicity collisions are dominated by soft, isotropic processes. On the contrary, strangeness production in events with jet-like processes is significantly reduced. The results presented in this article are compared with several QCD-inspired Monte Carlo event generators. Models that incorporate a two-component phenomenology, either through mechanisms accounting for string density, or thermal production, are able to describe the observed strangeness enhancement as a function of SpT=1O.