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Osteosarcomas are aggressive bone tumours with a high degree of genetic heterogeneity, which has historically complicated driver gene discovery. Here we sequence exomes of 31 tumours and decipher their evolutionary landscape by inferring clonality of the individual mutation events. Exome findings are interpreted in the context of mutation and SNP array data from a replication set of 92 tumours. We identify 14 genes as the main drivers, of which some were formerly unknown in the context of osteosarcoma. None of the drivers is clearly responsible for the majority of tumours and even TP53 mutations are frequently mapped into subclones. However, >80% of osteosarcomas exhibit a specific combination of single-base substitutions, LOH, or large-scale genome instability signatures characteristic of BRCA1/2-deficient tumours. Our findings imply that multiple oncogenic pathways drive chromosomal instability during osteosarcoma evolution and result in the acquisition of BRCA-like traits, which could be therapeutically exploited.
Background: Repetitive transcranial magnetic stimulation (rTMS) allows non-invasive stimulation of the human brain. However, no suitable marker has yet been established to monitor the immediate rTMS effects on cortical areas in children.
Objective: TMS-evoked EEG potentials (TEPs) could present a well-suited marker for real-time monitoring. Monitoring is particularly important in children where only few data about rTMS effects and safety are currently available.
Methods: In a single-blind sham-controlled study, twenty-five school-aged children with ADHD received subthreshold 1 Hz-rTMS to the primary motor cortex. The TMS-evoked N100 was measured by 64-channel-EEG pre, during and post rTMS, and compared to sham stimulation as an intraindividual control condition.
Results: TMS-evoked N100 amplitude decreased during 1 Hz-rTMS and, at the group level, reached a stable plateau after approximately 500 pulses. N100 amplitude to supra-threshold single pulses post rTMS confirmed the amplitude reduction in comparison to the pre-rTMS level while sham stimulation had no influence. EEG source analysis indicated that the TMS-evoked N100 change reflected rTMS effects in the stimulated motor cortex. Amplitude changes in TMS-evoked N100 and MEPs (pre versus post 1 Hz-rTMS) correlated significantly, but this correlation was also found for pre versus post sham stimulation.
Conclusion: The TMS-evoked N100 represents a promising candidate marker to monitor rTMS effects on cortical excitability in children with ADHD. TMS-evoked N100 can be employed to monitor real-time effects of TMS for subthreshold intensities. Though TMS-evoked N100 was a more sensitive parameter for rTMS-specific changes than MEPs in our sample, further studies are necessary to demonstrate whether clinical rTMS effects can be predicted from rTMS-induced changes in TMS-evoked N100 amplitude and to clarify the relationship between rTMS-induced changes in TMS-evoked N100 and MEP amplitudes. The TMS-evoked N100 amplitude reduction after 1 Hz-rTMS could either reflect a globally decreased cortical response to the TMS pulse or a specific decrease in inhibition.
Background: Previously, we used inhibitors blocking BET bromodomain binding proteins (BRDs) in Ewing sarcoma (EwS) and observed that long term treatment resulted in the development of resistance. Here, we analyze the possible interaction of BRD4 with cyclin-dependent kinase (CDK) 9. Methods: Co-immunoprecipitation experiments (CoIP) to characterize BRD4 interaction and functional consequences of inhibiting transcriptional elongation were assessed using drugs targeting of BRD4 or CDK9, either alone or in combination. Results: CoIP revealed an interaction of BRD4 with EWS-FLI1 and CDK9 in EwS. Treatment of EwS cells with CDKI-73, a specific CDK9 inhibitor (CDK9i), induced a rapid downregulation of EWS-FLI1 expression and block of contact-dependent growth. CDKI-73 induced apoptosis in EwS, as depicted by cleavage of Caspase 7 (CASP7), PARP and increased CASP3 activity, similar to JQ1. Microarray analysis following CDKI-73 treatment uncovered a transcriptional program that was only partially comparable to BRD inhibition. Strikingly, combined treatment of EwS with BRD- and CDK9-inhibitors re-sensitized cells, and was overall more effective than individual drugs not only in vitro but also in a preclinical mouse model in vivo. Conclusion: Treatment with BRD inhibitors in combination with CDK9i offers a new treatment option that significantly blocks the pathognomonic EWS-ETS transcriptional program and malignant phenotype of EwS.
Calreticulin is a Ca2+ -binding chaperone that resides in the lumen of the endoplasmic reticulum and is involved in the regulation of intracellular Ca2+ homeostasis and in the folding of newly synthesized glycoproteins. In this study, we have used site-specific mutagenesis to map amino acid residues that are critical in calreticulin function. We have focused on two cysteine residues (Cys(88) and Cys(120)), which form a disulfide bridge in the N-terminal domain of calreticulin, on a tryptophan residue located in the carbohydrate binding site (Trp(302)), and on certain residues located at the tip of the "hairpin-like" P-domain of the protein (Glu(238), Glu(239), Asp(241), Glu(243), and Trp(244)). Calreticulin mutants were expressed in crt(-/-) fibroblasts, and bradykinin-dependent Ca2+ release was measured as a marker of calreticulin function. Bradykinin-dependent Ca2+ release from the endoplasmic reticulum was rescued by wild-type calreticulin and by the Glu(238), Glu(239), Asp(241), and Glu(243) mutants. The Cys(88) and Cys(120) mutants rescued the calreticulin-deficient phenotype only partially ( approximately 40%), and the Trp(244) and Trp(302) mutants did not rescue it at all. We identified four amino acid residues (Glu(239), Asp(241), Glu(243), and Trp(244)) at the hairpin tip of the P-domain that are critical in the formation of a complex between ERp57 and calreticulin. Although the Glu(239), Asp(241), and Glu(243) mutants did not bind ERp57 efficiently, they fully restored bradykinin-dependent Ca2+ release in crt(-/-) cells. This indicates that binding of ERp57 to calreticulin may not be critical for the chaperone function of calreticulin with respect to the bradykinin receptor.
Background The inhibitor telaprevir (VX-950) of the hepatitis C virus (HCV) protease NS3-4A has been tested in a recent phase 1b clinical trial in patients infected with HCV genotype 1. This trial revealed residue mutations that confer varying degrees of drug resistance. In particular, two protease positions with the mutations V36A/G/L/M and T54A/S were associated with low to medium levels of drug resistance during viral breakthrough, together with only an intermediate reduction of viral replication fitness. These mutations are located in the protein interior and far away from the ligand binding pocket. Results Based on the available experimental structures of NS3-4A, we analyze the binding mode of different ligands. We also investigate the binding mode of VX-950 by protein-ligand docking. A network of non-covalent interactions between amino acids of the protease structure and the interacting ligands is analyzed to discover possible mechanisms of drug resistance. We describe the potential impact of V36 and T54 mutants on the side chain and backbone conformations and on the non-covalent residue interactions. We propose possible explanations for their effects on the antiviral efficacy of drugs and viral fitness. Molecular dynamics simulations of T54A/S mutants and rotamer analysis of V36A/G/L/M side chains support our interpretations. Experimental data using an HCV V36G replicon assay corroborate our findings. Conclusion T54 mutants are expected to interfere with the catalytic triad and with the ligand binding site of the protease. Thus, the T54 mutants are assumed to affect the viral replication efficacy to a larger degree than V36 mutants. Mutations at V36 and/or T54 result in impaired interaction of the protease residues with the VX-950 cyclopropyl group, which explains the development of viral breakthrough variants.
Background: Bipolar disorder is associated with circadian disruption and a high risk of suicidal behavior. In a previous exploratory study of patients with bipolar I disorder, we found that a history of suicide attempts was associated with differences between winter and summer levels of solar insolation. The purpose of this study was to confirm this finding using international data from 42% more collection sites and 25% more countries. Methods: Data analyzed were from 71 prior and new collection sites in 40 countries at a wide range of latitudes. The analysis included 4876 patients with bipolar I disorder, 45% more data than previously analyzed. Of the patients, 1496 (30.7%) had a history of suicide attempt. Solar insolation data, the amount of the sun’s electromagnetic energy striking the surface of the earth, was obtained for each onset location (479 locations in 64 countries). Results: This analysis confirmed the results of the exploratory study with the same best model and slightly better statistical significance. There was a significant inverse association between a history of suicide attempts and the ratio of mean winter insolation to mean summer insolation (mean winter insolation/mean summer insolation). This ratio is largest near the equator which has little change in solar insolation over the year, and smallest near the poles where the winter insolation is very small compared to the summer insolation. Other variables in the model associated with an increased risk of suicide attempts were a history of alcohol or substance abuse, female gender, and younger birth cohort. The winter/summer insolation ratio was also replaced with the ratio of minimum mean monthly insolation to the maximum mean monthly insolation to accommodate insolation patterns in the tropics, and nearly identical results were found. All estimated coefficients were significant at p < 0.01. Conclusion: A large change in solar insolation, both between winter and summer and between the minimum and maximum monthly values, may increase the risk of suicide attempts in bipolar I disorder. With frequent circadian rhythm dysfunction and suicidal behavior in bipolar disorder, greater understanding of the optimal roles of daylight and electric lighting in circadian entrainment is needed.
Endothelial cells can acquire a mesenchymal phenotype through a process called Endothelial-to-Mesenchymal transition (EndMT). This event is found in embryonic development, but also in pathological conditions. Blood vessels lose their ability to maintain vascular homeostasis and ultimately develop atherosclerosis, pulmonary hypertension, or fibrosis. An increase in inflammatory signals causes an upregulation of EndMT transcription factors, mesenchymal markers, and a decrease in endothelial markers. In our study, we show that the induction of EndMT results in an increase in long non-coding RNA AERRIE expression. JMJD2B, a known EndMT regulator, induces AERRIE and subsequently SULF1. Silencing of AERRIE shows a partial regulation of SULF1 but showed no effect on the endothelial and mesenchymal markers. Additionally, the overexpression of AERRIE results in no significant changes in EndMT markers, suggesting that AERRIE is marginally regulating mesenchymal markers and transcription factors. This study identifies AERRIE as a novel factor in EndMT, but its mechanism of action still needs to be elucidated.
Background: Intensive Care Resources are heavily utilized during the COVID-19 pandemic. However, risk stratification and prediction of SARS-CoV-2 patient clinical outcomes upon ICU admission remain inadequate. This study aimed to develop a machine learning model, based on retrospective & prospective clinical data, to stratify patient risk and predict ICU survival and outcomes. Methods: A Germany-wide electronic registry was established to pseudonymously collect admission, therapeutic and discharge information of SARS-CoV-2 ICU patients retrospectively and prospectively. Machine learning approaches were evaluated for the accuracy and interpretability of predictions. The Explainable Boosting Machine approach was selected as the most suitable method. Individual, non-linear shape functions for predictive parameters and parameter interactions are reported. Results: 1039 patients were included in the Explainable Boosting Machine model, 596 patients retrospectively collected, and 443 patients prospectively collected. The model for prediction of general ICU outcome was shown to be more reliable to predict “survival”. Age, inflammatory and thrombotic activity, and severity of ARDS at ICU admission were shown to be predictive of ICU survival. Patients’ age, pulmonary dysfunction and transfer from an external institution were predictors for ECMO therapy. The interaction of patient age with D-dimer levels on admission and creatinine levels with SOFA score without GCS were predictors for renal replacement therapy. Conclusions: Using Explainable Boosting Machine analysis, we confirmed and weighed previously reported and identified novel predictors for outcome in critically ill COVID-19 patients. Using this strategy, predictive modeling of COVID-19 ICU patient outcomes can be performed overcoming the limitations of linear regression models. Trial registration “ClinicalTrials” (clinicaltrials.gov) under NCT04455451.