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Zusammenfassung Hintergrund Eine angemessene, bedarfsgerechte psychoonkologische Versorgung reduziert Depressivität und Ängste von Krebserkrankten sowie ihren Angehörigen und erhöht die Lebensqualität. Psychisch belastete Krebserkrankte werden jedoch nicht flächendeckend identifiziert, um ihnen psychoonkologische Unterstützung anzubieten. Screeningfragebögen haben sich zur Identifikation bewährt, allerdings bestehen bei der Anwendung im klinischen Alltag Hürden. Pflegekräfte haben durch ihren kontinuierlichen Kontakt zu Patient_innen, die vielfältigen klinischen Eindrücke und ihre Verbindung zu anderen Berufsgruppen eine Schlüsselrolle. Ziele Die OptiScreen-Schulung soll Pflegekräfte in der Onkologie zur Durchführung des Distress-Screenings befähigen, entsprechendes Expert_innenwissen vermitteln und Hürden sowie Unsicherheiten im Screeningprozess abbauen, um somit belastete Erkrankte zielgerichtet identifizieren und einer psychoonkologischen Versorgung zuführen zu können. Das Training Die OptiScreen-Schulung gliedert sich in drei Module à 1,5–2 h zu den Themen psychische Störungen bei Krebs, psychoonkologische Versorgung, psychische Belastung, Distress-Screening, Kommunikation im Screeningprozess und Psychohygiene (vermittelt durch Vorträge, Videos, Rollenspiele, Übungen). Ergebnisse und Diskussion Erste praktische Erfahrungen weisen auf eine erfolgreiche Umsetzung des Schulungskonzepts hin. Weitere Ziele sind es, den Wissenszuwachs und die zunehmende Sicherheit der Pflegekräfte im Screeningprozess zu stärken sowie die Effekte langfristig aufrechtzuerhalten. Zusätzlich soll die Schulung in verschiedenen Settings etabliert und die Auswirkungen der Schulung in Bezug auf die Informiertheit und Zufriedenheit der Patient_innen mit dem Screeningprozess untersucht werden.

Current capabilities in genomic sequencing outpace functional interpretations. Our previous work showed that 3D protein structure calculations enhance mechanistic understanding of genetic variation in sequenced tumors and patients with rare diseases. The KRAS GTPase is among the critical genetic factors driving cancer and germline conditions. Because KRAS-altered tumors frequently harbor one of three classic hotspot mutations, nearly all studies have focused on these mutations, leaving significant functional ambiguity across the broader KRAS genomic landscape observed in cancer and non-cancer diseases. Herein, we extend structural bioinformatics with molecular simulations to study an expanded landscape of 86 KRAS mutations. We identify multiple coordinated changes strongly associated with experimentally established KRAS biophysical and biochemical properties. The patterns we observe span hotspot and non-hotspot alterations, which can all dysregulate Switch regions, producing mutation-restricted conformations with different effector binding propensities. We experimentally measured mutation thermostability and identified shared and distinct patterns with simulations. Our results indicate mutation-specific conformations which show potential for future research into how these alterations reverberate into different molecular and cellular functions. The data we present is not predictable using current genomic tools, demonstrating the added functional information derived from molecular simulations for interpreting human genetic variation.Key PointsPlease provide 3 bullet points summarizing the manuscript’s contribution to the field (100 characters max per point)1)We functionally grouped 86 distinct KRAS mutations using MD scores, demonstrating scalability for genomics2)MD-based groups explain experimental differences and mechanistic information about mutant proteins3)Demonstrated added functional information from simulations for interpreting human genetic variation

The histone demethylase KDM6A has recently elicited significant attention because its mutations are associated with a rare congenital disorder (Kabuki syndrome) and various types of human cancers. However, distinguishing KDM6A mutations that are deleterious to the enzyme and their underlying mechanisms of dysfunction remain to be fully understood. Here, we report the results from a multi-tiered approach evaluating the impact of 197 KDM6A somatic mutations using information derived from combining conventional genomics data with computational biophysics. This comprehensive approach incorporates multiple scores derived from alterations in protein sequence, structure, and molecular dynamics. Using this method, we classify the KDM6A mutations into 136 damaging variants (69.0%), 32 tolerated variants (16.2%), and 29 variants of uncertain significance (VUS, 14.7%), which is a significant improvement from the previous classification based on the conventional tools (over 40% VUS). We further classify the damaging variants into 15 structural variants (SV), 88 dynamic variants (DV), and 33 structural and dynamic variants (SDV). Comparison with variant scoring methods used in current clinical diagnosis guidelines demonstrates that our approach provides a more comprehensive evaluation of damaging potential and reveals mechanisms of dysfunction. Thus, these results should be taken into consideration for clinical assessment of the damaging potential of each mutation, as they provide hypotheses for experimental validation and critical information for the development of mutant-specific drugs to fight diseases caused by KDM6A dysfunctions.

Pancreatic cancer is characterized by abundant desmoplasia, a dense stroma composed of extra-cellular and cellular components, with cancer associated fibroblasts (CAFs) being the major cellular component. However, the tissue(s) of origin for CAFs remains controversial. Here we determine the tissue origin of pancreatic CAFs through comprehensive lineage tracing studies in mice. We find that the splanchnic mesenchyme, the fetal cell layer surrounding the endoderm from which the pancreatic epithelium originates, gives rise to the majority of resident fibroblasts in the normal pancreas. In a genetic mouse model of pancreatic cancer, resident fibroblasts expand and constitute the bulk of CAFs. Single cell RNA profiling identifies gene expression signatures that are shared among the fetal splanchnic mesenchyme, adult fibroblasts and CAFs, suggesting a persistent transcriptional program underlies splanchnic lineage differentiation. Together, this study defines the phylogeny of the mesenchymal component of the pancreas and provides insights into pancreatic morphogenesis and tumorigenesis.

Author(s): Cottrell, Catherine E; Bender, Nicole R; Zimmermann, Michael T; Heusel, Jonathan W; Corliss, Meagan; Evenson, Michael J; Magrini, Vincent; Corsmeier, Donald J; Avenarius, Matthew; Dudley, Jeffrey N; Johnston, Jennifer J; Lindhurst, Marjorie J; Vigh-Conrad, Katinka; Davies, Olivia MT; Coughlin, Carrie C; Frieden, Ilona J; Tollefson, Megha; Zaenglein, Andrea L; Ciliberto, Heather; Tosi, Laura L; Semple, Robert K; Biesecker, Leslie G; Drolet, Beth A | Abstract: PurposeSomatic activating variants in the PI3K-AKT pathway cause vascular malformations with and without overgrowth. We previously reported an individual with capillary and lymphatic malformation harboring a pathogenic somatic variant in PIK3R1, which encodes three PI3K complex regulatory subunits. Here, we investigate PIK3R1 in a large cohort with vascular anomalies and identify an additional 16 individuals with somatic mosaic variants in PIK3R1.MethodsAffected tissue from individuals with vascular lesions and overgrowth recruited from a multisite collaborative network was studied. Next-generation sequencing targeting coding regions of cell-signaling and cancer-associated genes was performed followed by assessment of variant pathogenicity.ResultsThe phenotypic and variant spectrum associated with somatic variation in PIK3R1 is reported herein. Variants occurred in the inter-SH2 or N-terminal SH2 domains of all three PIK3R1 protein products. Phenotypic features overlapped those of the PIK3CA-related overgrowth spectrum (PROS). These overlapping features included mixed vascular malformations, sandal toe gap deformity with macrodactyly, lymphatic malformations, venous ectasias, and overgrowth of soft tissue or bone.ConclusionSomatic PIK3R1 variants sharing attributes with cancer-associated variants cause complex vascular malformations and overgrowth. The PIK3R1-associated phenotypic spectrum overlaps with PROS. These data extend understanding of the diverse phenotypic spectrum attributable to genetic variation in the PI3K-AKT pathway.

DOT1L is the only non-SET domain histone lysine methyltransferase (KMT) and writer of H3K79 methylation on nucleosomes marked by H2B ubiquitination. DOT1L has elicited significant attention because of its interaction or fusion with members of the AF protein family in blood cell biology and leukemogenic transformation. Here, our goal was to extend previous structural information by performing a robust molecular dynamic study of DOT1L and its leukemogenic partners combined with mutational analysis. We show that statically and dynamically, D161, G163, E186 and F223 make frequent time-dependent interactions with SAM, while additional residues T139, K187 and N241 interact with SAM only under dynamics. Dynamics models reveal DOT1L, SAM and H4 moving as one and show that more than twice the number of DOT1L residues interact with these partners, relative to the static structure. Mutational analyses indicate that six of these residues are intolerant to substitution. We describe the dynamic behavior of DOT1L interacting with AF10 and AF9. Studies on the dynamics of a heterotrimeric complex of DOT1L1-AF10 illuminated describes coordinated motions that impact the relative position of the DOT1L HMT domain to the nucleosome. The molecular motions of the DOT1L-AF9 complex are less extensive and highly dynamic, resembling a swivel-like mechanics. Through molecular dynamics and mutational analysis, we extend the knowledge previous provided by static measurements. These results are important to consider when describing the biochemical properties of DOT1L, under normal and in disease conditions, as well as for the development of novel therapeutic agents. This article is protected by copyright. All rights reserved.

Background The clinical performance of the novel high-sensitivity cardiac troponin I EXL (hs-cTnI-EXL) assay is unknown so far. Purpose We aimed to validate the clinical performance of the hs-cTnI-EXL assay and to derive and validate an hs-cTnI-EXL-specific 0/1h-algorithm for the early diagnosis of myocardial infarction (MI). Methods This multicenter study included patients presenting to the emergency department with symptoms suggestive of myocardial infarction. Central adjudication of final diagnoses was performed by two independent cardiologists using all clinical information including cardiac imaging twice: first, using serial hs-cTnI-Architect (primary analysis) and second, using serial hs-cTnT-Elecsys (secondary analysis) concentrations in addition to those clinically used (hs)-cTn. Hs-cTnI-EXL was measured at presentation and at 1h. The primary objective was to directly compare diagnostic accuracy quantified by the area under the receiver-operating-characteristic curve (AUC) of hs-cTnI-EXL, hs-cTnI-Architect and hs-cTnT-Elecsys. Secondary objectives included the derivation and validation of an hs-cTnI- EXL-specific 0/1h-algorithm. Results MI was the adjudicated final diagnosis in 204/1454 (14%) patients. At presentation, the AUC for hs-cTnI-EXL was 0.94 (95% CI, 0.93–0.96), being comparable to hs-cTnI-Architect (0.95; 95% CI, 0.93–0.96) and hs-cTnT-Elecsys (0.93; 95% CI, 0.91–0.95; Figure 1). In the derivation cohort (n=813), an optimal hs-cTnI-EXL-0/1h-algorithm was rule-out of MI with 3h or Conclusions Hs-cTnI-EXL has comparable diagnostic performance to the currently best-validated hs-cTnT/I assays. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Swiss National Foundation, Swiss Heart Foundation

Background The rapid and safe exclusion of functionally relevant coronary artery disease (CAD) is a crucial, yet unmet clinical need. High-sensitivity cardiac troponin (hs-cTn) may be an attractive strategy, particularly in patients without previously known CAD. Purpose To derive and internally validate optimal rule-out cutoffs for an early and safe exclusion of functionally relevant CAD in symptomatic patients without previously known CAD. Methods In an ongoing single-center, prospective, cohort study, we enrolled consecutive patients without previously known CAD that were referred with symptoms possibly related to functionally relevant CAD. Cardiac troponin concentrations were measured at presentation using two high-sensitivity assays (Elecsys hs-cTnT and Architect hs-cTnI). Presence of functionally relevant CAD was adjudicated by 2 independent cardiologists, blinded to hs-cTn measurements, using MPI-SPECT/CT in all patients, as well as coronary angiography and fractional flow reserve measurements, whenever available. The primary diagnostic outcome was safety for early rule-out of functionally relevant CAD, quantified by sensitivity and the negative predictive value (NPV). The co-primary prognostic outcomes were cumulative incidences of cardiovascular death and all-cause death after 5 years. A NPV ≥90% and sensitivity ≥90% were predefined as acceptable performance criteria. The derived cutoffs were further evaluated in pre-specified subgroups. Internal validity was assessed with a bootstrapping procedure for a realistic estimate in similar future patients. Cumulative incidence curves stratified by the presence of functionally relevant CAD and hs-cTn concentrations below and above the derived cutoffs were constructed. Results Among 2111 eligible patients, 498 (23.6%) had a final diagnosis of functionally relevant CAD. Median age was 68 years and 938 (44.4%) were female. For ruling out functionally relevant CAD, a hs-cTnT concentration Conclusion In symptomatic patients without previously known CAD, very low hs-cTn concentrations may generally allow to safely and effectively exclude functionally relevant CAD. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Swiss National Science FoundationSwiss Heart Foundation

Background Clinical practice guidelines assume that both cardiac troponin (cTn) T and cTnI concentrations reflect identical pathophysiological processes and are equally effective in the detection of myocardial injury. However, there are differences between cTnT and cTnI that have been reported. Purpose The aim of this study was to directly compare the diagnostic performance of high-sensitivity cardiac troponin (hs-cTn) T versus hs-cTnI for the early diagnosis of acute myocardial infarction (MI). Methods In a prospective multicentre study, diagnostic and prognostic accuracies of hs-cTnT and I were analyzed in consecutive patients presenting to the emergency department with acute chest pain. The final diagnosis was adjudicated by two independent cardiologists using all information pertaining to the individual patient according to the fourth universal definition of MI. Adjudication of the final diagnoses was performed twice: once using serial measurements of hs-cTnT and once using hs-cTnI. Furthermore, the clinical performance of hs-cTnT/I when embedded in the European Society of Cardiology (ESC) 0/1h-algorithm was assessed. Results Among 5087 consecutive patients (median [Interquartile range, IQR] age 61 [49.0, 74.0] years, 33.2% female), 951 (18.7%) and 901 patients (17.7%) had an adjudicated final diagnosis of non-ST-segment elevation myocardial infarction (NSTEMI) when using serial measurements of hs-cTnT and hs-cTnI for adjudication, respectively. Diagnostic accuracy was very high for both hs-cTnT and hs-cTnI and comparable when using hs-cTnT for adjudication (hs-cTnT: area under the curve [AUC] 0.93 [95% CI 0.92–0.94] versus hs-cTnI AUC 0.93 [95% CI 0.92–0.94]; p=0.891). However, when using serial measurements of hs-cTnI for adjudication, diagnostic accuracy was significantly higher for hs-cTnI (AUC 0.93 [95% CI 0.92–0.94] versus AUC 0.94 [95% CI 0.94–0.95], p Conclusions While there seem to be differences between hs-cTnT and hs-cTnI in their diagnostic and prognostic performance, clinical relevance needs to be further evaluated since both assays performed excellent when embedded in their respective early triage algorithms. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Swiss National Foundation, Swiss Heart Foundation

Background Clinical practice and guidelines assume that cardiac troponin I (cTnI) and cTnT are interchangeable, reflecting identical pathophysiological processes. However, it is unknown if cTnI and cTnT really are equivalent measures in different pathophysiological settings. Purpose To highlight potential differences in the release of cTnI and cTnT. Methods Large pooled cohort analysis including extensively characterized individuals, stratified into three groups: no cardiac disease (normal aging), chronic cardiac disease, and acute cardiac disease. Circulating cTnI and cTnT concentrations were measured blinded to clinical data using high-sensitivity assays (hs-cTnI-Architect, hs-cTnT-Elecsys) and their ratio calculated. Findings were validated using a second hs-cTnI assay (hs-cTnI-Clarity). Results Among 8719 individuals, 29% female, 10% had no known cardiac disease, 71% chronic cardiac disease, and 20% acute cardiac disease. Baseline characteristics including renal function were comparable between individuals with chronic and acute cardiac disease. Normal aging (without cardiac disease) was associated with a disproportional increase in cTnT versus cTnI (low cTnI/cTnT ratio, median 0.50, IQR 0.38–0.68). Although older, patients with chronic cardiac disease had a slightly higher cTnI/cTnT ratio (median 0.53, IQR 0.37–0.79, p Conclusion These findings suggest relevant differences in the release of cTnI and cTnT with a greater release of cTnT versus cTnI in normal aging and a disproportional increase in cTnI versus cTnT in acute cardiac disease. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Swiss National Science Foundation

Background Short-term forecasts of infectious disease burden can contribute to situational awareness and aid capacity planning. Based on best practice in other fields and recent insights in infectious disease epidemiology, one can maximise the predictive performance of such forecasts if multiple models are combined into an ensemble. Here we report on the performance of ensembles in predicting COVID-19 cases and deaths across Europe between 08 March 2021 and 07 March 2022. Methods We used open-source tools to develop a public European COVID-19 Forecast Hub. We invited groups globally to contribute weekly forecasts for COVID-19 cases and deaths reported from a standardised source over the next one to four weeks. Teams submitted forecasts from March 2021 using standardised quantiles of the predictive distribution. Each week we created an ensemble forecast, where each predictive quantile was calculated as the equally-weighted average (initially the mean and then from 26th July the median) of all individual models’ predictive quantiles. We measured the performance of each model using the relative Weighted Interval Score (WIS), comparing models’ forecast accuracy relative to all other models. We retrospectively explored alternative methods for ensemble forecasts, including weighted averages based on models’ past predictive performance. Results Over 52 weeks we collected and combined up to 28 forecast models for 32 countries. We found a weekly ensemble had a consistently strong performance across countries over time. Across all horizons and locations, the ensemble performed better on relative WIS than 84% of participating models’ forecasts of incident cases (with a total N=862), and 92% of participating models’ forecasts of deaths (N=746). Across a one to four week time horizon, ensemble performance declined with longer forecast periods when forecasting cases, but remained stable over four weeks for incident death forecasts. In every forecast across 32 countries, the ensemble outperformed most contributing models when forecasting either cases or deaths, frequently outperforming all of its individual component models. Among several choices of ensemble methods we found that the most influential and best choice was to use a median average of models instead of using the mean, regardless of methods of weighting component forecast models. Conclusions Our results support the use of combining forecasts from individual models into an ensemble in order to improve predictive performance across epidemiological targets and populations during infectious disease epidemics. Our findings further suggest that median ensemble methods yield better predictive performance more than ones based on means. Our findings also highlight that forecast consumers should place more weight on incident death forecasts than incident case forecasts at forecast horizons greater than two weeks. Code and data availability All data and code are publicly available on Github: covid19-forecast-hub-europe/euro-hub-ensemble.

Objectives Otitis media (OM) is the most common pediatric diagnosis in the United States. However, our understanding of the molecular pathogenesis of OM remains relatively poor. Investigation of molecular pathways involved in OM may improve the understanding of this disease process and elucidate novel therapeutic targets. In this study, RNA sequencing (RNA-Seq) was used to discern cellular changes associated with OME compared to healthy middle ear epithelium (MEE). Study design Ex vivo case-control translational. Methods Middle ear epithelia was collected from five pediatric patients diagnosed with OME undergoing tympanostomy tube placement and five otherwise healthy pediatric patients undergoing cochlear implantation. Specimens underwent RNA-Seq and pathways analyses. Results A total of 1,292 genes exhibited differential expression in MEE from OME patients compared to controls including genes involved in inflammation, immune response to bacterial OM pathogens, mucociliary clearance, regulation of proliferation and transformation, and auditory cell differentiation. Top networks identified in OME were organismal injury and abnormalities, cell morphology, and auditory disease. Top Ingenuity canonical pathways identified were axonal guidance signaling, which contains genes associated with auditory development and disease and nicotine degradation II and III pathways. Associated upstream regulators included β-estradiol, dexamethasone, and G-protein-coupled estrogen receptor-1 (GPER1), which are associated with otoprotection or inflammation during insult. Conclusions RNA-Seq demonstrates differential gene expression in MEE from patients with OME compared to healthy controls with important implications for infection susceptibility, hearing loss, and a role for tobacco exposure in the development and/or severity of OME in pediatric patients. Level of evidence 4 Laryngoscope, 2021.

Background Kabuki syndrome is a genetic disorder that affects several body systems and presents with variations in symptoms and severity. The syndrome is named for a common phenotype of faces resembling stage makeup used in a Japanese traditional theatrical art named kabuki. The most frequent cause of this syndrome is mutations in the H3K4 family of histone methyltransferases while a smaller percentage results from genetic alterations affecting the histone demethylase, KDM6A. Because of the rare presentation of the latter form of the disease, little is known about how missense changes in the KDM6A protein sequence impact protein function. Results In this study, we use molecular mechanic and molecular dynamic simulations to enhance the annotation and mechanistic interpretation of the potential impact of eleven KDM6A missense variants found in Kabuki syndrome patients. These variants (N910S, D980V, S1025G, C1153R, C1153Y, P1195L, L1200F, Q1212R, Q1248R, R1255W, and R1351Q) are predicted to be pathogenic, likely pathogenic or of uncertain significance by sequence-based analysis. Here, we demonstrate, for the first time, that although Kabuki syndrome missense variants are found outside the functionally critical regions, they could affect overall function by significantly disrupting global and local conformation (C1153R, C1153Y, P1195L, L1200F, Q1212R, Q1248R, R1255W and R1351Q), chemical environment (C1153R, C1153Y, P1195L, L1200F, Q1212R, Q1248R, R1255W and R1351Q), and/or molecular dynamics of the catalytic domain (all variants). In addition, our approaches predict that many mutations, in particular C1153R, could allosterically disrupt the key enzymatic interactions of KDM6A. Conclusions Our study demonstrates that the KDM6A Kabuki syndrome variants may impair histone demethylase function through various mechanisms that include altered protein integrity, local environment, molecular interactions and protein dynamics. Molecular dynamics simulations of the wild type and the variants are critical to gain a better understanding of molecular dysfunction. This type of comprehensive structure- and MD-based analyses should help develop improved impact scoring systems to interpret the damaging effects of variants in this protein and other related proteins as well as provide detailed mechanistic insight that is not currently predictable from sequence alone.

Motivation Protein-coding genetic alterations are frequently observed in Clinical Genetics, but the high yield of variants of uncertain significance remains a limitation in decision making. RAS-family GTPases are cancer drivers, but only 54 variants, across all family members, fall within well-known hotspots. However, extensive sequencing has identified 881 non-hotspot variants for which significance remains to be investigated. Results Here, we evaluate 935 missense variants from seven RAS genes, observed in cancer, RASopathies and the healthy adult population. We characterized hotspot variants, previously studied experimentally, using 63 sequence- and 3D structure-based scores, chosen by their breadth of biophysical properties. Applying scores that display best correlation with experimental measures, we report new valuable mechanistic inferences for both hot-spot and non-hotspot variants. Moreover, we demonstrate that 3D scores have little-to-no correlation with those based on DNA sequence, which are commonly used in Clinical Genetics. Thus, combined, these new knowledge bear significant relevance. Availability and implementation All genomic and 3D scores, and markdown for generating figures, are provided in our supplemental data. Supplementary information Supplementary data are available at Bioinformatics online.

Hundreds of human membrane proteins form channels that transport necessary ions and compounds, including drugs and metabolites, yet details of their normal function or how function is altered by genetic variants to cause diseases are often unknown. Without this knowledge, researchers are less equipped to develop approaches to diagnose and treat channelopathies. High-resolution computational approaches such as molecular modeling enable researchers to investigate channelopathy protein function, facilitate detailed hypothesis generation, and produce data that is difficult to gather experimentally. Molecular modeling can be tailored to each physiologic context that a protein may act within, some of which may currently be difficult or impossible to assay experimentally. Because many genomic variants are observed in channelopathy proteins from high-throughput sequencing studies, methods with mechanistic value are needed to interpret their effects. The eminent field of structural bioinformatics integrates techniques from multiple disciplines including molecular modeling, computational chemistry, biophysics, and biochemistry, to develop mechanistic hypotheses and enhance the information available for understanding function. Molecular modeling and simulation access 3D and time-dependent information, not currently predictable from sequence. Thus, molecular modeling is valuable for increasing the resolution with which the natural function of protein channels can be investigated, and for interpreting how genomic variants alter them to produce physiologic changes that manifest as channelopathies. © 2022 American Physiological Society. Compr Physiol 12:3141-3166, 2022.

BACKGROUND: Impaired endothelium-dependent vasodilation has been suggested to be a key component of coronary microvascular dysfunction (CMD). A better understanding of endothelial pathways involved in vasodilation in human arterioles may provide new insight into the mechanisms of CMD. OBJECTIVE: To investigate the role of TRPV4, NOX4, and their interaction in human arterioles and examine the underlying mechanisms. METHODS AND RESULTS: Arterioles were freshly isolated from adipose and heart tissues obtained from 71 patients without coronary artery disease, and vascular reactivity was studied by videomicroscopy. In human adipose arterioles (HAA), ACh-induced dilation was significantly reduced by TRPV4 inhibitor HC067047 and by NOX 1/4 inhibitor GKT137831, but GKT137831 did not further affect the dilation in the presence of TRPV4 inhibitors. GKT137831 also inhibited TRPV4 agonist GSK1016790A-induced dilation in HAA and human coronary arterioles (HCA). NOX4 transcripts and proteins were detected in endothelial cells of HAA and HCA. Using fura-2 imaging, GKT137831 significantly reduced GSK1016790A-induced Ca(2+) influx in the primary culture of endothelial cells and TRPV4-WT-overexpressing human coronary artery endothelial cells (HCAEC). However, GKT137831 did not affect TRPV4-mediated Ca(2+) influx in non-phosphorylatable TRPV4-S823A/S824A-overexpressing HCAEC. In addition, treatment of HCAEC with GKT137831 decreased the phosphorylation level of Ser824 in TRPV4. Finally, proximity ligation assay (PLA) revealed co-localization of NOX4 and TRPV4 proteins. CONCLUSION: Both TRPV4 and NOX4 contribute to ACh-induced dilation in human arterioles from patients without coronary artery disease. NOX4 increases TRPV4 phosphorylation in endothelial cells, which in turn enhances TRPV4-mediated Ca(2+) entry and subsequent endothelium-dependent dilation in human arterioles.

This article presents results of a research carried out with the objective of analyzing the participation of Social Control in the sphere of Public Health Policy of a small municipality, located in the Extreme West of Santa Catarina. This is a qualitative study, carried out with the participation of twelve directors, six representatives of non-governmental entities and six of governmental entities. Data were collected between August and December 2019, through interviews with open and closed questions. The research was approved by the Ethics and Research Committee. All requirements contained in the Resolution of the National Health Council - CNS 466/2012 were observed and respected. The limits for the participation of members in the Council, time and lack of knowledge in the health area were found as limits, and with regard to the possibilities, a broader view of the health services offered, and within this, the prioritization of actions and actions. It is observed that Social Control is very important and necessarily needs to be maintained and strengthened. The Federal Constitution of 1988 consolidated and predicted in its devices the participation of citizens in the formulation and implementation of Social Control, public health policies. It is hoped that the result of the research can boost proposals that are configured as responses to the strengthening of participation in Social Control, recognizing the Health Councils are deliberative and permanent collegiate organs of the SUS, existing in each sphere of government and members of the basic structure of the health system.

Kurzfassung Das Konzept des Digital Twins nutzt Modelle über die Entwicklung und Absicherung hinaus auch für Betriebsoptimierungen. Modellbasiertes Systems Engineering (MBSE) ermöglicht eine durchgängige Systemmodellierung mit offenen Schnittstellen für die Systementwicklung. Dieser Beitrag stellt an einem beispielhaften IoT-Pumpensystem Schnittstellenerweiterungen vor, um MBSE für eine durchgängige Modellierung, von der Entwicklung über den Betrieb bis End-of-Life, nutzen zu können. Die gewonnenen Informationen können zurückgeführt und für zukünftige Produktneuentwicklungen genutzt werden.

OBJECTIVE Laryngopharyngeal reflux (LPR) is a common affliction that contributes to laryngeal inflammation, symptoms that impact quality of life, and life-threatening illnesses such as cancer. Effective treatment strategies for LPR are lacking. Pepsin is a proinflammatory and carcinogenic element of refluxate. Investigation of molecular pathways involved in pepsin-mediated damage may lead to identification of novel biomarkers and therapeutic targets for LPR. In this study, RNA sequencing was used to examine changes in human laryngeal epithelial cells following brief pepsin insult. Cells were immortalized to generate a model to aid future study of laryngeal injury and therapeutics. STUDY DESIGN In vitro translational. METHODS Laryngeal epithelial cells were cultured from a patient without signs or symptoms of LPR or laryngeal cancer. Cells were treated with 0.1 mg/ml pepsin for 1 hour or normal growth media (control) prior to RNA sequencing. Cells were immortalized via HPV E6/7 and characterized by microscopy, immunohistochemistry, G-banding, and soft agar assay. RESULTS Three hundred ninety-seven genes exhibited differences in expression with pepsin treatment (P

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which upper and lower motor neuron loss is the primary phenotype, leading to muscle weakness and wasting, respiratory failure, and death. Although a portion of ALS cases are linked to one of over 50 unique genes, the vast majority of cases are sporadic in nature. However, the mechanisms underlying the motor neuron loss in either familial or sporadic ALS are not entirely clear. Here we used induced pluripotent stem cells derived from a set of identical twin brothers discordant for ALS to assess the role of astrocytes and microglia on the expression and accumulation of neurofilament proteins in motor neurons. We found that motor neurons derived from the affected twin exhibited increased transcript levels of all three neurofilament isoforms and increased expression of phosphorylated neurofilament puncta. We further found that treatment of the motor neurons with astrocyte conditioned medium and microglial conditioned medium significantly impacted neurofilament deposition. Together, these data suggest that glial-secreted factors can alter neurofilament pathology in ALS iPSC-derived motor neurons.

Background Genomics enables individualized diagnosis and treatment, but large challenges remain to functionally interpret rare variants. To date, only one causative variant has been described for KCNK9 imprinting syndrome (KIS). The genotypic and phenotypic spectrum of KIS has yet to be described and the precise mechanism of disease fully understood. Methods This study discovers mechanisms underlying KCNK9 imprinting syndrome (KIS) by describing 15 novel KCNK9 alterations from 47 KIS-affected individuals. We use clinical genetics and computer-assisted facial phenotyping to describe the phenotypic spectrum of KIS. We then interrogate the functional effects of the variants in the encoded TASK3 channel using sequence-based analysis, 3D molecular mechanic and dynamic protein modeling, and in vitro electrophysiological and functional methodologies. Results We describe the broader genetic and phenotypic variability for KIS in a cohort of individuals identifying an additional mutational hotspot at p.Arg131 and demonstrating the common features of this neurodevelopmental disorder to include motor and speech delay, intellectual disability, early feeding difficulties, muscular hypotonia, behavioral abnormalities, and dysmorphic features. The computational protein modeling and in vitro electrophysiological studies discover variability of the impact of KCNK9 variants on TASK3 channel function identifying variants causing gain and others causing loss of conductance. The most consistent functional impact of KCNK9 genetic variants, however, was altered channel regulation. Conclusions This study extends our understanding of KIS mechanisms demonstrating its complex etiology including gain and loss of channel function and consistent loss of channel regulation. These data are rapidly applicable to diagnostic strategies, as KIS is not identifiable from clinical features alone and thus should be molecularly diagnosed. Furthermore, our data suggests unique therapeutic strategies may be needed to address the specific functional consequences of KCNK9 variation on channel function and regulation.

ChemCatChem (2022). doi:10.1002/cctc.202101878, Published by WILEY-VCH Verlag, Weinheim

Environmental pollution 302, 119040 (2022). doi:10.1016/j.envpol.2022.119040, Published by Elsevier Science, Amsterdam [u.a.]