Incomplete-penetrant hypertrophic cardiomyopathy MYH7 G256E mutation causes hypercontractility and elevated mitochondrial respiration.

Determining the pathogenicity of hypertrophic cardiomyopathy-associated mutations in the ß-myosin heavy chain (MYH7) can be challenging due to its variable penetrance and clinical severity. This study investigates the early pathogenic effects of the incomplete-penetrant MYH7 G256E mutation on myosin function that may trigger pathogenic adaptations and hypertrophy. We hypothesized that the G256E mutation would alter myosin biomechanical function, leading to changes in cellular functions. We developed a collaborative pipeline to characterize myosin function across protein, myofibril, cell, and tissue levels to determine the multiscale effects on structure-function of the contractile apparatus and its implications for gene regulation and metabolic state. The G256E mutation disrupts the transducer region of the S1 head and reduces the fraction of myosin in the folded-back state by 33%, resulting in more myosin heads available for contraction. Myofibrils from gene-edited MYH7WT/G256E human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exhibited greater and faster tension development. This hypercontractile phenotype persisted in single-cell hiPSC-CMs and engineered heart tissues. We demonstrated consistent hypercontractile myosin function as a primary consequence of the MYH7 G256E mutation across scales, highlighting the pathogenicity of this gene variant. Single-cell transcriptomic and metabolic profiling demonstrated upregulated mitochondrial genes and increased mitochondrial respiration, indicating early bioenergetic alterations. This work highlights the benefit of our multiscale platform to systematically evaluate the pathogenicity of gene variants at the protein and contractile organelle level and their early consequences on cellular and tissue function. We believe this platform can help elucidate the genotype-phenotype relationships underlying other genetic cardiovascular diseases.

The cardiac conduction system: History, development, and disease.

The heart is the first organ to form during embryonic development, establishing the circulatory infrastructure necessary to sustain life and enable downstream organogenesis. Critical to the heart's function is its ability to initiate and propagate electrical impulses that allow for the coordinated contraction and relaxation of its chambers, and thus, the movement of blood and nutrients. Several specialized structures within the heart, collectively known as the cardiac conduction system (CCS), are responsible for this phenomenon. In this review, we discuss the discovery and scientific history of the mammalian cardiac conduction system as well as the key genes and transcription factors implicated in the formation of its major structures. We also describe known human diseases related to CCS development and explore existing challenges in the clinical context.

Bacteria and bacteriophage consortia are associated with protective intestinal metabolites in patients receiving stem cell transplantation.

The microbiome is a predictor of clinical outcome in patients receiving allogeneic hematopoietic stem cell transplantation (allo-SCT). Microbiota-derived metabolites can modulate these outcomes. How bacteria, fungi and viruses contribute to the production of intestinal metabolites is still unclear. We combined amplicon sequencing, viral metagenomics and targeted metabolomics from stool samples of patients receiving allo-SCT (n = 78) and uncovered a microbiome signature of Lachnospiraceae and Oscillospiraceae and their associated bacteriophages, correlating with the production of immunomodulatory metabolites (IMMs). Moreover, we established the IMM risk index (IMM-RI), which was associated with improved survival and reduced relapse. A high abundance of short-chain fatty acid-biosynthesis pathways, specifically butyric acid via butyryl-coenzyme A (CoA):acetate CoA-transferase (BCoAT, which catalyzes EC 2.8.3.8) was detected in IMM-RI low-risk patients, and virome genome assembly identified two bacteriophages encoding BCoAT as an auxiliary metabolic gene. In conclusion, our study identifies a microbiome signature associated with protective IMMs and provides a rationale for considering metabolite-producing consortia and metabolite formulations as microbiome-based therapies.

The microbial metabolite desaminotyrosine enhances T-cell priming and cancer immunotherapy with immune checkpoint inhibitors.

BACKGROUND: Inter-individual differences in response to immune checkpoint inhibitors (ICI) remain a major challenge in cancer treatment. The composition of the gut microbiome has been associated with differential ICI outcome, but the underlying molecular mechanisms remain unclear, and therapeutic modulation challenging. METHODS: We established an in vivo model to treat C57Bl/6j mice with the type-I interferon (IFN-I)-modulating, bacterial-derived metabolite desaminotyrosine (DAT) to improve ICI therapy. Broad spectrum antibiotics were used to mimic gut microbial dysbiosis and associated ICI resistance. We utilized genetic mouse models to address the role of host IFN-I in DAT-modulated antitumour immunity. Changes in gut microbiota were assessed using 16S-rRNA sequencing analyses. FINDINGS: We found that oral supplementation of mice with the microbial metabolite DAT delays tumour growth and promotes ICI immunotherapy with anti-CTLA-4 or anti-PD-1. DAT-enhanced antitumour immunity was associated with more activated T cells and natural killer cells in the tumour microenvironment and was dependent on host IFN-I signalling. Consistent with this, DAT potently enhanced expansion of antigen-specific T cells following vaccination with an IFN-I-inducing adjuvant. DAT supplementation in mice compensated for the negative effects of broad-spectrum antibiotic-induced dysbiosis on anti-CTLA-4-mediated antitumour immunity. Oral administration of DAT altered the gut microbial composition in mice with increased abundance of bacterial taxa that are associated with beneficial response to ICI immunotherapy. INTERPRETATION: We introduce the therapeutic use of an IFN-I-modulating bacterial-derived metabolite to overcome resistance to ICI. This approach is a promising strategy particularly for patients with a history of broad-spectrum antibiotic use and associated loss of gut microbial diversity. FUNDING: Melanoma Research Alliance, Deutsche Forschungsgemeinschaft, German Cancer Aid, Wilhelm Sander Foundation, Novartis Foundation.

Multi-scale models reveal hypertrophic cardiomyopathy MYH7 G256E mutation drives hypercontractility and elevated mitochondrial respiration.

Rationale: Over 200 mutations in the sarcomeric protein ß-myosin heavy chain (MYH7) have been linked to hypertrophic cardiomyopathy (HCM). However, different mutations in MYH7 lead to variable penetrance and clinical severity, and alter myosin function to varying degrees, making it difficult to determine genotype-phenotype relationships, especially when caused by rare gene variants such as the G256E mutation. Objective: This study aims to determine the effects of low penetrant MYH7 G256E mutation on myosin function. We hypothesize that the G256E mutation would alter myosin function, precipitating compensatory responses in cellular functions. Methods: We developed a collaborative pipeline to characterize myosin function at multiple scales (protein to myofibril to cell to tissue). We also used our previously published data on other mutations to compare the degree to which myosin function was altered. Results: At the protein level, the G256E mutation disrupts the transducer region of the S1 head and reduces the fraction of myosin in the folded-back state by 50.9%, suggesting more myosins available for contraction. Myofibrils isolated from hiPSC-CMs CRISPR-edited with G256E (MYH7 WT/G256E ) generated greater tension, had faster tension development and slower early phase relaxation, suggesting altered myosin-actin crossbridge cycling kinetics. This hypercontractile phenotype persisted in single-cell hiPSC-CMs and engineered heart tissues. Single-cell transcriptomic and metabolic profiling demonstrated upregulation of mitochondrial genes and increased mitochondrial respiration, suggesting altered bioenergetics as an early feature of HCM. Conclusions: MYH7 G256E mutation causes structural instability in the transducer region, leading to hypercontractility across scales, perhaps from increased myosin recruitment and altered crossbridge cycling. Hypercontractile function of the mutant myosin was accompanied by increased mitochondrial respiration, while cellular hypertrophy was modest in the physiological stiffness environment. We believe that this multi-scale platform will be useful to elucidate genotype-phenotype relationships underlying other genetic cardiovascular diseases.

Generation of a CRISPR/Cas edited human induced pluripotent stem cell line DHMi005-A-1 carrying a patient-specific disease-causing point mutation in the TBX5 gene.

A number of mutations in the human TBX5 gene have been described which cause Holt-Oram syndrome, a severe congenital disease associated with abnormalities in heart and upper limb development. We have used a prime-editing approach to introduce a patient-specific disease-causing TBX5 mutation (c.920_C > A) into an induced pluripotent stem cell (iPSC) line from a healthy donor. The resulting iPSC line provides a powerful tool to identify and analyze the biological and molecular impact of this specific TBX5 mutation in comparison to the isogenic control iPSC line during cardiac development.

Health economic impact of first-pass success among patients with acute ischemic stroke treated with mechanical thrombectomy: a United States and European perspective.

BACKGROUND: First-pass effect (FPE), restoring complete or near complete reperfusion (modified Thrombolysis in Cerebral Infarction (mTICI) 2c-3) in a single pass, is an independent predictor for good functional outcomes in the endovascular treatment of acute ischemic stroke. The economic implications of achieving FPE have not been assessed. OBJECTIVE: To assess the economic impact of achieving complete or near complete reperfusion after the first pass. METHODS: Post hoc analyses were conducted using ARISE II study data. The target population consisted of patients in whom mTICI 2c-3 was achieved, stratified into two groups: (1) mTICI 2c-3 achieved after the first pass (FPE group) or (2) after multiple passes (non-FPE group). Baseline characteristics, clinical outcomes, and healthcare resource use were compared between groups. Costs from peer-reviewed literature were applied to assess cost consequences from the perspectives of the United States (USA), France, Germany, Italy, Spain, Sweden, and United Kingdom (UK). RESULTS: Among patients who achieved mTICI 2c-3 (n=172), FPE was achieved in 53% (n=91). A higher proportion of patients in the FPE group reached good functional outcomes (90-day modified Rankin Scale score 0-2 80.46% vs 61.04%, p<0.01). The patients in the FPE group had a shorter mean length of stay (6.10 vs 9.48 days, p<0.01) and required only a single stent retriever, whereas 35% of patients in the non-FPE group required at least one additional device. Driven by improvement in clinical outcomes, the FPE group had lower procedural/hospitalization-related (24-33% reduction) and annual care (11-27% reduction) costs across all countries. CONCLUSIONS: FPE resulted in improved clinical outcomes, translating into lower healthcare resource use and lower estimated costs.

Benefit of endovascular thrombectomy for M2 middle cerebral artery occlusion in the ARISE II study.

BACKGROUND: The benefit of endovascular thrombectomy for acute ischemic stroke with M2 segment middle cerebral artery occlusion remains controversial, with uncertainty and paucity of data specific to this population. OBJECTIVE: To compare outcomes between M1 and M2 occlusions in the Analysis of Revascularization in Ischemic Stroke with EmboTrap (ARISE II) trial. METHODS: We performed a prespecified analysis of the ARISE II trial with the primary outcome of 90-day modified Rankin Scale score of 0-2, which we termed good outcome. Secondary outcomes included reperfusion rates and major adverse events. The primary predictor was M2 occlusion, which we compared with M1 occlusion. RESULTS: We included 183 patients, of whom 126 (69%) had M1 occlusion and 57 (31%) had M2 occlusion. There was no difference in the reperfusion rates or adverse events between M2 and M1 occlusions. The rate of good outcome was not different in M2 versus M1 occlusions (70.2% vs 69.7%, p=0.946). In a logistic regression model adjusted for age, sex, and baseline National Institutes of Health Stroke Scale score, M2 occlusions did not have a significantly different odds of good outcome compared with M1 occlusions (OR 0.94, 95% CI 0.47 to 1.88, p=0.87). CONCLUSION: In ARISE II, M2 occlusions achieved a 70.2% rate of good outcome at 90 days, which is above published rates for untreated M2 occlusions and superior to prior reports of M2 occlusions treated with endovascular thrombectomy. We also report similar rates of good outcome, successful reperfusion, death, and other adverse events when comparing the M1 and M2 occlusions.

Optimized Protocol for the In Situ Derivatization of Glutathione with N-Ethylmaleimide in Cultured Cells and the Simultaneous Determination of Glutathione/Glutathione Disulfide Ratio by HPLC-UV-QTOF-MS.

Glutathione (GSH) and glutathione disulfide (GSSG) are commonly used to assess the oxidative status of a biological system. Various protocols are available for the analysis of GSH and GSSG in biomedical specimens. In this study, we present an optimized protocol for the in situ derivatization of GSH with N-ethylmaleimide (NEM) to prevent GSH autooxidation, and thus to preserve the GSH/GSSG ratio during sample preparation. The protocol comprises the incubation of cells in NEM containing phosphate buffered saline (PBS), followed by metabolite extraction with 80% methanol. Further, to preserve the use of QTOF-MS, which may lack the linear dynamic range required for the simultaneous quantification of GSH and GSSG in non-targeted metabolomics, we combined liquid chromatographic separation with the online monitoring of UV absorbance of GS-NEM at 210 nm and the detection of GSSG and its corresponding stable isotope-labeled internal standard by QTOF-MS operated with a 10 Da Q1 window. The limit of detection (LOD) for GS-NEM was 7.81 µM and the linear range extended from 15.63 µM to 1000 µM with a squared correlation coefficient R2 of 0.9997. The LOD for GSSG was 0.001 µM, and the lower limit of quantification (LLOQ) was 0.01 µM, with the linear (R2 = 0.9994) range extending up to 10 µM. The method showed high repeatability with intra-run and inter-run coefficients of variation of 3.48% and 2.51% for GS-NEM, and 3.11% and 3.66% for GSSG, respectively. Mean recoveries of three different spike-in levels (low, medium, high) of GSSG and GS-NEM were above 92%. Finally, the method was applied to the determination of changes in the GSH/GSSG ratio either in response to oxidative stress in cells lacking one or both monocarboxylate transporters MCT1 and MCT4, or in adaptation to the NADPH (nicotinamide adenine dinucleotide phosphate) consuming production of D-2-hydroxyglutarate in cells carrying mutations in the isocitrate dehydrogenase genes IDH1 and IDH2.

DTD: An R Package for Digital Tissue Deconvolution.

Digital tissue deconvolution (DTD) estimates the cellular composition of a tissue from its bulk gene-expression profile. For this, DTD approximates the bulk as a mixture of cell-specific expression profiles. Different tissues have different cellular compositions, with cells in different activation states, and embedded in different environments. Consequently, DTD can profit from tailoring the deconvolution model to a specific tissue context. Loss-function learning adapts DTD to a specific tissue context, such as the deconvolution of blood, or a specific type of tumor tissue. We provide software for loss-function learning, for its validation and visualization, and for applying the DTD models to new data.

Quantification and 13C-Tracer analysis of total reduced glutathione by HPLC-QTOFMS/MS.

Glutathione is an essential intra- and extracellular antioxidant. The level of glutathione in the body is highly related to different disease states and is a useful indicator of disease risk and oxidative stress status. We have developed a sensitive, selective, and comprehensive LC-MS/MS method for the absolute quantification and 13C-tracer analysis of total glutathione using dithiothreitol for the reduction of glutathione disulfide. The limit of detection (LOD) was 0.01 µM, while the lower limit of quantification (LLOQ) was 0.78 µM, with the linear (R = 0.9997) range extending up to 100 µM. The intra-run and inter-run coefficients of variation of 2.49% and 2.04%, respectively, attest to high repeatability. Mean (±SD) recoveries of three different concentrations (low, medium, high) of GSH spiked into aliquots of HCT116 cells prior to cell extraction were 108.9% (±2.1), 100.8% (±8.3), and 99.9% (±7.1), respectively. Finally, using a 20 Da wide Q1 window in MRM mode, we were able to detect and relatively quantify all isotopic labeling states of GSH extracted from HCT116 cells fed with either 13C-labeled glucose or glutamine.

Correcting for natural isotope abundance and tracer impurity in MS-, MS/MS- and high-resolution-multiple-tracer-data from stable isotope labeling experiments with IsoCorrectoR.

Experiments with stable isotope tracers such as 13C and 15N are increasingly used to gain insights into metabolism. However, mass spectrometric measurements of stable isotope labeling experiments should be corrected for the presence of naturally occurring stable isotopes and for impurities of the tracer substrate. Here, we analyzed the effect that such correction has on the data: omitting correction or performing invalid correction can result in largely distorted data, potentially leading to misinterpretation. IsoCorrectoR is the first R-based tool to offer said correction capabilities. It is easy-to-use and comprises all correction features that comparable tools can offer in a single solution: correction of MS and MS/MS data for natural stable isotope abundance and tracer impurity, applicability to any tracer isotope and correction of multiple-tracer data from high-resolution measurements. IsoCorrectoR's correction performance agreed well with manual calculations and other available tools including Python-based IsoCor and Perl-based ICT. IsoCorrectoR can be downloaded as an R-package from: http://bioconductor.org/packages/release/bioc/html/IsoCorrectoR.html .

Cooperative STAT/NF-κB signaling regulates lymphoma metabolic reprogramming and aberrant GOT2 expression.

Knowledge of stromal factors that have a role in the transcriptional regulation of metabolic pathways aside from c-Myc is fundamental to improvements in lymphoma therapy. Using a MYC-inducible human B-cell line, we observed the cooperative activation of STAT3 and NF-κB by IL10 and CpG stimulation. We show that IL10 + CpG-mediated cell proliferation of MYClow cells depends on glutaminolysis. By 13C- and 15N-tracing of glutamine metabolism and metabolite rescue experiments, we demonstrate that GOT2 provides aspartate and nucleotides to cells with activated or aberrant Jak/STAT and NF-κB signaling. A model of GOT2 transcriptional regulation is proposed, in which the cooperative phosphorylation of STAT3 and direct joint binding of STAT3 and p65/NF-κB to the proximal GOT2 promoter are important. Furthermore, high aberrant GOT2 expression is prognostic in diffuse large B-cell lymphoma underscoring the current findings and importance of stromal factors in lymphoma biology.

Srebp-controlled glucose metabolism is essential for NK cell functional responses.

Activated natural killer (NK) cells engage in a robust metabolic response that is required for normal effector function. Using genetic, pharmacological and metabolic analyses, we demonstrated an essential role for Srebp transcription factors in cytokine-induced metabolic reprogramming of NK cells that was independent of their conventional role in the control of lipid synthesis. Srebp was required for elevated glycolysis and oxidative phosphorylation and promoted a distinct metabolic pathway configuration in which glucose was metabolized to cytosolic citrate via the citrate-malate shuttle. Preventing the activation of Srebp or direct inhibition of the citrate-malate shuttle inhibited production of interferon-γ and NK cell cytotoxicity. Thus, Srebp controls glucose metabolism in NK cells, and this Srebp-dependent regulation is critical for NK cell effector function.

Doctors' experience of stress during simulated bad news consultations.

OBJECTIVE: Breaking bad news (BBN) is a core component of medicine. Psychophysiological studies confirm the subjective reports of doctors that BBN is a stressful experience. This study investigated doctors' physiological stress responses prior to and during two simulated bad news consultations. METHODS: Thirty-one doctors participated in a speech-interaction task and two simulated BBN consultations. Heart rate (HR) and skin conductance (SC) were recorded using consecutive 30-s epochs during each of the interactions. The simulations were video recorded. RESULTS: Most doctors showed an early anticipatory increase in HR and SC that peaked during the reading of the case history prior to the BBN consultations. Most doctors then experienced a brief and relatively small stress response. However, about one-third of the doctors showed a significant and sustained stress response. CONCLUSIONS: The results suggest that most doctors were cognitively engaged with the BBN tasks, however, a small proportion of doctors might have focused more on their own internal feelings and less on these contextual features. PRACTICE IMPLICATIONS: In regards to training medical students and doctors, these results suggest that there is a need to focus more on the impact of these encounters on the doctors, not just their performance during these encounters.

Atorvastatin added to interferon ß for relapsing multiple sclerosis: a randomized controlled trial.

Statins have anti-inflammatory and immunomodulatory properties in addition to lipid-lowering effects. The present study evaluated the effect of atorvastatin added to interferon beta-1b in multiple sclerosis (MS) in a multicenter, randomized, parallel-group, rater-blinded study performed in eight Swiss hospitals. Seventy-seven patients with relapsing-remitting MS started interferon beta-1b every other day. After 3 months, they were randomized 1:1 to receive atorvastatin 40 mg/day or not in addition to interferon beta-1b until month 15. The primary endpoint was the proportion of patients with new lesions on T2-weighted images at month 15 compared to baseline at month three. At study end, the proportion of patients with new lesions on T2-weighted images was equal in both groups (odds ratio 1.14; 95 % CI 0.36-3.56; p = 0.81). All predefined secondary endpoints including number of new lesions and total lesion volume on T2-weighted images, total number of new Gd-enhancing lesions on T1-weighted images, total brain volume, volume of grey matter, volume of white matter, EDSS, MSFC, relapse rate, time to first relapse, number of relapse-free patients and neutralizing antibodies did not show any significant differences (all p values >0.1). Transient elevations of liver enzymes were more frequent with atorvastatin (p = 0.02). In conclusion, atorvastatin 40 mg/day in addition to interferon beta-1b did not have a beneficial effect on relapsing-remitting MS compared to interferon beta-1b monotherapy over a 12-month period.

Managing the delivery of bad news: an in-depth analysis of doctors' delivery style.

OBJECTIVE: The purpose of this study was to identify and describe the delivery styles doctors typically use when breaking bad news (BBN). METHODS: Thirty one doctors were recruited to participate in two standardised BBN consultations involving a sudden death. Delivery styles were determined using time to deliver the bad news as a standardised differentiation as well as qualitative analysis of interaction content and language style. Communication performance was also assessed. RESULTS: Analysis of BBN interactions revealed three typical delivery styles. A blunt style characterised by doctors delivering news within the first 30 s of the interaction; Forecasting, a staged delivery of the news within the first 2 min and a stalling approach, delaying news delivery for more than 2 min. This latter avoidant style relies on the news recipient reaching a conclusion about event outcome without the doctor explicitly conveying the news. CONCLUSION: Three typical bad news delivery styles used by doctors when BBN were confirmed both semantically and operationally in the study. The relationship between delivery style and the overall quality of BBN interactions was also investigated. PRACTICE IMPLICATIONS: This research provides a new template for approaching BBN training and provides evidence for a need for greater flexibility when communicating bad news.

The development of novel interventions to assist the leaders of cancer support groups.

PURPOSE: The literature on cancer support groups supports the provision of ongoing education and training for cancer support group leaders, with evidence suggesting that more skilled and experienced leaders create better outcomes for group members. To address support and training needs reported by leaders, three novel interventions were developed and pilot-tested. These included a leaders' website and discussion forum, DVD and manual, and a 2-day training workshop. METHODS: The interventions were developed using a combination of literature review, expert consensus, and consumer feedback. A convenience sample of ten leaders pilot-tested the Website and discussion forum. Using a mixed-method approach, evaluation of the workshop and the DVD and manual was conducted with 35 leaders. RESULTS: Overall, satisfaction with all aspects of the Website and discussion forum was high. Analysis of the quantitative data revealed extremely high satisfaction with the workshop and DVD and manual. The qualitative responses of workshop participants further supported the quantitative findings with enhanced knowledge, understanding, and confidence reported by leaders. CONCLUSIONS: All three interventions exhibited a high degree of user acceptance, regardless of the skill or experience of the cancer support group leader. The overall positive findings from the evaluation of the leader Website and discussion forum, the DVD and manual, and the workshop for cancer support group leaders provides evidence to support more rigorous evaluation of these resources in a randomized controlled trial.

A model of professional development for practicing genetic counselors: adaptation of communication skills training in oncology.

Ongoing professional development for practicing genetic counselors is critical in maintaining best practice. Communication skills training (CST) workshops for doctors in oncology, utilizing trained actors in role plays, have been implemented for many years to improve patient-centred communication. This model was adapted to provide professional development in counseling skills for practicing genetic counselors, already highly trained in counseling skills. Detailed evidence based scenarios were developed. Evaluation of participants' experience and perceived outcomes on practice included surveys immediately post workshops (2002, 2004, 2005, 2008 (×2); n = 88/97), 2-5 years later (2007; n = 21/38) and a focus group (2007; n = 7). All rated workshops as effective training. Aspects highly valued included facilitator feedback, actors rather than role-playing with peers and being able to stop and try doing things differently. Perceived outcomes included the opportunity to reflect on practice; bring focus to communication; motivation and confidence. The high level of satisfaction is a strong endorsement for ongoing communication skills training in this format as part of professional development.