Recent advances in the understanding and use of pluripotent stem cells have produced major changes in approaches to the diagnosis and treatment of human disease. An obstacle to the use of human ...induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for regenerative medicine, disease modeling and drug discovery is their immature state relative to adult myocardium. We show the effects of a combination of biochemical factors, thyroid hormone, dexamethasone, and insulin-like growth factor-1 (TDI) on the maturation of hiPSC-CMs in 3D cardiac microtissues (CMTs) that recapitulate aspects of the native myocardium. Based on a comparison of the gene expression profiles and the structural, ultrastructural, and electrophysiological properties of hiPSC-CMs in monolayers and CMTs, and measurements of the mechanical and pharmacological properties of CMTs, we find that TDI treatment in a 3D tissue context yields a higher fidelity adult cardiac phenotype, including sarcoplasmic reticulum function and contractile properties consistent with promotion of the maturation of hiPSC derived cardiomyocytes.
•3D microtissues used to probe biochemical maturation of hiPSC-cardiomyocytes (CMs).•Thyroid hormone, dexamethasone, and IGF-1 (TDI) improve CM maturation in 3D.•Gene expression and ultrastructure show improved adult cardiac phenotype.•Enhanced electrophysiological properties, contraction and ionotropic response
Cancer cells develop mechanisms to escape immunosurveillance, among which modulating the expression of immune suppressive messenger RNAs is most well-documented. However, how this is molecularly ...achieved remains largely unresolved. Here, we develop an in vivo mouse model of liver cancer to study oncogene cooperation in immunosurveillance. We show that MYC overexpression (MYC
) synergizes with KRAS
to induce an aggressive liver tumor leading to metastasis formation and reduced mouse survival compared with KRAS
alone. Genome-wide ribosomal footprinting of MYC
;KRAS
tumors compared with KRAS
revealed potential alterations in translation of mRNAs, including programmed-death-ligand 1 (PD-L1). Further analysis revealed that PD-L1 translation is repressed in KRAS
tumors by functional, non-canonical upstream open reading frames in its 5' untranslated region, which is bypassed in MYC
;KRAS
tumors to evade immune attack. We show that this mechanism of PD-L1 translational upregulation was effectively targeted by a potent, clinical compound that inhibits eIF4E phosphorylation, eFT508, which reverses the aggressive and metastatic characteristics of MYC
;KRAS
tumors. Together, these studies reveal how immune-checkpoint proteins are manipulated by distinct oncogenes at the level of mRNA translation, which can be exploited for new immunotherapies.
An ensemble Kalman filter for convective‐scale data assimilation (KENDA) has been developed for the COnsortium for Small‐scale MOdelling (COSMO) model. The KENDA system comprises a local ensemble ...transform Kalman filter (LETKF) and a deterministic analysis based on the Kalman gain for the analysis ensemble mean. The KENDA software suite includes tools for adaptive localization, multiplicative covariance inflation, relaxation to prior perturbations and adaptive observation errors. In the version introduced here, conventional data (radiosonde, aircraft, wind profiler, surface station data) are assimilated. Latent heat nudging of radar precipitation has also been added to the KENDA system to be applied to the deterministic analysis only or additionally to all ensemble members. The performance of different system components is investigated in a quasi‐operational setting using a basic cycling environment (BACY) for a period of six days with 24 h forecasts. For this period and an additional 28 day period, deterministic KENDA forecasts are compared with forecasts based on the observation nudging data assimilation scheme, which is currently operational at the German Weather Service (Deutscher Wetterdienst, DWD). For our experiments, lateral boundary conditions for the regional model are given by a global ensemble Kalman filter for the ICOsahedral Nonhydrostatic (ICON) model. The performance of the KENDA system proves overall to be superior to the forecast quality of the operational nudging scheme, in particular with regard to precipitation. Latent heat nudging improves precipitation forecasts in both systems and has slightly more benefit in combination with the LETKF than with observation nudging.
After cardiac injury, activated cardiac myofibroblasts can influence tissue electrophysiology. Because mechanical coupling through adherens junctions provides a route for intercellular communication, ...we tested the hypothesis that myofibroblasts exert tonic contractile forces on the cardiomyocytes and affect electric propagation via a process of mechanoelectric feedback.
The role of mechanoelectric feedback was examined in transforming growth factor-β-treated monolayers of cocultured myofibroblasts and neonatal rat ventricular cells by inhibiting myofibroblast contraction and blocking mechanosensitive channels. Untreated (control) and transforming growth factor-β-treated (fibrotic) anisotropic monolayers were optically mapped for electrophysiological comparison. Longitudinal conduction velocity, transverse conduction velocity, and normalized action potential upstroke velocity (dV/dt(max)) significantly decreased in fibrotic monolayers (14.4 ± 0.7 cm/s mean ± SEM, 4.1 ± 0.3 cm/s n=53, and 3.1 ± 0.2% per ms n=14, respectively) compared with control monolayers (27.2 ± 0.8 cm/s, 8.5 ± 0.4 cm/s n=40, and 4.9 ± 0.1% per ms n=12, respectively). Application of the excitation-contraction uncoupler blebbistatin or the mechanosensitive channel blocker gadolinium or streptomycin dramatically increased longitudinal conduction velocity, transverse conduction velocity, and dV/dt(max) in fibrotic monolayers (35.9 ± 1.5 cm/s, 10.3 ± 0.6 cm/s n=17, and 4.5 ± 0.1% per ms n=14, respectively). Similar results were observed with connexin43-silenced cardiac myofibroblasts. Spiral-wave induction in fibrotic monolayers also decreased after the aforementioned treatments. Finally, traction force measurements of individual myofibroblasts showed a significant increase with transforming growth factor-β, a decrease with blebbistatin, and no change with mechanosensitive channel blockers.
These observations suggest that myofibroblast-myocyte mechanical interactions develop during cardiac injury, and that cardiac conduction may be impaired as a result of increased mechanosensitive channel activation owing to tension applied to the myocyte by the myofibroblast.
Dysregulated translation of mRNA plays a major role in tumorigenesis. Mitogen-activated protein kinase interacting kinases (MNK)1/2 are key regulators of mRNA translation integrating signals from ...oncogenic and immune signaling pathways through phosphorylation of eIF4E and other mRNA binding proteins. Modulation of these key effector proteins regulates mRNA, which controls tumor/stromal cell signaling. Compound 23 (eFT508), an exquisitely selective, potent dual MNK1/2 inhibitor, was designed to assess the potential for control of oncogene signaling at the level of mRNA translation. The crystal structure-guided design leverages stereoelectronic interactions unique to MNK culminating in a novel pyridone-aminal structure described for the first time in the kinase literature. Compound 23 has potent in vivo antitumor activity in models of diffuse large cell B-cell lymphoma and solid tumors, suggesting that controlling dysregulated translation has real therapeutic potential. Compound 23 is currently being evaluated in Phase 2 clinical trials in solid tumors and lymphoma. Compound 23 is the first highly selective dual MNK inhibitor targeting dysregulated translation being assessed clinically.
Interactions between cardiac myofibroblasts and myocytes may slow conduction and generate spontaneous beating in fibrosis, increasing the chance of life-threatening arrhythmia. While co-culture ...studies have shown that myofibroblasts can affect cardiomyocyte electrophysiology in vitro, the extent of myofibroblast-myocyte electrical conductance in a syncytium is unknown. In this neonatal rat study, cardiac myofibroblasts were transduced with Channelrhodopsin-2, which allowed acute and selective increase of myofibroblast current, and plated on top of cardiomyocytes. Optical mapping revealed significantly decreased conduction velocity (- 27 ± 6%, p < 10
), upstroke rate (- 13 ± 4%, p = 0.002), and action potential duration (- 14 ± 7%, p = 0.004) in co-cultures when 0.017 mW/mm
light was applied, as well as focal spontaneous beating in 6/7 samples and a decreased cycle length (- 36 ± 18%, p = 0.002) at 0.057 mW/mm
light. In silico modeling of the experiments reproduced the experimental findings and suggested the light levels used in experiments produced excess current similar in magnitude to endogenous myofibroblast current. Fitting the model to experimental data predicted a tissue-level electrical conductance across the 3-D interface between myofibroblasts and cardiomyocytes of ~ 5 nS/cardiomyocyte, and showed how increased myofibroblast-myocyte conductance, increased myofibroblast/myocyte capacitance ratio, and increased myofibroblast current, which occur in fibrosis, can work in tandem to produce pro-arrhythmic increases in conduction and spontaneous beating.
Cells respond to mechanical forces whether applied externally or generated internally via the cytoskeleton. To study the cellular response to forces separately, we applied external forces to cells ...via microfabricated magnetic posts containing cobalt nanowires interspersed among an array of elastomeric posts, which acted as independent sensors to cellular traction forces. A magnetic field induced torque in the nanowires, which deflected the magnetic posts and imparted force to individual adhesions of cells attached to the array. Using this system, we examined the cellular reaction to applied forces and found that applying a step force led to an increase in local focal adhesion size at the site of application but not at nearby nonmagnetic posts. Focal adhesion recruitment was enhanced further when cells were subjected to multiple force actuations within the same time interval. Recording the traction forces in response to such force stimulation revealed two responses: a sudden loss in contractility that occurred within the first minute of stimulation or a gradual decay in contractility over several minutes. For both types of responses, the subcellular distribution of loss in traction forces was not confined to locations near the actuated micropost, nor uniformly across the whole cell, but instead occurred at discrete locations along the cell periphery. Together, these data reveal an important dynamic biological relationship between external and internal forces and demonstrate the utility of this microfabricated system to explore this interaction.
Most adolescents and young adults who experience psychological distress do
not
seek professional help. This study aims to enhance the understanding of sociodemographic, psychological, and clinical ...characteristics associated with the underuse of health services by adolescents and young adults with mental disorders. Data from a cross-sectional, epidemiological study with a population-based sample (
N
= 1180 participants, 14–21 years old) were used. Participants completed a fully standardized, computer-assisted diagnostic interview (DIA-X-5/D-CIDI) administered by trained clinical interviewers to assess lifetime mental disorders according to DSM-5 as well as lifetime health service use for mental health problems, and completed self-report questionnaires to assess various psychological variables (e.g., stigma). Predictors of health service use were examined using univariate and multiple logistic regression analyses, data were weighted for age and sex to improve representativeness Of
n
= 597 participants with any lifetime mental disorder, 32.4% 95% CI 28.4; 36.7 had ever used any health services because of a mental health, psychosomatic, or substance use problem. Even less had received psychotherapeutic or pharmacological treatment (Cognitive Behavioral Therapy: 12.1% 9.5; 15.2; other psychotherapy: 10.7% 8.4; 13.7; medication: 5.4% 3.7; 7.8). High education was associated with less health service use (low/ middle/ other vs. high education: 53.8% vs. 26.9%; OR = 0.26,
p
< .001). In the multiple regression model, stigma toward mental disorders was the single psychological variable associated with a reduced likelihood of using health services (OR = 0.69 0.52; 0.90,
p
< .01). These findings draw attention to the treatment gap for mental disorders during adolescence and highlight related factors to be addressed in public health contexts.
An ensemble data assimilation system for 3D radar reflectivity data is introduced for the convection‐permitting numerical weather prediction model of the COnsortium for Small‐scale MOdelling (COSMO) ...based on the Kilometre‐scale ENsemble Data Assimilation system (KENDA), developed by Deutscher Wetterdienst and its partners. KENDA provides a state‐of‐the‐art ensemble data assimilation system on the convective scale for operational data assimilation and forecasting based on the Local Ensemble Transform Kalman Filter (LETKF). In this study, the Efficient Modular VOlume RADar Operator is applied for the assimilation of radar reflectivity data to improve short‐term predictions of precipitation. Both deterministic and ensemble forecasts have been carried out. A case‐study shows that the assimilation of 3D radar reflectivity data clearly improves precipitation location in the analysis and significantly improves forecasts for lead times up to 4 h, as quantified by the Brier Score and the Continuous Ranked Probability Score. The influence of different update rates on the noise in terms of surface pressure tendencies and on the forecast quality in general is investigated. The results suggest that, while high update rates produce better analyses, forecasts with lead times of above 1 h benefit from less frequent updates. For a period of seven consecutive days, assimilation of radar reflectivity based on the LETKF is compared to that of DWD's current operational radar assimilation scheme based on latent heat nudging (LHN). It is found that the LETKF competes with LHN, although it is still in an experimental phase.
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