The role of junctophilin proteins in cellular function.

Junctophilins (JPHs) comprise a family of structural proteins that connect the plasma membrane to intracellular organelles such as the endo/sarcoplasmic reticulum (ER/SR). Tethering of these membrane structures results in the formation of highly organized subcellular junctions that play important signaling roles in all excitable cell types. There are four JPH isoforms, expressed primarily in muscle and neuronal cell types. Each JPH protein consists of six membrane occupation and recognition nexus (MORN) motifs, a joining region connecting these to another set of two MORN motifs, a putative alpha-helical region, a divergent region exhibiting low homology between JPH isoforms, and a carboxy-terminal transmembrane region anchoring into the ER/SR membrane. JPH isoforms play essential roles in developing and maintaining subcellular membrane junctions. Conversely, inherited mutations in JPH2 cause hypertrophic or dilated cardiomyopathy, while trinucleotide expansions in the JPH3 gene cause Huntington Disease-Like 2. Loss of JPH1 protein levels can cause skeletal myopathy, while loss of cardiac JPH2 levels causes heart failure and atrial fibrillation, among other disease. This review will provide a comprehensive overview of the JPH gene family, phylogeny, and evolutionary analysis of JPH genes and other MORN domain proteins. JPH biogenesis, membrane tethering, and binding partners will be discussed, as well as functional roles of JPH isoforms in excitable cells. Finally, potential roles of JPH isoform deficits in human disease pathogenesis will be reviewed.

Structural insights into the regulation of RyR1 by S100A1.

S100A1, a small homodimeric EF-hand Ca2+-binding protein (~21 kDa), plays an important regulatory role in Ca2+ signaling pathways involved in various biological functions including Ca2+ cycling and contractile performance in skeletal and cardiac myocytes. One key target of the S100A1 interactome is the ryanodine receptor (RyR), a huge homotetrameric Ca2+ release channel (~2.3 MDa) of the sarcoplasmic reticulum. Here, we report cryoelectron microscopy structures of S100A1 bound to RyR1, the skeletal muscle isoform, in absence and presence of Ca2+. Ca2+-free apo-S100A1 binds beneath the bridging solenoid (BSol) and forms contacts with the junctional solenoid and the shell-core linker of RyR1. Upon Ca2+-binding, S100A1 undergoes a conformational change resulting in the exposure of the hydrophobic pocket known to serve as a major interaction site of S100A1. Through interactions of the hydrophobic pocket with RyR1, Ca2+-bound S100A1 intrudes deeper into the RyR1 structure beneath BSol than the apo-form and induces sideways motions of the C-terminal BSol region toward the adjacent RyR1 protomer resulting in tighter interprotomer contacts. Interestingly, the second hydrophobic pocket of the S100A1-dimer is largely exposed at the hydrophilic surface making it prone to interactions with the local environment, suggesting that S100A1 could be involved in forming larger heterocomplexes of RyRs with other protein partners. Since S100A1 interactions stabilizing BSol are implicated in the regulation of RyR-mediated Ca2+ release, the characterization of the S100A1 binding site conserved between RyR isoforms may provide the structural basis for the development of therapeutic strategies regarding treatments of RyR-related disorders.

Dysferlin Enables Tubular Membrane Proliferation in Cardiac Hypertrophy.

BACKGROUND: Cardiac hypertrophy compensates for increased biomechanical stress of the heart induced by prevalent cardiovascular pathologies but can result in heart failure if left untreated. Here, we hypothesized that the membrane fusion and repair protein dysferlin is critical for the integrity of the transverse-axial tubule (TAT) network inside cardiomyocytes and contributes to the proliferation of TAT endomembranes during pressure overload-induced cardiac hypertrophy. METHODS: Stimulated emission depletion and electron microscopy were used to localize dysferlin in mouse and human cardiomyocytes. Data-independent acquisition mass spectrometry revealed the cardiac dysferlin interactome and proteomic changes of the heart in dysferlin-knockout mice. After transverse aortic constriction, we compared the hypertrophic response of wild-type versus dysferlin-knockout hearts and studied TAT network remodeling mechanisms inside cardiomyocytes by live-cell membrane imaging. RESULTS: We localized dysferlin in a vesicular compartment in nanometric proximity to contact sites of the TAT network with the sarcoplasmic reticulum, a.k.a. junctional complexes for Ca2+-induced Ca2+ release. Interactome analyses demonstrated a novel protein interaction of dysferlin with the membrane-tethering sarcoplasmic reticulum protein juncophilin-2, a putative interactor of L-type Ca2+ channels and ryanodine receptor Ca2+ release channels in junctional complexes. Although the dysferlin-knockout caused a mild progressive phenotype of dilated cardiomyopathy, global proteome analysis revealed changes preceding systolic failure. Following transverse aortic constriction, dysferlin protein expression was significantly increased in hypertrophied wild-type myocardium, while dysferlin-knockout animals presented markedly reduced left-ventricular hypertrophy. Live-cell membrane imaging showed a profound reorganization of the TAT network in wild-type left-ventricular myocytes after transverse aortic constriction with robust proliferation of axial tubules, which critically depended on the increased expression of dysferlin within newly emerging tubule components. CONCLUSIONS: Dysferlin represents a new molecular target in cardiac disease that protects the integrity of tubule-sarcoplasmic reticulum junctional complexes for regulated excitation-contraction coupling and controls TAT network reorganization and tubular membrane proliferation in cardiomyocyte hypertrophy induced by pressure overload.

Impaired Intracellular Calcium Buffering Contributes to the Arrhythmogenic Substrate in Atrial Myocytes From Patients With Atrial Fibrillation.

BACKGROUND: Alterations in the buffering of intracellular Ca2+, for which myofilament proteins play a key role, have been shown to promote cardiac arrhythmia. It is interesting that although studies report atrial myofibrillar degradation in patients with persistent atrial fibrillation (persAF), the intracellular Ca2+ buffering profile in persAF remains obscure. Therefore, we aimed to investigate the intracellular buffering of Ca2+ and its potential arrhythmogenic role in persAF. METHODS: Transmembrane Ca2+ fluxes (patch-clamp) and intracellular Ca2+ signaling (fluo-3-acetoxymethyl ester) were recorded simultaneously in myocytes from right atrial biopsies of sinus rhythm (Ctrl) and patients with persAF, alongside human atrial subtype induced pluripotent stem cell-derived cardiac myocytes (iPSC-CMs). Protein levels were quantified by immunoblotting of human atrial tissue and induced pluripotent stem cell-derived cardiac myocytes. Mouse whole heart and atrial electrophysiology were measured on a Langendorff system. RESULTS: Cytosolic Ca2+ buffering was decreased in atrial myocytes of patients with persAF because of a depleted amount of Ca2+ buffers. In agreement, protein levels of selected Ca2+ binding myofilament proteins, including cTnC (cardiac troponin C), a major cytosolic Ca2+ buffer, were significantly lower in patients with persAF. Small interfering RNA (siRNA)-mediated knockdown of cTnC (si-cTNC) in atrial iPSC-CM phenocopied the reduced cytosolic Ca2+ buffering observed in persAF. Si-cTnC treated atrial iPSC-CM exhibited a higher predisposition to spontaneous Ca2+ release events and developed action potential alternans at low stimulation frequencies. Last, indirect reduction of cytosolic Ca2+ buffering using blebbistatin in an ex vivo mouse whole heart model increased vulnerability to tachypacing-induced atrial arrhythmia, validating the direct mechanistic link between impaired cytosolic Ca2+ buffering and atrial arrhythmogenesis. CONCLUSIONS: Our findings suggest that loss of myofilament proteins, particularly reduced cTnC protein levels, causes diminished cytosolic Ca2+ buffering in persAF, thereby potentiating the occurrence of spontaneous Ca2+ release events and atrial fibrillation susceptibility. Strategies targeting intracellular buffering may represent a promising therapeutic lead in persAF management.

An Alternative Mechanism of Subcellular Iron Uptake Deficiency in Cardiomyocytes.

BACKGROUND: Systemic defects in intestinal iron absorption, circulation, and retention cause iron deficiency in 50% of patients with heart failure. Defective subcellular iron uptake mechanisms that are independent of systemic absorption are incompletely understood. The main intracellular route for iron uptake in cardiomyocytes is clathrin-mediated endocytosis. METHODS: We investigated subcellular iron uptake mechanisms in patient-derived and CRISPR/Cas-edited induced pluripotent stem cell-derived cardiomyocytes as well as patient-derived heart tissue. We used an integrated platform of DIA-MA (mass spectrometry data-independent acquisition)-based proteomics and signaling pathway interrogation. We employed a genetic induced pluripotent stem cell model of 2 inherited mutations (TnT [troponin T]-R141W and TPM1 [tropomyosin 1]-L185F) that lead to dilated cardiomyopathy (DCM), a frequent cause of heart failure, to study the underlying molecular dysfunctions of DCM mutations. RESULTS: We identified a druggable molecular pathomechanism of impaired subcellular iron deficiency that is independent of systemic iron metabolism. Clathrin-mediated endocytosis defects as well as impaired endosome distribution and cargo transfer were identified as a basis for subcellular iron deficiency in DCM-induced pluripotent stem cell-derived cardiomyocytes. The clathrin-mediated endocytosis defects were also confirmed in the hearts of patients with DCM with end-stage heart failure. Correction of the TPM1-L185F mutation in DCM patient-derived induced pluripotent stem cells, treatment with a peptide, Rho activator II, or iron supplementation rescued the molecular disease pathway and recovered contractility. Phenocopying the effects of the TPM1-L185F mutation into WT induced pluripotent stem cell-derived cardiomyocytes could be ameliorated by iron supplementation. CONCLUSIONS: Our findings suggest that impaired endocytosis and cargo transport resulting in subcellular iron deficiency could be a relevant pathomechanism for patients with DCM carrying inherited mutations. Insight into this molecular mechanism may contribute to the development of treatment strategies and risk management in heart failure.

Targeting calpain-2-mediated junctophilin-2 cleavage delays heart failure progression following myocardial infarction.

Coronary heart disease (CHD) is a prevalent cardiac disease that causes over 370,000 deaths annually in the USA. In CHD, occlusion of a coronary artery causes ischemia of the cardiac muscle, which results in myocardial infarction (MI). Junctophilin-2 (JPH2) is a membrane protein that ensures efficient calcium handling and proper excitation-contraction coupling. Studies have identified loss of JPH2 due to calpain-mediated proteolysis as a key pathogenic event in ischemia-induced heart failure (HF). Our findings show that calpain-2-mediated JPH2 cleavage yields increased levels of a C-terminal cleaved peptide (JPH2-CTP) in patients with ischemic cardiomyopathy and mice with experimental MI. We created a novel knock-in mouse model by removing residues 479-SPAGTPPQ-486 to prevent calpain-2-mediated cleavage at this site. Functional and molecular assessment of cardiac function post-MI in cleavage site deletion (CSD) mice showed preserved cardiac contractility and reduced dilation, reduced JPH2-CTP levels, attenuated adverse remodeling, improved T-tubular structure, and normalized SR Ca2+-handling. Adenovirus mediated calpain-2 knockdown in mice exhibited similar findings. Pulldown of CTP followed by proteomic analysis revealed valosin-containing protein (VCP) and BAG family molecular chaperone regulator 3 (BAG3) as novel binding partners of JPH2. Together, our findings suggest that blocking calpain-2-mediated JPH2 cleavage may be a promising new strategy for delaying the development of HF following MI.

Noninvasive analysis of contractility during identical maturations revealed two phenotypes in ventricular but not in atrial iPSC-CM.

Patient-derived induced pluripotent stem cells (iPSCs) can be differentiated into atrial and ventricular cardiomyocytes to allow for personalized drug screening. A hallmark of differentiation is the manifestation of spontaneous beating in a two-dimensional (2-D) cell culture. However, an outstanding observation is the high variability in this maturation process. We valued that contractile parameters change during differentiation serving as an indicator of maturation. Consequently, we recorded noninvasively spontaneous motion activity during the differentiation of male iPSC toward iPSC cardiomyocytes (iPSC-CMs) to further analyze similar maturated iPSC-CMs. Surprisingly, our results show that identical differentiations into ventricular iPSC-CMs are variable with respect to contractile parameters resulting in two distinct subpopulations of ventricular-like cells. In contrast, differentiation into atrial iPSC-CMs resulted in only one phenotype. We propose that the noninvasive and cost-effective recording of contractile activity during maturation using a smartphone device may help to reduce the variability in results frequently reported in studies on ventricular iPSC-CMs.NEW & NOTEWORTHY Differentiation of induced pluripotent stem cells (iPSCs) into iPSC-derived cardiomyocytes (iPSC-CMs) exhibits a high variability in mature parameters. Here, we monitored noninvasively contractile parameters of iPSC-CM during full-time differentiation using a smartphone device. Our results show that parallel maturations of iPSCs into ventricular iPSC-CMs, but not into atrial iPSC-CMs, resulted in two distinct subpopulations of iPSC-CMs. These findings suggest that our cost-effective method may help to compare iPSC-CMs at the same maturation level.

Genome mining leads to the identification of a stable and promiscuous Baeyer-Villiger monooxygenase from a thermophilic microorganism.

Baeyer-Villiger monooxygenases (BVMOs) are NAD(P)H-dependent flavoproteins that convert ketones to esters and lactones. While these enzymes offer an appealing alternative to traditional Baeyer-Villiger oxidations, these proteins tend to be either too unstable or exhibit too narrow of a substrate scope for implementation as industrial biocatalysts. Here, sequence similarity networks were used to search for novel BVMOs that are both stable and promiscuous. Our genome mining led to the identification of an enzyme from Chloroflexota bacterium (strain G233) dubbed ssnBVMO that exhibits i) the highest melting temperature of any naturally sourced BVMO (62.5 ºC), ii) a remarkable kinetic stability across a wide range of conditions, similar to those of PAMO and PockeMO, iii) optimal catalysis at 50 °C, and iv) a broad substrate scope that includes linear aliphatic, aromatic, and sterically bulky ketones. Subsequent quantitative assays using propiophenone demonstrated >95% conversion. Several fusions were also constructed that linked ssnBVMO to a thermostable phosphite dehydrogenase. These fusions can recycle NADPH and catalyze oxidations with sub-stoichiometric quantities of this expensive cofactor. Characterization of these fusions permitted identification of PTDH-L1-ssnBVMO as the most promising protein that could have utility as a seed sequence for enzyme engineering campaigns aiming to develop biocatalysts for Baeyer-Villiger oxidations.

Human prostate MRI at ultrahigh-performance gradient: A feasibility study.

PURPOSE: To demonstrate the technical feasibility and the value of ultrahigh-performance gradient in imaging the prostate in a 3T MRI system. METHODS: In this local institutional review board-approved study, prostate MRI was performed on 4 healthy men. Each subject was scanned in a prototype 3T MRI system with a 42-cm inner-diameter gradient coil that achieves a maximum gradient amplitude of 200 mT/m and slew rate of 500 T/m/s. PI-RADS V2.1-compliant axial T2 -weighted anatomical imaging and single-shot echo planar DWI at standard gradient of 70 mT/m and 150 T/m/s were obtained, followed by DWI at maximum performance (i.e., 200 mT/m and 500 T/m/s). In comparison to state-of-the-art clinical whole-body MRI systems, the high slew rate improved echo spacing from 1020 to 596 µs and, together with a high gradient amplitude for diffusion encoding, TE was reduced from 55 to 36 ms. RESULTS: In all 4 subjects (waist circumference = 81-91 cm, age = 45-65 years), no peripheral nerve stimulation sensation was reported during DWI. Reduced image distortion in the posterior peripheral zone prostate gland and higher signal intensity, such as in the surrounding muscle of high-gradient DWI, were noted. CONCLUSION: Human prostate MRI at simultaneously high gradient amplitude of 200 mT/m and slew rate of 500 T/m/s is feasible, demonstrating that improved gradient performance can address image distortion and T2 decay-induced SNR issues for in vivo prostate imaging.

Regulation of ADAM10 activity through microdomain-dependent intracellular calcium changes.

A disintegrin and metalloproteinases (ADAMs) are transmembrane proteases that cleave other proteins close to the surface in a process called shedding. The prominent member ADAM10 has been linked to several pathologies such as Alzheimer's disease, bacterial infection, cancer development and metastasis. Although the regulation of the ADAM10 activity by calcium influx and calmodulin inhibition has been reported, the spatiotemporal regulation of Ca2+-dependent ADAM10 activation and the required source of Ca2+ ions have not been thoroughly studied. In the present study, we observed the rapid Ca2+-dependent activation of ADAM10 in A549 lung carcinoma cells upon stimulation with ionomycin. The calmodulin-inhibitors trifluoperazine and ophiobolin A mediated delayed activation of ADAM10, which apparently did not depend on intracellular Ca2+ in the case of trifluoperazine. Furthermore, the surface translocation and release of ADAM10 in extracellular vesicles exhibited different kinetics and were only partially linked to catalytic activation. Finally, ADAM10 activation was observed after the entry of Ca2+ through certain channels, such as canonical members of transient receptor potential (TRP) channels. Therefore, the opening of particular channels for Ca2+ entry points and subsequent Ca2+ flux as well as the temporal aspects of the consequent increase in Ca2+ levels, must be considered for future therapeutic options involving the increasing or decreasing ADAM10 activity.

Manual prostate MRI segmentation by readers with different experience: a study of the learning progress.

OBJECTIVE: To evaluate the learning progress of less experienced readers in prostate MRI segmentation. MATERIALS AND METHODS: One hundred bi-parametric prostate MRI scans were retrospectively selected from the Göteborg Prostate Cancer Screening 2 Trial (single center). Nine readers with varying degrees of segmentation experience were involved: one expert radiologist, two experienced radiology residents, two inexperienced radiology residents, and four novices. The task was to segment the whole prostate gland. The expert's segmentations were used as reference. For all other readers except three novices, the 100 MRI scans were divided into five rounds (cases 1-10, 11-25, 26-50, 51-76, 76-100). Three novices segmented only 50 cases (three rounds). After each round, a one-on-one feedback session between the expert and the reader was held, with feedback on systematic errors and potential improvements for the next round. Dice similarity coefficient (DSC) > 0.8 was considered accurate. RESULTS: Using DSC > 0.8 as the threshold, the novices had a total of 194 accurate segmentations out of 250 (77.6%). The residents had a total of 397/400 (99.2%) accurate segmentations. In round 1, the novices had 19/40 (47.5%) accurate segmentations, in round 2 41/60 (68.3%), and in round 3 84/100 (84.0%) indicating learning progress. CONCLUSIONS: Radiology residents, regardless of prior experience, showed high segmentation accuracy. Novices showed larger interindividual variation and lower segmentation accuracy than radiology residents. To prepare datasets for artificial intelligence (AI) development, employing radiology residents seems safe and provides a good balance between cost-effectiveness and segmentation accuracy. Employing novices should only be considered on an individual basis. CLINICAL RELEVANCE STATEMENT: Employing radiology residents for prostate MRI segmentation seems safe and can potentially reduce the workload of expert radiologists. Employing novices should only be considered on an individual basis. KEY POINTS: • Using less experienced readers for prostate MRI segmentation is cost-effective but may reduce quality. • Radiology residents provided high accuracy segmentations while novices showed large inter-reader variability. • To prepare datasets for AI development, employing radiology residents seems safe and might provide a good balance between cost-effectiveness and segmentation accuracy while novices should only be employed on an individual basis.

Quantitative Prostate MRI, From the AJR Special Series on Quantitative Imaging.

Prostate MRI has traditionally relied on qualitative interpretation. However, quantitative components hold the potential to markedly improve performance. The ADC from DWI is probably the most widely recognized quantitative MRI biomarker and has shown strong discriminatory value for clinically significant prostate cancer (csPCa) as well as for recurrent cancer after treatment. Advanced diffusion techniques, including intravoxel incoherent motion, diffusion kurtosis, diffusion tensor imaging, and specific implementations such as restriction spectrum imaging, purport even better discrimination, but are more technically challenging. The inherent T1 and T2 of tissue also provide diagnostic value, with more advanced techniques deriving luminal water imaging and hybrid-multidimensional MRI. Dynamic contrast-enhanced imaging, primarily using a modified Tofts model, also shows independent discriminatory value. Finally, quantitative size and shape features can be combined with the aforementioned techniques and be further refined using radiomics, texture analysis, and artificial intelligence. Which technique will ultimately find widespread clinical use will depend on validation across a myriad of platforms use-cases.

Blood trauma in veno-venous extracorporeal membrane oxygenation: low pump pressures and low circuit resistance matter.

BACKGROUND: Veno-venous extracorporeal membrane oxygenation (VV ECMO) has become standard of care in patients with the most severe forms of acute respiratory distress syndrome. However, hemolysis and bleeding are one of the most frequent side effects, affecting mortality. Despite the widespread use of VV ECMO, current protocols lack detailed, in-vivo data-based recommendations for safe ECMO pump operating conditions. This study aims to comprehensively analyze the impact of VV ECMO pump operating conditions on hemolysis by combining in-silico modeling and clinical data analysis. METHODS: We combined data from 580 patients treated with VV ECMO in conjunction with numerical predictions of hemolysis using computational fluid dynamics and reduced order modeling of the Rotaflow (Getinge) and DP3 (Xenios) pumps. Blood trauma parameters across 94,779 pump operating points were associated with numerical predictions of shear induced hemolysis. RESULTS: Minimal hemolysis was observed at low pump pressures and low circuit resistance across all flow rates, whereas high pump pressures and circuit resistance consistently precipitated substantial hemolysis, irrespective of flow rate. However, the lower the flow rate, the more pronounced the influence of circuit resistance on hemolysis became. Numerical models validated against clinical data demonstrated a strong association (Spearman's r = 0.8) between simulated and observed hemolysis, irrespective of the pump type. CONCLUSIONS: Integrating in-silico predictions with clinical data provided a novel approach in understanding and potentially reducing blood trauma in VV ECMO. This study further demonstrated that a key factor in lowering side effects of ECMO support is the maintenance of low circuit resistance, including oxygenators with the lowest possible resistance, the shortest feasible circuit tubing, and cannulae with an optimal diameter.

3D Super-Resolution Nuclear Q-FISH Imaging Reveals Cell-Cycle-Related Telomere Changes.

We present novel workflows for Q-FISH nanoscopy with the potential for prognostic applications and resolving novel chromatin compaction changes. DNA-fluorescence in situ hybridization (DNA-FISH) is a routine application to visualize telomeres, repetitive terminal DNA sequences, in cells and tissues. Telomere attrition is associated with inherited and acquired diseases, including cancer and cardiomyopathies, and is frequently analyzed by quantitative (Q)-FISH microscopy. Recently, nanoscopic imaging techniques have resolved individual telomere dimensions and their compaction as a prognostic marker, in part leading to conflicting conclusions still unresolved to date. Here, we developed a comprehensive Q-FISH nanoscopy workflow to assess telomeres with PNA telomere probes and 3D-Stimulated Emission Depletion (STED) microscopy combined with Dynamic Intensity Minimum (DyMIN) scanning. We achieved single-telomere resolution at high, unprecedented telomere coverage. Importantly, our approach revealed a decrease in telomere signal density during mitotic cell division compared to interphase. Innovatively expanding FISH-STED applications, we conducted double FISH targeting of both telomere- and chromosome-specific sub-telomeric regions and accomplished FISH-STED in human cardiac biopsies. In summary, this work further advanced Q-FISH nanoscopy, detected a new aspect of telomere compaction related to the cell cycle, and laid the groundwork for future applications in complex cell types such as post-mitotic neurons and muscle cells.

Cytokine-directed cellular cross-talk imprints synovial pathotypes in rheumatoid arthritis.

INTRODUCTION: Structural reorganisation of the synovium with expansion of fibroblast-like synoviocytes (FLS) and influx of immune cells is a hallmark of rheumatoid arthritis (RA). Activated FLS are increasingly recognised as a critical component driving synovial tissue remodelling by interacting with immune cells resulting in distinct synovial pathotypes of RA. METHODS: Automated high-content fluorescence microscopy of co-cultured cytokine-activated FLS and autologous peripheral CD4+ T cells from patients with RA was established to quantify cell-cell interactions. Phenotypic profiling of cytokine-treated FLS and co-cultured T cells was done by flow cytometry and RNA-Seq, which were integrated with publicly available transcriptomic data from patients with different histological synovial pathotypes. Computational prediction and knock-down experiments were performed in FLS to identify adhesion molecules for cell-cell interaction. RESULTS: Cytokine stimulation, especially with TNF-α, led to enhanced FLS-T cell interaction resulting in cell-cell contact-dependent activation, proliferation and differentiation of T cells. Signatures of cytokine-activated FLS were significantly enriched in RA synovial tissues defined as lymphoid-rich or leucocyte-rich pathotypes, with the most prominent effects for TNF-α. FLS cytokine signatures correlated with the number of infiltrating CD4+ T cells in synovial tissue of patients with RA. Ligand-receptor pair interaction analysis identified ICAM1 on FLS as an important mediator in TNF-mediated FLS-T cell interaction. Both, ICAM1 and its receptors were overexpressed in TNF-treated FLS and co-cultured T cells. Knock-down of ICAM1 in FLS resulted in reduced TNF-mediated FLS-T cell interaction. CONCLUSION: Our study highlights the role of cytokine-activated FLS in orchestrating inflammation-associated synovial pathotypes providing novel insights into disease mechanisms of RA.

Truly reproducible uniform estimation of the ADC with multi-b diffusion data- Application in prostate diffusion imaging.

PURPOSE: The ADC is a well-established parameter for clinical diagnostic applications, but lacks reproducibility because it is also influenced by the choice diffusion weighting level. A framework is evaluated that is based on multi-b measurement over a wider range of diffusion-weighting levels and higher order tissue diffusion modeling with retrospective, fully reproducible ADC calculation. METHODS: Averaging effect from curve fitting for various model functions at 20 linearly spaced b-values was determined by means of simulations and theoretical calculations. Simulation and patient multi-b image data were used to compare the new approach for diffusion-weighted image and ADC map reconstruction with and without Rician bias correction to an active clinical trial protocol probing three non-zero b-values. RESULTS: Averaging effect at a certain b-value varies for model function and maximum b-value used. Images and ADC maps from the novel procedure are on-par with the clinical protocol. Higher order modeling and Rician bias correction is feasible, but comes at the cost of longer computation times. CONCLUSIONS: Application of the new framework makes higher order modeling more feasible in a clinical setting while still providing patient images and reproducible ADC maps of adequate quality.

Enforced viral replication activates adaptive immunity and is essential for the control of a cytopathic virus.

The innate immune system limits viral replication via type I interferon and also induces the presentation of viral antigens to cells of the adaptive immune response. Using infection of mice with vesicular stomatitis virus, we analyzed how the innate immune system inhibits viral propagation but still allows the presentation of antigen to cells of the adaptive immune response. We found that expression of the gene encoding the inhibitory protein Usp18 in metallophilic macrophages led to lower type I interferon responsiveness, thereby allowing locally restricted replication of virus. This was essential for the induction of adaptive antiviral immune responses and, therefore, for preventing the fatal outcome of infection. In conclusion, we found that enforced viral replication in marginal zone macrophages was an immunological mechanism that ensured the production of sufficient antigen for effective activation of the adaptive immune response.

cAMP Imaging at Ryanodine Receptors Reveals ß2-Adrenoceptor Driven Arrhythmias.

[Figure: see text].

Caveolin3 Stabilizes McT1-Mediated Lactate/Proton Transport in Cardiomyocytes.

[Figure: see text].

Evidence for liquid-liquid phase separation during the early stages of Mg-struvite formation.

The precipitation of struvite, a magnesium ammonium phosphate hexahydrate (MgNH4PO4 · 6H2O) mineral, from wastewater is a promising method for recovering phosphorous. While this process is commonly used in engineered environments, our understanding of the underlying mechanisms responsible for the formation of struvite crystals remains limited. Specifically, indirect evidence suggests the involvement of an amorphous precursor and the occurrence of multi-step processes in struvite formation, which would indicate non-classical paths of nucleation and crystallization. In this study, we use synchrotron-based in situ x-ray scattering complemented by cryogenic transmission electron microscopy to obtain new insights from the earliest stages of struvite formation. The holistic scattering data captured the structure of an entire assembly in a time-resolved manner. The structural features comprise the aqueous medium, the growing struvite crystals, and any potential heterogeneities or complex entities. By analysing the scattering data, we found that the onset of crystallization causes a perturbation in the structure of the surrounding aqueous medium. This perturbation is characterized by the occurrence and evolution of Ornstein-Zernike fluctuations on a scale of about 1 nm, suggesting a non-classical nature of the system. We interpret this phenomenon as a liquid-liquid phase separation, which gives rise to the formation of the amorphous precursor phase preceding actual crystal growth of struvite. Our microscopy results confirm that the formation of Mg-struvite includes a short-lived amorphous phase, lasting >10 s.