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.

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.

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 cardiac failure if left untreated. We hypothesized that the tail-anchored protein dysferlin with multiple Ca2+-binding C2-domains 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. OBJECTIVE: To reveal the impact of the membrane fusion and repair protein dysferlin on TAT network stabilization and proliferation necessary for the hypertrophic growth of cardiomyocytes. METHODS AND RESULTS: Super-resolution light and electron microscopy of mouse cardiomyocytes identified a specific localization of 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. Mass spectrometry was used to characterize the cardiac dysferlin interactome, thereby identifying a novel protein interaction 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. While the dysferlin knockout caused a mild progressive phenotype of dilated cardiomyopathy in the mouse heart, 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 demonstrated a profound reorganization of the TAT network in wild-type left-ventricular myocytes post-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.

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.

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 aim to investigate the intracellular buffering of calcium and its potential arrhythmogenic role in persAF. METHODS: Simultaneous transmembrane fluxes (patch-clamp) and intracellular Ca2+ signaling (fluo-3-acetoxymethyl ester) were recorded in myocytes from right atrial biopsies of sinus rhythm (control) 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 was 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 in induced pluripotent stem cell-derived cardiac myocytes (si-cTnC) phenocopied the reduced cytosolic Ca2+ buffering observed in persAF. Si-cTnC induced pluripotent stem cell-derived cardiac myocytes 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 AF susceptibility. Strategies targeting intracellular buffering may represent a promising therapeutic lead in AF management.

A bicarbonate-rich liquid condensed phase in non-saturated solutions in the absence of divalent cations.

Bicarbonate (HCO3 -) and sodium (Na+)-containing solutions contain droplets of a separate, bicarbonate-rich liquid condensed phase (LCP) that have higher concentrations of HCO3 - relative to the bulk solution in which they reside. The existence and composition of the LCP droplets has been investigated by nanoparticle tracking analysis, nuclear magnetic resonance spectroscopy, refractive index measurements and X-ray pair distribution function analysis. The bicarbonate-rich LCP species is a previously unaccounted-for, ionic phenomenon which occurs even in solutions with solely monovalent cations. Its existence requires re-evaluation of models used to describe and model aqueous solution physicochemistry, especially those used to describe and model carbonate mineral formation.

CaV1.3 channel clusters characterized by live-cell and isolated plasma membrane nanoscopy.

A key player of excitable cells in the heart and brain is the L-type calcium channel CaV1.3. In the heart, it is required for voltage-dependent Ca2+-signaling, i.e., for controlling and modulating atrial cardiomyocyte excitation-contraction coupling. The clustering of CaV1.3 in functionally relevant channel multimers has not been addressed due to a lack of stoichiometric labeling combined with high-resolution imaging. Here, we developed a HaloTag-labeling strategy to visualize and quantify CaV1.3 clusters using STED nanoscopy to address the questions of cluster size and intra-cluster channel density. Channel clusters were identified in the plasma membrane of transfected live HEK293 cells as well as in giant plasma membrane vesicles derived from these cells that were spread on modified glass support to obtain supported plasma membrane bilayers (SPMBs). A small fraction of the channel clusters was colocalized with early and recycling endosomes at the membranes. STED nanoscopy in conjunction with live-cell and SPMB imaging enabled us to quantify CaV1.3 cluster sizes and their molecular density revealing significantly lower channel densities than expected for dense channel packing. CaV1.3 channel cluster size and molecular density were increased in SPMBs after treatment of the cells with the sympathomimetic compound isoprenaline, suggesting a regulated channel cluster condensation mechanism.

HER2 Low Expression in Primary Male Breast Cancer.

Purpose: The introduction of HER2-targeting antibody drug conjugates (ADCs) offers new treatment options for female breast cancer patients (FBC) expressing low levels of HER2 (HER2 low). No evidence was found that HER2 low describes a new FBC subtype. There is a lack of studies determining the impact of HER2 low in male breast cancer (MBC). In this study, we evaluate the prevalence of HER2 low in primary MBC and correlate the results with patient characteristics. Patients and Methods: In this study, histological specimens were obtained from 120 male patients diagnosed and treated for primary invasive breast cancer from 1995 to 2022 at Breast Cancer Units in Bergisch Gladbach, Chemnitz, and Zwickau, Germany. HER2 immunostaining and in situ hybridization were performed by central pathology and evaluated based on the ASCO/CAP guidelines. The correlation of expression of HER2 low with tumor biological characteristics and patient outcomes was investigated. Results: Out of all cases, four patients (3.3%) showed HER2 positivity (3+), 39 (32.5%) patients were classified as HER2 low, 7 (5.8%) were HER2 2+ (no amplification), 32 (26.7%) were HER2 1+, and 77 (64.2%) were classified as HER2 zero. Out of 77 HER2 zero cases, 47 tumors (61.0%) showed incomplete staining, with <10% of tumor cells classified as HER2 ultralow. No statistical correlation between HER2 low and tumor biological characteristics and patients' survival was found. Conclusion: Our findings show a notable, albeit lower, prevalence of HER2 low expression in primary MBC. However, tumors expressing HER2 low do not show specific tumor biological features to define a new breast cancer subtype in MBC. Our results suggest that a significant number of MBC patients could benefit from ADCs, as shown in FBC. Further studies are required to better understand HER2 low breast cancer, both generally and in MBC.

Ca2+/calmodulin-dependent kinase IIδC-induced chronic heart failure does not depend on sarcoplasmic reticulum Ca2+ leak.

AIMS: Hyperactivity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) has emerged as a central cause of pathologic remodelling in heart failure. It has been suggested that CaMKII-induced hyperphosphorylation of the ryanodine receptor 2 (RyR2) and consequently increased diastolic Ca2+ leak from the sarcoplasmic reticulum (SR) is a crucial mechanism by which increased CaMKII activity leads to contractile dysfunction. We aim to evaluate the relevance of CaMKII-dependent RyR2 phosphorylation for CaMKII-induced heart failure development in vivo. METHODS AND RESULTS: We crossbred CaMKIIδC overexpressing [transgenic (TG)] mice with RyR2-S2814A knock-in mice that are resistant to CaMKII-dependent RyR2 phosphorylation. Ca2+-spark measurements on isolated ventricular myocytes confirmed the severe diastolic SR Ca2+ leak previously reported in CaMKIIδC TG [4.65 ± 0.73 mF/F0 vs. 1.88 ± 0.30 mF/F0 in wild type (WT)]. Crossing in the S2814A mutation completely prevented SR Ca2+-leak induction in the CaMKIIδC TG, both regarding Ca2+-spark size and frequency, demonstrating that the CaMKIIδC-induced SR Ca2+ leak entirely depends on the CaMKII-specific RyR2-S2814 phosphorylation. Yet, the RyR2-S2814A mutation did not affect the massive contractile dysfunction (ejection fraction = 12.17 ± 2.05% vs. 45.15 ± 3.46% in WT), cardiac hypertrophy (heart weight/tibia length = 24.84 ± 3.00 vs. 9.81 ± 0.50 mg/mm in WT), or severe premature mortality (median survival of 12 weeks) associated with cardiac CaMKIIδC overexpression. In the face of a prevented SR Ca2+ leak, the phosphorylation status of other critical CaMKII downstream targets that can drive heart failure, including transcriptional regulator histone deacetylase 4, as well as markers of pathological gene expression including Xirp2, Il6, and Col1a1, was equally increased in hearts from CaMKIIδC TG on a RyR WT and S2814A background. CONCLUSIONS: S2814 phosphoresistance of RyR2 prevents the CaMKII-dependent SR Ca2+ leak induction but does not prevent the cardiomyopathic phenotype caused by enhanced CaMKIIδC activity. Our data indicate that additional mechanisms-independent of SR Ca2+ leak-are critical for the maladaptive effects of chronically increased CaMKIIδC activity with respect to heart failure.

Normal Brain and Brain Tumor ADC: Changes Resulting From Variation of Diffusion Time and/or Echo Time in Pulsed-Gradient Spin Echo Diffusion Imaging.

OBJECTIVES: Increasing gradient performance on modern magnetic resonance imaging scanners has profoundly reduced the attainable diffusion and echo times for clinically available pulsed-gradient spin echo (PGSE) sequences. This study investigated how this may impact the measured apparent diffusion coefficient (ADC), which is considered an important diagnostic marker for differentiation between normal and abnormal brain tissue and for therapeutic follow-up. MATERIALS AND METHODS: Diffusion time and echo time dependence of the ADC were evaluated on a high-performance 3 T magnetic resonance imaging scanner. Diffusion PGSE brain scans were performed in 10 healthy volunteers and in 10 brain tumor patients using diffusion times of 16, 40, and 70 ms, echo times of 60, 75, and 104 ms at 3 b-values (0, 100, and 1000 s/mm 2 ), and a maximum gradient amplitude of 68 mT/m. A low gradient performance system was also emulated by reducing the diffusion encoding gradient amplitude to 19 mT/m. In healthy subjects, the ADC was measured in 6 deep gray matter regions and in 6 white matter regions. In patients, the ADC was measured in the solid part of the tumor. RESULTS: With increasing diffusion time, a small but significant ADC increase of up to 2.5% was observed for 6 aggregate deep gray matter structures. With increasing echo time or reduced gradient performance, a small but significant ADC decrease of up to 2.6% was observed for 6 aggregate white matter structures. In tumors, diffusion time-related ADC changes were inconsistent without clear trend. For tumors with diffusivity above 1.0 µm 2 /ms, with prolonged echo time, there was a pronounced ADC increase of up to 12%. Meanwhile, for tumors with diffusivity at or below 1.0 µm 2 /ms, no change or a reduction was observed. Similar results were observed for gradient performance reduction, with an increase of up to 21%. The coefficient of variation determined in repeat experiments was 2.4%. CONCLUSIONS: For PGSE and the explored parameter range, normal tissue ADC changes seem negligible. Meanwhile, observed tumor ADC changes can be relevant if ADC is used as a quantitative biomarker and not merely assessed by visual inspection. This highlights the importance of reporting all pertinent timing parameters in ADC studies and of considering these effects when building scan protocols for use in multicenter investigations.

Diagnostic yield of cone beam CT based navigation bronchoscopy in patients with metastatic lesions: A propensity score matched case-control study.

BACKGROUND: Cone beam CT based Navigation Bronchoscopy (CBCT-NB) has predominantly been investigated as a diagnostic tool in (suspected) primary lung cancers. Small metastatic lesions are clinically considered more challenging to diagnose, but no study has explored the yield of navigation bronchoscopy in patients with pulmonary metastatic lesions (ML) compared to primary lung cancers (PL), correcting for known lesion characteristics affecting diagnostic yield. MATERIALS AND METHODS: This is a single-center, retrospective, propensity score-matched case-control study. We matched a subset of patients who underwent CBCT-NB and received a final diagnosis of pulmonary metastases of solid tumors between December 2017 and 2021 against confirmed primary lung cancer lesions subjected to CBCT-NB in the same time period. The lesions were propensity score matched based on known characteristics affecting yield, including location (upper lobe, lower lobe), size, bronchus sign, and lesion solidity. RESULTS: Fifty-six metastatic pulmonary lesions (mean size 14.7 mm) were individually case-matched to a selection of 297 available primary lung cancer lesions. Case-matching revealed non-significant differences in navigation success rate (PL: 89.3 % vs. ML: 82.1 %, 95%CI on differences: -21.8 to +7.5) and yield (PL: 60.7 % vs. ML: 55.4 %, 95%CI on differences: -25.4 to +14.7). The overall complication rate was comparable (5.4 % in PL vs. 5,4 % in ML). CONCLUSION: After matching primary and metastatic lesions based on CT assessable lesions characteristics, CBCT-NB showed no clinically relevant or significantly different navigation success or yield in either group. We recommend a careful assessment of CT characteristics to determine procedural difficulty rather than selecting based on the suspicion of lesion origin.

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.

Electrophysiological correlates of symbolic numerical order processing.

Determining if a sequence of numbers is ordered or not is one of the fundamental aspects of numerical processing linked to concurrent and future arithmetic skills. While some studies have explored the neural underpinnings of order processing using functional magnetic resonance imaging, our understanding of electrophysiological correlates is comparatively limited. To address this gap, we used a three-item symbolic numerical order verification task (with Arabic numerals from 1 to 9) to study event-related potentials (ERPs) in 73 adult participants in an exploratory approach. We presented three-item sequences and manipulated their order (ordered vs. unordered) as well as their inter-item numerical distance (one vs. two). Participants had to determine if a presented sequence was ordered or not. They also completed a speeded arithmetic fluency test, which measured their arithmetic skills. Our results revealed a significant mean amplitude difference in the grand average ERP waveform between ordered and unordered sequences in a time window of 500-750 ms at left anterior-frontal, left parietal, and central electrodes. We also identified distance-related amplitude differences for both ordered and unordered sequences. While unordered sequences showed an effect in the time window of 500-750 ms at electrode clusters around anterior-frontal and right-frontal regions, ordered sequences differed in an earlier time window (190-275 ms) in frontal and right parieto-occipital regions. Only the mean amplitude difference between ordered and unordered sequences showed an association with arithmetic fluency at the left anterior-frontal electrode. While the earlier time window for ordered sequences is consistent with a more automated and efficient processing of ordered sequential items, distance-related differences in unordered sequences occur later in time.

Rapid on-site evaluation of touch imprint cytology in navigation bronchoscopy for small peripheral pulmonary nodules.

BACKGROUND: Rapid on-site evaluation (ROSE) of cytopathology plays an important role in determining whether representative samples have been taken during navigation bronchoscopy. With touch imprint cytology (TIC), histologic samples can be assessed using ROSE. Although advised by guidelines, there have been almost no studies on the performance of TIC during navigation bronchoscopy. The objective of this study was to evaluate the value of TIC-ROSE (forceps/cryobiopsy) in combination with conventional ROSE (cytology needle/brush). METHODS: In this single-center, prospective cohort study, patients who had pulmonary nodules with an indication for navigation bronchoscopy were consecutively included. The primary outcome of the study was the concordance of ROSE and the procedural outcome. The concordance rates of TIC-ROSE and the combination of TIC-ROSE plus conventional ROSE were compared. RESULTS: Fifty-eight patients with 66 nodules were included. Conventional ROSE and TIC-ROSE were assessable in 61 nodules (90.9%) each. By combining both ROSE techniques, all sampled lesions were assessable. Combining conventional ROSE with TIC-ROSE showed concordant results in 51 of 66 cases (77.3%) versus 44 of 66 (66.7%) and 48 of 66 (72.8%) concordant results for conventional ROSE and TIC-ROSE alone, respectively, compared with the procedural outcome. There was no indication of tissue depletion as a result of TIC. The combined ROSE approach had a statistically significant higher concordance rate compared with conventional ROSE alone. CONCLUSIONS: TIC-ROSE is a cheap, easily implementable technique that can result in higher concordant ROSE outcomes. This could lead to more efficient procedures and possibly higher diagnostic results. In a monomodality sampling setting with only histologic samples, TIC can provide ROSE.

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.

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.

Cardiac multiscale bioimaging: from nano- through micro- to mesoscales.

Cardiac multiscale bioimaging is an emerging field that aims to provide a comprehensive understanding of the heart and its functions at various levels, from the molecular to the entire organ. It combines both physiologically and clinically relevant dimensions: from nano- and micrometer resolution imaging based on vibrational spectroscopy and high-resolution microscopy to assess molecular processes in cardiac cells and myocardial tissue, to mesoscale structural investigations to improve the understanding of cardiac (patho)physiology. Tailored super-resolution deep microscopy with advanced proteomic methods and hands-on experience are thus strategically combined to improve the quality of cardiovascular research and support future medical decision-making by gaining additional biomolecular information for translational and diagnostic applications.

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.

A dual-targeted drug inhibits cardiac ryanodine receptor Ca2+ leak but activates SERCA2a Ca2+ uptake.

In the heart, genetic or acquired mishandling of diastolic [Ca2+] by ryanodine receptor type 2 (RyR2) overactivity correlates with risks of arrhythmia and sudden cardiac death. Strategies to avoid these risks include decrease of Ca2+ release by drugs modulating RyR2 activity or increase in Ca2+ uptake by drugs modulating SR Ca2+ ATPase (SERCA2a) activity. Here, we combine these strategies by developing experimental compounds that act simultaneously on both processes. Our screening efforts identified the new 1,4-benzothiazepine derivative GM1869 as a promising compound. Consequently, we comparatively studied the effects of the known RyR2 modulators Dantrolene and S36 together with GM1869 on RyR2 and SERCA2a activity in cardiomyocytes from wild type and arrhythmia-susceptible RyR2R2474S/+ mice by confocal live-cell imaging. All drugs reduced RyR2-mediated Ca2+ spark frequency but only GM1869 accelerated SERCA2a-mediated decay of Ca2+ transients in murine and human cardiomyocytes. Our data indicate that S36 and GM1869 are more suitable than dantrolene to directly modulate RyR2 activity, especially in RyR2R2474S/+ mice. Remarkably, GM1869 may represent a new dual-acting lead compound for maintenance of diastolic [Ca2+].