Kinesin-14 HSET and KlpA are non-processive microtubule motors with load-dependent power strokes.

Accurate chromosome segregation during cell division relies on coordinated actions of microtubule (MT)-based motor proteins in the mitotic spindle. Kinesin-14 motors play vital roles in spindle assembly and maintenance by crosslinking antiparallel MTs at the spindle midzone and anchoring spindle MTs' minus ends at the poles. In this study, we investigate the force generation and motility of the Kinesin-14 motors HSET and KlpA. Our findings reveal that both motors are non-processive, producing single load-dependent power strokes per MT encounter, with estimated load-free power strokes of ~30 and ~35 nm, respectively. Each homodimeric motor generates forces of ~0.5 pN, but when assembled in teams, they cooperate to generate forces of 1 pN or more. Notably, the cooperative activity among multiple motors leads to increased MT-sliding velocities. These results quantitatively elucidate the structure-function relationship of Kinesin-14 motors and underscore the significance of cooperative behavior in their cellular functions.

Optogenetic control of kinesin-1, -2, -3 and dynein reveals their specific roles in vesicular transport.

Each cargo in a cell employs a unique set of motor proteins for its transport. To dissect the roles of each type of motor, we developed optogenetic inhibitors of endogenous kinesin-1, -2, -3 and dynein motors and examined their effect on the transport of early endosomes, late endosomes, and lysosomes. While kinesin-1, -3, and dynein transport vesicles at all stages of endocytosis, kinesin-2 primarily drives late endosomes and lysosomes. Transient optogenetic inhibition of kinesin-1 or dynein causes both early and late endosomes to move more processively by relieving competition with opposing motors. Kinesin-2 and -3 support long-range transport, and optogenetic inhibition reduces the distances that their cargoes move. These results suggest that the directionality of transport is controlled through regulating kinesin-1 and dynein activity. On vesicles transported by several kinesin and dynein motors, modulating the activity of a single type of motor on the cargo is sufficient to direct motility.

Lead and generator dysfunction in children and adolescents with epicardial pacemaker and implantable cardioverter defibrillator systems: The challenge of early recognition.

BACKGROUND: A major issue of cardiac implantable electronic device therapy in pediatric patients is the high incidence of lead dysfunctions and associated reinterventions. This study aims to analyze the timing and mode of generator and lead dysfunction. METHODS: Retrospective single-center analysis of 283 children and young adults with an epicardial pacemaker or implantable cardioverter defibrillator therapy from 1998 to 2018. RESULTS: Mean age at implant was 6.1 years (SD ± 5.8 years) and median follow-up 6.4 years (IQR, 3.4-10.4 years) with a total of 1998.1 patient-years of cardiac device therapy. A total of 120 lead-related complications were observed in 82 patients (29.0%). They were detected by device interrogation (n = 86), symptoms (n = 13), intraoperative findings (n = 7), routine chest radiography (n = 5), routine ECG (n = 4), patient alert sound by device (n = 3), and physical examination (n = 2). It was possible to find the date of the event on the device memory in 21 out of 120 lead dysfunctions (18%) with a median time interval between occurrence and detection of 1.3 months (IQR, 0.2-5.0 months). Moreover, 20 generator-related complications were found in 13 patients. CONCLUSIONS: Early recognition of lead and generator dysfunction remains challenging in pediatric patients. As symptoms are relatively rare conditions in the context of PM and ICD dysfunction, close patient monitoring is mandatory, even in asymptomatic patients with a good clinical course. To further improve the safety of pediatric pacing systems, more durable epicardial electrodes are desirable.

Successful reduction of indoor radon activity concentration via cross-ventilation: experimental data and CFD simulations.

Advanced computational fluid dynamics (CFD) simulations are essential for predicting airflow in ventilated spaces and assessing indoor air quality. In this study, a focus was set on techniques for the reduction of indoor radon-222 activity concentration [Rn], and it is demonstrated how true-to-scale 3D CFD models can predict the evolution of complex ventilation experiments. A series of ventilation experiments in an unoccupied flat on the ground floor of a residential block in Bad Schlema (Saxony, Germany) were performed. Specifically, the 'Cross-ventilation 100 %' experiment resulted in room-specific [Rn] reductions from ∼3000 to ∼300 Bq m-3. We quantitatively interpreted the results of the ventilation experiment using a CFD model with a k-ϵ turbulent stationary flow model characterised by the used decentralised ventilation system. The model was coupled with a transient transport model simulating indoor [Rn]. In a first approach, the model overestimated the decrease in the starting of the experiment and the steady state. Adjusting the model parameters inflowing radon and inlet velocity the model results are in a good agreement with the experimental values. In conclusion, this paper demonstrates the potential of CFD modelling as a suitable tool in evaluating and optimising ventilation systems for an effective reduction of elevated [Rn].

Transseptal Puncture Guided by Three-Dimensional Electroanatomical Mapping: Early Experience Using a Simplified Approach in Adults with Congenital Heart Disease.

AIMS: The widespread use of three-dimensional (3D) mapping systems and echocardiography in the field of cardiac electrophysiology has made it possible to perform transseptal punctures (TSP) with low or no fluoroscopy. However, such attempts in adults with congenital heart disease (ACHD) who have previously undergone surgical or interventional treatment are limited. Therefore, we sought to explore the feasibility and safety of an approach to perform zero- or low-fluoroscopy TSP in ACHD patients undergoing left atrial cardiac ablation procedures. METHODS AND RESULTS: This study included 45 ACHD patients who underwent TSP for ablation of left-sided tachycardias (left atrium or pulmonary venous atrium). Computed tomography (CT) of the heart was performed in all patients prior to ablation. 3D mapping of the right-sided heart chambers before TSP was used to superimpose the registered anatomy, which was subsequently used for the mapping-guided TSP technique. TSP was performed with zero-fluoroscopy in 27 patients, and the remaining 18 patients had a mean fluoroscopy exposure of 315.88 ± 598.43 µGy.m2 and a mean fluoroscopy duration of 1.9 ± 5.4 min. No patient in this cohort experienced TSP-related complications. CONCLUSION: Our study describes a fluoroscopy-free or low-dose fluoroscopy approach for TSP in ACHD patients undergoing catheter ablation of left-sided tachyarrhythmias who had been previously treated surgically or interventionally due to congenital heart defects. By superimposing 3D electroanatomic mapping with cardiac CT anatomy, this protocol proved to be highly effective, feasible and safe.

Use of implantable cardioverter-defibrillator in children supported with ventricular assist device: An analysis of data from the EUROMACS registry.

BACKGROUND: Data on the use and outcome of children on ventricular assist device (VAD) support provided with an implantable cardioverter-defibrillator (ICD) remains poor. METHODS: A retrospective analysis of the EUROMACS database on children supported with VAD < 19 years of age from January 1, 2009 to April 1, 2020. Patients with missing data on status of ICD, missing baseline and/or follow up information were excluded. The primary independent variable of interest was the concomitant presence or absence of an ICD at the time of VAD placement. Kaplan-Meier survival analysis was performed to evaluate survival differences between children on VAD with and without an ICD. RESULTS: Out of 303 patients provided with a VAD, 7% (7♀, 15♂) had an ICD implanted and formed the study group. Median age was 14 years, median weight was 43.5 kg, and median BSA was 1.39. Median Intermacs stage was 2 (range: 1-7). Seventeen patients (77%) were transplanted, 4 (18%) died while on support, and 1 (5%) was weaned from device after myocardial recovery. Median time on support was 68 days compared to 361 days in the control group (p: 0.01). Three patients underwent device exchange due to thrombus formation in the pump. There was no difference in survival between groups (p = 0.342). CONCLUSION: The presence of ICD in pediatric patients supported with a VAD is low (7%). Children on VAD support provided with an ICD do not have a survival benefit compared to children without an ICD.

Catecholaminergic Polymorphic Ventricular Tachycardia: Multiple Clinical Presentations of a Genetically Determined Disease.

BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare, inherited heart rhythm disorder that is caused by variants in genes responsible for cardiac calcium homeostasis. The aim of this study was to analyze different genotype-specific clinical manifestations of this disease. METHODS AND RESULTS: We analyzed five CPVT cases from our institution in the context of specific patient characteristics and genotype-phenotype correlations. In this cohort, three of the index patients were male. The median age at diagnosis was 11 (11-30) years, and median age at disease onset was 12 (12-33) years. Four index patients suffered from syncope, while one female index patient suffered from out-of-hospital cardiac arrest. Two index patients experienced concomitant atrial flutter and atrial fibrillation. Three patients received an implantable cardioverter defibrillator and one patient received an event recorder. All index patients had causative genetic variants in the RYR2-gene. CONCLUSIONS: This study presents various phenotypic presentations of patients with CPVT harboring different pathogenic variants in the RYR2 gene, some of which have not previously been described in published studies. Syncope was the most prevalent symptom on admission. Adjustment of beta-blocker therapy may be necessary due to side effects. Moreover, our work further highlights the common occurrence of atrial tachyarrhythmias in these patients.

Physical Activity in Fontan Patients Relates to Quality of Life and Sleep Quality.

Background and Aim: Fontan patients tend to have reduced physical exercise capacity. This study investigates physical activity (PA) and its relationship to exercise capacity, heart rates, cardiac function, biomarkers, health-related quality of life (HRQoL), and sleep quality. Methods: Cardiovascular magnetic resonance (CMR), exercise testing (CPET), 24 h-ECG, and blood samples were prospectively performed in 38 patients, age 13 (11-16) years. PA was assessed by accelerometer during 7 consecutive days. HRQoL was self-assessed with KIDSCREEN-27 and SF-36 according to patients' age; sleep quality with Pediatric Sleep Questionnaire (PSQ) and Pittsburgh Sleep Quality Index (PSQI). Results: Daily moderate to vigorous physical activity (MVPA) was in median (IQR) 40 (28-57) mins; 7/38 (18%) patients reached the recommended 60 mins/day of MVPA. MVPA did not correlate with gender, age, single ventricle morphology, time from Fontan, heart rate, ventricular volumes, and ejection fraction at CMR, biomarkers, or CPET. Physical wellbeing (r = 0.33, p = 0.04), autonomy (r = 0.39, p = 0.03), and social support (r = 0.43, p = 0.009) assessed using the KIDSCREEN-27, and both physical (r = 0.57, p = 0.03) and mental (r = 0.54, p = 0.04) domains of the SF-36 questionnaire correlated with daily minutes of MVPA. PSQI global sleeping score (r = -0.7, p = 0.007), and PSQ scales for behavior (r = -0.36; p = 0.03) correlated with daily minutes of MVPA. Conclusion: Only 18% of the Fontan patients meet the recommendation for daily MVPA. Measures of exercise capacity, cardiac function or chronotropic competence are not correlated to daily physical activity. In contrast, HRQoL and sleep quality seem to be associated with regular physical activity.

Self-assembly of pericentriolar material in interphase cells lacking centrioles.

The major microtubule-organizing center (MTOC) in animal cells, the centrosome, comprises a pair of centrioles surrounded by pericentriolar material (PCM), which nucleates and anchors microtubules. Centrosome assembly depends on PCM binding to centrioles, PCM self-association and dynein-mediated PCM transport, but the self-assembly properties of PCM components in interphase cells are poorly understood. Here, we used experiments and modeling to study centriole-independent features of interphase PCM assembly. We showed that when centrioles are lost due to PLK4 depletion or inhibition, dynein-based transport and self-clustering of PCM proteins are sufficient to form a single compact MTOC, which generates a dense radial microtubule array. Interphase self-assembly of PCM components depends on γ-tubulin, pericentrin, CDK5RAP2 and ninein, but not NEDD1, CEP152, or CEP192. Formation of a compact acentriolar MTOC is inhibited by AKAP450-dependent PCM recruitment to the Golgi or by randomly organized CAMSAP2-stabilized microtubules, which keep PCM mobile and prevent its coalescence. Linking of CAMSAP2 to a minus-end-directed motor leads to the formation of an MTOC, but MTOC compaction requires cooperation with pericentrin-containing self-clustering PCM. Our data reveal that interphase PCM contains a set of components that can self-assemble into a compact structure and organize microtubules, but PCM self-organization is sensitive to motor- and microtubule-based rearrangement.

Inducibility of atrioventricular nodal reentrant tachycardia and ectopic atrial tachycardia in children under general anesthesia.

BACKGROUND: In children, invasive electrophysiological studies (EPS) and radiofrequency catheter ablations (RFA) of supraventricular tachycardia (SVT) are often performed under general anesthesia. Atrioventricular nodal reentrant tachycardia (AVNRT) and ectopic atrial tachycardia (EAT) must be inducible during EPS as reliable diagnosis and subsequent therapy are not possible in sinus rhythm. This study aims to assess the problem of noninducible AVNRT and EAT under general anesthesia. METHODS AND RESULTS: Anesthesia protocols of 166 patients undergoing EPS were retrospectively analyzed. 122 AVNRT patients were compared to 22 whose tachycardia was not inducible but probably due to an AVNRT mechanism. Another 16 patients with inducible EAT were compared to 6 whose EAT appeared on surface ECG but not during EPS. Demographic characteristics were similar among all groups. Inducibility did not differ (p = .42) between AVNRT patients with inhalational anesthesia (sevoflurane and/or nitrous oxide) and patients with intravenous anesthesia (propofol with/without remifentanil). The EAT group exhibited lower inducibility under intravenous anesthesia (64%) than under inhalational (88%), however without significance (p = .35). CONCLUSION: Tachycardia induction succeeds with similar frequency under both inhalational and intravenous general anesthesia in children with AVNRT. In children with EAT, inhalational anesthesia is associated with a trend towards better inducibility.

Subsynaptic mobility of presynaptic mGluR types is differentially regulated by intra- and extracellular interactions.

Presynaptic metabotropic glutamate receptors (mGluRs) are essential for the control of synaptic transmission. However, how the subsynaptic dynamics of these receptors is controlled and contributes to synaptic signaling remain poorly understood quantitatively. Particularly, since the affinity of individual mGluR subtypes for glutamate differs considerably, the activation of mGluR subtypes critically depends on their precise subsynaptic distribution. Here, using superresolution microscopy and single-molecule tracking, we unravel novel molecular mechanisms that control the nanoscale distribution and mobility of presynaptic mGluRs in hippocampal neurons. We demonstrate that the high-affinity group II receptor mGluR2 localizes diffusely along the axon, and is highly mobile, while the low-affinity group III receptor mGluR7 is stably anchored at the active zone. We demonstrate that intracellular interactions modulate surface diffusion of mGluR2, while immobilization of mGluR7 at the active zone relies on its extracellular domain. Receptor activation or increases in synaptic activity do not alter the surface mobility of presynaptic mGluRs. Finally, computational modeling of presynaptic mGluR activity revealed that this particular nanoscale arrangement directly impacts their ability to modulate neurotransmitter release. Altogether, this study demonstrates that distinct mechanisms control surface mobility of presynaptic mGluRs to contribute differentially to glutamatergic synaptic transmission.

Quantitative Mapping of Flavor and Pharmacologically Active Compounds in European Licorice Roots (Glycyrrhiza glabra L.) in Response to Growth Conditions and Arbuscular Mycorrhiza Symbiosis.

Application of a sensitive UHPLC-MS/MSMRM method enabled the simultaneous quantitation of 23 sweet-, licorice-, and bitter-tasting saponins in Glycyrrhiza glabra L., Glycyrrhiza uralensis Fisch., different licorice plants and root compartments, processed licorice, as well as different Glycyrrhiza spp. The combination of quantitative data with sweet, licorice, and bitter taste thresholds led to the determination of dose-over-threshold factors to elucidate the sweet, licorice, and bitter impact of the individual saponins with and without mycorrhiza symbiosis to evaluate the licorice root quality. Aside from glycyrrhizin (1), which is the predominant sweet- and licorice-tasting saponin in all licorice samples, 20 out of 22 quantitated saponins contributed to the taste profile of licorice roots. Next to sweet-/licorice-tasting glycyrrhizin (1), 24-hydroxy-glycyrrhizin (9), 30-hydroxy-glycyrrhizin (11), and 11-deoxo-24-hydroxy-glycyrrhizin (14) as well as licorice tasting saponins 20α-galacturonic acid glycyrrhizin (17), 24-hydroxy-20α-glycyrrhizin (21), and 11-deoxo-glycyrrhizin (12) were determined as key contributors to licorice root's unique taste profile. A quantitative comparison of 23 saponins as well as 28 polyphenols between licorice roots inoculated with arbuscular mycorrhiza fungi and controls showed that important taste-mediating saponins were increased in mycorrhizal roots, and these alterations depended on the growth substrate and the level of phosphate fertilization.

High Site Selectivity in Electrophilic Aromatic Substitutions: Mechanism of C-H Thianthrenation.

The introduction of thianthrene as a linchpin has proven to be a versatile strategy for the C-H functionalization of aromatic compounds, featuring a broad scope and fast diversification. The synthesis of aryl thianthrenium salts has displayed an unusually high para regioselectivity, notably superior to those observed in halogenation or borylation reactions for various substrates. We report an experimental and computational study on the mechanism of aromatic C-H thianthrenation reactions, with an emphasis on the elucidation of the reactive species and the nature of the exquisite site selectivity. Mechanisms involving a direct attack of arene to the isolated O-trifluoracetylthianthrene S-oxide (TT+-TFA) or to the thianthrene dication (TT2+) via electron transfer under acidic conditions are identified. A reversible interconversion of the different Wheland-type intermediates before a subsequent, irreversible deprotonation is proposed to be responsible for the exceptional para selectivity of the reaction.

Beyond the visible spectrum - applying 3D multispectral full-body imaging to the VirtoScan system.

Multispectral photography offers a wide range of applications for forensic investigations. It is commonly used to detect latent evidence and to enhance the visibility of findings. Additionally, three-dimensional (3D) full-body documentation has become much easier and more affordable in recent years. However, the benefits of performing 3D imaging beyond the visible (VIS) spectrum are not well known, and the technique has not been widely used in forensic medical investigations. A multicamera setup was used to employ multispectral photogrammetry between 365 and 960 nm in postmortem investigations. The multicamera setup included four modified digital cameras, ultraviolet (UV) and near-infrared (NIR) light sources and supplemental lens filters. Full-body documentation was performed in conjunction with the use of a medical X-ray computed tomography (CT) scanner to automate the imaging procedure. Textured 3D models based on multispectral datasets from four example cases were reconstructed successfully. The level of detail and overall quality of the 3D reconstructions varied depending on the spectral range of the image data. Generally, the NIR datasets showed enhanced visibility of vein patterns and specific injuries, whereas the UV-induced datasets highlighted foreign substances on the skin. Three-dimensional multispectral full-body imaging enables the detection of latent evidence that is invisible to the naked eye and allows visualization, documentation and analysis of evidence beyond the VIS spectrum.

Trifluoromethyl Thianthrenium Triflate: A Readily Available Trifluoromethylating Reagent with Formal CF3+, CF3•, and CF3- Reactivity.

Here we report the synthesis and application of trifluoromethyl thianthrenium triflate (TT-CF3+OTf-) as a novel trifluoromethylating reagent, which is conveniently accessible in a single step from thianthrene and triflic anhydride. We demonstrate the use of TT-CF3+OTf- in electrophilic, radical, and nucleophilic trifluoromethylation reactions.

Late-Stage Heteroarylation of Hetero(aryl)sulfonium Salts Activated by α-Amino Alkyl Radicals.

We report a late-stage heteroarylation of aryl sulfonium salts through activation with α-amino alkyl radicals in a mechanistically distinct approach from previously reported halogen-atom transfer (XAT). The new mode of activation of aryl sulfonium salts proceeds in the absence of light and photoredox catalysts, engaging a wide range of hetarenes. Furthermore, we demonstrate the applicability of this methodology in synthetically useful cross-coupling transformations.

Dynein self-organizes while translocating the centrosome in T-cells.

T-cells massively restructure their internal architecture upon reaching an antigen-presenting cell (APC) to form the immunological synapse (IS), a cell-cell interface necessary for efficient elimination of the APC. This reorganization occurs through tight coordination of cytoskeletal processes: actin forms a peripheral ring, and dynein motors translocate the centrosome toward the IS. A recent study proposed that centrosome translocation involves a microtubule (MT) bundle that connects the centrosome perpendicularly to dynein at the synapse center: the "stalk." The synapse center, however, is actin-depleted, while actin was assumed to anchor dynein. We propose that dynein is attached to mobile membrane anchors, and investigate this model with computer simulations. We find that dynein organizes into a cluster in the synapse when translocating the centrosome, aligning MTs into a stalk. By implementing both a MT-capture-shrinkage and a MT-sliding mechanism, we explicitly demonstrate that this organization occurs in both systems. However, results obtained with MT-sliding dynein are more robust and display a stalk morphology consistent with our experimental data obtained with expansion microscopy. Thus, our simulations suggest that actin organization in T-cells during activation defines a specific geometry in which MT-sliding dynein can self-organize into a cluster and cause stalk formation.