Large-Area Layered Membranes with Precisely Controlled Nano-Confined Channels.

Two-dimensional (2D) nanosheets-based membranes, which have controlled 2D nano-confined channels, are highly desirable for molecular/ionic sieving and confined reactions. However, it is still difficult to develop an efficient method to prepare large-area membranes with high stability, high orientation, and accurately adjustable interlayer spacing. Here, we present a strategy to produce metal ion cross-linked membranes with precisely controlled 2D nano-confined channels and high stability in different solutions using superspreading shear-flow-induced assembly strategy. For example, membranes based on graphene oxide (GO) exhibit interlayer spacing ranging from 8.0 ± 0.1 Å to 10.3 ± 0.2 Å, with a precision of down to 1 Å. At the same time, the value of the orientation order parameter (f) of GO membranes is up to 0.95 and GO membranes exhibit superb stability in different solutions. The strategy we present, which can be generalized to the preparation of 2D nano-confined channels based on a variety of 2D materials, will expand the application scope and provide better performances of membranes.

High ambient temperature may induce presbyopia via TRPV1 activation.

The prevalence of presbyopia and nuclear cataracts (NUC) is reported to be higher in tropical areas than that in other regions, suggesting a potential influence of high temperatures on lens health. Transient receptor potential vanilloid (TRPV) channels play a crucial role in detecting ambient temperatures across various species, with TRPV1 and TRPV4 expressed in lens epithelial cells. In this study, we investigated whether ambient temperatures affect TRPV1 and TRPV4 activity in the lens, potentially contributing to the development of presbyopia and NUC. We conducted experiments using cultured human lens epithelial cell lines under different temperature conditions. Our results revealed that the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) and p38 pathways, downstream molecules of TRPV1, were activated, while Src family kinase, a downstream molecule of TRPV4, was inhibited at 37.5 °C culture compared to 35.0 °C. Confocal microscope images demonstrated higher expression of TRPV1 in 3D-structured cells under high-temperature culture conditions. Additionally, in organ culture lenses, higher elasticity was observed at elevated temperatures compared to that at lower temperatures. These results suggest that high ambient temperatures may induce lens sclerosis via TRPV1 activation, potentially contributing to the development of presbyopia and NUC.

Microglial Piezo1 mechanosensitive channel as a therapeutic target in Alzheimer's disease.

Microglia are the resident macrophages of the central nervous system (CNS) that control brain development, maintain neural environments, respond to injuries, and regulate neuroinflammation. Despite their significant impact on various physiological and pathological processes across mammalian biology, there remains a notable gap in our understanding of how microglia perceive and transmit mechanical signals in both normal and diseased states. Recent studies have revealed that microglia possess the ability to detect changes in the mechanical properties of their environment, such as alterations in stiffness or pressure. These changes may occur during development, aging, or in pathological conditions such as trauma or neurodegenerative diseases. This review will discuss microglial Piezo1 mechanosensitive channels as potential therapeutic targets for Alzheimer's disease (AD). The structure, function, and modulation of Piezo1 will be discussed, as well as its role in facilitating microglial clearance of misfolded amyloid-ß (Aß) proteins implicated in the pathology of AD.

Kv beta complex facilitates exercise-induced augmentation of myocardial perfusion and cardiac growth.

The oxygen sensitivity of voltage-gated potassium (Kv) channels regulates cardiovascular physiology. Members of the Kv1 family interact with intracellular Kvß proteins, which exhibit aldo-keto reductase (AKR) activity and confer redox sensitivity to Kv channel gating. The Kvß proteins contribute to vasoregulation by controlling outward K+ currents in smooth muscle upon changes in tissue oxygen consumption and demand. Considering exercise as a primary physiological stimulus of heightened oxygen demand, the current study tested the role of Kvß proteins in exercise performance, exercise-induced adaptations in myocardial perfusion, and physiological cardiac growth. Our findings reveal that genetic ablation of Kvß2 proteins diminishes baseline exercise capacity in mice and attenuates the enhancement in exercise performance observed after long-term training. Moreover, we demonstrate that Kvß2 proteins are critical for exercise-mediated enhancement in myocardial perfusion during cardiac stress as well as adaptive changes in cardiac structure. Our results underscore the importance of Kvß proteins in metabolic vasoregulation, highlighting their role in modulating both exercise capacity and cardiovascular benefits associated with training. Furthermore, our study sheds light on a novel molecular target for enhancing exercise performance and improving the health benefits associated with exercise training in patients with limited capacity for physical activity.

Assessing expert reliability in determining intracranial EEG channel quality and introducing the Automated Bad Channel Detection algorithm.

OBJECTIVES: To evaluate the inter- and intra-rater reliability for the identification of bad channels among neurologists, EEG Technologists, and naïve research personnel, and to compare their performance with the automated bad channel detection (ABCD) algorithm for detecting bad channels. Methods: Six Neurologists, ten EEG Technologists, and six naïve research personnel (22 raters in total) were asked to rate 1440 real intracranial EEG channels as good or bad. Intra- and interrater kappa statistics were calculated for each group. We then compared each group to the ABCD algorithm which uses spectral and temporal domain features to classify channels as good or bad. Results: Analysis of channel ratings from our participants revealed variable intra-rater reliability within each group, with no significant differences across groups. Inter-rater reliability was moderate among neurologists and EEG Technologists but minimal among naïve participants. Neurologists demonstrated a slightly higher consistency in ratings than EEG Technologists. Both groups occasionally misclassified flat channels, and participants generally focused on low-frequency content for their assessments. The ABCD algorithm, in contrast, relied more on high-frequency content. A logistic regression model showed a linear relationship between the algorithm's ratings and user responses for predominantly good channels, but less so for channels rated as bad. Sensitivity and specificity analyses further highlighted differences in rating patterns among the groups, with neurologists showing higher sensitivity and naïve personnel higher specificity. Significance: Our study reveals the bias in human assessments of iEEG data quality and the tendency of even experienced professionals to overlook certain bad channels, highlighting the need for standardized, unbiased methods. The ABCD algorithm, outperforming human raters, suggests the potential of automated solutions for more reliable iEEG interpretation and seizure characterization, offering a reliable approach free from human biases. .

Piezo2 voltage-block regulates mechanical pain sensitivity.

PIEZO2 is a trimeric mechanically-gated ion channel expressed by most sensory neurones in the dorsal root ganglia. Mechanosensitive PIEZO2 channels are also genetically required for normal touch sensation in both mice and humans. We previously showed that PIEZO2 channels are also strongly modulated by membrane voltage. Specifically, it is only at very positive voltages that all channels are available for opening by mechanical force. Conversely, most PIEZO2 channels are blocked at normal negative resting membrane potentials. The physiological function of this unusual biophysical property of PIEZO2 channels, however, remained unknown. We characterized the biophysical properties of three PIEZO2 ion channel mutations at an evolutionarily conserved Arginine (R2756). Using genome engineering in mice we generated Piezo2R2756H/R2756H and Piezo2R2756K/R2756K knock-in mice to characterize the physiological consequences of altering PIEZO2 voltage sensitivity in vivo. We measured endogenous mechanosensitive currents in sensory neurones isolated from the dorsal root ganglia and characterized mechanoreceptor and nociceptor function using electrophysiology. Mice were also assessed behaviourally and morphologically. Mutations at the conserved Arginine (R2756) dramatically changed the biophysical properties of the channel relieving voltage block and lowering mechanical thresholds for channel activation. Piezo2R2756H/R2756H and Piezo2R2756K/R2756K knock-in mice that were homozygous for gain of function mutations were viable and were tested for sensory changes. Surprisingly, mechanosensitive currents in nociceptors, neurones that detect noxious mechanical stimuli, were substantially sensitized in Piezo2 knock-in mice, but mechanosensitive currents in most mechanoreceptors that underlie touch sensation were only mildly affected by the same mutations. Single-unit electrophysiological recordings from sensory neurones innervating the glabrous skin revealed that rapidly-adapting mechanoreceptors that innervate Meissner's corpuscles exhibited slightly decreased mechanical thresholds in Piezo2 knock-in mice. Consistent with measurements of mechanically activated currents in isolated sensory neurones essentially all cutaneous nociceptors, both fast conducting Aδ-mechanonociceptors and unmyelinated C-fibre nociceptors were substantially more sensitive to mechanical stimuli and indeed acquired receptor properties similar to ultrasensitive touch receptors in Piezo2 knock-in mice. Mechanical stimuli also induced enhanced ongoing activity in cutaneous nociceptors in Piezo2 knock-in mice and hyper-sensitive PIEZO2 channels were sufficient alone to drive ongoing activity, even in isolated nociceptive neurones. Consistently, Piezo2 knock-in mice showed substantial behaviourally hypersensitivity to noxious mechanical stimuli. Our data indicate that ongoing activity and sensitization of nociceptors, phenomena commonly found in human chronic pain syndromes, can be driven by relieving the voltage-block of PIEZO2 ion channels. Indeed, membrane depolarization caused by multiple noxious stimuli may sensitize nociceptors by relieving voltage-block of PIEZO2 channels.

Identification and properties of TRPV4 mutant channels present in polycystic kidney disease patients.

Polycystic kidney disease (PKD), a disease characterized by enlargement of the kidney through cystic growth is the fourth leading cause of end-stage kidney disease world-wide. TRPV4, a calcium-permeable TRP, channel participates in kidney cell physiology and since TRPV4 forms complexes with another channel whose malfunction is associated to PKD, TRPP2 (or PKD2), we sought to determine whether patients with PKD, exhibit previously unknown mutations in TRPV4. Here, we report the presence of mutations in the TRPV4 gene in patients diagnosed with PKD and determine that they produce gain-of-function (GOF). Mutations in the sequence of the TRPV4 gene have been associated to a broad spectrum of neuropathies and skeletal dysplasias but not PKD, and their biophysical effects on channel function have not been elucidated. We identified and examined the functional behavior of a novel E6K mutant and of the previously known S94L and A217S mutant TRVP4 channels. The A217S mutation has been associated to mixed neuropathy and/or skeletal dysplasia phenotypes, however, the PKD carriers of these variants had not been diagnosed with these reported clinical manifestations. The presence of certain mutations in TRPV4 may influence the progression and severity of PKD through GOF mechanisms. PKD patients carrying TRVP4 mutations are putatively more likely to require dialysis or renal transplant as compared to those without these mutations.

PAR1-mediated Non-periodical Synchronized Calcium Oscillations in human Mesangial Cells.

Mesangial cells offer structural support to the glomerular tuft and regulate glomerular capillary flow through their contractile capabilities. These cells undergo phenotypic changes, such as proliferation and mesangial expansion, resulting in abnormal glomerular tuft formation and reduced capillary loops. Such adaptation to the changing environment is commonly associated with various glomerular diseases, including diabetic nephropathy and glomerulonephritis. Thrombin-induced mesangial remodeling was found in diabetic patients, and expression of the corresponding protease-activated receptors (PARs) in the renal mesangium was reported. However, the functional PAR-mediated signaling in mesangial cells was not examined. This study investigated protease-activated mechanisms regulating mesangial cell calcium waves that may play an essential role in the mesangial proliferation or constriction of the arteriolar cells. Our results indicate that coagulation proteases like thrombin induce synchronized oscillations in cytoplasmic Ca2+ concentration of mesangial cells. The oscillations required PAR1 GPCRs-related activation, but not a PAR4, and were further mediated presumably through store-operated calcium entry and TRPC3 channel activity. Understanding thrombin signaling pathways and their relation to mesangial cells' contractile or synthetic (proliferative) phenotype may play a role in the development of chronic kidney disease and requires further investigation.

Multisite Validation of a Functional Assay to Adjudicate SCN5A Brugada Syndrome-Associated Variants.

BACKGROUND: Brugada syndrome is an inheritable arrhythmia condition that is associated with rare, loss-of-function variants in SCN5A. Interpreting the pathogenicity of SCN5A missense variants is challenging, and ≈79% of SCN5A missense variants in ClinVar are currently classified as variants of uncertain significance. Automated patch clamp technology enables high-throughput functional studies of ion channel variants and can provide evidence for variant reclassification. METHODS: An in vitro SCN5A-Brugada syndrome automated patch clamp assay was generated and independently studied at Vanderbilt University Medical Center and Victor Chang Cardiac Research Institute. The assay was calibrated according to ClinGen Sequence Variant Interpretation recommendations using high-confidence variant controls (n=49). Normal and abnormal ranges of function were established based on the distribution of benign variant assay results. Odds of pathogenicity values were derived from the experimental results according to ClinGen Sequence Variant Interpretation recommendations. The calibrated assay was then used to study SCN5A variants of uncertain significance observed in 4 families with Brugada syndrome and other arrhythmia phenotypes associated with SCN5A loss-of-function. RESULTS: Variant channel parameters generated independently at the 2 research sites showed strong correlations, including peak INa density (R2=0.86). The assay accurately distinguished benign controls (24/25 concordant variants) from pathogenic controls (23/24 concordant variants). Odds of pathogenicity values yielded 0.042 for normal function and 24.0 for abnormal function, corresponding to strong evidence for both American College of Medical Genetics and Genomics/Association for Molecular Pathology benign and pathogenic functional criteria (BS3 and PS3, respectively). Application of the assay to 4 clinical SCN5A variants of uncertain significance revealed loss-of-function for 3/4 variants, enabling reclassification to likely pathogenic. CONCLUSIONS: This validated high-throughput assay provides clinical-grade functional evidence to aid the classification of current and future SCN5A-Brugada syndrome variants of uncertain significance.

Lead and calcium crosstalk tempted acrosome damage and hyperpolarization of spermatozoa: signaling and ultra-structural evidences.

BACKGROUND: Exposure of humans and animals to heavy metals is increasing day-by-day; thus, lead even today remains of significant public health concern. According to CDC, blood lead reference value (BLRV) ranges from 3.5 µg/dl to 5 µg/dl in adults. Recently, almost 2.6% decline in male fertility per year has been reported but the cause is not well established. Lead (Pb2+) affects the size of testis, semen quality, and secretory functions of prostate. But the molecular mechanism(s) of lead toxicity in sperm cells is not clear. Thus, present study was undertaken to evaluate the adverse effects of lead acetate at environmentally relevant exposure levels (0.5, 5, 10 and 20 ppm) on functional and molecular dynamics of spermatozoa of bucks following in vitro exposure for 15 min and 3 h. RESULTS: Lead significantly decreased motility, viable count, and motion kinematic patterns of spermatozoa like curvilinear velocity, straight-line velocity, average path velocity, beat cross frequency and maximum amplitude of head lateral displacement even at 5 ppm concentration. Pb2+ modulated intracellular cAMP and Ca2+ levels in sperm cells through L-type calcium channels and induced spontaneous or premature acrosome reaction (AR) by increasing tyrosine phosphorylation of sperm proteins and downregulated mitochondrial transmembrane potential. Lead significantly increased DNA damage and apoptosis as well. Electron microscopy studies revealed Pb2+ -induced deleterious effects on plasma membrane of head and acrosome including collapsed cristae in mitochondria. CONCLUSIONS: Pb2+ not only mimics Ca2+ but also affects cellular targets involved in generation of cAMP, mitochondrial transmembrane potential, and ionic exchange. Lead seems to interact with Ca2+ channels because of charge similarity and probably enters the sperm cell through these channels and results in hyperpolarization. Our findings also indicate lead-induced TP and intracellular Ca2+ release in spermatozoa which in turn may be responsible for premature acrosome exocytosis which is essential feature of capacitation for fertilization. Thus, lead seems to reduce the fertilizing capacity of spermatozoa even at 0.5 ppm concentrations.

Kv7 Channel Activators Flupirtine and ML213 Alleviate Neuropathic Pain Behavior in the Streptozotocin Rat Model of Diabetic Neuropathy.

Background & Objective: Chronic peripheral neuropathic pain (PNP) is a debilitating condition that is associated with many types of injury/diseases, including diabetes mellitus. Patients with longstanding diabetes develop diabetic PNP (DPNP), which is resilient to currently available drugs. The underlying molecular mechanisms of DPNP are still illusive, but Kv7 channels that have been implicated in the pathogenesis of various types of chronic pain are likely to be involved. Indeed, using the streptozotocin (STZ) rat model of DPNP, we have previously shown that Kv7 activation with their non-selective activator retigabine attenuated neuropathic pain behavior suggesting that these channels are implicated in DPNP pathogenesis. Here, we evaluated, in the same STZ model, whether the more potent and more selective Kv7 channel openers flupirtine and ML213 attenuate STZ-induced pain hypersensitivity. Methods: Male Sprague Dawley rats (250-300 g) were used. The STZ model involved a single injection of STZ (60 mg/kg, i.p.). Behavioral testing for mechanical and heat pain sensitivity was performed using a dynamic plantar aesthesiometer and Hargreaves analgesiometer, respectively. Results: STZ rats exhibited behavioral signs of mechanical and heat hypersensitivity as indicated by significant decreases in the mean paw withdrawal threshold (PWT) and mean paw withdrawal latency (PWL), respectively, at 35 days post-STZ treatment. Single injections of flupirtine (10 mg/kg, i.p.) and ML213 (5 mg/kg, i.p.) to STZ rats (35-days after STZ treatment) caused significant increases in the mean PWT, but not PWL, indicating attenuation of mechanical, but not heat hypersensitivity. Both flupirtine and ML213 were as effective as the positive control gabapentin (10/kg, i.p.), and their anti-allodynic effects were prevented by the Kv7 channel-specific blocker XE991 (3 mg/kg, i.p.). Conclusion: The findings suggest that Kv7 channels are involved in the mechanisms of mechanical but not heat hypersensitivity associated with DPNP, and that their activation may prove to be effective in alleviating DPNP symptoms.

C. elegans touch receptor neurons direct mechanosensory complex organization via repurposing conserved basal lamina proteins.

The sense of touch is conferred by the conjoint function of somatosensory neurons and skin cells. These cells meet across a gap filled by a basal lamina, an ancient structure found in metazoans. Using Caenorhabditis elegans, we investigate the composition and ultrastructure of the extracellular matrix at the epidermis and touch receptor neuron (TRN) interface. We show that membrane-matrix complexes containing laminin, nidogen, and the MEC-4 mechano-electrical transduction channel reside at this interface and are central to proper touch sensation. Interestingly, the dimensions and spacing of these complexes correspond with the discontinuous beam-like extracellular matrix structures observed in serial-section transmission electron micrographs. These complexes fail to coalesce in touch-insensitive extracellular matrix mutants and in dissociated neurons. Loss of nidogen reduces the density of mechanoreceptor complexes and the amplitude of the touch-evoked currents they carry. Thus, neuron-epithelium cell interfaces are instrumental in mechanosensory complex assembly and function. Unlike the basal lamina ensheathing the pharynx and body wall muscle, nidogen recruitment to the puncta along TRNs is not dependent upon laminin binding. MEC-4, but not laminin or nidogen, is destabilized by point mutations in the C-terminal Kunitz domain of the extracellular matrix component, MEC-1. These findings imply that somatosensory neurons secrete proteins that actively repurpose the basal lamina to generate special-purpose mechanosensory complexes responsible for vibrotactile sensing.

Corrigendum: Localization of hyperpolarization-activated cyclic nucleotide-gated channels in the vertebrate retinas across species and their physiological roles.

[This corrects the article DOI: 10.3389/fnana.2024.1385932.].

Surface Cleaning of Oil Contaminants Using Bulk Nanobubbles.

The removal of oil from solid surfaces, such as textiles and plates, remains a challenge due to the strong binding affinity of the oil. Conventional methods for surface cleaning often require surfactants and mechanical abrasion to enhance the cleaning process. However, in excess, these can pose adverse effects on the environment and to the material. This study investigated how bulk nanobubble water can clean oil microdroplets deposited on surfaces like glass coverslips and dishes. Microscopy imaging and further image analysis clearly revealed that these microdroplets detached from both hydrophobic and hydrophilic surfaces when washed with bulk nanobubble water within a fluidic microchannel. Oil contaminant cleaning was also conducted in water as mobile phase to mimic the circumstances that occur in a dishwasher and washing machine. Cleaning on a larger scale also proved very successful in the removal of oil from a porcelain bowl. These results indicate that nanobubble water can easily remove oil contaminants from glass and porcelain surfaces without the assistance of surfactants. This is in stark contrast to negligible results obtained with a control solution without nanobubbles. This study indicates that nanobubble technology is an innovative, low-cost, eco-friendly approach for oil removal, demonstrating its potential for broad practical applications.

Crystal structure of tris-{N,N-diethyl-N'-[(4-nitro-phen-yl)(oxo)meth-yl]carbamimido-thio-ato}cobalt(III).

The synthesis, crystal structure, and a Hirshfeld surface analysis of tris-{N,N-diethyl-N'-[(4-nitro-phen-yl)(oxo)meth-yl]carbamimido-thio-ato}cobalt(III) conducted at 180 K are presented. The complex consists of three N,N-diethyl-N'-[(4-nitro-benzene)(oxo)meth-yl]carbamimido-thio-ato ligands, threefold sym-metric-ally bonded about the CoIII ion, in approximately octa-hedral coordination, which generates a triple of individually near planar metallacyclic (Co-S-C-N-C-O) rings. The overall geometry of the complex is determined by the mutual orientation of each metallacycle about the crystallographically imposed threefold axis [dihedral angles = 81.70 (2)°] and by the dihedral angles between the various planar groups within each asymmetric unit [metallacycle to benzene ring = 13.83 (7)°; benzene ring to nitro group = 17.494 (8)°]. The complexes stack in anti-parallel columns about the axis of the space group (P), generating solvent-accessible channels along [001]. These channels contain ill-defined, multiply disordered, partial-occupancy solvent. Atom-atom contacts in the crystal packing predominantly (∼96%) involve hydrogen, the most abundant types being H⋯H (36.6%), H⋯O (31.0%), H⋯C (19.2%), H⋯N (4.8%), and H⋯S (4.4%).

Kv7/M channel dysfunction produces hyperexcitability in hippocampal CA1 pyramidal cells of Fmr1 knockout mice.

Fragile X syndrome (FXS), the most frequent monogenic form of intellectual disability, is caused by transcriptional silencing of the FMR1 gene that could render neuronal hyperexcitability. Here we show that pyramidal cells (PCs) in the dorsal CA1 region of the hippocampus elicited a larger action potential (AP) number in response to suprathreshold stimulation in juvenile Fmr1 knockout (KO) than wild-type (WT) mice. Because Kv7/M channels modulate CA1 PC excitability in rats, we investigated if their dysfunction produces neuronal hyperexcitability in Fmr1 KO mice. Immunohistochemical and western blot analyses showed no differences in the expression of Kv7.2 and Kv7.3 channel subunits between genotypes; however, the current mediated by Kv7/M channels was reduced in Fmr1 KO mice. In both genotypes, bath application of XE991 (10 µM), a blocker of Kv7/M channels: produced an increased AP number, produced an increased input resistance, produced a decreased AP voltage threshold and shaped AP medium afterhyperpolarization by increasing mean velocities. Retigabine (10 µM), an opener of Kv7/M channels, produced opposite effects to XE991. Both XE991 and retigabine abolished differences in all these parameters found in control conditions between genotypes. Furthermore, a low concentration of retigabine (2.5 µM) normalized CA1 PC excitability of Fmr1 KO mice. Finally, ex vivo seizure-like events evoked by 4-aminopyiridine (200 µM) in the dorsal CA1 region were more frequent in Fmr1 KO mice, and were abolished by retigabine (5-10 µM). We conclude that CA1 PCs of Fmr1 KO mice exhibit hyperexcitability, caused by Kv7/M channel dysfunction, and increased epileptiform activity, which were abolished by retigabine. KEY POINTS: Dorsal pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice exhibit hyperexcitability. Kv7/M channel activity, but not expression, is reduced in pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice. Kv7/M channel dysfunction causes hyperexcitability in pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice by increasing input resistance, decreasing AP voltage threshold and shaping medium afterhyperpolarization. A Kv7/M channel opener normalizes neuronal excitability in pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice. Ex vivo seizure-like events evoked in the dorsal CA1 region were more frequent in Fmr1 KO mice, and such an epileptiform activity was abolished by a Kv7/M channel opener depending on drug concentration. Kv7/M channels may represent a therapeutic target for treating symptoms associated with hippocampal alterations in fragile X syndrome.

Deconvoluting and derisking QRS complex widening to improve cardiac safety profile of novel plasmepsin X antimalarials.

Quinoline-related antimalarial drugs have been associated with cardiotoxicity risk, in particular QT prolongation and QRS complex widening. In collaboration with Medicines for Malaria Venture (MMV), we discovered novel plasmepsin X (PMX) inhibitors for malaria treatment. The first lead compounds tested in anesthetized guinea pigs (GP) induced profound QRS widening, although exhibiting weak inhibition of NaV1.5-mediated currents in standard patch clamp assays. To understand the mechanism(s) underlying QRS widening to identify further compounds devoid of such liability, we established a set of in vitro models including CaV1.2, NaV1.5 rate-dependence and NaV1.8 patch clamp assays, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM), and Langendorff-perfused isolated GP hearts. Six compounds were tested in all models including anesthetized GP, and 8 additional compounds were tested in vitro only. All compounds tested in anesthetized GP and isolated hearts showed a similar cardiovascular profile, consisting of QRS widening, bradycardia, negative inotropy, hypotension, and for some, QT prolongation. However, a left shift of the concentration-response curves was noted from in vitro to in vivo GP data. When comparing in vitro models, there was a good consistency between decrease in sodium spike amplitude in hiPSC-CM and QRS widening in isolated hearts. Patch clamp assay results showed that the QRS widening observed with PMX inhibitors is likely multifactorial, primarily due to NaV1.8 and NaV1.5 rate-dependent sodium blockade and/or calcium channel-mediated mechanisms. In conclusion, early de-risking of QRS widening using a set of different in vitro assays allowed to identify novel PMX inhibitors with improved cardiac safety profile.

Contributions of brain regions to machine learning-based classifications of attention deficit hyperactivity disorder (ADHD) utilizing EEG signals.

OBJECTIVE: The study presented focuses on the creation of a machine learning (ML) model that uses electrophysiological (EEG) data to identify kids with attention deficit hyperactivity disorder (ADHD) from healthy controls. The EEG signals are acquired during cognitive tasks to distinguish children with ADHD from their counterparts. METHODOLOGY: The EEG data recorded in cognitive exercises was filtered using low pass Bessel filter and notch filters to remove artifacts, by the data set owners. To identify unique EEG patterns, we used many well-known classifiers, including Naïve Bayes (NB), Random Forest, Decision Tree (DT), K-Nearest Neighbors (KNN), Support Vector Machine (SVM), AdaBoost and Linear Discriminant Analysis (LDA), to identify distinct EEG patterns. Input features comprised EEG data from nineteen channels, individually and in combination. FINDINGS: Study indicates that EEG-based categorization can differentiate between individuals with ADHD and healthy individuals with accuracy of 84%. The RF classifier achieved a maximum accuracy of 0.84 when particular region combinations were used. Evaluation of classification performance utilizing hemisphere-specific EEG data yielded promising outcomes, particularly in the right hemisphere channels. NOVELTY: The study goes beyond traditional methodologies by investigating the effect of regional data on categorization results. The contributions of various brain regions to these classifications are being extensively researched. Understanding the role of different brain regions in ADHD can lead to better diagnosis and treatment options for individuals with ADHD. The study of categorization ability, utilizing EEG data specific to each hemisphere, particularly channels in the right hemisphere region, provides further granularity to the findings.

MRS2 missense variation at Asp216 abrogates inhibitory Mg2+ binding, potentiating cell migration and apoptosis resistance.

Mitochondrial magnesium (Mg2+) is a crucial modulator of protein stability, enzymatic activity, ATP synthesis, and cell death. Mitochondrial RNA splicing protein 2 (MRS2) is the main Mg2+ channel in the inner mitochondrial membrane that mediates influx into the matrix. Recent cryo-electron microscopy (cryo-EM) human MRS2 structures exhibit minimal conformational changes at high and low Mg2+, yet the regulation of human MRS2 and orthologues by Mg2+ binding to analogous matrix domains has been well established. Further, a missense variation at D216 has been identified associated with malignant melanoma and MRS2 expression and activity is implicated in gastric cancer. Thus, to gain more mechanistic and functional insight into Mg2+ sensing by the human MRS2 matrix domain and the association with proliferative disease, we assessed the structural, biophysical, and functional effects of a D216Q mutant. We show that the D216Q mutation is sufficient to abrogate Mg2+-binding and associated conformational changes including increased α-helicity, stability, and monomerization. Further, we reveal that the MRS2 matrix domains interact with ~µM affinity, which is weakened by up to two orders of magnitude in the presence of Mg2+ for wild-type but unaffected for D216Q. Finally, we demonstrate the importance of Mg2+ sensing by MRS2 to prevent matrix Mg2+ overload as HeLa cells overexpressing MRS2 show enhanced Mg2+ uptake, cell migration, and resistance to apoptosis while MRS2 D216Q robustly potentiates these cancer phenotypes. Collectively, our findings further define the MRS2 matrix domain as a critical Mg2+ sensor that undergoes conformational and assembly changes upon Mg2+ interactions dependent on D216 to temper matrix Mg2+ overload.

Why are continent catheterizable channels continent? A stomal pressure profilometry feasibility study.

INTRODUCTION: A continent catheterizable channel (CCC) may be a solution for patients with impaired bladder emptying and difficult transurethral access. Leakage of the CCC is a common complication. To prevent leakage, the pressure in the CCC has to be higher than the reservoir (bladder/pouch) pressure in at least one location. It has not been clearly defined through which mechanism(s) the CCC achieves continence. In this feasibility study, we measured the CCC pressure profile in adult patients with various types of CCC's with and without stomal leakage. METHODS: Adult patients with a CCC on a (augmented) bladder or pouch who underwent a urodynamic investigation between January and March 2023 were included. Next to the standard urodynamic investigation, a continuous stomal pressure measurement (CSP) and stomal pressure profilometry with empty bladder (SPP-1) and with filled bladder (SPP-2) of the CCC were performed. RESULTS: A total of 17 patients were included. It was technically possible to perform SPP-1 and SPP-2 in all patients, and to measure the CSP in 16/17 patients. The median maximum stomal pressures in SPP-1 and SPP-2 were 112 (interquartile range [IQR], 76-140) cmH2O and 120 (IQR, 92-140) cmH2O, respectively. Nine patients had stomal leakage during the urodynamic investigation. In five patients, the detrusor leak point pressure (dLPP) was low (<20 cmH2O). A pressure peak at the beginning of SPP-2 was absent in all patients with stomal leakage at low dLPP. CONCLUSION: SPP and CSP measurement in CCCs are feasible. We found differences in SPP-2 between patients with and without leakage at low dLPP, indicative of a role of the intravesical tunnel in continence or high dLPP. The results of this study may improve our understanding of the physiology and dynamics of CCCs as well as the management of CCC-related complications.