Generation of 3D lacrimal gland organoids from human pluripotent stem cells.

Lacrimal glands are the main exocrine glands of the eyes. Situated within the orbit, behind the upper eyelid and towards the temporal side of each eye, they secrete lacrimal fluid as a major component of the tear film. Here we identify cells with characteristics of lacrimal gland primordia that emerge in two-dimensional eye-like organoids cultured from human pluripotent stem cells1. When isolated by cell sorting and grown under defined conditions, the cells form a three-dimensional lacrimal-gland-like tissue organoid with ducts and acini, enabled by budding and branching. Clonal colony analyses indicate that the organoids originate from multipotent ocular surface epithelial stem cells. The organoids exhibit notable similarities to native lacrimal glands on the basis of their morphology, immunolabelling characteristics and gene expression patterns, and undergo functional maturation when transplanted adjacent to the eyes of recipient rats, developing lumina and producing tear-film proteins.

Approaching the intrinsic exciton physics limit in two-dimensional semiconductor diodes.

Two-dimensional (2D) semiconductors have attracted intense interest for their unique photophysical properties, including large exciton binding energies and strong gate tunability, which arise from their reduced dimensionality1-5. Despite considerable efforts, a disconnect persists between the fundamental photophysics in pristine 2D semiconductors and the practical device performances, which are often plagued by many extrinsic factors, including chemical disorder at the semiconductor-contact interface. Here, by using van der Waals contacts with minimal interfacial disorder, we suppress contact-induced Shockley-Read-Hall recombination and realize nearly intrinsic photophysics-dictated device performance in 2D semiconductor diodes. Using an electrostatic field in a split-gate geometry to independently modulate electron and hole doping in tungsten diselenide diodes, we discover an unusual peak in the short-circuit photocurrent at low charge densities. Time-resolved photoluminescence reveals a substantial decrease of the exciton lifetime from around 800 picoseconds in the charge-neutral regime to around 50 picoseconds at high doping densities owing to increased exciton-charge Auger recombination. Taken together, we show that an exciton-diffusion-limited model well explains the charge-density-dependent short-circuit photocurrent, a result further confirmed by scanning photocurrent microscopy. We thus demonstrate the fundamental role of exciton diffusion and two-body exciton-charge Auger recombination in 2D devices and highlight that the intrinsic photophysics of 2D semiconductors can be used to create more efficient optoelectronic devices.

Translation reinitiation in c.453delC frameshift mutation of KCNH2 producing functional hERG K+ channels with mild dominant negative effect in the heterozygote patient-derived iPSC cardiomyocytes.

The c.453delC (p.Thr152Profs*14) frameshift mutation in KCNH2 is associated with an elevated risk of Long QT syndrome (LQTS) and fatal arrhythmia. Nevertheless, the loss-of-function mechanism underlying this mutation remains unexplored and necessitates an understanding of electrophysiology. To gain insight into the mechanism of the LQT phenotype, we conducted whole-cell patch-clamp and immunoblot assays, utilizing both a heterologous expression system and patient-derived induced pluripotent stem cell-cardiomyocytes (iPSC-CMs) with 453delC-KCNH2. We also explored the site of translational reinitiation by employing LC/MS mass spectrometry. Contrary to the previous assumption of early termination of translation, the findings of this study indicate that the 453delC-KCNH2 leads to an N-terminally truncated hERG channel, a potential from a non-canonical start codon, with diminished expression and reduced current (IhERG). The co-expression with wildtype KCNH2 produced heteromeric hERG channel with mild dominant-negative effect. Additionally, the heterozygote patient-derived iPSC-CMs exhibited prolonged action potential duration and reduced IhERG, which was ameliorated with the use of a hERG activator, PD-118057. The results of our study offer novel insights into the mechanisms involved in congenital LQTS associated with the 453delC mutation of KCNH2. The mutant results in the formation of less functional N-terminal-truncated channels with reduced amount of membrane expression. A hERG activator is capable of correcting abnormalities in both the heterologous expression system and patient-derived iPSC-CMs.

HyGAnno: hybrid graph neural network-based cell type annotation for single-cell ATAC sequencing data.

Reliable cell type annotations are crucial for investigating cellular heterogeneity in single-cell omics data. Although various computational approaches have been proposed for single-cell RNA sequencing (scRNA-seq) annotation, high-quality cell labels are still lacking in single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq) data, because of extreme sparsity and inconsistent chromatin accessibility between datasets. Here, we present a novel automated cell annotation method that transfers cell type information from a well-labeled scRNA-seq reference to an unlabeled scATAC-seq target, via a parallel graph neural network, in a semi-supervised manner. Unlike existing methods that utilize only gene expression or gene activity features, HyGAnno leverages genome-wide accessibility peak features to facilitate the training process. In addition, HyGAnno reconstructs a reference-target cell graph to detect cells with low prediction reliability, according to their specific graph connectivity patterns. HyGAnno was assessed across various datasets, showcasing its strengths in precise cell annotation, generating interpretable cell embeddings, robustness to noisy reference data and adaptability to tumor tissues.

Enhancing Single Photon Emission Purity via Design of van der Waals Heterostructures.

Quantum emitters are essential components of quantum photonic circuitry envisioned beyond the current optoelectronic state-of-the-art. Two dimensional materials are attractive hosts for such emitters. However, the high single photon purity required is rarely realized due to the presence of spectrally degenerate classical light originating from defects. Here, we show that design of a van der Waals heterostructure effectively eliminates this spurious light, resulting in purities suitable for a variety of quantum technological applications. Single photon purity from emitters in monolayer WSe2 increases from 60% to 92% by incorporating this monolayer in a simple graphite/WSe2 heterostructure. Fast interlayer charge transfer quenches a broad photoluminescence background by preventing radiative recombination through long-lived defect bound exciton states. This approach is generally applicable to other 2D emitter materials, circumvents issues of material quality, and offers a path forward to achieve the ultrahigh single photon purities ultimately required for photon-based quantum technologies.

Inhibition of Ca2+-permeable TRPV3 and inflammatory cytokine release by honokiol and magnolol in human epidermal keratinocytes.

Transient receptor potential vanilloid-3 (TRPV3) ion channels are prominently expressed in keratinocytes, playing a vital role in skin functions. Honokiol and magnolol (H&M) the primary bioactive constituents in Magnolia officinalis extract, demonstrate anti-inflammatory and skin-protective properties. Nevertheless, the underlying mechanism regarding their effect on Ca2+-permeable ion channels remain unclear. Our purpose in this study is to investigate the effect of H&M on TRPV3 and cytokine release in normal human epidermal keratinocytes (NHEKs), including its gain-of-function (GOF) mutants (G573S and G573C) associated with Olmstead syndrome. We performed whole-cell patch-clamp, fura-2 spectrofluorimetry to investigate channels activity, CCK-8 assay to analyze cell death and enzyme-linked immunosorbent assay to assess the cytokine release from NHEKs. H&M inhibited the TRPV3 current (ITRPV3) and cytosolic calcium increase in NHEKs, HEK293T cells overexpressing hTRPV3 and its GOF mutants. Moreover, the release of pro-inflammatory cytokines (interleukin-6 and -8) from keratinocytes stimulated by TRPV3 agonist was effectively suppressed by H&M. Our findings provide insights into the mechanism underlying the anti-inflammatory effects of H&M, highlighting their potential in treating skin diseases.

Calcium homeostasis modulator 2 (Calhm2) as slowly activating membrane current channel in mouse B cells.

In mouse B lymphocytes, an unidentified slow-activating voltage-dependent current resembling the characteristics of the Calhm family ion channel (ICalhm-L) was investigated. RT-PCR analysis revealed the presence of Calhm2 and 6 transcripts, with subsequent whole-cell patch-clamp studies indicating that the ICalhm-L is augmented by heat, alkaline pH, and low extracellular [Ca2+]. Overexpression of Calhm2, but not Calhm6, in N2A cells recapitulated ICalhm-L. Moreover, Calhm2 knockdown in Bal-17 cells abolished ICalhm-L. We firstly identify the voltage-dependent ion channel function of the Calhm2 in the mouse immune cells. ATP release assays in primary mouse B cells suggested a significant contribution of Calhm2 for purinergic signaling at physiological temperature.

Synergistic Effect of Ferroptosis-Inducing Nanoparticles and X-Ray Irradiation Combination Therapy.

Ferroptosis, characterized by the induction of cell death via lipid peroxidation, has been actively studied over the last few years and has shown the potential to improve the efficacy of cancer nanomedicine in an iron-dependent manner. Radiation therapy, a common treatment method, has limitations as a stand-alone treatment due to radiation resistance and safety as it affects even normal tissues. Although ferroptosis-inducing drugs help alleviate radiation resistance, there are no safe ferroptosis-inducing drugs that can be considered for clinical application and are still in the research stage. Here, the effectiveness of combined treatment with radiotherapy with Fe and hyaluronic acid-based nanoparticles (FHA-NPs) to directly induce ferroptosis, considering the clinical applications is reported. Through the induction of ferroptosis by FHA-NPs and apoptosis by X-ray irradiation, the therapeutic efficiency of cancer is greatly improved both in vitro and in vivo. In addition, Monte Carlo simulations are performed to assess the physical interactions of the X-rays with the iron-oxide nanoparticle. The study provides a deeper understanding of the synergistic effect of ferroptosis and X-ray irradiation combination therapy. Furthermore, the study can serve as a valuable reference for elucidating the role and mechanisms of ferroptosis in radiation therapy.

Neuronal STING activation in amyotrophic lateral sclerosis and frontotemporal dementia.

The stimulator of interferon genes (STING) pathway has been implicated in neurodegenerative diseases, including Parkinson's disease and amyotrophic lateral sclerosis (ALS). While prior studies have focused on STING within immune cells, little is known about STING within neurons. Here, we document neuronal activation of the STING pathway in human postmortem cortical and spinal motor neurons from individuals affected by familial or sporadic ALS. This process takes place selectively in the most vulnerable cortical and spinal motor neurons but not in neurons that are less affected by the disease. Concordant STING activation in layer V cortical motor neurons occurs in a mouse model of C9orf72 repeat-associated ALS and frontotemporal dementia (FTD). To establish that STING activation occurs in a neuron-autonomous manner, we demonstrate the integrity of the STING signaling pathway, including both upstream activators and downstream innate immune response effectors, in dissociated mouse cortical neurons and neurons derived from control human induced pluripotent stem cells (iPSCs). Human iPSC-derived neurons harboring different familial ALS-causing mutations exhibit increased STING signaling with DNA damage as a main driver. The elevated downstream inflammatory markers present in ALS iPSC-derived neurons can be suppressed with a STING inhibitor. Our results reveal an immunophenotype that consists of innate immune signaling driven by the STING pathway and occurs specifically within vulnerable neurons in ALS/FTD.

Ultrasound-guided genitofemoral nerve block for femoral arterial access gain and closure: a randomized controlled trial.

OBJECTIVES: This study aimed to compare the analgesic efficacy and safety of the femoral branch block of the genitofemoral nerve (FBB) versus local infiltration anesthesia (LIA) for femoral arterial access gain and closure. METHODS: Eighty-two patients (age, 64.8 ± 10.9 years; female, 30.5%) undergoing endovascular procedures using 5-Fr femoral sheath were assigned to either FBB (n = 41) or LIA (n = 41). In both groups, 2% lidocaine HCL with 1:100,000 epinephrine was used as an anesthetic solution. Pain scores during access gain and closure were evaluated using a visual analog scale (score 0-10), patient satisfaction levels with the quality of anesthesia were scored on a 7-point Likert scale, and adverse events were recorded. RESULTS: The primary endpoint, pain scores during access closure, was significantly lower in the FBB group than in the LIA group (0.1 ± 0.37 vs 1.73 ± 0.92; p < 0.001). The FBB group also had significantly lower pain scores during access gain compared to the LIA group (0.83 ± 0.83 vs 2.78 ± 1.26; p < 0.001). There was an inverse relationship between pain scores and FBB after adjustment for age, gender, and body mass index (p < 0.001). FBB group reported significantly higher satisfaction with anesthesia quality compared to the LIA group (6.49 ± 0.64 vs 4.05 ± 1.05; p < 0.001). No complications were recognized in either group. CONCLUSIONS: Ultrasound-guided genitofemoral nerve blocks offered better acute pain relief and higher patient satisfaction than LIA during femoral arterial access gain and closure. CLINICAL RELEVANCE STATEMENT: In this prospective randomized controlled trial, ultrasound-guided genitofemoral nerve blocks offered better acute pain relief than local infiltration anesthesia, resulting in enhanced patient satisfaction. KEY POINTS: • FBB provided better pain relief during access gain and closure than LIA. • FBB offered higher patient satisfaction with the quality of anesthesia than LIA. • No anesthesia-related or access site complications were recognized in either treatment group.

Approaching the Schottky-Mott limit in van der Waals metal-semiconductor junctions.

The junctions formed at the contact between metallic electrodes and semiconductor materials are crucial components of electronic and optoelectronic devices 1 . Metal-semiconductor junctions are characterized by an energy barrier known as the Schottky barrier, whose height can, in the ideal case, be predicted by the Schottky-Mott rule2-4 on the basis of the relative alignment of energy levels. Such ideal physics has rarely been experimentally realized, however, because of the inevitable chemical disorder and Fermi-level pinning at typical metal-semiconductor interfaces2,5-12. Here we report the creation of van der Waals metal-semiconductor junctions in which atomically flat metal thin films are laminated onto two-dimensional semiconductors without direct chemical bonding, creating an interface that is essentially free from chemical disorder and Fermi-level pinning. The Schottky barrier height, which approaches the Schottky-Mott limit, is dictated by the work function of the metal and is thus highly tunable. By transferring metal films (silver or platinum) with a work function that matches the conduction band or valence band edges of molybdenum sulfide, we achieve transistors with a two-terminal electron mobility at room temperature of 260 centimetres squared per volt per second and a hole mobility of 175 centimetres squared per volt per second. Furthermore, by using asymmetric contact pairs with different work functions, we demonstrate a silver/molybdenum sulfide/platinum photodiode with an open-circuit voltage of 1.02 volts. Our study not only experimentally validates the fundamental limit of ideal metal-semiconductor junctions but also defines a highly efficient and damage-free strategy for metal integration that could be used in high-performance electronics and optoelectronics.

Pioneering gut health improvements in piglets with phytogenic feed additives.

This research investigates the effects of phytogenic feed additives (PFAs) on the growth performance, gut microbial community, and microbial metabolic functions in weaned piglets via a combined 16S rRNA gene amplicon and shotgun metagenomics approach. A controlled trial was conducted using 200 pigs to highlight the significant influence of PFAs on gut microbiota dynamics. Notably, the treatment group revealed an increased gut microbiota diversity, as measured with the Shannon and Simpson indices. The increase in diversity is accompanied by an increase in beneficial bacterial taxa, such as Roseburia, Faecalibacterium, and Prevotella, and a decline in potential pathogens like Clostridium sensu stricto 1 and Campylobacter. Shotgun sequencing at the species level confirmed these findings. This modification in microbial profile was coupled with an altered profile of microbial metabolic pathways, suggesting a reconfiguration of microbial function under PFA influence. Significant shifts in overall microbial community structure by week 8 demonstrate PFA treatment's temporal impact. Histomorphological examination unveiled improved gut structure in PFA-treated piglets. The results of this study indicate that the use of PFAs as dietary supplements can be an effective strategy, augmenting gut microbiota diversity, reshaping microbial function, enhancing gut structure, and optimising intestinal health of weaned piglets providing valuable implications for swine production. KEY POINTS: • PFAs significantly diversify the gut microbiota in weaned piglets, aiding balance. • Changes in gut structure due to PFAs indicate improved resistance to weaning stress. • PFAs show potential to ease weaning stress, offering a substitute for antibiotics in piglet diets.

Two-in-one strategy for optimizing chemical and structural properties of carbon felt electrodes for vanadium redox flow batteries.

Vanadium redox flow batteries (VRFBs) have received significant attention for use in large-scale energy storage systems (ESSs) because of their long cycle life, flexible capacity, power design, and safety. However, the poor electrochemical activity of the conventionally used carbon felt electrode results in low energy efficiency of the VRFBs and consequently impedes their commercialization. In this study, a carbon felt (CF) electrode with numerous nanopores and robust oxygen-containing functional groups at its edge sites is designed to improve the electrochemical activity of a carbon felt electrode. To achieve this, Ni metal nanoparticles were initially precipitated on the surface of the CF electrode, followed by etching of the precipitated Ni nanoparticles on the CF electrode using sulfuric acid. The resulting CF electrode had a specific surface area eight times larger than that of the pristine CF electrode. In addition, the oxygen-containing functional groups anchored at the graphite edge sites of the nanopores can act as robust electrocatalysts for VO2+/VO2+ and V2+/V3+ redox reactions. Consequently, the VRFB cell with the resulting carbon felt electrode can deliver a high energy efficiency of 86.2% at the current density of 60 mA cm-2, which is 20% higher than that of the VRFB cell with the conventionally heat-treated CF electrode. Furthermore, the VRFB cell with the resultant carbon felt electrodes showed stable cycling performance with no considerable energy efficiency loss over 200 charge-discharge cycles. In addition, even at a high current density of 160 mA cm-2 , the developed carbon felt electrode can achieve an energy efficiency of 70.1%.

This work reveals the importance of the robust graphite edge-site oxygen functional group and the holey structure of the ET-CF electrode, emphasizing that high VRFB efficiency can be achieved by engineering both the structure and surface properties of the carbon felt electrode.

Autonomous Interfacial Assembly of Polymer Nanofilms via Surfactant-Regulated Marangoni Instability.

Interfacial polymerization (IP) provides a versatile platform for fabricating defect-free functional nanofilms for various applications, including molecular separation, energy, electronics, and biomedical materials. Unfortunately, coupled with complex natural instability phenomena, the IP mechanism and key parameters underlying the structural evolution of nanofilms, especially in the presence of surfactants as an interface regulator, remain puzzling. Here, we interfacially assembled polymer nanofilm membranes at the free water-oil interface in the presence of differently charged surfactants and comprehensively characterized their structure and properties. Combined with computational simulations, an in situ visualization of interfacial film formation discovered the critical role of Marangoni instability induced by the surfactants via various mechanisms in structurally regulating the nanofilms. Despite their different instability-triggering mechanisms, the delicate control of the surfactants enabled the fabrication of defect-free, ultra-permselective nanofilm membranes. Our study identifies critical IP parameters that allow us to rationally design nanofilms, coatings, and membranes for target applications.

Outcomes of Aural Rehabilitation Provided in Person or by Telehealth Among Deaf/Hard of Hearing Young Children with Cochlear Implants or Hearing Aids.

Background: Cochlear implants and hearing aids may facilitate the development of listening and spoken language (LSL) in deaf/hard of hearing young children, but they require aural rehabilitation therapy-often unavailable outside urban areas-for optimal outcomes. This trial assessed the relative effectiveness of LSL therapy delivered either in person or by interactive video. The hypothesis was that telehealth service delivery would be noninferior to in-person therapy. Methods: Most parents refused randomization of their children to telehealth or in-person conditions; therefore, randomization was impossible. In consultation with the funder (NIDCD), the study design was modified. Parents were allowed to select their preferred study condition, and the study team was blinded to group membership. Forty-two families were in the in-person group and 35 in telehealth (40 and 30, respectively, after attrition). Primary endpoints were total score, auditory comprehension, and expressive communication on the Preschool Language Scale, 5th edition. There were several secondary speech, hearing, and language outcome measures. Assessments occurred at baseline and at follow-up after 6 months of LSL therapy. Results: Propensity scores were used to create two matched groups. At baseline, groups did not differ on PLS-5 scores. Change from baseline to F/U on age-equivalents for all three scores was nearly identical for both groups, although the telehealth group was younger, on average, than the in-person group. Discussion: Telehealth was noninferior to in-person services for all primary endpoints. For secondary outcomes, neither group demonstrated a significant advantage. Magnitudes of estimated group differences were small, suggesting nonsignificant differences not predominantly because of sample size. The telehealth group showed greater improvement on 15/24 of secondary language outcome measures. The findings provide evidence that telehealth is equivalent to in-person care for providing LSL therapy to young children with cochlear implants and hearing aids.

The Anticancer Activities of Natural Terpenoids That Inhibit Both Melanoma and Non-Melanoma Skin Cancers.

The prevalence of two major types of skin cancer, melanoma and non-melanoma skin cancer, has been increasing worldwide. Skin cancer incidence is estimated to rise continuously over the next 20 years due to ozone depletion and an increased life expectancy. Chemotherapeutic agents could affect healthy cells, and thus may be toxic to them and cause numerous side effects or drug resistance. Phytochemicals that are naturally occurring in fruits, plants, and herbs are known to possess various bioactive properties, including anticancer properties. Although the effects of phytochemicals are relatively milder than chemotherapeutic agents, the long-term intake of phytochemicals may be effective and safe in preventing tumor development in humans. Diverse phytochemicals have shown anti-tumorigenic activities for either melanoma or non-melanoma skin cancer. In this review, we focused on summarizing recent research findings of the natural and dietary terpenoids (eucalyptol, eugenol, geraniol, linalool, and ursolic acid) that have anticancer activities for both melanoma and non-melanoma skin cancers. These terpenoids may be helpful to protect skin collectively to prevent tumorigenesis of both melanoma and nonmelanoma skin cancers.

Unveiling the Potential of Natural Compounds: A Comprehensive Review on Adipose Thermogenesis Modulation.

The process of adipocyte browning has recently emerged as a novel therapeutic target for combating obesity and obesity-related diseases. Non-shivering thermogenesis is the process of biological heat production in mammals and is primarily mediated via brown adipose tissue (BAT). The recruitment and activation of BAT can be induced through chemical drugs and nutrients, with subsequent beneficial health effects through the utilization of carbohydrates and fats to generate heat to maintain body temperature. However, since potent drugs may show adverse side effects, nutritional or natural substances could be safe and effective as potential adipocyte browning agents. This review aims to provide an extensive overview of the natural food compounds that have been shown to activate brown adipocytes in humans, animals, and in cultured cells. In addition, some key genetic and molecular targets and the mechanisms of action of these natural compounds reported to have therapeutic potential to combat obesity are discussed.

Therapeutic Efficacy of Ultrasound-Guided Selective Nerve Block on Chronic Cervical Radiculopathy.

Background and Objectives: Cervical radiculopathy (CR) manifests as pain and sensorimotor disturbances in the upper extremities, often resulting from nerve root compression due to intervertebral disc herniation, degenerative changes, or trauma. While conservative treatments are initially preferred, persistent or severe cases may require surgical intervention. Ultrasound-guided selective nerve root block (SNRB) has emerged as a promising intervention for alleviating symptoms and potentially obviating the need for surgery. This study evaluates the therapeutic efficacy of ultrasound-guided SNRB in managing chronic CR, aiming to determine its potential in symptom relief and delaying or avoiding surgical procedures. Materials and Methods: A retrospective analysis was conducted on 720 outpatients treated for CR between October 2019 and March 2022. After excluding patients with traumatic CR, previous surgeries, malignancies, progressive neurological symptoms requiring immediate surgery, or inadequate conservative treatment, 92 patients who had experienced cervical radicular pain for more than three months and had failed to improve after more than six weeks of conservative treatment with VAS scores ≥ 5 were included. The patients underwent single or multiple ultrasound-guided SNRB procedures, involving the injection of dexamethasone and lidocaine under real-time ultrasound guidance. Symptom severity was assessed at the baseline, and at 4, 8, and 12 weeks post-procedure using the Visual Analog Scale (VAS). The data collected included age, sex, presence of neck and/or radicular pain, physical examination findings, recurrence of symptoms, improvement in symptoms, and whether surgical intervention was ultimately required. Statistical analyses were performed to identify the factors associated with symptom improvement or recurrence. Results: Significant symptom improvement was observed in 69 (75.0%) participants post-SNRB, with 55 (79.7%) showing improvement at 4 weeks, 11 (15.9%) at 8 weeks, and 3 (4.4%) at 12 weeks. Symptom recurrence, defined by an increase in VAS score accompanied by a pain flare lasting at least 24 h after a pain-free interval of at least one month, was noted in 48 (52.2%) patients. The presence of combined neck and radicular pain was a significant predictor of recurrence (p = 0.008). No significant associations were found between symptom relief and factors such as age, gender, initial pain severity, or MRI findings. Conclusions: Ultrasound-guided SNRB effectively manages chronic CR, providing substantial symptom relief and potentially reducing the need for surgical intervention. This technique offers a promising conservative treatment option, especially given its real-time visualization advantages and minimal radiation exposure.

Differential expression of the enzymes regulating myosin light chain phosphorylation are responsible for the slower relaxation of pulmonary artery than mesenteric artery in rats.

While arterial tone is generally determined by the phosphorylation of Ser19 in myosin light chain (p-MLC2), Thr18/Ser19 diphosphorylation of MLC2 (pp-MLC2) has been suggested to hinder the relaxation of smooth muscle. In a dual-wire myography of rodent pulmonary artery (PA) and mesenteric artery (MA), we noticed significantly slower relaxation in PA than in MA after 80 mM KCl-induced condition (80K-contraction). Thus, we investigated the MLC2 phosphorylation and the expression levels of its regulatory enzymes; soluble guanylate cyclase (sGC), Rho-A dependent kinase (ROCK) and myosin light chain phosphatase target regulatory subunit (MYPT1). Immunoblotting showed higher sGC-α and ROCK2 in PA than MA, while sGC-ß and MYPT1 levels were higher in MA than in PA. Interestingly, the level of pp-MLC2 was higher in PA than in MA without stimulation. In the 80K-contraction state, the levels of p-MLC2 and pp-MLC2 were commonly increased. Treatment with the ROCK inhibitor (Y27632, 10 µM) reversed the higher pp-MLC2 in PA. In the myography study, pharmacological inhibition of sGC (ODQ, 10 µM) slowed relaxation during washout, which was more pronounced in PA than in MA. The simultaneous treatment of Y27632 and ODQ reversed the impaired relaxation in PA and MA. Although treatment of PA with Y27632 alone could increase the rate of relaxation, it was still slower than that of MA without Y27632 treatment. Taken together, we suggest that the higher ROCK and lower MYPT in PA would have induced the higher level of MLC2 phosphorylation, which is responsible for the characteristic slow relaxation in PA.

Requirement of ß subunit for the reduced voltage-gated Na+ current of a Brugada syndrome patient having novel double missense mutation (p.A385T/R504T) of SCN5A.

Mutations within the SCN5A gene, which encodes the α-subunit 5 (NaV1.5) of the voltage-gated Na+ channel, have been linked to three distinct cardiac arrhythmia disorders: long QT syndrome type 3, Brugada syndrome (BrS), and cardiac conduction disorder. In this study, we have identified novel missense mutations (p.A385T/R504T) within SCN5A in a patient exhibiting overlap arrhythmia phenotypes. This study aims to elucidate the functional consequences of SCN5A mutants (p.A385T/R504T) to understand the clinical phenotypes. Whole-cell patch-clamp technique was used to analyze the NaV1.5 current (INa) in HEK293 cells transfected with the wild-type and mutant SCN5A with or without SCN1B co-expression. The amplitude of INa was not altered in mutant SCN5A (p.A385T/R504T) alone. Furthermore, a rightward shift of the voltage-dependent inactivation and faster recovery from inactivation was observed, suggesting a gain-of-function state. Intriguingly, the coexpression of SCN1B with p.A385T/R504T revealed significant reduction of INa and slower recovery from inactivation, consistent with the loss-of-function in Na+ channels. The SCN1B dependent reduction of INa was also observed in a single mutation p.R504T, but p.A385T co-expressed with SCN1B showed no reduction. In contrast, the slower recovery from inactivation with SCN1B was observed in A385T while not in R504T. The expression of SCN1B is indispensable for the electrophysiological phenotype of BrS with the novel double mutations; p.A385T and p.R504T contributed to the slower recovery from inactivation and reduced current density of NaV1.5, respectively.