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Purpose: We aim to explore a potential treatment strategy for hair loss., Materials and Methods: A male 6-year-old child was diagnosed with hidrotic ectodermal dysplasia 2 (HED2) caused by GJB6 (p.G11R) mutations. He presented at our clinic with diffuse thinning and fine and brittle hair since birth. Additionally, the child exhibited abnormal development of teeth, fingernails, and toenails. The condition of the child's hair had not improved significantly with age. He was treated with botanical extracts combined with Minoxidil., Results: After one and a half months of treatment, the patient showed remarkable hair growth., Conclusions: Our team has previously used botanical extracts in combination for the treatment of autosomal recessive wooly hair in children. In the present case, treatment with botanical extract combined with minoxidil was found to be equally efficacious. This case report provides valuable information for future studies on the use of botanical extracts in treating hair loss, as well as a safe and effective potential treatment strategy for children with congenital alopecia.

Hearing loss is one of the most prevalent genetic disorders in humans. Locus and allelic heterogeneity cause fundamental challenges in hearing loss genetic diagnosis and management of patients and their families. This study examined the genetic profile of patients with prelingual hearing loss who were referred to the Genetic Foundation of Khorasan Razavi spanning over a decade. Deleterious variants in GJB2 were evaluated through Sanger sequencing among 745 non-syndromic hearing loss patients. Furthermore, exome sequencing was applied in 250 patients with negative GJB2 sequencing results and 30 patients with syndromic hearing loss. The findings revealed a relatively low frequency of GJB2 variants among the studied patients. Exome sequencing successfully identified the genetic causes of hearing loss in 70% of the patients. Moreover, variants in 10 genes, namely SLC26A4, MYO15A, TMPRSS3, TMC1, OTOF, CDH23, PJVK, MYO7A, TECTA, and PCDH15, accounted for 66% of the positive exome sequencing findings in this study. At least three prevalent founder alleles in the hearing-impaired population of eastern Iran were identified. This study emphasizes the efficiency of exome sequencing as a powerful tool for determining the etiology of prelingual hearing loss in the eastern Iranian population., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Erythrokeratodermia variabilis et progressiva (EKVP) is a rare hereditary skin disorder characterized by hyperkeratotic plaques and erythematous patches that progressively worsen with age. This disorder has been associated with variants in three connexin encoding genes (GJA1, GJB3, GJB4) and four unrelated genes (KRT83, KDSR, TRPM4, PERP). Most cases of connexin-linked EKVP exhibit an autosomal dominant mode of inheritance, with rare autosomal recessive cases. Collectively, evidence suggests that connexin variants associated with EKVP elicit a plethora of molecular defects including impaired gap junction (GJ) formation, dysregulated hemichannel and/or GJ channel function, cytotoxicity, dominant disruption of co-expressed connexins, and/or altered turnover kinetics. Here, we review the progress made in understanding the genetic and molecular basis of EKVP associated with connexin gene variants. We also discuss the landscape of treatment options used for this disorder and the future directions for research into this rare condition., (© 2024 The Author(s).)

Intracranial angioleiomyoma (IALM) is a rare neoplasm mimicking meningioma. We aimed to explore the clinical and molecular characteristics of IALMs. We included 40 patients with IALMs who had tumor resection at our center from 2009 to 2022. Clinicopathological and radiological characteristics were extracted and analyzed thoroughly. GJA4 mutation status was detected and correlated with clinical characteristics. IALMs accounted for about 10% of all angioleiomyoma and had a significant male predominance compared to extracranial angioleiomyoma (p=0.04). However, there was no age difference between extracranial angioleiomyomas and IALMs. In our IALM cohort, orbital (35%) were the most common tumor location, followed by tentorium (25.0%), cavernous sinuous (17.5%). Vision decrease, diplopia and exophthalmos (40%) were the most common symptom. The radiological characteristics of ILAMs were similar to meningiomas; 20 patients (57.1%) in our cohort were misdiagnosed as meningioma preoperatively. Gross total resection was achieved in all patients. The postoperative pathology showed median Ki-67 index was 1% (range: 0-10%). Vision improvement or exophthalmos relief was achieved in 12 of 16 patients (75.0%). During the long-term follow-up (mean 70.3 months, range 30 - 128 months), no patient experienced tumor recurrence or died of tumor progression, indicating that IALM was clinically benign. GJA4 mutation (p. Gly41Cys) was detected in 17 (42.5%) patients. Of note, the correlations analysis revealed that orbital and cavernous sinus areas (64.7%) were the leading location that harbor GJA4 mutations. Tumors with mutant GJA4 were associated with positive progesterone receptor (PR) expression (p=0.02). This first large case series demonstrated that IALM predominantly affected males, was located in the cavernous sinus and orbital areas, and often manifested visual impairment and diplopia. IALM had favorable outcome. Notably, IALMs frequently exhibited a GJA4 mutation, which was linked to the cavernous sinus and orbital locations, as well as PR expression., Competing Interests: Declarations Ethics approval and consent to participate This study used publicly available datasets and the ethics approval and consent to participate was not applicable. Consent for publication The authors consent for publication. Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Two closely related connexins, Cx26 and Cx30, share widespread expression in the cochlear cellular networks. Gap junction channels formed by these connexins have been shown to have different permeability profiles, with Cx30 showing a strongly reduced preference for anionic tracers. The pore-forming segment of the first extracellular loop, E1, identified by computational studies of the Cx26 crystal structure to form a parahelix and a narrowed region of the pore, differs at a single residue at position 49. Cx26 contains an Ala and Cx30, a charged Glu at this position, and cysteine scanning in hemichannels identified this position to be pore-lining. To assess whether the Ala/Glu difference affects permeability, we modeled and quantified Lucifer Yellow transfer between HeLa cell pairs expressing WT Cx26 and Cx30 and variants that reciprocally substituted Glu and Ala at position 49. Cx26(A49E) and Cx30(E49A) substitutions essentially reversed the Lucifer Yellow permeability profile when accounting for junctional conductance. Moreover, by using a calcein efflux assay in single cells, we observed a similar reduced anionic preference in undocked Cx30 hemichannels and a reversal with reciprocal Ala/Glu substitutions. Thus, our data indicate that Cx26 and Cx30 gap junction channels and undocked hemichannels retain similar permeability characteristics and that a single residue difference in their E1 domains can largely account for their differential permeabilities to anionic tracers. The higher anionic permeability of Cx26 compared with Cx30 suggests that these connexins may serve distinct signaling functions in the cochlea, perhaps reflected in the vastly higher prevalence of Cx26 mutations in human deafness., (© 2024 Kraujaliene et al.)

Connexins (Cxs) function as gap junction (GJ) channels and hemichannels that mediate intercellular and transmembrane signaling, respectively. Here, we investigated the proximal segment of the first extracellular loop, E1, of two closely related Cxs, Cx26 and Cx30, that share widespread expression in the cochlea. Computational studies of Cx26 proposed that this segment of E1 contains a parahelix and functions in gating. The sequence of the parahelix is identical between Cx26 and Cx30 except for an Ala/Glu difference at position 49. We show through cysteine-scanning and mutational analyses that position 49 is pore-lining and interacts with the adjacent Asp50 residue to impact hemichannel functionality. When both positions 49 and 50 are charged, as occurs naturally in Cx30, the hemichannel function is dampened. Co-expression of Cx30 with Cx26(D50N), the most common mutation associated with keratitis-ichthyosis-deafness syndrome, results in robust hemichannel currents indicating that position 49-50 interactions are relevant in heteromerically assembled hemichannels. Cysteine substitution at position 49 in either Cx26 or Cx30 results in tonic inhibition of hemichannels, both through disulfide formation and high-affinity metal coordination, suggestive of a flexible region of the pore that can narrow substantially. These effects are absent in GJ channels, which exhibit wild-type functionality. Examination of postnatal cochlear explants suggests that Cx30 expression is associated with reduced propagation of Ca2+ waves. Overall, these data identify a pore locus in E1 of Cx26 and Cx30 that impacts hemichannel functionality and provide new considerations for understanding the roles of these connexins in cochlear function., (© 2024 Sanchez et al.)

Objective: By conducting genetic testing of hereditary hearing loss in pregnant women within 17 weeks of gestation in Dali areas, the importance of genetic testing and genetic counseling during pregnancy was emphasized. Methods: Twenty-one mutation sites of 4 hearing loss genes, including GJB2 , GJB3 , SLC26A4 and mtDNA , were detected by PCR amplification technology. The positive ratio, mutation ratio and ethnic distribution of positive samples were statistically described. Results: The positive ratios of GJB2 and SLC26A4 genes were 1.24% and 1.43%, respectively, with mutation rates of 40.62% and 46.88% in the positive samples, respectively. The positive ratio of GJB3 gene was 0.19%, and mtDNA mutation genes accounted for 0.14%, and all of them were mtDNA （Heterozygous）. There was only one case of GJB2 / SLC26A4 double positive multi-gene mutation, with a positive ratio of 0.05%. The frequency of GJB2 c. 235delC site was the highest, accounting for 65.38% of GJB2 mutation genes and 26.56% of mutation gene samples. Conclusion: GJB2 and SLC26A4 are the most common genes of hearing loss, and GJB2 c. 235delC site is the most common mutation site. Identifying the hearing loss mutation site is of great importance to prevent the birth of hereditary hearing loss children, and genetic diagnosis, genetic counseling, and appropriate intervention are crucial to alleviate congenital problems., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Clinical Otorhinolaryngology Head and Neck Surgery.)

The relation of astrocytic endfeet to the vasculature plays a key functional role in the neuro-glia-vasculature unit. We characterize the spatial organization of astrocytes and the structural aspects that facilitate their involvement in molecular exchanges. Using double transgenic mice, we performed co-immunostaining, confocal microscopy, and three-dimensional digital segmentation to investigate the biophysical and molecular organization of astrocytes and their intricate endfoot network at the micrometer level in the isocortex and hippocampus. The results showed that hippocampal astrocytes had smaller territories, reduced endfoot dimensions, and fewer contacts with blood vessels compared with those in the isocortex. Additionally, we found that both connexins 43 and 30 have a higher density in the endfoot and the former is overexpressed relative to the latter. However, due to the limitations of the method, further studies are needed to determine the exact localization on the endfoot. The quantitative information obtained in this study will be useful for modeling the interactions of astrocytes with the vasculature., (© 2024 The Author(s). GLIA published by Wiley Periodicals LLC.)

The audiological features of hearing loss (HL) in patients with autosomal recessive deafness type 1A (DFNB1A) caused by splice site variants of the GJB2 gene are less studied than those of patients with other variants of this gene. In this study, we present the audiological features of DFNB1A in a large cohort of 134 patients with the homozygous splice site variant c.-23+1G>A and 34 patients with other biallelic GJB2 genotypes (n = 168 patients with DFNB1A). We found that the preservation of hearing thresholds in the speech frequency range (PTA0.5,1.0,2.0,4.0 kHz) in patients with the c.[-23+1G>A];[-23+1G>A] genotype is significantly better than in patients with the "severe" c.[35delG];[35delG] genotype (p = 0.005) and significantly worse than in patients with the "mild" c.[109G>A];[109G>A] genotype (p = 0.041). This finding indicates a "medium" pathological effect of this splice site variant on hearing function. A detailed clinical and audiological analysis showed that in patients with the c.[-23+1G>A];[-23+1G>A] genotype, HL is characterized as congenital or early onset (57.5% onset before 12 months), sensorineural (97.8%), bilateral, symmetrical (82.8%), variable in severity (from mild to profound HL, median hearing threshold in PTA0.5,1.0,2.0,4.0 kHz is 86.73±21.98 dB), with an extremely "flat" audioprofile, and with a tendency toward slow progression (a positive correlation of hearing thresholds with age, r = 0.144, p = 0.041). In addition, we found that the hearing thresholds in PTA0.5,1.0,2.0,4.0 kHz were significantly better preserved in females (82.34 dB) than in males (90.62 dB) (p = 0.001). We can conclude that in patients with DFNB1A caused by the c.-23+1G>A variant, male sex is associated with deteriorating auditory function; in contrast, female sex is a protective factor., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Teryutin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Patients who aged≥6 years and had hearing loss due to GJB2 or SLC26A4 mutation from the Chinese Deafness Genome Project between January 2020 and December 2023 were included. A total of 43 patients with GJB2 mutation were detected, including 25 males and 18 females, with a mean age of (20.4±11.4) years. There were 20 patients with SLC26A4 mutation, including 9 males and 11 females, with a mean age of (15.7±9.1) years. The mean hearing threshold of GJB2 and SLC26A4 mutation patients was 54.1 (95% CI : 49.1-59.1) decibel hearing level (dB HL) and 66.1 (95% CI : 58.6-73.6) dB HL, respectively. The hearing threshold of GJB2 and SLC26A4 mutation patients increased at a rate of 0.21 (95% CI :-0.15-0.57) dB HL/year and 2.22 (95% CI : 1.46-2.99) dB HL/year, respectively. The current study indicates that patients with SLC26A4 mutation show a progressive trend of hearing decline with the increase of age.

Background: Cat eye syndrome (CES) is a rare congenital disease frequently caused by a partial tetrasomy of the proximal long (q) arm of chromosome 22, due to a small supernumerary marker chromosome (sSMC). CES patients show remarkable phenotypic variability. Despite the progress of molecular cytogenetic technology, the cause of phenotypic variability and the genotype-phenotype correlations remain unknown., Methods: We analyzed clinical and genetic data of a new patient with CES together with 27 previously reported ones with a confirmed genomic gain in the PubMed database between 2012 and 2023., Results: We reported a boy with CES carrying a 22q11.1-q11.21 duplication of 1.76 Mb tetrasomy (16888900_18644241, hg19) who presented currently rare or unreported clinical findings such as congenital aural atresia, hearing loss, PLSVC, and IVC. The results of the whole exome sequencing (WES) showed a heterozygous mutation of the GJB2 gene (NM_004004.6: exon2: c.109G > A). In addition, the results of our literature review showed that the presence of a classical sSMC was the most frequent cytogenetic abnormality in CES (82%). 63% of cases were in a homogenous state and 37% of cases were in a mosaic state. 72% of cases had a 1-2 Mb duplication. In the majority of CES patients the breakpoints in chromosome 22 are localized to a 50 kb region (18610000_18660000 bp). The CES critical region (CESCR) may be further delimited to a 0.3 Mb region (17799398_18111588 bp). Within this region CECR2, SLC25A18, ATP6V1E1, and BCL2L13 are strong candidate genes for causing the main CES phenotype. The ear anomalies are the most frequent features in CES patients (89%) and hearing loss was present in 36% of CES patients., Conclusions: The phenotypic features in CES are highly variable. Our findings expand the symptom spectrum of CES and lay the foundation for better delineating the clinical phenotype, molecular cytogenetic features associated with CES and genotype-phenotype correlations. We recommend performing WES to rule out the involvement of other genetic factors in the patient's phenotype. In addition, our findings also highlight the need for genetic counseling and recurrence risk assessment., (© 2024. The Author(s).)

Gap junctions formed by the major neuronal connexin Cx36 function as electrical synapses in the nervous system and provide unique functions such as synchronizing neuron activities or supporting network oscillations. Although the physiological significance of electrical synapses for neuronal networks is well established, little is known about the pathways that regulate the transport of its main component: Cx36. Here we have used HEK293T cells as an expression system in combination with siRNA and BioID screens to study the transition of Cx36 from the ER to the cis Golgi. Our data indicate that the C-terminal tip of Cx36 is a key factor in this process, mediating binding interactions with two distinct components in the early secretory pathway: the COPII complex and the Golgi stacking protein Grasp55. The C-terminal amino acid valine serves as an ER export signal to recruit COPII cargo receptors Sec24A/B/C at ER exit sites, whereas the PDZ binding motif "SAYV" mediates an interaction with Grasp55. These two interactions have opposing effects in their respective compartments. While Sec24 subunits carry Cx36 out of the ER, Grasp55 stabilizes Cx36 in the Golgi as shown in over expression experiments. These early regulatory steps of Cx36 are expected to be essential for the formation, function, regulation and plasticity of electrical synapses in the developing and mature nervous system., (© 2024. The Author(s).)

Colorectal cancer (CRC) is the third deadliest cancer in the world, with a high incidence, aggressiveness, poor prognosis, and resistant to drugs. 5-fluorouracil (5-FU) is the most commonly used drug for the chemotherapeutic of CRC, however, CRC is resistant to 5-FU after a period of treatment. Therefore, there is an urgent need to explore the underlying molecular mechanisms of CRC resistance to 5-FU. In the present study, we found that the expression of PANX2 was increased in CRC tissues and metastatic tissues from the TCGA database. The K-M survival curve showed that the high expression of PANX2 was associated with poor cancer prognosis. GDSC database showed that the IC50 of 5-Fu in the PANX2 high expression group was significantly higher, and the results were verified in CRC cells. In vitro cell function and in vivo tumorigenesis experiments showed that PANX2 promoted CRC cell proliferation, clone formation, migration and tumorigenesis in vivo. WB result revealed that PANX2 may lead to resistance to 5-Fu in CRC by affecting the PI3K-AKT signaling pathway. Overall, PANX2 regulates CRC proliferation, clone formation, migration, and 5-Fu resistance by PI3K-AKT signaling pathway., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In avascular wound repair, calcium signaling events are the predominant mechanism cells use to transduce information about stressors in the environment into an effective and coordinated migratory response. Live cell imaging and computational analysis of corneal epithelial wound healing revealed that signal initiation and propagation at the wound edge are highly ordered, with groups of cells engaging in cyclical patterns of initiation and propagation. The cells in these groups exhibit a diverse range of signaling behavior, and dominant "conductor cells" drive activity in groups of lower-signaling neighbors. Ex vivo model systems reveal that conductor cells are present in wing cell layers of the corneal epithelium and that signaling propagates both within and between wing and basal layers. There are significant aberrations in conductor phenotype and interlayer propagation in type II diabetic murine models, indicating that signal hierarchy breakdown is an early indicator of disease. In vitro models reveal that signaling profile diversity and conductor cell phenotype is eliminated with P2X7 inhibition and is altered in Pannexin-1 or P2Y2 but not Connexin-43 inhibition. Conductor cells express significantly less P2X7 than their lower-signaling neighbors and exhibit significantly less migratory behavior after injury. Together, our results show that the postinjury calcium signaling cascade exhibits significantly more ordered and hierarchical behavior than previously thought, that proteins previously shown to be essential for regulating motility are also essential for determining signaling phenotype, and that loss of signal hierarchy integrity is an early indicator of disease state. NEW & NOTEWORTHY Calcium signaling in corneal epithelial cells after injury is highly ordered, with groups of cells engaged in cyclical patterns of event initiation and propagation driven by high-signaling cells. Signaling behavior is determined by P2X7, Pannexin-1, and P2Y2 and influences migratory behavior. Signal hierarchy is observed in healthy ex vivo models after injury and becomes aberrant in diabetes. This represents a paradigm shift, as signaling was thought to be random and determined by factors in the environment.

This study aimed to explore how Dab1 functional silencing influences the expression patterns of different connexins in the developing yotari ( yot ) mice eyes as potential determinants of retinogenesis. Using immunofluorescence staining, the protein expression of Dab1, Reelin, and connexin 37, 40, 43, and 45 (Cx37, Cx40, Cx43, and Cx45) in the wild-type (wt) and yot eyes at embryonic days 13.5 and 15.5 (E13.5 and E15.5) were analyzed. Different expression patterns of Cx37 were seen between the wt and yot groups. The highest fluorescence intensity of Cx37 was observed in the yot animals at E15.5. Cx40 had higher expression at the E13.5 when differentiation of retinal layers was still beginning, whereas it decreased at the E15.5 when differentiation was at the advanced stage. Higher expression of Cx43 was found in the yot group at both time points. Cx45 was predominantly expressed at E13.5 in both groups. Our results reveal the altered expression of connexins during retinogenesis in yot mice and their potential involvement in retinal pathology, where they might serve as prospective therapeutic targets.

Genetic screening of newborns for deafness plays an important role in elucidating the etiology of deafness, diagnosing it early, and intervening in it. Genetic screening of newborns has been conducted for 11 years in Beijing. It started with a chip to screen for 9 variants of 4 genes in 2012; the chip screened for 15 variants of those genes in 2018, and it now screens for 23 variants of those genes. In the current study, a comparative analysis of three screening protocols and follow-up for infants with pathogenic variants was performed. The rates of detection and hearing test results of infants with pathogenic variants were analyzed. Subjects were 493,821 infants born at 122 maternal and child care centers in Beijing from April 2012 to August 2023. Positivity increased from 4.599% for the chip to screen for 9 variants to 4.971% for the chip to screen for 15 variants, and further to 11.489% for the chip to screen for 23 variants. The carrier frequency of the GJB2 gene increased from 2.489% for the chip to screen for 9 variants and 2.422% for the chip to screen for 15 variants to 9.055% for the chip to screen for 23 variants. The carrier frequency of the SLC26A4 gene increased from 1.621% for the chip to screen for 9 variants to 2.015% for the chip to screen for 15 variants and then to 2.151% for the chip to screen for 23 variants. According to the chip to screen for 9 variants and the chip to screen for 15 variants, the most frequent mutant allele was c.235delC. According to the chip to screen for 23 variants, the most frequent mutant allele was c.109G>A. The chip to screen for 15 variants was used to screen 66.67% (14/21) of newborns with biallelic variants in the SLC26A4 gene for newly added mutations. The chip to screen for 23 variants was used to screen 92.98% (53/57) of newborns with biallelic variants in the GJB2 gene (52 cases were biallelic c.109G>A) and 25% (1/4) of newborns with biallelic variants in the SLC26A4 gene for newly added mutations. Among the infants with pathogenic variants (biallelic variants in GJB2 or SLC26A4), 20.66% (25/121) currently have normal hearing. In addition, 34.62% (9/26) of newborns who passed the hearing screening were diagnosed with hearing loss. Findings indicate that a growing number of newborns have benefited, and especially in the early identification of potential late-onset hearing loss, as the number of screening sites has increased. Conducting long-term audiological monitoring for biallelic variants in individuals with normal hearing is of paramount significance.

Hearing loss affects around 5% of the global population. Two preliminary studies have described genetic variants in sporadic individuals with hearing loss from Pakistan. Here we extend these studies to determine the spectrum of variants in a cohort of individuals with no previous history of hearing loss. Individuals with hearing loss born to consanguineous couples were identified from special schools. Audiograms were assessed. DNA from participants negative for GJB2 pathogenic variants was subjected to exome sequencing. Data were filtered to include variants with frequencies < 0.01 in the public databases. The effects of the missense variants on respective amino acids were analyzed by using PyMol software. Among the 44 participants, hearing loss was moderate for two individuals; 14 exhibited moderately-severe hearing loss while 25 had a severe degree of hearing loss. Hearing loss was reported to have been progressive in four participants and was currently profound in three participants. Variants were unambiguously identified in 17 genes, of which the majority affected SLC26A4. CDH23, MYO15A and OTOF were other significant contributors. Deleterious variants detected in two genes suggest new associations for hearing loss. Molecular characterization of hearing loss in our cohort revealed high genetic heterogeneity with a 75% diagnostic rate., (© 2024. The Author(s).)

Background: The formation of stem cell clones enables close contact of stem cells inside. The gap junctions in such clone spheres establish a microenvironment that allows frequent intercellular communication to maintain self-renewal and functions of stem cells. Nevertheless, the essential gap junction protein for molecular signaling in clones is poorly known., Methods: Primary human airway basal cells (hBCs) were isolated from brushing samples through bronchoscopy and then cultured. A tightly focused femtosecond laser was used to excite the local Ca 2+ in an individual cell to initiate an internal Ca 2+ wave in a clone to screen gap junction proteins. Immunoflourescence staining and clonogenicity assay were used to evaluate self-renewal and functions. RNA and protein levels were assessed by PCR and Western blot. Air-liquid interface assay was conducted to evaluate the differentiation potential. A Naphthalene injury mouse model was used to assess the regeneration potential., Results: Herein, we identify Connexin 25 (Cx25) dominates intercellular Ca 2+ communications in clones of hBCs in vitro to maintain the self-renewal and pluripotency of them. The self-renewal and in vitro differentiation functions and in vivo regeneration potential of hBCs in an airway damage model are both regulated by Cx25. The abnormal expression of Cx25 is validated in several diseases including IPF, Covid-19 and bronchiectasis., Conclusion: Cx25 is essential for hBC clones in maintaining self-renewal and functions of hBCs via gap junctions., (© 2024. The Author(s).)

The chemical gating of gap junction channels is mediated by cytosolic calcium (Ca 2+ i ) at concentrations ([Ca 2+ ] i ) ranging from high nanomolar (nM) to low micromolar (µM) range. Since the proteins of gap junctions, connexins/innexins, lack high-affinity Ca 2+ -binding sites, most likely gating is mediated by a Ca 2+ -binding protein, calmodulin (CaM) being the best candidate. Indeed, the role of Ca 2+ -CaM in gating is well supported by studies that have tested CaM blockers, CaM expression inhibition, testing of CaM mutants, co-localization of CaM and connexins, existence of CaM-binding sites in connexins/innexins, and expression of connexins (Cx) mutants, among others. Based on these data, since 2000, we have published a Ca 2+ -CaM-cork gating model. Despite convincing evidence for the Ca 2+ -CaM role in gating, a recent study has proposed an alternative gating model that would involve a direct electrostatic Ca 2+ -connexin interaction. However, this study, which tested the effect of unphysiologically high [Ca 2+ ] i on the structure of isolated junctions, reported that neither changes in the channel's pore diameter nor connexin conformational changes are present, in spite of exposure of isolated gap junctions to [Ca 2+ ] i as high at the 20 mM. In conclusion, data generated in the past four decades by multiple experimental approaches have clearly demonstrated the direct role of Ca 2+ -CaM in gap junction channel gating.

Background: Pathogenic variants in Gap junction protein beta 1 (GJB1), which encodes Connexin 32, are known to cause X-linked Charcot-Marie-Tooth disease (CMTX), the second most common form of CMT. CMTX presents with the following five central nervous systems (CNS) phenotypes: subclinical electrophysiological abnormalities, mild fixed abnormalities on neurological examination and/or imaging, transient CNS dysfunction, cognitive impairment, and persistent CNS manifestations., Case Presentation: A 40-year-old Japanese male showed CNS symptoms, including nystagmus, prominent spastic paraplegia, and mild cerebellar ataxia, accompanied by subclinical peripheral neuropathy. Brain magnetic resonance imaging revealed hyperintensities in diffusion-weighted images of the white matter, particularly along the pyramidal tract, which had persisted since childhood. Nerve conduction assessment showed a mild decrease in motor conduction velocity, and auditory brainstem responses beyond wave II were absent. Peripheral and central conduction times in somatosensory evoked potentials elicited by stimulation of the median nerve were prolonged. Genetic analysis identified a hemizygous GJB1 variant, NM&#95;000166.6:c.520C > T p.Pro174Ser., Conclusions: The patient in the case described here, with a GJB1 p.Pro174Ser variant, presented with a unique CNS-dominant phenotype, characterized by spastic paraplegia and persistent extensive leukoencephalopathy, rather than CMTX. Similar phenotypes have also been observed in patients with GJC2 and CLCN2 variants, likely because of the common function of these genes in regulating ion and water balance, which is essential for maintaining white matter function. CMTX should be considered within the spectrum of GJB1-related disorders, which can include patients with predominant CNS symptoms, some of which can potentially be classified as a new type of spastic paraplegia., (© 2024. The Author(s).)

Pannexin1 (PANX1) is a highly glycosylated membrane channel-forming protein, which has been found to implicate in multiple physiological and pathophysiological functions. Variants in the PANX1 gene have been reported to be associated with oocyte death and recurrent in vitro fertilization failure. In this study, we identified a novel heterozygous PANX1 variant (NM&#95;015368.4 c.410 C > T (p.Ser137Leu)) associated with the phenotype of oocyte death in a non-consanguineous family, followed by an autosomal dominant (AD) mode. We explored the molecular mechanism of the novel variant and the variant c.976&#95;978del (p.Asn326del) that we reported previously. Both of the variants altered the PANX1 glycosylation pattern in cultured cells, led to aberrant PANX1 channel activation, affected ATP release and membrane electrophysiological properties, which resulted in mouse and human oocyte death in vitro. For the first time, we presented the direct evidence of the effect of the PANX1 variants on human oocyte development. Our findings expand the variant spectrum of PANX1 genes associated with oocyte death and provide new support for the genetic diagnosis of female infertility., (© 2024. The Author(s).)

Purpose: To characterize the heterogeneity and cell clusters of postnatal lens epithelial cells (LECs) and to investigate the downstream targets of connexin 50 (Cx50) in the regulation of lens homeostasis and lens growth. To determine differentially expressed genes (DEGs) in the connexin 50 knockout (Cx50KO) lens epithelial cells that shed light on novel mechanism underlying the cataract and small size of the Cx50KO lenses., Methods: Single-cell RNA sequencing (scRNA-seq) of lens epithelial cells isolated from one-month-old Cx50KO and wild-type (WT) mice were performed. Differentially expressed genes were identified, and selected DEGs were further studied by quantitative real-time PCR (RT-qPCR) analysis and Western blot analysis., Results: The expression profiles of several thousand genes were identified by scRNA-seq data analysis. In comparison to the WT control, many DEGs were identified in the Cx50KO lens epithelial cells, including growth regulating transcriptional factors and genes encoding water channels. Significantly upregulated aquaporin 1 (Aqp1) gene expression was confirmed by RT-qPCR, and upregulated AQP1 protein expression was confirmed by Western blot analysis and immunostaining both in vivo and in vitro., Conclusions: Lens epithelial cells exhibit an intrinsic heterogeneity of different cell clusters in regulating lens homeostasis and lens growth. Upregulated Aqp1 in Cx50KO lens epithelial cells suggests that both connexin 50 and AQP1 likely play important roles in regulating water homeostasis in lens epithelial cells.

Charcot-Marie-Tooth disease (CMT) is a genetically heterogeneous disorder. As more genes are identified and overlapping of neuropathy phenotypes, the traditional classification of CMT often proves inadequate. A young male with typical clinical features was suspected to have CMT, but family history about the inheritance pattern and distinct features suggestive of particular subtypes were lacking. He was directly evaluated by whole exome sequencing (WES). The libraries from extracted DNA were sequenced on Illumina sequencing platform. The variant detected in WES was confirmed by Sanger sequencing. WES shows a likely pathogenic hemizygous missense variation in exon 2 of the gap junction protein beta 1(GJB1) gene (chrX: 70444104; C > T; Depth: 66x) that results in the substitution of cysteine for arginine at codon 183 (p.Arg183Cys). This novel variation expands the spectrum of GJB1 mutations associated with X-linked CMT (CMTX) to establish a specific genetic cause., (Copyright © 2024 Copyright: © 2024 Neurology India, Neurological Society of India.)

Purkinje-related ventricular arrhythmias have been increasingly reported, and with the development of catheter ablation techniques, intervention for Purkinje-related arrhythmias has been shown to be effective. The characteristics of Purkinje fibres orientation in the 12 canine left ventricles were observed at a gross level by staining the endocardium with Lugol's solution. Purkinje fibres were observed microscopically by HE, Masson's, PAS glycogen, and Cx40 immunohistochemical staining. Staining was successful, and the transverse orientation characteristics of Purkinje fibres were observed by Lugol's staining, and the longitudinal distribution was observed microscopically. The distribution of Purkinje fibres in the canine left ventricle is 'graded', 'layered', and 'networked', which can guide catheter ablation of Purkinje-related ventricular arrhythmia., (© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background: Pyroptosis has been implicated in the advancement of various cancers. Triggering pyroptosis within tumors amplifies the immune response, thereby fostering an antitumor immune environment. Nonetheless, few published studies have evaluated associations between functional variants in the pyroptosis-related genes and clinical outcomes of patients with non-oropharyngeal head and neck squamous cell carcinoma (NON-ORO HNSCC)., Methods: We conducted an association study of 985 NON-ORO HNSCC patients who were randomly divided into two groups: the discovery group of 492 patients and the replication group of 493 patients. We used Cox proportional hazards regression analysis to examine associations between genetic variants of the pyroptosis-related genes and survival of patients with NON-ORO HNSCC. Bayesian false discovery probability (BFDP) was used for multiple testing correction. Functional annotation was applied to the identified survival-associated genetic variants., Results: There are 8254 single-nucleotide polymorphisms (SNPs) located in 82 pyroptosis-related genes, of which 202 SNPs passed multiple testing correction with BFDP < 0.8 in the discovery and six SNPs retained statistically significant in the replication. In subsequent stepwise multivariable Cox regression analysis, two independent SNPs (CHMP4A rs1997996 G > A and PANX1 rs56175344 C > G) remained significant with an adjusted hazard ratios (HR) of 1.31 (95% confidence interval [CI] = 1.09-1.57, p = 0.004) and 0.65 (95% CI = 0.51-0.83, p = 0.0005) for overall survival (OS), respectively. Further analysis of the combined genotypes revealed progressively worse OS associated with the number of unfavorable genotypes (p trend  < 0.0001 and 0.021 for OS and disease-specific survival, respectively). Moreover, both PANX1 rs56175344G and CHMP4A rs1997996A alleles were correlated with reduced mRNA expression levels., Conclusions: Genetic variants in the pyroptosis pathway genes may predict the survival of NON-ORO HNSCC patients, likely by reducing the gene expression, but our findings need to be replicated by larger studies., (© 2024 Wiley Periodicals LLC.)

Pannexin1 (Panx1) ATP channels are important in adipocyte biology, potentially influencing energy storage and expenditure. We compared the metabolic phenotype of young (14 weeks old) and mature (20 weeks old) wild-type (WT) and Panx1 -/- mice exposed or not to cold (6 °C) during 28 days, a condition promoting adipocyte browning. Young Panx1 -/- mice weighed less and exhibited increased fat mass, improved glucose tolerance, and lower insulin sensitivity than WT mice. Their energy expenditure and respiratory exchange ratio (RER) were increased, and their fatty acid oxidation decreased. These metabolic effects were no longer observed in mature Panx1 -/- mice. The exposure of mature mice to cold exacerbated their younger metabolic phenotype. The white adipose tissue (WAT) of cold-exposed Panx1 -/- mice contained more small-sized adipocytes, but, in contrast to WT mice, white adipocytes did not increase their expression of Ucp1 nor of other markers of browning adipocytes. Interestingly, Glut4 expression was already enhanced in the WAT of young Panx1 -/- mice kept at 22 °C as compared to WT mice. Thus, Panx1 deletion exerts overall beneficial metabolic effects in mice that are pre-adapted to chronic cold exposure. Panx1 -/- mice show morphological characteristics of WAT browning, which are exacerbated upon cold exposure, an effect that appears to be associated with Ucp1-independent thermogenesis.

Rationale: Congenital sensorineural hearing loss is a significant global health issue, primarily driven by genetic factors, such as mutations in the GJB2 gene. This report presents a Chinese girl with congenital deafness and a novel mutation of the GJB2 gene., Patient Concerns: A newborn Chinese girl exhibited signs of congenital deafness., Diagnosis: Congenital deafness was confirmed through comprehensive newborn hearing screenings that included otologic, audiologic, and physical examinations. Genetic analysis revealed a compound heterozygous mutation involving c.188delT and c.235delC in the GJB2 gene, indicating a genetic basis for her hearing loss., Interventions: The patient underwent cochlear implantation, which resulted in stable auditory outcomes., Outcomes: Despite follow-up difficulties, stable auditory outcomes were achieved post-cochlear implantation, highlighting the potential efficacy of this intervention in GJB2-related hearing loss., Lessons: This case study enriches our understanding of GJB2 mutations and underscores the critical role of genetic testing in diagnosing congenital sensorineural hearing loss. It emphasizes the necessity for early intervention and sustained interdisciplinary care to enhance the quality of life for patients with genetic hearing impairment., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.)

Aims: Pannexin-1 (PANX1) is a hemichannel that releases ATP upon opening, initiating inflammation, cell proliferation, and migration. However, the role of PANX1 channels in colon cancer remains poorly understood, thus constituting the focus of this study., Main Methods: PANX1 mRNA expression was analyzed using multiple cancer databases. PANX1 protein expression and distribution were evaluated by immunohistochemistry on primary tumor tissue and non-tumor colonic mucosa from colon cancer patients. PANX1 inhibitors (probenecid or 10 Panx) were used to assess colon cancer cell lines viability. To study the role of PANX1 in vivo, a subcutaneous xenograft model using HCT116 cells was performed in BALB/c NOD/SCID immunodeficient mice to evaluate tumor growth under PANX1 inhibition using probenecid., Key Findings: PANX1 mRNA was upregulated in colon cancer tissue compared to non-tumor colonic mucosa. Elevated PANX1 mRNA expression in tumors correlated with worse disease-free survival. PANX1 protein abundance was increased on tumor cells compared to epithelial cells in paired samples, in a cancer stage-dependent manner. In vitro and in vivo experiments indicated that blocking PANX1 reduced cell viability and tumor growth., Significance: PANX1 can be used as a biomarker of colon cancer progression and blocking PANX1 channel opening could be used as a potential therapeutic strategy against this disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Connexin hemichannels were identified as the first members of the eukaryotic large-pore channel family that mediate permeation of both atomic ions and small molecules between the intracellular and extracellular environments. The conventional view is that their pore is a large passive conduit through which both ions and molecules diffuse in a similar manner. In stark contrast to this notion, we demonstrate that the permeation of ions and of molecules in connexin hemichannels can be uncoupled and differentially regulated. We find that human connexin mutations that produce pathologies and were previously thought to be loss-of-function mutations due to the lack of ionic currents are still capable of mediating the passive transport of molecules with kinetics close to those of wild-type channels. This molecular transport displays saturability in the micromolar range, selectivity, and competitive inhibition, properties that are tuned by specific interactions between the permeating molecules and the N-terminal domain that lies within the pore-a general feature of large-pore channels. We propose that connexin hemichannels and, likely, other large-pore channels, are hybrid channel/transporter-like proteins that might switch between these two modes to promote selective ion conduction or autocrine/paracrine molecular signaling in health and disease processes., Competing Interests: Competing interests statement:The authors declare no competing interest.

Background: PANX1 (pannexin 1), a ubiquitously expressed ATP release membrane channel, has been shown to play a role in inflammation, blood pressure regulation, and myocardial infarction. However, the possible role of PANX1 in cardiomyocytes in the progression of heart failure has not yet been investigated., Method: We generated a novel mouse line with constitutive deletion of PANX1 in cardiomyocytes (Panx1 MyHC6 )., Results: PANX1 deletion in cardiomyocytes had no effect on unstressed heart function but increased the glycolytic metabolism and resulting glycolytic ATP production, with a concurrent decrease in oxidative phosphorylation, both in vivo and in vitro. In vitro, treatment of H9c2 (H9c2 rat myoblast cell line) cardiomyocytes with isoproterenol led to PANX1-dependent release of ATP and Yo-Pro-1 uptake, as assessed by pharmacological blockade with spironolactone and siRNA-mediated knockdown of PANX1. To investigate nonischemic heart failure and the preceding cardiac hypertrophy, we administered isoproterenol, and we demonstrated that Panx1 MyHC6 mice were protected from systolic and diastolic left ventricle volume increases as a result of cardiomyocyte hypertrophy. Moreover, we found that Panx1 MyHC6 mice showed decreased isoproterenol-induced recruitment of immune cells (CD45 + ), particularly neutrophils (CD11b + [integrin subunit alpha M], Ly6g + [lymphocyte antigen 6 family member G]), to the myocardium., Conclusions: Together, these data demonstrate that PANX1 deficiency in cardiomyocytes increases glycolytic metabolism and protects against cardiac hypertrophy in nonischemic heart failure at least in part by reducing immune cell recruitment. Our study implies PANX1 channel inhibition as a therapeutic approach to ameliorate cardiac dysfunction in patients with heart failure., Competing Interests: None.

Porokeratotic adnexal ostial nevus (PAON) is a rare adnexal hamartoma characterized by keratotic papules following Blaschko's lines, typically located on the unilateral distal extremities. Cutaneous somatic GJB2 mutations have been linked to the pathogenesis of PAON. However, the genetic mechanism underlying bilateral or extended forms, which are less documented, remains unknown. In this study, we presented two cases of PAON with widespread cutaneous lesions and scalp involvement, and demonstrated the presence of GJB2 mosaic mutations in both patients. We further investigated the mosaic frequency in different tissues to gain insights into the mutation events contributing to the phenotype of widespread PAON. Our findings suggest that early postzygotic mutation causing mosaic GJB2 mutations may contribute to the widespread phenotype of PAON, thereby enriching the disease spectrum and mutation profile of PAON., (© 2024 Japanese Dermatological Association.)

The lymphatic network of capillaries and collecting vessels ensures tissue fluid homeostasis, absorption of dietary fats and trafficking of immune cells. Pannexin1 (Panx1) channels allow for the passage of ions and small metabolites between the cytosol and extracellular environment. Panx1 channels regulate the pathophysiological function of several tissues in a sex-dependent manner. Here, we studied the role of Panx1 in lymphatic function, and potential sex-dependent differences therein, in Prox1-CreER T2 Panx1 fl/fl and Panx1 fl/fl control mice. Panx1 expression was higher in lymphatic endothelial cells (LECs) of male mice. Lymphatic vessel morphology was not affected in Prox1-CreER T2 Panx1 fl/fl male and female mice. Lymphatic drainage was decreased by 25% in male Prox1-CreER T2 Panx1 fl/fl mice, but was similar in females of both genotypes. Accordingly, only male Prox1-CreER T2 Panx1 fl/fl mice exhibited tail swelling, pointing to interstitial fluid accumulation in males upon Panx1 deletion in LECs. Moreover, serum triglyceride and free fatty acid levels raised less in Prox1-CreER T2 Panx1 fl/fl mice of both sexes in an oral lipid tolerance test. Finally, the percentage of migratory dendritic cells arriving in draining lymph nodes was increased in Prox1-CreER T2 Panx1 fl/fl female mice, but was comparable between male mice of both genotypes. Our results point to a LEC-specific role for Panx1 in the functions of the lymphatic system., (© 2024 The Author(s). Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Background: Congenital cataracts, which can arise due to a combination of factors like environmental influences and genetic predisposition, significantly impact children's visual health globally. The occurrence rate of congenital cataracts varies from 0. 63 to 9.74 per 10,000 births. There are 7.4 instances per 10,000 children, with the highest occurrence seen in Asia. Symptoms of the disease include clouding of the lens and visual impairment. Timely identification of the condition plays a crucial role in the management and outlook of pediatric patients., Objective: This investigation aimed to discover causative mutations in four separate Chinese family lineages., Methods: The detailed clinical data and family history of four Chinese families with autosomal dominant congenital cataracts were carefully documented. Examination of the Whole Exome Sequencing was utilized to identify the genetic anomalies present in the familial cases. Subsequent validation of the identified mutations was carried out using PCR and Sanger sequencing. Following this, various computational predictive programs were utilized to evaluate how the mutations impact the structure and function of the protein., Results: The sequencing results reveal four potential disease-causing mutations: c.436G > A (p.V146M) of CRYBB2 Family 1, c.26G > T (p.R9I) of GJA3 in family 2, c.227G > A (p.R76H) of GJA8 in family 3, c.-168G > T of FTL in family 4. Among them, the causative mutation in Family GJA3 is novel, and Family FTL is a rare cataract syndrome. These familial mutations showed complete co-segregation with the affected individuals, with no presence in unaffected family members or the 100 controls. Several bioinformatic prediction tools also support the likely pathogenicity of these mutations., Conclusion: Our findings expand the mutational and phenotypic spectrum of genes associated with congenital cataracts and provide clues to the pathogenesis of congenital cataracts. These data also demonstrate the importance of NGS technology for the molecular diagnosis of congenital cataract patients., (© 2024. The Author(s) under exclusive licence to The Genetics Society of Korea.)

The involvement of inwardly rectifying potassium channel 4.1 (Kir4.1) in neuropathic pain has been established. However, there is limited understanding of the downstream mechanism through which Kir4.1 contributes to orofacial neuropathic pain. The objective of this study was to examine the regulation of Kir4.1 on the expression of pannexin 3 (Panx3) in the trigeminal ganglion (TG) and the underlying mechanism in the context of orofacial neuropathic pain caused by chronic constriction injury of the infraorbital nerve (CCI-ION). The study observed a significant increase in Panx3 expression in the TG of mice with CCI-ION. Inhibition of Panx3 in the TG of CCI-ION mice resulted in alleviation of orofacial mechanical allodynia. Furthermore, conditional knockdown (CKD) of Kir4.1 in the TG of both male and female mice led to mechanical allodynia and upregulation of Panx3 expression. Conversely, overexpression of Kir4.1 decreased Panx3 levels in the TG and relieved mechanical allodynia in CCI-ION mice. In addition, silencing Kir4.1 in satellite glial cells (SGCs) decreased Panx3 expression and increased the phosphorylation of P38 MAPK. Moreover, silencing Kir4.1 in SGCs increased the levels of reactive oxygen species (ROS). The elevated phosphorylation of P38 MAPK resulting from Kir4.1 silencing was inhibited by using a superoxide scavenger known as the tempol. Silencing Panx3 in the TG in vivo attenuated the mechanical allodynia caused by Kir4.1 CKD. In conclusion, these findings suggest that the reduction of Kir4.1 promotes the expression of Panx3 by activating the ROS-P38 MAPK signalling pathway, thus contributing to the development of orofacial neuropathic pain., (© 2024 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Pannexin 3 (Panx3) is a glycoprotein that forms mechanosensitive channels expressed in chondrocytes and annulus fibrosus cells of the intervertebral disc (IVD). Evidence suggests Panx3 plays contrasting roles in traumatic versus aging osteoarthritis (OA) and intervertebral disc degeneration (IDD). However, whether its deletion influences the response of joint tissue to forced use is unknown. The purpose of this study was to determine if Panx3 deletion in mice causes increased knee joint OA and IDD after forced treadmill running. Male and female wildtype (WT) and Panx3 knockout (KO) mice were randomized to either a no-exercise group (sedentary; SED) or daily forced treadmill running (forced exercise; FEX) from 24 to 30 weeks of age. Knee cartilage and IVD histopathology were evaluated by histology, while tibial secondary ossification centers were analyzed using microcomputed tomography (µCT). Both male and female Panx3 KO mice developed larger superficial defects of the tibial cartilage after forced treadmill running compared with SED WT mice. Additionally, Panx3 KO mice developed reduced bone volume, and female PANX3 KO mice had lengthening of the lateral tubercle at the intercondylar eminence. In the lower lumbar spine, both male and female Panx3 KO mice developed histopathological features of IDD after running compared to SED WT mice. These findings suggest that the combination of deleting Panx3 and forced treadmill running induces OA and causes histopathological changes associated with the degeneration of the IVDs in mice., (© 2024 The Authors. Journal of Orthopaedic Research ® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

GJB2 gene mutations are the most common causes of autosomal recessive non-syndromic hereditary deafness. For individuals suffering from severe to profound GJB2-related deafness, cochlear implants have emerged as the sole remedy for auditory improvement. Some previous studies have highlighted the crucial role of preserving cochlear neural components in achieving favorable outcomes after cochlear implantation. Thus, we generated a conditional knockout mouse model (Cx26-CKO) in which Cx26 was completely deleted in the cochlear supporting cells driven by the Sox2 promoter. The Cx26-CKO mice showed severe hearing loss and massive loss of hair cells and Deiter's cells, which represented the extreme form of human deafness caused by GJB2 gene mutations. In addition, multiple pathological changes in the peripheral auditory nervous system were found, including abnormal innervation, demyelination, and degeneration of spiral ganglion neurons as well as disruption of heminodes in Cx26-CKO mice. These findings provide invaluable insights into the deafness mechanism and the treatment for severe deafness in Cx26-null mice., (© 2024. Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences.)

Gain of function mutations in the pore forming Kir6 subunits of the ATP sensitive K + channels (K(ATP) channels) of pancreatic β-cells are the major cause of neonatal diabetes in humans. In this study, we show that in insulin secreting mouse β-cell lines, gain of function mutations in Kir6.1 result in a significant connexin36 (Cx36) overexpression, which form gap junctional connections and mediate electrical coupling between β-cells within pancreatic islets. Using computational modeling, we show that upregulation in Cx36 might play a functional role in the impairment of glucose stimulated Ca 2+ oscillations in a cluster of β-cells with Kir6.1 gain of function mutations in their K(ATP) channels (GoF-K(ATP) channels). Our results show that without an increase in Cx36 expression, a gain of function mutation in Kir6.1 might not be sufficient to diminish glucose stimulated Ca 2+ oscillations in a β-cell cluster. We also show that a reduced Cx36 expression, which leads to loss of coordination in a wild-type β-cell cluster, restores coordinated Ca 2+ oscillations in a β-cell cluster with GoF-K(ATP) channels. Our results indicate that in a heterogenous β-cell cluster with GoF-K(ATP) channels, there is an inverted u-shaped nonmonotonic relation between the cluster activity and Cx36 expression. These results show that in a neonatal diabetic β-cell model, gain of function mutations in the Kir6.1 cause Cx36 overexpression, which aggravates the impairment of glucose stimulated Ca 2+ oscillations., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Vehpi Yildirim reports financial support was provided by Scientific and Technological Research Council of Turkey. Richard Bertram reports financial support was provided by National Science Foundation. Co-authors Omer Faruk Karatas and Elanur Aydin Karatas hold stocks in EcoTech Biotechnology. The terms of this arrangement have been reviewed and approved by Erzurum Technical University in accordance with its policy on objectivity in research. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Although hyperactivity is associated with a wide variety of neurodevelopmental disorders, the early embryonic origins of locomotion have hindered investigation of pathogenesis of these debilitating behaviors. The earliest motor output in vertebrate animals is generated by clusters of early-born motor neurons (MNs) that occupy distinct regions of the spinal cord, innervating stereotyped muscle groups. Gap junction electrical synapses drive early spontaneous behavior in zebrafish, prior to the emergence of chemical neurotransmitter networks. We use a genetic model of hyperactivity to gain critical insight into the consequences of errors in motor circuit formation and function, finding that Fragile X syndrome model mutant zebrafish are hyperexcitable from the earliest phases of spontaneous behavior, show altered sensitivity to blockade of electrical gap junctions, and have increased expression of the gap junction protein Connexin 34/35. We further show that this hyperexcitable behavior can be rescued by pharmacological inhibition of electrical synapses. We also use functional imaging to examine MN and interneuron (IN) activity in early embryogenesis, finding genetic disruption of electrical gap junctions uncouples activity between mnx1 + MNs and INs. Taken together, our work highlights the importance of electrical synapses in motor development and suggests that the origins of hyperactivity in neurodevelopmental disorders may be established during the initial formation of locomotive circuits., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

Opioid withdrawal is a liability of chronic opioid use and misuse, impacting people who use prescription or illicit opioids. Hyperactive autonomic output underlies many of the aversive withdrawal symptoms that make it difficult to discontinue chronic opioid use. The locus coeruleus (LC) is an important autonomic centre within the brain with a poorly defined role in opioid withdrawal. We show here that pannexin-1 (Panx1) channels expressed on microglia critically modulate LC activity during opioid withdrawal. Within the LC, we found that spinally projecting tyrosine hydroxylase (TH)-positive neurons (LC spinal ) are hyperexcitable during morphine withdrawal, elevating cerebrospinal fluid (CSF) levels of norepinephrine. Pharmacological and chemogenetic silencing of LC spinal neurons or genetic ablation of Panx1 in microglia blunted CSF NE release, reduced LC neuron hyperexcitability, and concomitantly decreased opioid withdrawal behaviours in mice. Using probenecid as an initial lead compound, we designed a compound (EG-2184) with greater potency in blocking Panx1. Treatment with EG-2184 significantly reduced both the physical signs and conditioned place aversion caused by opioid withdrawal in mice, as well as suppressed cue-induced reinstatement of opioid seeking in rats. Together, these findings demonstrate that microglial Panx1 channels modulate LC noradrenergic circuitry during opioid withdrawal and reinstatement. Blocking Panx1 to dampen LC hyperexcitability may therefore provide a therapeutic strategy for alleviating the physical and aversive components of opioid withdrawal., (© 2024. The Author(s).)

Bioelectric signaling, intercellular communication facilitated by membrane potential and electrochemical coupling, is emerging as a key regulator of animal development. Gap junction (GJ) channels can mediate bioelectric signaling by creating a fast, direct pathway between cells for the movement of ions and other small molecules. In vertebrates, GJ channels are formed by a highly conserved transmembrane protein family called the connexins. The connexin gene family is large and complex, creating challenges in identifying specific connexins that create channels within developing and mature tissues. Using the embryonic zebrafish neuromuscular system as a model, we identify a connexin conserved across vertebrate lineages, gjd4, which encodes the Cx46.8 protein, that mediates bioelectric signaling required for slow muscle development and function. Through mutant analysis and in vivo imaging, we show that gjd4/Cx46.8 creates GJ channels specifically in developing slow muscle cells. Using genetics, pharmacology, and calcium imaging, we find that spinal-cord-generated neural activity is transmitted to developing slow muscle cells, and synchronized activity spreads via gjd4/Cx46.8 GJ channels. Finally, we show that bioelectrical signal propagation within the developing neuromuscular system is required for appropriate myofiber organization and that disruption leads to defects in behavior. Our work reveals a molecular basis for GJ communication among developing muscle cells and reveals how perturbations to bioelectric signaling in the neuromuscular system may contribute to developmental myopathies. Moreover, this work underscores a critical motif of signal propagation between organ systems and highlights the pivotal role of GJ communication in coordinating bioelectric signaling during development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: We aimed to analyse the efficacy and added value of a targeted Israeli expanded carrier screening panel (IL-ECSP), beyond the first-tier test covered by the Israeli Ministry of Health (IMOH) and the second-tier covered by the Health Maintenance Organisations (HMOs)., Methods: A curated variant-based IL-ECSP, tailored to the uniquely diverse Israeli population, was offered at two tertiary hospitals and a major genetics laboratory. The panel includes 1487 variants in 357 autosomal recessive and X-linked genes., Results: We analysed 10 115 Israeli samples during an 18-month period. Of these, 6036 (59.7%) were tested as couples and 4079 (40.3%) were singles. Carriers were most frequently identified with mutations in the following genes: GJB2/GJB6 (1:22 allele frequency), CFTR (1:28), GBA (1:34), TYR (1:39), PAH (1:50), SMN1 (1:52) and HEXA (1:56). Of 3018 couples tested, 753 (25%) had no findings, in 1464 (48.5%) only one partner was a carrier, and in 733 (24.3%) both were carriers of different diseases. We identified 79 (2.6%) at-risk couples, where both partners are carriers of the same autosomal recessive condition, or the female carries an X-linked disease. Importantly, 48.1% of these would not have been detected by ethnically-based screening tests currently provided by the IMOH and HMOs, for example, variants in GBA, TYR, PAH and GJB2/GJB6 ., Conclusion: This is the largest cohort of targeted ECSP testing, tailored to the diverse Israeli population. The IL-ECSP expands the identification of couples at risk and empowers their reproductive choices. We recommend endorsing an expanded targeted panel to the National Genetic Carrier Screening programme., Competing Interests: Competing interests: DMB was involved in the design of the CarrierScan array and is entitled to certain royalties., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

The expression and functional relevance of the gap junction molecule connexin-45 (Cx45; GJC1) in lymphatic endothelium were not previously known. We found that Cx45 was expressed widely in the endothelium of murine lymphatics, in both valve and nonvalve regions. Cell-specific deletion of Cx45, driven by a constitutive Cre line (Lyve1-Cre) or an inducible Cre line (Prox1-CreERT2), compromised the function of lymphatic valves, as assessed by physiological tests (back leak and closure) of isolated, single-valve vessel segments. The defects were comparable to those previously reported for loss of Cx43, and as with Cx43, deletion of Cx45 resulted in shortening or increased asymmetry of lymphatic valve leaflets, providing an explanation for the compromised valve function. In contrast with Cx43, lymphatic endothelial cell-specific (LEC-specific) deletion of Cx45 did not alter the number of valves in mesenteric or dermal lymphatic networks or the expression patterns of the canonical valve-associated proteins PROX1, ITGA9, or CLAUDIN5. Constitutive deletion of Cx45 from LECs resulted in increased backflow of injected tracer in popliteal networks in vivo and compromised the integrity of the LEC permeability barrier in a subset of collecting vessels. These findings provide evidence for an unexpected role of Cx45 in the development and maintenance of lymphatic valves.

Noninvasive prenatal diagnosis (NIPD) for autosomal recessive nonsyndromic hearing loss (ARNSHL) has been rarely reported until recent years. Additionally, the existing method can not be used for challenging genome loci (eg, copy number variations, deletions, inversions, or gene recombinants) or on families without proband genotype. This study assessed the performance of relative haplotype dosage analysis (RHDO)-based NIPD for identifying fetal genotyping in pregnancies at risk of ARNSHL. Fifty couples carrying pathogenic variants associated with ARNSHL in either GJB2 or SLC26A4 were recruited. The RHDO-based targeted linked-read sequencing combined with whole gene coverage probes was used to genotype the fetal cell-free DNA of 49 families who met the quality control standard. Fetal amniocyte samples were genotyped using invasive prenatal diagnosis (IPD) to assess the performance of NIPD. The NIPD results showed 100% (49/49) concordance with those obtained through IPD. Two families with copy number variation and recombination were also successfully identified. Sufficient specific informative single-nucleotide polymorphisms for haplotyping, as well as the fetal cell-free DNA concentration and sequencing depth, are prerequisites for RHDO-based NIPD. This method has the merits of covering the entire genes of GJB2 and SLC26A4, qualifying for copy number variation and recombination analysis with remarkable sensitivity and specificity. Therefore, it has clinical potential as an alternative to traditional IPD for ARNSHL., (Copyright © 2024 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Sterol-regulatory element binding proteins (SREBPs) are a conserved transcription factor family governing lipid metabolism. When cellular cholesterol level is low, SREBP2 is transported from the endoplasmic reticulum to the Golgi apparatus where it undergoes proteolytic activation to generate a soluble N-terminal fragment, which drives the expression of lipid biosynthetic genes. Malfunctional SREBP activation is associated with various metabolic abnormalities. In this study, we find that overexpression of the active nuclear form SREBP2 (nSREBP2) causes caspase-dependent lytic cell death in various types of cells. These cells display typical pyroptotic and necrotic signatures, including plasma membrane ballooning and release of cellular contents. However, this phenotype is independent of the gasdermin family proteins or mixed lineage kinase domain-like (MLKL). Transcriptomic analysis identifies that nSREBP2 induces expression of p73, which further activates caspases. Through whole-genome CRISPR-Cas9 screening, we find that Pannexin-1 (PANX1) acts downstream of caspases to promote membrane rupture. Caspase-3 or 7 cleaves PANX1 at the C-terminal tail and increases permeability. Inhibition of the pore-forming activity of PANX1 alleviates lytic cell death. PANX1 can mediate gasdermins and MLKL-independent cell lysis during TNF-induced or chemotherapeutic reagents (doxorubicin or cisplatin)-induced cell death. Together, this study uncovers a noncanonical function of SREBPs as a potentiator of programmed cell death and suggests that PANX1 can directly promote lytic cell death independent of gasdermins and MLKL., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: The purpose of this study was to utilize multi-parametric magnetic resonance imaging (MRI) to investigate in vivo age-related changes in the physiology and optics of mouse lenses where Connexin 50 has been deleted (Cx50KO) or replaced by Connexin 46 (Cx50KI46)., Methods: The lenses of transgenic Cx50KO and Cx50KI46 mice were imaged between 3 weeks and 6 months of age using a 7T MRI. Measurements of lens geometry, the T2 (water-bound protein ratios), the refractive index (n), and T1 (free water content) values were calculated by processing the acquired images. The lens power was calculated from an optical model that combined the geometry and the n. All transgenic mice were compared with control mice at the same age., Results: Cx50KO and Cx50KI46 mice developed smaller lenses compared with control mice. The lens thickness, volume, and surface radii of curvatures all increased with age but were limited to the size of the lenses. Cx50KO lenses exhibited higher lens power than Cx50KI46 lenses at all ages, and this was correlated with significantly lower water content in these lenses, which was probably modulated by the gap junction coupling. The refractive power tended to a steady state with age, similar to the control mice., Conclusions: The modification of Cx50 gap junctions significantly impacted lens growth and physiological optics as the mouse aged. The lenses showed delayed development growth, and altered optics governed by different lens physiology. This research provides new insights into how gap junctions regulate the development of the lens's physiological optics.

Long-lived lens fiber cells require a robust cellular protective function against oxidative insults to maintain their hemostasis and viability; however, the underlying mechanism is largely obscure. In this study, we unveiled a new mechanism that protects lens fiber cells against oxidative stress-induced cell death. We found that mechano-activated connexin (Cx) hemichannels (HCs) mediate the transport of glutathione (GSH) into chick embryonic fibroblasts (CEF) and primary lens fiber cells, resulting in a decrease in the accumulation of intracellular reactive oxygen species induced by both H 2 O 2 and ultraviolet B, providing protection to lens fiber cells against cell apoptosis and necrosis. Furthermore, HCs formed by both homomeric Cx50 or Cx46 and heteromeric Cx50/Cx46 were mechanosensitive and could transport GSH into CEF cells. Notably, mechano-activated Cx50 HCs exhibited a greater capacity to transport GSH than Cx46 HCs. Consistently, the deficiency of Cx50 in single lens fiber cells led to a higher level of oxidative stress. Additionally, outer cortical short lens fiber cells expressing full length Cxs demonstrated greater resistance to oxidative injury compared to central core long lens fibers. Taken together, our results suggest that the activation of Cx HCs by interstitial fluid flow in cultured epithelial cells and isolated fiber cells shows that HCs can serve as a pathway for moving GSH across the cell membrane to offer protection against oxidative stress., Competing Interests: Declaration of competing interest Declarations of interest for all authors: none., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: To investigate the epidemiological characteristics and mutation spectrum of monogenic diseases in Chinese population through a large-scale, multicenter carrier screening., Methods: This study was conducted among a total of 33 104 participants (16 610 females) from 12 clinical centers across China.Carrier status for 223 genes was analyzed using high-throughput sequencing and different PCR methods., Results: The overall combined carrier frequency was 55.58% for 197 autosomal genes and 1.84% for 26 X-linked genes in these participants.Among the 16 669 families, 874 at-risk couples (5.24%) were identified.Specifically, 584 couples (3.50%) were at risk for autosomal genes, 306(1.84%) for X-linked genes, and 16 for both autosomal and X-linked genes.The most frequently detected autosomal at-risk genes included GJB2(autosomal recessive deafness type 1A, 393 couples), HBA1/HBA2(α-thalassemia, 36 couples), PAH (phenylketonuria, 14 couples), and SMN1(spinal muscular atrophy, 14 couples).The most frequently detected X-linked at-risk genes were G6PD (G6PD deficiency, 236 couples), DMD (Duchenne muscular dystrophy, 23 couples), and FMR1(fragile X syndrome, 17 couples).After excluding GJB2 c.109G>A, the detection rate of at-risk couples was 3.91%(651/16 669), which was lowered to 1.72%(287/16 669) after further excluding G6PD.The theoretical incidence rate of severe monogenic birth defects was approximately 4.35‰(72.5/16 669).Screening for a battery of the top 22 most frequent genes in the at-risk couples could detect over 95% of at-risk couples, while screening for the top 54 genes further increased the detection rate to over 99%., Conclusion: This study reveals the carrier frequencies of 223 monogenic genetic disorders in the Chinese population and provides evidence for carrier screening strategy development and panel design tailored to the Chinese population.In carrier testing, genetic counseling for specific genes or gene variants can be challenging, and the couples need to be informed of these difficulties before testing and provided with options for not screening these genes or gene variants.

Mutations in more than half of human connexin genes encoding gap junction (GJ) subunits have been linked to inherited human diseases. Functional studies of human GJ channels are essential for revealing mechanistic insights into the etiology of disease-linked connexin mutants. However, the commonly used Xenopus oocytes, N2A, HeLa, and other model cells for recombinant expression of human connexins have different and significant limitations. Here we developed a human cell line (HEK293) with each of the endogenous connexins (Cx43 and Cx45) knocked out using the CRISPR-Cas9 system. Double knockout HEK293 cells showed no background GJ coupling, were easily transfected with several human connexin genes (such as those encoding Cx46, Cx50, Cx37, Cx45, Cx26, and Cx36) which successfully formed functional GJs and were readily accessible for dual patch clamp analysis. Single knockout Cx43 or Cx45 HEK cell lines could also be used to characterize human GJ channels formed by Cx45 or Cx43, respectively, with an expression level suitable for studying macroscopic and single channel GJ channel properties. A cardiac arrhythmia linked Cx45 mutant R184G failed to form functional GJs in DKO HEK293 cells with impaired localizations. These genetically engineered HEK293 cells are well suited for patch clamp study of human GJ channels., (© 2024 The Author(s).)

Pannexin1 hemichannels (Panx1 HCs) are found in the membrane of most mammalian cells and communicate the intracellular and extracellular spaces, enabling the passive transfer of ions and small molecules. They are involved in physiological and pathophysiological conditions. During apoptosis, the C-terminal tail of Panx1 is proteolytically cleaved, but the permeability features of hemichannels and their role in cell death remain elusive. To address these topics, HeLa cells transfected with full-length human Panx1 (fl-hPanx1) or C-terminal truncated hPanx1 (Δ371hPanx1) were exposed to alkaline extracellular saline solution, increasing the activity of Panx1 HCs. The Δ371hPanx1 HC was permeable to DAPI and Etd + , but not to propidium iodide, whereas fl-hPanx1 HC was only permeable to DAPI. Furthermore, the cytoplasmic Ca 2+ signal increased only in Δ371hPanx1 cells, which was supported by bioinformatics approaches. The influx of Ca 2+ through Δ371hPanx1 HCs was necessary to promote cell death up to about 95% of cells, whereas the exposure to alkaline saline solution without Ca 2+ failed to induce cell death, and the Ca 2+ ionophore A23187 promoted more than 80% cell death even in fl-hPanx1 transfectants. Moreover, cell death was prevented with carbenoxolone or 10 Panx1 in Δ371hPanx1 cells, whereas it was undetectable in HeLa Panx1 -/- cells. Pretreatment with Ferrostatin-1 and necrostatin-1 did not prevent cell death, suggesting that ferroptosis or necroptosis was not involved. In comparison, zVAD-FMK, a pancaspase inhibitor, reduced death by ~60%, suggesting the involvement of apoptosis. Therefore, alkaline pH increases the activity of Δ371hPanx1HCs, leading to a critical intracellular free-Ca 2+ overload that promotes cell death., Competing Interests: Competing interests statement:V.M.B., a reviewer, and J.C.S. were on a retrospective for Michael Bennett together this year (https://pubmed.ncbi.nlm.nih.gov/38278514/).

Connexin hemichannels (HCs) expressed at the plasma membrane of mammalian cells are of paramount importance for intercellular communication. In physiological conditions, HCs can form gap junction (GJ) channels, providing a direct diffusive path between neighbouring cells. In addition, unpaired HCs provide conduits for the exchange of solutes between the cytoplasm and the extracellular milieu, including messenger molecules involved in paracrine signalling. The synergistic action of membrane potential and Ca 2+ ions controls the gating of the large and relatively unselective pore of connexin HCs. The four orders of magnitude difference in gating sensitivity to the extracellular ([Ca 2+ ] e ) and the cytosolic ([Ca 2+ ] c ) Ca 2+ concentrations suggests that at least two different Ca 2+ sensors may exist. While [Ca 2+ ] e acts as a spatial modulator of the HC opening, which is most likely dependent on the cell layer, compartment, and organ, [Ca 2+ ] c triggers HC opening and the release of extracellular bursts of messenger molecules. Such molecules include ATP, cAMP, glutamate, NAD + , glutathione, D-serine, and prostaglandins. Lost or abnormal HC regulation by Ca 2+ has been associated with several diseases, including deafness, keratitis ichthyosis, palmoplantar keratoderma, Charcot-Marie-Tooth neuropathy, oculodentodigital dysplasia, and congenital cataracts. The fact that both an increased and a decreased Ca 2+ sensitivity has been linked to pathological conditions suggests that Ca 2+ in healthy cells finely tunes the normal HC function. Overall, further investigation is needed to clarify the structural and chemical modifications of connexin HCs during [Ca 2+ ] e and [Ca 2+ ] c variations. A molecular model that accounts for changes in both Ca 2+ and the transmembrane voltage will undoubtedly enhance our interpretation of the experimental results and pave the way for developing therapeutic compounds targeting specific HC dysfunctions.