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Pathogenic mutations in the Gjb2 and Gjb6 genes, encoding connexin 26 (Cx26) and connexin 30 (Cx30), respectively, have been linked to the most frequent monogenic hearing impairment, nonsyndromic hearing loss, and deafness DFNB1. It is known that Cx26 plays an important role in auditory development, while the role of Cx30 in hearing remains controversial. Previous studies found that partial deletion of Cx26 can accelerate age-related hearing loss (ARHL), a multifactorial complex disorder, with both environmental and genetic factors contributing to the etiology of the disease. Here, we investigated the role of Cx30 in cochlear-aging processes using a transgenic mouse model with total deletion of Cx30 (Cx30 ΔΔ mice), in which Cx30 was removed without perturbing the surrounding sequences. We show that these mice are affected by exacerbated ARHL, with increased morphological cochlear damage, oxidative stress, inflammation, and vascular dysfunctions. Overall, our data demonstrate that Cx30 deletion can be considered a genetic risk factor for ARHL, making cochlear structures more susceptible to aging processes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Paciello, Zorzi, Raspa, Scavizzi, Grassi, Mammano and Fetoni.)

Severe SARS-CoV-2 infections are characterized by lymphopenia, but the mechanisms involved are still elusive. Based on our knowledge of HIV pathophysiology, we hypothesized that SARS-CoV-2 infection-mediated lymphopenia could also be related to T cell apoptosis. By comparing intensive care unit (ICU) and non-ICU COVID-19 patients with age-matched healthy donors, we found a strong positive correlation between plasma levels of soluble FasL (sFasL) and T cell surface expression of Fas/CD95 with the propensity of T cells to die and CD4 T cell counts. Plasma levels of sFasL and T cell death are correlated with CXCL10 which is part of the signature of 4 biomarkers of disease severity (ROC, 0.98). We also found that members of the Bcl-2 family had modulated in the T cells of COVID-19 patients. More importantly, we demonstrated that the pan-caspase inhibitor, Q-VD, prevents T cell death by apoptosis and enhances Th1 transcripts. Altogether, our results are compatible with a model in which T-cell apoptosis accounts for T lymphopenia in individuals with severe COVID-19. Therefore, a strategy aimed at blocking caspase activation could be beneficial for preventing immunodeficiency in COVID-19 patients., (© 2022. The Author(s).)

Connexin (Cx) hemichannels (HCs) are large pore hexameric structures that allow the exchange of ions, metabolites and a variety of other molecules between the cell cytoplasm and extracellular milieu. HC inhibitors are attracting growing interest as drug candidates because deregulated fluxes through HCs have been implicated in a plethora of genetic conditions and other diseases. HC activity has been mainly investigated by electrophysiological methods and/or using HC-permeable dye uptake measurements. Here, we present an all-optical assay based on fluorometric measurements of ionized calcium (Ca 2+ ) uptake with a Ca 2+ -selective genetically encoded indicator (GCaMP6s) that permits the optical tracking of cytosolic Ca 2+ concentration ([Ca 2+ ] cyt ) changes with high sensitivity. We exemplify use of the assay in stable pools of HaCaT cells overexpressing human Cx26, Cx46, or the pathological mutant Cx26G45E, under control of a tetracycline (Tet) responsive element (TRE) promoter (Tet-on). We demonstrate the usefulness of the assay for the characterization of new monoclonal antibodies (mAbs) targeting the extracellular domain of the HCs. Although we developed the assay on a spinning disk confocal fluorescence microscope, the same methodology can be extended seamlessly to high-throughput high-content platforms to screen other kinds of inhibitors and/or to probe HCs expressed in primary cells and microtissues.

Mutations in the GJB2 gene that encodes connexin 26 (Cx26) are the predominant cause of prelingual hereditary deafness, and the most frequently encountered variants cause complete loss of protein function. To investigate how Cx26 deficiency induces deafness, we examined the levels of apoptosis and autophagy in Gjb2 loxP/loxP ; ROSA26 CreER mice injected with tamoxifen on the day of birth. After weaning, these mice exhibited severe hearing impairment and reduced Cx26 expression in the cochlear duct. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive cells were observed in apical, middle, and basal turns of Kölliker's organ at postnatal (P) day 1 (P1), associated with increased expression levels of cleaved caspase 3, but decreased levels of autophagy-related proteins LC3-II, P62, and Beclin1. In Kölliker's organ cells with decreased Cx26 expression, we also found significantly reduced levels of intracellular ATP and hampered Ca 2+ responses evoked by extracellular ATP application. These results offer novel insight into the mechanisms that prevent hearing acquisition in mouse models of non-syndromic hearing impairment due to Cx26 loss of function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sun, Gao, Chen, Hou, Li, Huang, Mammano, Chen and Yang.)

Multimodal microscopy combines multiple non-linear techniques that take advantage of different optical processes to generate contrast and increase the amount of information that can be obtained from biological samples. However, the most advanced optical architectures are typically custom-made and often require on-site adjustment of optical components performed by trained personnel for optimal performance. Here, we describe a hybrid system we built based on a commercial upright microscope. We show that our multimodal imaging platform can be used to seamlessly perform two-photon STED, wavelength mixing and label-free microscopy in both ex vivo and in vivo turbid samples. The system is stable and endowed with remote alignment hardware that ensures long-term operability also for non-expert users, using the alignment protocol described in this article and in the related material. This optical architecture is an important step forward towards a wider practical applicability of non-linear optics to bioimaging., Competing Interests: The authors declare no conflicts of interest., (© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement.)

Clinical presentation of congenital heart disease is heterogeneous, making identification of the disease-causing genes and their genetic pathways and mechanisms of action challenging. By using in vivo electrocardiography, transthoracic echocardiography and microcomputed tomography imaging to screen 3,894 single-gene-null mouse lines for structural and functional cardiac abnormalities, here we identify 705 lines with cardiac arrhythmia, myocardial hypertrophy and/or ventricular dilation. Among these 705 genes, 486 have not been previously associated with cardiac dysfunction in humans, and some of them represent variants of unknown relevance (VUR). Mice with mutations in Casz1, Dnajc18, Pde4dip, Rnf38 or Tmem161b genes show developmental cardiac structural abnormalities, with their human orthologs being categorized as VUR. Using UK Biobank data, we validate the importance of the DNAJC18 gene for cardiac homeostasis by showing that its loss of function is associated with altered left ventricular systolic function. Our results identify hundreds of previously unappreciated genes with potential function in congenital heart disease and suggest causal function of five VUR in congenital heart disease., (© 2022. The Author(s).)

GJB2 and GJB6 are adjacent genes encoding connexin 26 (Cx26) and connexin 30 (Cx30), respectively, with overlapping expressions in the inner ear. Both genes are associated with the commonest monogenic hearing disorder, recessive isolated deafness DFNB1. Cx26 plays an important role in auditory development, while the role of Cx30 in hearing remains controversial. Previous studies found that Cx30 knockout mice had severe hearing loss along with a 90% reduction in Cx26, while another Cx30 knockout mouse model showed normal hearing with nearly half of Cx26 preserved. In this study, we used CRISPR/Cas9 technology to establish a new Cx30 knockout mouse model (Cx30 -/- ), which preserves approximately 70% of Cx26. We found that the 1, 3, and 6-month-old Cx30 -/- mice showed mild hearing loss at full frequency. Immunofluorescence and HE staining suggested no significant differences in microstructure of the cochlea between Cx30 -/- mice and wild-type mice. However, transmission electron microscopy showed slight cavity-like damage in the stria vascularis of Cx30 -/- mice. And Cx30 deficiency reduced the production of endocochlear potential (EP) and the release of ATP, which may have induced hearing loss. Taken together, this study showed that lack of Cx30 can lead to hearing loss with an approximately 30% reduction of Cx26 in the present Cx30 knockout model. Hence, Cx30 may play an important rather than redundant role in hearing development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chen, Chen, He, Gong, Li, Zhang, Lv, Mammano, Hou and Yang.)

The epidermis forms an essential barrier against a variety of insults. The overall goal of this study was to shed light not only on the effects of accidental epidermal injury, but also on the mechanisms that support laser skin resurfacing with intra-epidermal focal laser-induced photodamage, a widespread medical practice used to treat a range of skin conditions. To this end, we selectively photodamaged a single keratinocyte with intense, focused and pulsed laser radiation, triggering Ca 2+ waves in the epidermis of live anesthetized mice with ubiquitous expression of a genetically encoded Ca 2+ indicator. Waves expanded radially and rapidly, reaching up to eight orders of bystander cells that remained activated for tens of minutes, without displaying oscillations of the cytosolic free Ca 2+ concentration ([Formula: see text]). By combining in vivo pharmacological dissection with mathematical modeling, we demonstrate that Ca 2+ wave propagation depended primarily on the release of ATP, a prime damage-associated molecular patterns (DAMPs), from the hit cell. Increments of the [Formula: see text] in bystander cells were chiefly due to Ca 2+ release from the endoplasmic reticulum (ER), downstream of ATP binding to P2Y purinoceptors. ATP-dependent ATP release though connexin hemichannels (HCs) affected wave propagation at larger distances, where the extracellular ATP concentration was reduced by the combined effect of passive diffusion and hydrolysis due to the action of ectonucleotidases, whereas pannexin channels had no role. Bifurcation analysis suggests basal keratinocytes have too few P2Y receptors (P2YRs) and/or phospholipase C (PLC) to transduce elevated extracellular ATP levels into inositol trisphosphate (IP 3 ) production rates sufficiently large to sustain [Formula: see text] oscillations., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Physiological Society.)

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease characterized by depletion of motor neurons (MNs), for which effective medical treatments are still required. Previous transcriptomic analysis revealed the up-regulation of C-X-C motif chemokine receptor 2 (CXCR2)-mRNA in a subset of sporadic ALS patients and SOD1G93A mice. Here, we confirmed the increase of CXCR2 in human ALS cortex, and showed that CXCR2 is mainly localized in cell bodies and axons of cortical neurons. We also investigated the effects of reparixin, an allosteric inhibitor of CXCR2, in degenerating human iPSC-derived MNs and SOD1G93A mice. In vitro, reparixin rescued MNs from apoptotic cell death, preserving neuronal morphology, mitochondrial membrane potential and cytoplasmic membrane integrity, whereas in vivo it improved neuromuscular function of SOD1G93A mice. Altogether, these data suggest a role for CXCR2 in ALS pathology and support its pharmacological inhibition as a candidate therapeutic strategy against ALS at least in a specific subgroup of patients., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

In this study, we used B16-F10 cells grown in the dorsal skinfold chamber (DSC) preparation that allowed us to gain optical access to the processes triggered by photodynamic therapy (PDT). Partial irradiation of a photosensitized melanoma triggered cell death in non-irradiated tumor cells. Multiphoton intravital microscopy with genetically encoded fluorescence indicators revealed that bystander cell death was mediated by paracrine signaling due to adenosine triphosphate (ATP) release from connexin (Cx) hemichannels (HCs). Intercellular calcium (Ca 2+ ) waves propagated from irradiated to bystander cells promoting intracellular Ca 2+ transfer from the endoplasmic reticulum (ER) to mitochondria and rapid activation of apoptotic pathways. Combination treatment with S-nitrosoglutathione (GSNO), an endogenous nitric oxide (NO) donor that biases HCs towards the open state, greatly potentiated anti-tumor bystander killing via enhanced Ca 2+ signaling, leading to a significant reduction of post-irradiation tumor mass. Our results demonstrate that HCs can be exploited to dramatically increase cytotoxic bystander effects and reveal a previously unappreciated role for HCs in tumor eradication promoted by PDT.

Greater epithelial ridge cells, a transient neonatal cell group in the cochlear duct, which plays a crucial role in the functional maturation of hair cell, structural development of tectorial membrane, and refinement of audio localization before hearing. Greater epithelial ridge cells are methodologically homogeneous, while whether different cell subtypes are existence in this intriguing region and the degeneration mechanism during postnatal cochlear development are poorly understood. In the present study, single-cell RNA sequencing was performed on the cochlear duct of postnatal rats at day 1 (P1) and day 7 (P7) to identify subsets of greater epithelial ridge cell and progression. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were used to examine genes enriched biological processes in these clusters. We identified a total of 26 clusters at P1 and P7 rats and found that the cell number of five cell clusters decreased significantly, while four clusters had similar gene expression patterns and biological properties. The genes of these four cell populations were mainly enriched in Ribosome and P13K-Akt signal pathway. Among them, Rps16, Rpsa, Col4a2, Col6a2, Ctsk, and Jun are particularly interesting as their expression might contribute to the greater epithelial ridge cells degeneration. In conclusion, our study provides an important reference resource of greater epithelial ridge cells landscape and mechanism insights for further understanding greater epithelial ridge cells degeneration during postnatal rat cochlear development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chen, Gao, Chen, Hou, He, Li, Li, Zhang, Sun, Mammano, Sun, Yang and Zheng.)

Type I interferons (IFNs) are a family of cytokines that represent a first line of defense against virus infections. The 12 different IFN-α subtypes share a receptor on target cells and trigger similar signaling cascades. Several studies have collectively shown that this apparent redundancy conceals qualitatively different responses induced by individual subtypes, which display different efficacies of inhibition of HIV replication. Some studies, however, provided evidence that the disparities are quantitative rather than qualitative. Since RNA expression analyses show a large but incomplete overlap of the genes induced, they may support both models. To explore if the IFN-α subtypes induce functionally relevant different anti-HIV activities, we have compared the efficacies of inhibition of all 12 subtypes on HIV spread and on specific steps of the viral replication cycle, including viral entry, reverse transcription, protein synthesis, and virus release. Finding different hierarchies of inhibition would validate the induction of qualitatively different responses. We found that while most subtypes similarly inhibit virus entry, they display distinctive potencies on other early steps of HIV replication. In addition, only some subtypes were able to target effectively the late steps. The extent of induction of known anti-HIV factors helps to explain some, but not all differences observed, confirming the participation of additional IFN-induced anti-HIV effectors. Our findings support the notion that different IFN-α subtypes can induce the expression of qualitatively different antiviral activities. IMPORTANCE The initial response against viruses relies in large part on type I interferons, which include 12 subtypes of IFN-α. These cytokines bind to a common receptor on the cell surface and trigger the expression of incompletely overlapping sets of genes. Whether the anti-HIV responses induced by IFN-α subtypes differ in the extent of expression or in the nature of the genes involved remains debated. Also, RNA expression profiles led to opposite conclusions, depending on the importance attributed to the induction of common or distinctive genes. To explore if relevant anti-HIV activities can be differently induced by the IFN-α subtypes, we compared their relative efficacies on specific steps of the replication cycle. We show that the hierarchy of IFN potencies depends on the step analyzed, supporting qualitatively different responses. This work will also prompt the search for novel IFN-induced anti-HIV factors acting on specific steps of the replication cycle.

Background: Connexin hemichannels have been implicated in pathology-promoting conditions, including inflammation, numerous widespread human diseases, including cancer and diabetes, and several rare diseases linked to pathological point mutations., Methods: We analysed the literature focusing on antibodies capable of modulating hemichannel function, highlighting generation methods, applications to basic biomedical research and translational potential., Results: Anti-hemichannel antibodies generated over the past 3 decades targeted mostly connexin 43, with a focus on cancer treatment. A slow transition from relatively unselective polyclonal antibodies to more selective monoclonal antibodies resulted in few products with interesting characteristics that are under evaluation for clinical trials. Selection of antibodies from combinatorial phage-display libraries, has permitted to engineer a monoclonal antibody that binds to and blocks pathological hemichannels formed by connexin 26, 30 and 32., Conclusions: All known antibodies that modulate connexin hemichannels target the two small extracellular loops of the connexin proteins. The extracellular region of different connexins is highly conserved, and few residues of each connexins are exposed. The search for new antibodies may develop an unprecedented potential for therapeutic applications, as it may benefit tremendously from novel whole-cell screening platforms that permit in situ selection of antibodies against membrane proteins in native state. The demonstrated efficacy of mAbs in reaching and modulating hemichannels in vivo, together with their relative specificity for connexins overlapping epitopes, should hopefully stimulate an interest for widening the scope of anti-hemichannel antibodies. There is no shortage of currently incurable diseases for which therapeutic intervention may benefit from anti-hemichannel antibodies capable of modulating hemichannel function selectively and specifically., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Pathogenic mutations in the non-syndromic hearing loss and deafness 1 (DFNB1) locus are the primary cause of monogenic inheritance for prelingual hearing loss. To unravel molecular pathways involved in etiopathology and look for early degeneration biomarkers, we used a system biology approach to analyze Cx30 -/- mice at an early cochlear post-natal developmental stage. These mice are a DFNB1 mouse model with severely reduced expression levels of two connexins in the inner ear, Cx30, and Cx26. Integrated analysis of miRNA and mRNA expression profiles in the cochleae of Cx30 -/- mice at post-natal day 5 revealed the overexpression of five miRNAs (miR-34c, miR-29b, miR-29c, miR-141, and miR-181a) linked to apoptosis, oxidative stress, and cochlear degeneration, which have Sirt1 as a common target of transcriptional and/or post-transcriptional regulation. In young adult Cx30 -/- mice (3 months of age), these alterations culminated with blood barrier disruption in the Stria vascularis ( SV ), which is known to have the highest aerobic metabolic rate of all cochlear structures and whose microvascular alterations contribute to age-related degeneration and progressive decline of auditory function. Our experimental validation of selected targets links hearing acquisition failure in Cx30 -/- mice, early oxidative stress, and metabolic dysregulation to the activation of the Sirt1-p53 axis. This is the first integrated analysis of miRNA and mRNA in the cochlea of the Cx30 -/- mouse model, providing evidence that connexin downregulation determines a miRNA-mediated response which leads to chronic exhaustion of cochlear antioxidant defense mechanisms and consequent SV dysfunction. Our analyses support the notion that connexin dysfunction intervenes early on during development, causing vascular damage later on in life. This study identifies also early miRNA-mediated biomarkers of hearing impairment, either inherited or age related., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gentile, Paciello, Zorzi, Spampinato, Guarnaccia, Crispino, Tettey-Matey, Scavizzi, Raspa, Fetoni, Cavallaro and Mammano.)

There is an urgent need for the development of new anti-HIV drugs that can complement existing medicines to be used against resistant strains. Here, we report the anti-HIV-1 peptide pepRF1, a human serum-resistant peptide derived from the Dengue virus capsid protein. In vitro , pepRF1 shows a 50% inhibitory concentration of 1.5 nM with a potential therapeutic window higher than 53 000. This peptide is specific for CXCR4-tropic strains, preventing viral entry into target cells by binding to the viral coreceptor CXCR4, acting as an antagonist of this receptor. pepRF1 is more effective than T20, the only peptide-based HIV-1 entry inhibitor approved, and excels in inhibiting a HIV-1 strain resistant to T20. Potentially, pepRF1 can be used alone or in combination with other anti-HIV drugs. Furthermore, one can also envisage its use as a novel therapeutic strategy for other CXCR4-related diseases.

Objective: HIV-1 transmission leads to a genetic bottleneck, with one or a few variants of the donor quasispecies establishing an infection in the new host. We aimed to characterize this bottleneck in more detail, by comparing the properties of HIV envelope glycoproteins from acute and chronic infections within the particular context of a male-to-male transmission cluster., Design: We compared the genotypic and phenotypic properties of envelope glycoproteins from viral variants derived from five study participants from the same transmission cluster., Methods: We used single-genome amplification to generate a collection of full-length env sequences. We then constructed pseudotyped viruses expressing selected Env variants from the quasispecies infecting each study participant and compared their infectivities and sensitivities to various entry inhibitors., Results: The genotypic analyses confirmed the genetic bottleneck expected after HIV transmission, with a limited number of variants identified in four study participants during acute infection. However, the transmitted sequences harbored no evident common signature and belonged to various genetic lineages. The phenotypic analyses revealed no difference in infectivity, susceptibility to the CCR5 antagonist maraviroc, the fusion inhibitor enfurvitide or type-I interferon between viruses from participants with acute and chronic infections. The key property distinguishing transmitted viruses was a higher resistance to soluble CD4, correlated with greater sensitivity to occupation of the CD4 receptor by the anti-CD4 antibodies LM52 and SK3., Conclusion: These results suggest that envelope glycoproteins from transmitted/founder viruses bind CD4 less efficiently than those of viruses from chronic infections.

Despite early antiretroviral therapy (ART), treatment interruption is associated with viral rebound, indicating early viral reservoir (VR) seeding and absence of full eradication of human immunodeficiency virus type 1 (HIV-1) that may persist in tissues. Herein, we address the contributing role of monocytes in maintaining VRs under ART, since these cells may represent a source of viral dissemination due to their ability to replenish mucosal tissues in response to injury. To this aim, monocytes with classical (CD14 + ), intermediate (CD14 + CD16 + ), and nonclassical (CD16 + ) phenotypes and CD4 + T cells were sorted from the blood, spleen, and intestines of untreated and early-ART-treated simian immunodeficiency virus (SIV)-infected rhesus macaques (RMs) before and after ART interruption. Cell-associated SIV DNA and RNA were quantified. We demonstrated that in the absence of ART, monocytes were productively infected with replication-competent SIV, especially in the spleen. Reciprocally, early ART efficiently (i) prevented the establishment of monocyte VRs in the blood, spleen, and intestines and (ii) reduced systemic inflammation, as indicated by changes in interleukin-18 (IL-18) and IL-1 receptor antagonist (IL-1Ra) plasma levels. ART interruption was associated with a rebound in viremia that led to the rapid productive infection of both CD4 + T cells and monocytes. Altogether, our results reveal the benefits of early ART initiation in limiting the contribution of monocytes to VRs and SIV-associated inflammation. IMPORTANCE Despite the administration of antiretroviral therapy (ART), HIV persists in treated individuals and ART interruption is associated with viral rebound. Persistent chronic immune activation and inflammation contribute to disease morbidity. Whereas monocytes are infected by HIV/SIV, their role as viral reservoirs (VRs) in visceral tissues has been poorly explored. Our work demonstrates that monocyte cell subsets in the blood, spleen, and intestines do not significantly contribute to the establishment of early VRs in SIV-infected rhesus macaques treated with ART. By preventing the infection of these cells, early ART reduces systemic inflammation. However, following ART interruption, monocytes are rapidly reinfected. Altogether, our findings shed new light on the benefits of early ART initiation in limiting VR and inflammation., (Copyright © 2020 American Society for Microbiology.)

Autophagy and apoptosis have a complex interplay in the early embryo development. The development of spiral ganglion neurons (SGNs) in addition to Corti's organ in the mammalian cochlea remains crucial in the first two-week postnatal period. To investigate the roles of apoptosis and autophagy in the development of SGNs, light microscopy was used to observe the morphological changes of SGNs. The number of SGNs was decreased from P1 to P7 and plateaued from P10 to P14. Immunohistochemistry results revealed positive expression of cleaved-caspase3, bcl-2, microtubule-associated protein light chain 3-II (LC3-II), Beclin1, and sequestosome 1 (SQSTM1/P62) in SGNs. The apoptotic bodies and autophagosomes and autolysosomes were also identified by transmission electron microscopy at P1 and P7. Real-time PCR and western blotting results revealed that the apoptotic activity peaked at P7 and the autophagy activity was gradually upregulated along with the development. Taken together, our results for the first time showed that autophagy and apoptosis in SGNs play distinct roles during specific developmental phases in a time-dependent manner., Competing Interests: The authors declare no competing financial interests., (Copyright © 2020 Shule Hou et al.)

Prior work supports the hypothesis that ATP release through connexin hemichannels drives spontaneous Ca2+ signaling in non-sensory cells of the greater epithelial ridge (GER) in the developing cochlea; however, direct proof is lacking. To address this issue, we plated cochlear organotypic cultures (COCs) and whole cell-based biosensors with nM ATP sensitivity (ATP-WCBs) at the bottom and top of an ad hoc designed transparent microfluidic chamber, respectively. By performing dual multiphoton Ca2+ imaging, we monitored the propagation of intercellular Ca2+ waves in the GER of COCs and ATP-dependent Ca2+ responses in overlying ATP-WCBs. Ca2+ signals in both COCs and ATP-WCBs were inhibited by supplementing the extracellular medium with ATP diphosphohydrolase (apyrase). Spontaneous Ca2+ signals were strongly depressed in the presence of Gjb6-/- COCs, in which connexin 30 (Cx30) is absent and connexin 26 (Cx26) is strongly downregulated. In contrast, spontaneous Ca2+ signals were not affected by replacement of Panx1-/- with Panx1+/+ COCs in the microfluidic chamber. Similar results were obtained by estimating ATP release from COCs using a classical luciferin-luciferase bioluminescence assay. Therefore, connexin hemichannels and not pannexin 1 channels mediate the release of ATP that is responsible for Ca2+ wave propagation in the developing mouse cochlea. The technological advances presented here have the potential to shed light on a plethora of unrelated open issues that involve paracrine signaling in physiology and pathology and cannot be addressed with standard methods.

Background: Numerous currently incurable human diseases have been causally linked to mutations in connexin (Cx) genes. In several instances, pathological mutations generate abnormally active Cx hemichannels, referred to also as "leaky" hemichannels. The goal of this study was to assay the in vivo efficacy of a potent antagonist antibody targeting Cx hemichannels., Methods: We employed the antibody to treat Cx30 A88V/A88V adult mutant mice, the only available animal model of Clouston syndrome, a rare orphan disease caused by Cx30 p.A88V leaky hemichannels. To gain mechanistic insight into antibody action, we also performed patch clamp recordings, Ca 2+ imaging and ATP release assay in vitro., Findings: Two weeks of antibody treatment sufficed to repress cell hyperproliferation in skin and reduce hypertrophic sebaceous glands (SGs) to wild type (wt) levels. These effects were obtained whether mutant mice were treated topically, by application of an antibody cream formulation, or systemically, by intraperitoneal antibody injection. Experiments with mouse primary keratinocytes and HaCaT cells revealed the antibody blocked Ca 2+ influx and diminished ATP release through leaky Cx30 p.A88V hemichannels., Interpretation: Our results show anti-Cx antibody treatment was effective in vivo and sufficient to counteract the effects of pathological connexin expression in Cx30 A88V/A88V mice. In vitro experiments suggest antibodies gained control over leaky hemichannels and contributed to restoring epidermal homeostasis. Therefore, regulating cell physiology by antibodies targeting the extracellular domain of Cxs may enforce an entirely new therapeutic strategy. These findings support the further development of antibodies as drugs to address unmet medical needs for Cx-related diseases. FUND: Fondazione Telethon, GGP19148; University of Padova, SID/BIRD187130; Consiglio Nazionale delle Ricerche, DSB.AD008.370.003\TERABIO-IBCN; National Science Foundation of China, 31770776; Science and Technology Commission of Shanghai Municipality, 16DZ1910200., Competing Interests: Declaration of Competing Interest Drs. F. Mammano, G. Yang and F. Zonta report a patent: “Fully human antibody specifically inhibiting connexin 26”, Inventors: Qu Z, Yang G, Mammano F, Zonta F, International application number: PCT/CN2016/109847, pending to ShanghaiTech University; and a patent: “Composition and Methods to treat Ectodermal Dysplasia 2, Clouston Type”, Inventors: Mammano F, Yang G, Zonta F, International Application No.: PCT/CN2019/088689, International Filing Date: 2019-05-28, pending to ShanghaiTech University. All other Authors have nothing to declare., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

The identification of causal variants in sequencing studies remains a considerable challenge that can be partially addressed by new gene-specific knowledge. Here, we integrate measures of how essential a gene is to supporting life, as inferred from viability and phenotyping screens performed on knockout mice by the International Mouse Phenotyping Consortium and essentiality screens carried out on human cell lines. We propose a cross-species gene classification across the Full Spectrum of Intolerance to Loss-of-function (FUSIL) and demonstrate that genes in five mutually exclusive FUSIL categories have differing biological properties. Most notably, Mendelian disease genes, particularly those associated with developmental disorders, are highly overrepresented among genes non-essential for cell survival but required for organism development. After screening developmental disorder cases from three independent disease sequencing consortia, we identify potentially pathogenic variants in genes not previously associated with rare diseases. We therefore propose FUSIL as an efficient approach for disease gene discovery.

In cells, photosensitizer (PS) activation by visible light irradiation triggers reactive oxygen species (ROS) formation, followed by a cascade of cellular responses involving calcium (Ca 2+ ) and other second messengers, resulting in cell demise. Cytotoxic effects spread to nearby cells not exposed to light by poorly characterized so-called "bystander effects". To elucidate the mechanisms involved in bystander cell death, we used both genetically encoded biosensors and fluorescent dyes. In particular, we monitored the kinetics of interorganellar Ca 2+ transfer and the production of mitochondrial superoxide anion (O 2 - ∙ ) and hydrogen peroxide (H 2 O 2 ) in irradiated and bystander B16-F10 mouse melanoma cancer cells. We determined that focal PS photoactivation in a single cell triggers Ca 2+ release from the endoplasmic reticulum (ER) also in the surrounding nonexposed cells, paralleled by mitochondrial Ca 2+ uptake. Efficient Ca 2+ efflux from the ER was required to promote mitochondrial O 2 - ∙ production in these bystander cells. Our results support a key role for ER-mitochondria communication in the induction of ROS-mediated apoptosis in both direct and indirect photodynamical cancer cell killing.

Connexin 26 and connexin 30 are the prevailing isoforms in the epithelial and connective tissue gap junction systems of the developing and mature cochlea. The most frequently encountered variants of the genes that encode these connexins, which are transcriptionally coregulated, determine complete loss of protein function and are the predominant cause of prelingual hereditary deafness. Reducing connexin 26 expression by Cre/loxP recombination in the inner ear of adult mice results in a decreased endocochlear potential, increased hearing thresholds, and loss of >90% of outer hair cells, indicating that this connexin is essential for maintenance of cochlear function. In the developing cochlea, connexins are necessary for intercellular calcium signaling activity. Ribbon synapses and basolateral membrane currents fail to mature in inner hair cells of mice that are born with reduced connexin expression, even though hair cells do not express any connexin. In contrast, pannexin 1, an alternative mediator of intercellular signaling, is dispensable for hearing acquisition and auditory function., (Copyright © 2019 Cold Spring Harbor Laboratory Press; all rights reserved.)

Connexin hemichannels, which are plasma membrane hexameric channels (connexons) composed of connexin protein protomers, have been implicated in a host of physiological processes and pathological conditions. A number of single point pathological mutations impart a "leaky" character to the affected hemichannels, i.e., make them more active or hyperactive, suggesting that normal physiological condition could be recovered using selective hemichannel inhibitors. Recently, a human-derived monoclonal antibody named abEC1.1 has been shown to inhibit both wild type and hyperactive hemichannels composed of human (h) connexin 26 (hCx26) subunits. The aims of this work were (1) to characterize further the ability of abEC1.1 to selectively modulate connexin hemichannel function and (2) to assess its in vitro stability in view of future translational applications. In silico analysis of abEC1.1 interaction with the hCx26 hemichannel identified critically important extracellular domain amino acids that are conserved in connexin 30 (hCx30) and connexin 32 (hCx32). Patch clamp experiments performed in HeLa DH cells confirmed the inhibition efficiency of abEC1.1 was comparable for hCx26, hCx30 and hCx32 hemichannels. Of note, even a single amino acid difference in the putative binding region reduced drastically the inhibitory effects of the antibody on all the other tested hemichannels, namely hCx30.2/31.3, hCx30.3, hCx31, hCx31.1, hCx37, hCx43 and hCx45. Plasma membrane channels composed of pannexin 1 were not affected by abEC1.1. Finally, size exclusion chromatography assays showed the antibody does not aggregate appreciably in vitro . Altogether, these results indicate abEC1.1 is a promising tool for further translational studies.

Outer hair cells (OHCs) are highly specialized sensory cells conferring the fine-tuning and high sensitivity of the mammalian cochlea to acoustic stimuli. Here, by genetically manipulating spontaneous Ca 2+ signalling in mice in vivo, through a period of early postnatal development, we find that the refinement of OHC afferent innervation is regulated by complementary spontaneous Ca 2+ signals originating in OHCs and non-sensory cells. OHCs fire spontaneous Ca 2+ action potentials during a narrow period of neonatal development. Simultaneously, waves of Ca 2+ activity in the non-sensory cells of the greater epithelial ridge cause, via ATP-induced activation of P2X 3 receptors, the increase and synchronization of the Ca 2+ activity in nearby OHCs. This synchronization is required for the refinement of their immature afferent innervation. In the absence of connexin channels, Ca 2+ waves are impaired, leading to a reduction in the number of ribbon synapses and afferent fibres on OHCs. We propose that the correct maturation of the afferent connectivity of OHCs requires experience-independent Ca 2+ signals from sensory and non-sensory cells., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Objectives: Ultra-deep sequencing (UDS) is a powerful tool for exploring the impact on virological outcome of minority variants with low frequencies, some even <1% of the virus population. Here, we compared HIV-1 minority variants at baseline, through plasma RNA and PBMC DNA analyses, and the dominant variants at the virological failure (VF) point, to evaluate the impact of minority drug-resistant variants (MDRVs) on virological outcomes., Methods: Single-molecule real-time sequencing (SMRTS) was performed on baseline RNA and DNA. The Stanford HIV-1 drug resistance database was used for the identification and evaluation of drug resistance-associated mutations (DRAMs)., Results: We classified 50 patients into virological success (VS) and VF groups. We found that the rates of reverse transcriptase inhibitor (RTI) DRAMs determined by SMRTS did not differ significantly within or between groups, whether based on RNA or DNA analyses. There was no significant difference in the level of resistance to specific drugs between groups, in either DNA or RNA analyses, except for the DNA-based analysis of lamivudine, for which there was a trend towards a higher prevalence of intermediate/high-level resistance in the VF group. The RNA MDRVs corresponded to DNA MDRVs, except for M100I and Y188H. Sequencing from DNA appeared to be more sensitive than from RNA to detect MDRVs., Conclusions: Detection of pretreatment minority HIV-1 RTI-resistant variants by UDS showed that MDRVs at baseline were not significantly associated with virological outcome. However, HIV-1 DNA sequencing by UDS was useful for detecting pretreatment drug resistance mutations in patients, potentially affecting virological responses, suggesting a potential clinical relevance for ultra-deep DNA sequencing., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

HIV-1 mutations rapidly accumulate through genetic recombination events, which require the infection of a single cell by two virions (coinfection). Accumulation of mutations in the viral population may lead to immune escape and high-level drug resistance. The existence of cell subpopulations characterized by different susceptibility to HIV-1 infection has been proposed as an important parameter driving coinfection (Dang et al., 2004). While the mechanism and the quantification of HIV-1 coinfection have been recently investigated by mathematical models, the detailed dynamics of this process during cell-free infection remains elusive. In this study, we constructed ordinary differential equations considering the heterogeneity of target cell populations during cell-free infection in cell culture, and reproduced the cell culture experimental data. Our mathematical analyses showed that the presence of two differently susceptible target cell subpopulations could explain our experimental datasets, while increasing the number of subpopulations did not improve the fitting. In addition, we quantitatively demonstrated that cells infected by multiple viruses mainly accumulated from one cell subpopulation under cell-free infection conditions. In particular, the frequency of infection events in the more susceptible subpopulation was 6.11-higher than that from the other subpopulation, and 98.3% of coinfected cells emerged from the more susceptible subpopulation. Our mathematical-experimental approach is able to extract such a quantitative information, and can be easily applied to other virus infections., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Mutations in GJB2, the gene that encodes connexin 26 (Cx26), are the most common cause of sensorineural hearing impairment. The truncating variant 35delG, which determines a complete loss of Cx26 protein function, is the prevalent GJB2 mutation in several populations. Here, we generated and analyzed Gjb2 +/- mice as a model of heterozygous human carriers of 35delG. Compared to control mice, auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs) worsened over time more rapidly in Gjb2 +/- mice, indicating they were affected by accelerated age-related hearing loss (ARHL), or presbycusis. We linked causally the auditory phenotype of Gjb2 +/- mice to apoptosis and oxidative damage in the cochlear duct, reduced release of glutathione from connexin hemichannels, decreased nutrient delivery to the sensory epithelium via cochlear gap junctions and deregulated expression of genes that are under transcriptional control of the nuclear factor erythroid 2-related factor 2 (Nrf2), a pivotal regulator of tolerance to redox stress. Moreover, a statistically significant genome-wide association with two genes (PRKCE and TGFB1) related to the Nrf2 pathway (p-value < 4 × 10 -2 ) was detected in a very large cohort of 4091 individuals, originating from Europe, Caucasus and Central Asia, with hearing phenotype (including 1076 presbycusis patients and 1290 healthy matched controls). We conclude that (i) elements of the Nrf2 pathway are essential for hearing maintenance and (ii) their dysfunction may play an important role in the etiopathogenesis of human presbycusis., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Connexin channels play numerous essential roles in virtually every organ by mediating solute exchange between adjacent cells, or between cytoplasm and extracellular milieu. Our understanding of the structure-function relationship of connexin channels relies on X-ray crystallographic data for human connexin 26 (hCx26) intercellular gap junction channels. Comparison of experimental data and molecular dynamics simulations suggests that the published structures represent neither fully-open nor closed configurations. To facilitate the search for alternative stable configurations, we developed a coarse grained (CG) molecular model of the hCx26 hemichannel and studied its responses to external electric fields. When challenged by a field of 0.06 V/nm, the hemichannel relaxed toward a novel configuration characterized by a widened pore and an increased bending of the second transmembrane helix (TM2) at the level of the conserved Pro87. A point mutation that inhibited such transition in our simulations impeded hemichannel opening in electrophysiology and dye uptake experiments conducted on HeLa tranfectants. These results suggest that the hCx26 hemichannel uses a global degree of freedom to transit between different configuration states, which may be shared among the whole connexin family.

In mammals, the sense of hearing arises through a complex sequence of morphogenetic events that drive the sculpting of the auditory sensory epithelium into its terminally functional three-dimensional shape. While the majority of the underlying mechanisms remain unknown, it has become increasingly clear that Ca 2+ signaling is at center stage and plays numerous fundamental roles both in the sensory hair cells and in the matrix of non-sensory, epithelial and supporting cells, which embed them and are tightly interconnected by a dense network of gap junctions formed by connexin 26 (Cx26) and connexin 30 (Cx30) protein subunits. In this review, we discuss the intricate interplay between Ca 2+ signaling, connexin expression and function, apoptosis and autophagy in the crucial steps that lead to hearing acquisition., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

HIV-infected subjects under antiretroviral treatment (ART) harbor a persistent viral reservoir in resting CD4 + T cells, which accounts for the resurgence of HIV replication after ART interruption. A large majority of HIV reservoir genomes are genetically defective, but even among intact proviruses few seem able to generate infectious virus. To understand this phenomenon, we examined the function and expression of HIV envelope glycoproteins reactivated from the reservoir of four HIV-infected subjects under suppressive ART. We studied full-length genetically intact env sequences from both replicative viruses and cell-associated mRNAs. We found that these Env proteins varied extensively in fusogenicity and infectivity, with strongest functional defects found in Envs from cell-associated mRNAs. Env functional impairments were essentially explained by defects in Env protein expression. Our results support the idea that defects in HIV Env expression, preventing cytopathic or immune HIV clearance, contribute to the persistence of the HIV T-cell reservoir in vivo IMPORTANCE In most individuals, evolution of HIV infection is efficiently controlled on the long-term by combination antiviral therapies. These treatments, however, fail to eradicate HIV from the infected subjects, a failure that results both in resurgence of virus replication and in resumption of HIV pathogenicity when the treatment is stopped. HIV resurgence, in these instances, is widely assumed to emerge from a reservoir of silent virus integrated in the genomes of a small number of T lymphocytes. The silent HIV reservoir is mostly composed of heavily deleted or mutated HIV DNA. Moreover, among the seemingly intact remaining HIV, only very few are actually able to efficiently propagate in tissue culture. In this study, we find that intact HIV in the reservoir often carry strong defects in their capacity to promote fusion to neighboring cells and infection of target cells, a defect related to the function and expression of the HIV envelope glycoprotein. Impaired envelope glycoprotein expression and function could explain why cells harboring these viruses tend to remain undetected and unharmed in the reservoir., (Copyright © 2018 American Society for Microbiology.)

Hair cells of the inner ear detect sound stimuli, inertial or gravitational forces by deflection of their apical stereocilia. A small number of stereociliary cation-selective mechanotransduction (MET) channels admit K + and Ca 2+ ions into the cytoplasm promoting hair cell membrane depolarization and, consequently, neurotransmitter release at the cell basolateral pole. Ca 2+ influx into the stereocilia compartment is counteracted by the unusual w/a splicing variant of plasma-membrane calcium-pump isoform 2 (PMCA2) which, unlike other PMCA2 variants, increases only marginally its activity in response to a rapid variation of the cytoplasmic free Ca 2+ concentration ([Ca 2+ ] c ). Missense mutations of PMCA2w/a cause deafness and loss of balance in humans. Mouse models in which the pump is genetically ablated or mutated show hearing and balance impairment, which correlates with defects in homeostatic regulation of stereociliary [Ca 2+ ] c , decreased sensitivity of mechanotransduction channels to hair bundle displacement and progressive degeneration of the organ of Corti. These results highlight a critical role played by the PMCA2w/a pump in the control of hair cell function and survival, and provide mechanistic insight into the etiology of deafness and vestibular disorders., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Panx1 forms plasma membrane channels in brain and several other organs, including the inner ear. Biophysical properties, activation mechanisms and modulators of Panx1 channels have been characterized in detail, however the impact of Panx1 on auditory function is unclear due to conflicts in published results. To address this issue, hearing performance and cochlear function of the Panx1 -/- mouse strain, the first with a reported global ablation of Panx1 , were scrutinized. Male and female homozygous ( Panx1 -/-), hemizygous ( Panx1 +/-) and their wild type (WT) siblings ( Panx1 +/+) were used for this study. Successful ablation of Panx1 was confirmed by RT-PCR and Western immunoblotting in the cochlea and brain of Panx1 -/- mice. Furthermore, a previously validated Panx1-selective antibody revealed strong immunoreactivity in WT but not in Panx1 -/- cochleae. Hearing sensitivity, outer hair cell-based "cochlear amplifier" and cochlear nerve function, analyzed by auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) recordings, were normal in Panx1 +/- and Panx1 -/- mice. In addition, we determined that global deletion of Panx1 impacts neither on connexin expression, nor on gap-junction coupling in the developing organ of Corti. Finally, spontaneous intercellular Ca 2+ signal (ICS) activity in organotypic cochlear cultures, which is key to postnatal development of the organ of Corti and essential for hearing acquisition, was not affected by Panx1 ablation. Therefore, our results provide strong evidence that, in mice, Panx1 is dispensable for hearing acquisition and auditory function.

Background: Mutations leading to changes in properties, regulation, or expression of connexin-made channels have been implicated in 28 distinct human hereditary diseases. Eight of these result from variants of connexin 26 (Cx26), a protein critically involved in cell-cell signaling in the inner ear and skin. Lack of non-toxic drugs with defined mechanisms of action poses a serious obstacle to therapeutic interventions for diseases caused by mutant connexins. In particular, molecules that specifically modulate connexin hemichannel function without affecting gap junction channels are considered of primary importance for the study of connexin hemichannel role in physiological as well as pathological conditions. Monoclonal antibodies developed in the last three decades have become the most important class of therapeutic biologicals. Recombinant methods permit rapid selection and improvement of monoclonal antibodies from libraries with large diversity. Methods: By screening a combinatorial library of human single-chain fragment variable (scFv) antibodies expressed in phage, we identified a candidate that binds an extracellular epitope of Cx26. We characterized antibody action using a variety of biochemical and biophysical assays in HeLa cells, organotypic cultures of mouse cochlea and human keratinocyte-derived cells. Results: We determined that the antibody is a remarkably efficient, non-toxic, and completely reversible inhibitor of hemichannels formed by connexin 26 and does not affect direct cell-cell communication via gap junction channels. Importantly, we also demonstrate that the antibody efficiently inhibits hyperative mutant Cx26 hemichannels implicated in autosomal dominant non-syndromic hearing impairment accompanied by keratitis and hystrix-like ichthyosis-deafness (KID/HID) syndrome. We solved the crystal structure of the antibody, identified residues that are critical for binding and used molecular dynamics to uncover its mechanism of action. Conclusions: Although further studies will be necessary to validate the effect of the antibody in vivo , the methodology described here can be extended to select antibodies against hemichannels composed by other connexin isoforms and, consequently, to target other pathologies associated with hyperactive hemichannels. Our study highlights the potential of this approach and identifies connexins as therapeutic targets addressable by screening phage display libraries expressing human randomized antibodies.

We have previously shown that in vitro transduction with bovine adeno-associated viral (BAAV) vectors restores connexin expression and rescues gap junction coupling in cochlear organotypic cultures from connexin-deficient mice that are models DFNB1 nonsyndromic hearing loss and deafness. The aims of this study were to manipulate inner ear connexin expression in vivo using BAAV vectors, and to identify the optimal route of vector delivery. Injection of a BAAV vector encoding a bacterial Cre recombinase via canalostomy in adult mice with floxed connexin 26 (Cx26) alleles promoted Cre/LoxP recombination, resulting in decreased Cx26 expression, decreased endocochlear potential, increased hearing thresholds, and extensive loss of outer hair cells. Injection of a BAAV vector encoding GFP-tagged Cx30 via canalostomy in P4 mice lacking connexin 30 (Cx30) promoted formation of Cx30 gap junctions at points of contacts between adjacent non-sensory cells of the cochlear sensory epithelium. Levels of exogenous Cx30 decayed over time, but were still detectable four weeks after canalostomy. Our results suggest that persistence of BAAV-mediated gene replacement in the cochlea is limited by the extensive remodeling of the organ of Corti throughout postnatal development and associated loss of non-sensory cells.

HIV-1 accumulates changes in its genome through both recombination and mutation during the course of infection. For recombination to occur, a single cell must be infected by two HIV strains. These coinfection events were experimentally demonstrated to occur more frequently than would be expected for independent infection events and do not follow a random distribution. Previous mathematical modeling approaches demonstrated that differences in target cell susceptibility can explain the non-randomness, both in the context of direct cell-to-cell transmission, and in the context of free virus transmission (Q. Dang et al., Proc. Natl. Acad. Sci. USA 101:632-7, 2004: K. M. Law et al., Cell reports 15:2711-83, 2016). Here, we build on these notions and provide a more detailed and extensive quantitative framework. We developed a novel mathematical model explicitly considering the heterogeneity of target cells and analysed datasets of cell-free HIV-1 single and double infection experiments in cell culture. Particularly, in contrast to the previous studies, we took into account the different susceptibility of the target cells as a continuous distribution. Interestingly, we showed that the number of infection events per cell during cell-free HIV-1 infection follows a negative-binomial distribution, and our model reproduces these datasets.

Objectives: In the ANRS IPERGAY pre-exposure prophylaxis (PrEP) trial, a single dose of tenofovir disoproxil fumarate and emtricitabine was taken orally 2-24 h before sexual intercourse. A sub-study was conducted to assess the pharmacokinetics of tenofovir and emtricitabine in blood, saliva and rectal tissue following this initial oral intake., Methods: Plasma, PBMC, saliva and rectal tissue sampling was performed over 24 h in 12 seronegative men before enrolment in the ANRS IPERGAY trial, following a single dose of 600 mg tenofovir disoproxil fumarate/400 mg emtricitabine. Ex vivo HIV infectibility of rectal biopsies was also assessed., Results: The median plasma T max of tenofovir (median C max : 401 μg/L) and emtricitabine (median C max : 2868 μg/L) was obtained 1 h (range: 0.5-4) and 2 h (range: 1-4) after dosing, respectively. The median C 24 of tenofovir and emtricitabine was 40 and 63 μg/L, respectively. The median PBMC tenofovir diphosphate and emtricitabine triphosphate levels were 12.2 and 16.7 fmol/10 6 cells and 2800 and 2000 fmol/10 6 cells at 2 and 24 h after dosing, respectively. Saliva/plasma AUC 0-24 ratios were 2% and 17% for tenofovir and emtricitabine, respectively. Emtricitabine was detected in rectal tissue 30 min after dosing, whereas tenofovir was only detectable at 24 h. Ex vivo HIV infectibility assays of rectal biopsies showed partial protection after dosing (P < 0.07)., Discussion: A single high dose of oral tenofovir disoproxil fumarate/emtricitabine provides rapid and high blood levels of tenofovir and emtricitabine, with rapid diffusion of emtricitabine in saliva and rectal tissue., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Mutations in the genes encoding for gap junction proteins connexin 26 (Cx26) and connexin 30 (Cx30) have been linked to syndromic and nonsyndromic hearing loss in mice and humans. The release of ATP from connexin hemichannels in cochlear nonsensory cells has been proposed to be the main trigger for action potential activity in immature sensory inner hair cells (IHCs), which is crucial for the refinement of the developing auditory circuitry. Using connexin knock-out mice, we show that IHCs fire spontaneous action potentials even in the absence of ATP-dependent intercellular Ca 2+ signaling in the nonsensory cells. However, this signaling from nonsensory cells was able to increase the intrinsic IHC firing frequency. We also found that connexin expression is key to IHC functional maturation. In Cx26 conditional knock-out mice (Cx26 Sox10-Cre ), the maturation of IHCs, which normally occurs at approximately postnatal day 12, was partially prevented. Although Cx30 has been shown not to be required for hearing in young adult mice, IHCs from Cx30 knock-out mice exhibited a comprehensive brake in their development, such that their basolateral membrane currents and synaptic machinery retain a prehearing phenotype. We propose that IHC functional differentiation into mature sensory receptors is initiated in the prehearing cochlea provided that the expression of either connexin reaches a threshold level. As such, connexins regulate one of the most crucial functional refinements in the mammalian cochlea, the disruption of which contributes to the deafness phenotype observed in mice and DFNB1 patients., Significance Statement: The correct development and function of the mammalian cochlea relies not only on the sensory hair cells, but also on the surrounding nonsensory cells. Although the nonsensory cells have been largely implicated in the general homeostasis in the mature cochlea, their involvement in the initial functional differentiation of the sensory inner hair cells is less clear. Using mutant mouse models for the most common form of congenital deafness in humans, which are knock-outs for the gap-junction channels connexin 26 and connexin 30 genes, we show that defects in nonsensory cells prevented the functional maturation of inner hair cells. In connexin knock-outs, inner hair cells remained stuck at a prehearing stage of development and, as such, are unable to process sound information., (Copyright © 2017 Johnson, Ceriani et al.)

Treatment of HIV-infected patients with IFN-α results in significant, but clinically insufficient, reductions of viremia. IFN induces the expression of several antiviral proteins including BST-2, which inhibits HIV by multiple mechanisms. The viral protein Vpu counteracts different effects of BST-2. We thus asked if Vpu proteins from IFN-treated patients displayed improved anti-BST-2 activities as compared to Vpu from baseline. Deep-sequencing analyses revealed that in five of seven patients treated by IFN-α for a concomitant HCV infection in the absence of antiretroviral drugs, the dominant Vpu sequences differed before and during treatment. In three patients, vpu alleles that emerged during treatment improved virus replication in the presence of IFN-α, and two of them conferred improved virus budding from cells expressing BST-2. Differences were observed for the ability to down-regulate CD4, while all Vpu variants potently down-modulated BST-2 from the cell surface. This report discloses relevant consequences of IFN-treatment on HIV properties., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Introduction: Epidemiological studies suggest that statins may promote the development or exacerbation of diabetes, but whether this occurs through inhibition of insulin secretion is unclear. This lack of understanding is partly due to the cellular models used to explore this phenomenon (cell lines or pooled islets), which are non-physiologic and have limited clinical transferability., Methods: Here, we study the effect of simvastatin on insulin secretion using single-islet cultures, an optimal compromise between biological observability and physiologic fidelity. We develop and validate a microfluidic device to study single-islet function ex vivo, which allows for switching between media of different compositions with a resolution of seconds. In parallel, fluorescence imaging provides real-time analysis of the membrane voltage potential, cytosolic Ca 2+ dynamics, and insulin release during perfusion under 3 or 11 mM glucose., Results: We found that simvastatin reversibly inhibits insulin secretion, even in high-glucose. This phenomenon is very rapid (<60 s), occurs without affecting Ca 2+ concentrations, and is likely unrelated to cholesterol biosynthesis and protein isoprenylation, which occur on a time span of hours., Conclusions: Our data provide the first real-time live demonstration that a statin inhibits insulin secretion in intact islets and that single islets respond differently from cell lines on a short time scale., Funding: University of Padova, EASD Foundation.

Spatially and temporally coordinated variations of the cytosolic free calcium concentration ([Ca 2+ ] c ) play a crucial role in a variety of tissues. In the developing sensory epithelium of the mammalian cochlea, elevation of extracellular adenosine trisphosphate concentration ([ATP] e ) triggers [Ca 2+ ] c oscillations and propagation of intercellular inositol 1,4,5-trisphosphate (IP 3 )-dependent Ca 2+ waves. What remains uncertain is the relative contribution of gap junction channels and connexin hemichannels to these fundamental mechanisms, defects in which impair hearing acquisition. Another related open question is whether [Ca 2+ ] c oscillations require oscillations of the cytosolic IP 3 concentration ([IP 3 ] c ) in this system. To address these issues, we performed Ca 2+ imaging experiments in the lesser epithelial ridge of the mouse cochlea around postnatal day 5 and constructed a computational model in quantitative adherence to experimental data. Our results indicate that [Ca 2+ ] c oscillations are governed by Hopf-type bifurcations within the experimental range of [ATP] e and do not require [IP 3 ] c oscillations. The model replicates accurately the spatial extent and propagation speed of intercellular Ca 2+ waves and predicts that ATP-induced ATP release is the primary mechanism underlying intercellular propagation of Ca 2+ signals. The model also uncovers a discontinuous transition from propagating regimes (intercellular Ca 2+ wave speed > 11 μm⋅s -1 ) to propagation failure (speed = 0), which occurs upon lowering the maximal ATP release rate below a minimal threshold value. The approach presented here overcomes major limitations due to lack of specific connexin channel inhibitors and can be extended to other coupled cellular systems., Competing Interests: The authors declare no conflict of interest.

HIV-1 relies on the host-cell machinery to accomplish its replication cycle, and characterization of these helper factors contributes to a better understanding of HIV-host interactions and can identify potential novel antiviral targets. Here we explored the contribution of CIB2, previously identified by RNAi screening as a potential helper factor, and its homolog, CIB1. Knockdown of either CIB1 or CIB2 strongly impaired viral replication in Jurkat cells and in primary CD4+ T-lymphocytes, identifying these proteins as non-redundant helper factors. Knockdown of CIB1 and CIB2 impaired envelope-mediated viral entry for both for X4- and R5-tropic HIV-1, and both cell-free and cell-associated entry pathways were affected. In contrast, the level of CIB1 and CIB2 expression did not influence cell viability, cell proliferation, receptor-independent viral binding to the cell surface, or later steps in the viral replication cycle. CIB1 and CIB2 knockdown was found to reduce the expression of surface molecules implicated in HIV-1 infection, including CXCR4, CCR5 and integrin α4β7, suggesting at least one mechanism through which these proteins promote viral infection. Thus, this study identifies CIB1 and CIB2 as host helper factors for HIV-1 replication that are required for optimal receptor-mediated viral entry.

Upon activation, neutrophils undergo histone citrullination by protein arginine deiminase (PAD)4, exocytosis of chromatin and enzymes as neutrophil extracellular traps (NETs), and death. In diabetes, neutrophils are primed to release NETs and die by NETosis. Although this process is a defense against infection, NETosis can damage tissue. Therefore, we examined the effect of NETosis on the healing of diabetic foot ulcers (DFUs). Using proteomics, we found that NET components were enriched in nonhealing human DFUs. In an independent validation cohort, a high concentration of neutrophil elastase in the wound was associated with infection and a subsequent worsening of the ulcer. NET components (elastase, histones, neutrophil gelatinase-associated lipocalin, and proteinase-3) were elevated in the blood of patients with DFUs. Circulating elastase and proteinase-3 were associated with infection, and serum elastase predicted delayed healing. Neutrophils isolated from the blood of DFU patients showed an increased spontaneous NETosis but an impaired inducible NETosis. In mice, skin PAD4 activity was increased by diabetes, and FACS detection of histone citrullination, together with intravital microscopy, showed that NETosis occurred in the bed of excisional wounds. PAD4 inhibition by Cl-amidine reduced NETting neutrophils and rescued wound healing in diabetic mice. Cumulatively, these data suggest that NETosis delays DFU healing., (© 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Confocal imaging of fluorescent probes offers a powerful, non-invasive tool which enables data collection from vast population of cells at high spatial and temporal resolution. Spinning disk confocal microscopy parallelizes the imaging process permitting the study of dynamic events in populations of living cells on the millisecond time scale. Several spinning disk microscopy solutions are commercially available, however these are often poorly configurable and relatively expensive. This chapter describes a procedure to assemble a cost-effective homemade spinning disk system for fluorescence microscopy, which is highly flexible and easily configurable. We finally illustrate a reliable protocol to obtain high-quality Ca(2+) and voltage imaging data from cochlear preparations.

Viral interference defines the reduced susceptibility of an infected cell to reinfection. For HIV-1, both receptor-dependent and independent pathways were described. The relative importance of different receptor-independent pathways has not been addressed. We have used reporter viruses to quantify the percentage of single- and double-infected cells, as a function of the delay between the two infections. For co-infection experiments, the frequency of double infected cells was higher than expected for independent events. By delaying the second infection, this frequency progressively diminished, resulting in significant interference after 18h. Interference measured here was largely receptor-independent. By individually deleting viral genes or expressing them in isolation, we demonstrate that the viral protein Rev plays a dominant role, while other viral proteins contributes to optimal interference. Our study defines the kinetics of early HIV-1 interference, describing the transition from higher susceptibility to double-infection to viral interference, and identifies Rev as its dominant effector., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Cell-to-cell viral infection, in which viruses spread through contact of infected cell with surrounding uninfected cells, has been considered as a critical mode of virus infection. However, since it is technically difficult to experimentally discriminate the two modes of viral infection, namely cell-free infection and cell-to-cell infection, the quantitative information that underlies cell-to-cell infection has yet to be elucidated, and its impact on virus spread remains unclear. To address this fundamental question in virology, we quantitatively analyzed the dynamics of cell-to-cell and cell-free human immunodeficiency virus type 1 (HIV-1) infections through experimental-mathematical investigation. Our analyses demonstrated that the cell-to-cell infection mode accounts for approximately 60% of viral infection, and this infection mode shortens the generation time of viruses by 0.9 times and increases the viral fitness by 3.9 times. Our results suggest that even a complete block of the cell-free infection would provide only a limited impact on HIV-1 spread.

Type-I interferons (IFNs) induce the expression of hundreds of cellular genes, some of which have direct antiviral activities. Although IFNs restrict different steps of HIV replication cycle, their dominant antiviral effect remains unclear. We first quantified the inhibition of HIV replication by IFN in tissue culture, using viruses with different tropism and growth kinetics. By combining experimental and mathematical analyses, we determined quantitative estimates for key parameters of HIV replication and inhibition, and demonstrate that IFN mainly inhibits de novo infection (33% and 47% for a X4- and a R5-strain, respectively), rather than virus production (15% and 6% for the X4 and R5 strains, respectively). This finding is in agreement with patient-derived data analyses.