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The southern black-horned capuchin, Sapajus nigritus cucullatus, is considered Near Threatened on the IUCN Red List and Vulnerable in Argentina. The species is mainly threatened by habitat loss and fragmentation. The aim of this study was to compare range size, group size, and density in S. n. cucullatus groups between areas of continuous and fragmented habitat in the Atlantic Forest in Argentina. The study was carried out in two areas in northern Misiones province, one continuous and one anthropogenic fragment. Fieldwork was carried out for 5 days each month from November 2019 to March 2020 and from November 2020 to March 2021. SARS-CoV-2 restrictions meant we could not survey in the intervening period. Group counts were made on existing trails and subsequent group follows. We georeferenced encounters and follows to estimate home range sizes. We calculated density based on home range modeling using 100% minimum convex polygons (MCP), and compared these using generalized linear models (GLM). Smaller groups and lower density of S. n. cucullatus were found in continuous forest, with group sizes between 12 and 23 individuals, and density of 0.14 ind/ha, whereas in the fragmented forest, group sizes were between 32 and 36, with density of 0.62 ind/ha (n = 107; zero-inflated negative binomial regression [ZINB], p < 0.05). The higher density in forest fragments may be due to reduced dispersal ability. This work highlights data on species plasticity that could contribute to the development of conservation management strategies for S. n. cucullatus and its habitat., (© 2024. The Author(s), under exclusive licence to Japan Monkey Centre.)

The simultaneous use of UVA light irradiation coupled with low energy cold plasma generated by a dielectric barrier discharge prototype, results in significant enhancement of efficiency of the integrated process with respect to the sole plasma treatment. This effect has been demonstrated both on microbial inactivation of a food-borne pathogen, i.e. Listeria monocytogenes, and on the degradation of a compound of biological origin such as phenylalanine. In the latter case, the analysis of its reaction intermediates and the spectroscopic identification and quantification of peroxynitrites, allowed to propose mechanistic hypotheses on the nature of the observed synergistic effects. Moreover, it has been demonstrated that the process does not affect the quality of trout fillets, indicating its suitability as a chlorine-free, green, and sustainable tool for the decontamination of fish processing water., 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 © 2023 Elsevier Ltd. All rights reserved.)

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.)

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.)

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.)

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.)

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.

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.)

Objectives: The ultrasound investigation of carotid and vertebral arteries is routinely performed in stroke patients to determine the etiopathogenetic classification and possible need of revascularization. However, the medium and long-term prognostic implications of carotid and vertebral ultrasound in ischemic stroke patients are not yet known., Methods: This study included 309 ischemic stroke patients (mean age 76.3; 160 men). They all had undergone carotid and vertebral ultrasound (carotid stenoses were measured according to the European Carotid Surgery Trial [ECST] method). After a median interval of 9.4 months, a telephone follow-up was performed to determine their outcome. Dependency or death (modified Rankin scale-mRS >2) and all cause mortality were the study end-points., Results: At follow-up, 158 patients had a mRS >2. In multivariate analysis, of 13 variables univariately predictive of dependency or death, only National Institutes of Health Stroke Scale (NIHSS) score (P < 0.0001), age (P < 0.0001) and ipsi- or contralateral carotid stenosis ≥60% (O.R. 3.5, 95% C.I. 1.5-8.6, P = 0.006) remained associated with a mRS >2. Sixty-nine patients had died. In a Cox proportional hazards regression, of 10 variables univariately predictive of mortality, only NIHSS score (P < 0.0001), age (P = 0.003), total anterior circulation syndrome (P = 0.004), vertebral Doppler abnormalities (O.R. 2.2, 95% C.I. 1.3-3.6, P = 0.006), male sex (P = 0.02), and hypercholesterolemia (P = 0.04, inverse relationship) remained associated with mortality., Conclusions: In stroke patients, carotid stenoses ≥60%, ipsi- or contralateral to cerebral lesions, were associated with an increased medium and long-term probability of dependency or death, and abnormalities of vertebrobasilar flow were a significant indicator of death risk, independent of stroke severity and age.

Electrophysiological and psychophysical data indicate that grasping observation automatically orients attention toward the incoming interactions between the actor's hand and the object. The aim of the present study was to clarify if this effect facilitates the detection of a graspable object with the observed action as compared to an ungraspable one. We submitted participants to an object-identity probability cueing experiment in which the two possible targets were of the same dimensions but one of them presented sharp tips at one extreme while the other presented flat faces. At the beginning of each trial the most probable target was briefly shown. After a variable interval, at the same position, the same (75%) or a different target (25%) was presented. Participants had to press a key in response to target appearance. Superimposed to the video showing cue and target, an agent performing the reaching and grasping of the target was presented. The kinematics of the action was or was not suitable for grasping the cued target, according to the absence or presence of the sharp tips. Results showed that response was modulated by the probability of target identity but only when the observed kinematics was suitable to grasp the attended target. A further experiment clarified that response modulation was never present when the superimposed video always showed the agent at a rest position. These findings are discussed at the light of neurophysiological and psychophysical literature, considering the relationship between the motor system and the perception of objects and of others' actions. We conclude that the prediction of the mechanical events that arise from the interactions between the hand and the attended object is at the basis of the capability to select a graspable object in space.

Ionizing and nonionizing radiation affect not only directly targeted cells but also surrounding "bystander" cells. The underlying mechanisms and therapeutic role of bystander responses remain incompletely defined. Here we show that photosentizer activation in a single cell triggers apoptosis in bystander cancer cells, which are electrically coupled by gap junction channels and support the propagation of a Ca2+ wave initiated in the irradiated cell. The latter also acts as source of nitric oxide (NO) that diffuses to bystander cells, in which NO levels are further increased by a mechanism compatible with Ca(2+)-dependent enzymatic production. We detected similar signals in tumors grown in dorsal skinfold chambers applied to live mice. Pharmacological blockade of connexin channels significantly reduced the extent of apoptosis in bystander cells, consistent with a critical role played by intercellular communication, Ca2+ and NO in the bystander effects triggered by photodynamic therapy.

Notexin (Ntx) is a group I phospholipase A2 (PLA2) protein, main component of the Australian snake Notechis scutatus scutatus venom. It is both a presynaptic neurotoxin and a myotoxin. In this work, for the first time, a method for the production and folding of recombinant Ntx was developed. Ntx was produced with wild type sequence (rNtx), with an extra peptide (T7-Ntx) or a methionine (M-Ntx) before Asn-1, and with Asn-1 substituted by alanine (Ntx-A1) or by serine (Ntx-S1). The proteins were analyzed for their catalytic and toxic activities. rNtx activity resulted to be comparable to that of the venom extracted protein. The Ntx N-terminus was found to have a major influence on both the catalytic and toxic activities of the protein. The first amino acid of snake venom PLA2s is highly conserved: it is an asparagine in about all group I PLA2s, while in most (>70%) of group II PLA2s it is a serine or an asparagine. Interestingly, Ntx-S1 resulted to be, for both enzymatic and toxic activities, the mutant most similar to the wild type protein. The role of the catalytic activity of Ntx in its toxicity was investigated by replacing the aspartic acid 49, involved in the coordination of the cofactor calcium ion, by a lysine. The obtained mutant (Ntx-K49) is deprived of catalytic activity but possesses a residual toxicity., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Adipocyte-fatty acid binding protein (A-FABP) is an intracellular lipid transporter that mediates metabolically triggered inflammation, and it is associated with insulin resistance, atherogenic dyslipidemia, and cardiovascular risk., Aims: The aim of this study was to evaluate A-FABP behavior in elderly people, and especially its association with liver steatosis at abdominal ultrasound., Method: Cross-sectional study of two cohort of individuals with and without steatosis, with assessment of several clinical and laboratory variables. Prospective evaluation of liver steatosis remodeling after six years of follow-up. One hundred and fifty-six subjects aged over 65 years were enrolled., Results: Serum A-FABP positively correlated with body fat percentage, total cholesterol, serum triglycerides and erythrocyte sedimentation rate. Unlike expected, high A-FABP levels were associated with absence of liver steatosis, while there was no evidence of association with steatosis grade changes after 6 years of follow-up., Conclusion: Among individuals aging more than 65 years included in the study, A-FABP was inversely associated with liver steatosis. It can be argued, that still uncovered mechanisms modify A-FABP behavior in elderly people, especially its association with multifactorial diseases.

To verify if the link between observed hand actions and executed foot actions found in aplasics is essentially induced by the constant use of foot substituting the hand, we investigated if the vision of a grasping hand is able to prime a foot response in normals. Participants were required to detect the time-to-contact of a hand grasping an object either with a suitable or a less suitable movement, an experimental paradigm known to induce a priming effect. Participants responded either with the hand or the foot, while having free or bound hands. Results showed that for hand responses motor priming effect was stronger when the hands were free, whereas for foot responses it was stronger when the hands were bound. These data are interpreted as a further evidence that a difficulty to move affects specific cognitive functions and that the vision of a grasping hand may prime a foot response.