Hair cell maturation is differentially regulated along the tonotopic axis of the mammalian cochlea

Sound amplification within the mammalian cochlea depends upon specialized hair cells, the outer hair cells (OHCs), which possess both sensory and motile capabilities. In various altricial rodents, OHCs become functionally competent from around postnatal day 7 (P7), before the primary sensory inner hair cells (IHCs), which become competent at about the onset of hearing (P12). The mechanisms responsible for the maturation of OHCs and their synaptic specialization remain poorly understood. We report that spontaneous Ca2+ activity in the immature cochlea, which is generated by CaV1.3 Ca2+ channels, differentially regulates the maturation of hair cells along the cochlea. Under near‐physiological recording conditions we found that, similar to IHCs, immature OHCs elicited spontaneous Ca2+ action potentials (APs), but only during the first few postnatal days. Genetic ablation of these APs in vivo, using CaV1.3−/− mice, prevented the normal developmental acquisition of mature‐like basolateral membrane currents in low‐frequency (apical) hair cells, such as IK,n (carried by KCNQ4 channels), ISK2 and IACh (α9α10nAChRs) in OHCs and IK,n and IK,f (BK channels) in IHCs. Electromotility and prestin expression in OHCs were normal in CaV1.3−/− mice. The maturation of high‐frequency (basal) hair cells was also affected in CaV1.3−/− mice, but to a much lesser extent than apical cells. However, a characteristic feature in CaV1.3−/− mice was the reduced hair cell size irrespective of their cochlear location. We conclude that the development of low‐ and high‐frequency hair cells is differentially regulated during development, with apical cells being more strongly dependent on experience‐independent Ca2+ APs

Age-related changes in P2Y receptor signalling in mouse cochlear supporting cells

Our sense of hearing depends on the function of a specialised class of sensory cells, the hair cells, which are found in the organ of Corti of the mammalian cochlea. The unique physiological environment in which these cells operate is maintained by a syncitium of non-sensory supporting cells, which are crucial for regulating cochlear physiology and metabolic homeostasis. Despite their importance for cochlear function, the role of these supporting cells in age-related hearing loss, the most common sensory deficit in the elderly, is poorly understood. Here, we investigated the age-related changes in the expression and function of metabotropic purinergic receptors (P2Y1 , P2Y2 and P2Y4 ) in the supporting cells of the cochlear apical coil. Purinergic signalling in supporting cells is crucial during the development of the organ of Corti and purinergic receptors are known to undergo changes in expression during ageing in several tissues. Immunolabelling and Ca2+ imaging experiments revealed a downregulation of P2Y receptor expression and a decrease of purinergic-mediated calcium responses after early postnatal stages in the supporting cells. An upregulation of P2Y receptor expression was observed in the aged cochlea when compared to 1 month-old adults. The aged mice also had significantly larger calcium responses and displayed calcium oscillations during prolonged agonist applications. We conclude that supporting cells in the aged cochlea upregulate P2Y2 and P2Y4 receptors and display purinergic-induced Ca2+ responses that mimic those observed during pre-hearing stages of development, possibly aimed at limiting or preventing further damage to the sensory epithelium. KEY POINTS: Age-related hearing loss is associated with lower hearing sensitivity and decreased ability to understand speech. We investigated age-related changes in the expression and function of metabotropic purinergic (P2Y) receptors in cochlear non-sensory supporting cells of mice displaying early-onset (C57BL/6N) and late-onset (C3H/HeJ) hearing loss. The expression of P2Y1 , P2Y2 and P2Y4 receptors in the supporting cells decreased during cochlear maturation, but that of P2Y2 and P2Y4 was upregulated in the aged cochlea. P2Y2 and P2Y4 receptors were primarily responsible for the ATP-induced Ca2+ responses in the supporting cells. The degree of purinergic expression upregulation in aged supporting cells mirrored hearing loss progression in the different mouse strains. We propose that the upregulation of purinergic-mediated signalling in the aged cochlea is subsequent to age-related changes in the hair cells and may act as a protective mechanism to limit or to avoid further damage to the sensory epithelium

TMC2 modifies permeation properties of the mechanoelectrical transducer channel in early postnatal mouse cochlear outer hair cells

The ability of cochlear hair cells to convert sound into receptor potentials relies on the mechanoelectrical transducer (MET) channels present in their stereociliary bundles. There is strong evidence implying that transmembrane channel-like protein (TMC) 1 contributes to the pore-forming subunit of the mature MET channel, yet its expression is delayed (∼>P5 in apical outer hair cells, OHCs) compared to the onset of mechanotransduction (∼P1). Instead, the temporal expression of TMC2 coincides with this onset, indicating that it could be part of the immature MET channel. We investigated MET channel properties from OHCs of homo- and heterozygous Tmc2 knockout mice. In the presence of TMC2, the MET channel blocker dihydrostreptomycin (DHS) had a lower affinity for the channel, when the aminoglycoside was applied extracellularly or intracellularly, with the latter effect being more pronounced. In Tmc2 knockout mice OHCs were protected from aminoglycoside ototoxicity during the first postnatal week, most likely due to their small MET current and the lower saturation level for aminoglycoside entry into the individual MET channels. DHS entry through the MET channels of Tmc2 knockout OHCs was lower during the first than in the second postnatal week, suggestive of a developmental change in the channel pore properties independent of TMC2. However, the ability of TMC2 to modify the MET channel properties strongly suggests it contributes to the pore-forming subunit of the neonatal channel. Nevertheless, we found that TMC2, different from TMC1, is not necessary for OHC development. While TMC2 is required for mechanotransduction in mature vestibular hair cells, its expression in the immature cochlea may be an evolutionary remnant

Coordinated calcium signalling in cochlear sensory and non‐sensory cells refines afferent innervation of outer hair cells

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 Ca2+ 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 Ca2+ signals originating in OHCs and non‐sensory cells. OHCs fire spontaneous Ca2+ action potentials during a narrow period of neonatal development. Simultaneously, waves of Ca2+ activity in the non‐sensory cells of the greater epithelial ridge cause, via ATP‐induced activation of P2X3 receptors, the increase and synchronization of the Ca2+ activity in nearby OHCs. This synchronization is required for the refinement of their immature afferent innervation. In the absence of connexin channels, Ca2+ 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 Ca2+ signals from sensory and non‐sensory cells

Nuclear Export and Import of Human Hepatitis B Virus Capsid Protein and Particles

It remains unclear what determines the subcellular localization of hepatitis B virus (HBV) core protein (HBc) and particles. To address this fundamental issue, we have identified four distinct HBc localization signals in the arginine rich domain (ARD) of HBc, using immunofluorescence confocal microscopy and fractionation/Western blot analysis. ARD consists of four tight clustering arginine-rich subdomains. ARD-I and ARD-III are associated with two co-dependent nuclear localization signals (NLS), while ARD-II and ARD-IV behave like two independent nuclear export signals (NES). This conclusion is based on five independent lines of experimental evidence: i) Using an HBV replication system in hepatoma cells, we demonstrated in a double-blind manner that only the HBc of mutant ARD-II+IV, among a total of 15 ARD mutants, can predominantly localize to the nucleus. ii) These results were confirmed using a chimera reporter system by placing mutant or wild type HBc trafficking signals in the heterologous context of SV40 large T antigen (LT). iii) By a heterokaryon or homokaryon analysis, the fusion protein of SV40 LT-HBc ARD appeared to transport from nuclei of transfected donor cells to nuclei of recipient cells, suggesting the existence of an NES in HBc ARD. This putative NES is leptomycin B resistant. iv) We demonstrated by co-immunoprecipitation that HBc ARD can physically interact with a cellular factor TAP/NXF1 (Tip-associated protein/nuclear export factor-1), which is known to be important for nuclear export of mRNA and proteins. Treatment with a TAP-specific siRNA strikingly shifted cytoplasmic HBc to nucleus, and led to a near 7-fold reduction of viral replication, and a near 10-fold reduction in HBsAg secretion. v) HBc of mutant ARD-II+IV was accumulated predominantly in the nucleus in a mouse model by hydrodynamic delivery. In addition to the revised map of NLS, our results suggest that HBc could shuttle rapidly between nucleus and cytoplasm via a novel TAP-dependent NES

Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis

BACKGROUND: Rates of antimicrobial resistance (AMR) are rising globally and there is concern that increased migration is contributing to the burden of antibiotic resistance in Europe. However, the effect of migration on the burden of AMR in Europe has not yet been comprehensively examined. Therefore, we did a systematic review and meta-analysis to identify and synthesise data for AMR carriage or infection in migrants to Europe to examine differences in patterns of AMR across migrant groups and in different settings. METHODS: For this systematic review and meta-analysis, we searched MEDLINE, Embase, PubMed, and Scopus with no language restrictions from Jan 1, 2000, to Jan 18, 2017, for primary data from observational studies reporting antibacterial resistance in common bacterial pathogens among migrants to 21 European Union-15 and European Economic Area countries. To be eligible for inclusion, studies had to report data on carriage or infection with laboratory-confirmed antibiotic-resistant organisms in migrant populations. We extracted data from eligible studies and assessed quality using piloted, standardised forms. We did not examine drug resistance in tuberculosis and excluded articles solely reporting on this parameter. We also excluded articles in which migrant status was determined by ethnicity, country of birth of participants' parents, or was not defined, and articles in which data were not disaggregated by migrant status. Outcomes were carriage of or infection with antibiotic-resistant organisms. We used random-effects models to calculate the pooled prevalence of each outcome. The study protocol is registered with PROSPERO, number CRD42016043681. FINDINGS: We identified 2274 articles, of which 23 observational studies reporting on antibiotic resistance in 2319 migrants were included. The pooled prevalence of any AMR carriage or AMR infection in migrants was 25·4% (95% CI 19·1-31·8; I2 =98%), including meticillin-resistant Staphylococcus aureus (7·8%, 4·8-10·7; I2 =92%) and antibiotic-resistant Gram-negative bacteria (27·2%, 17·6-36·8; I2 =94%). The pooled prevalence of any AMR carriage or infection was higher in refugees and asylum seekers (33·0%, 18·3-47·6; I2 =98%) than in other migrant groups (6·6%, 1·8-11·3; I2 =92%). The pooled prevalence of antibiotic-resistant organisms was slightly higher in high-migrant community settings (33·1%, 11·1-55·1; I2 =96%) than in migrants in hospitals (24·3%, 16·1-32·6; I2 =98%). We did not find evidence of high rates of transmission of AMR from migrant to host populations. INTERPRETATION: Migrants are exposed to conditions favouring the emergence of drug resistance during transit and in host countries in Europe. Increased antibiotic resistance among refugees and asylum seekers and in high-migrant community settings (such as refugee camps and detention facilities) highlights the need for improved living conditions, access to health care, and initiatives to facilitate detection of and appropriate high-quality treatment for antibiotic-resistant infections during transit and in host countries. Protocols for the prevention and control of infection and for antibiotic surveillance need to be integrated in all aspects of health care, which should be accessible for all migrant groups, and should target determinants of AMR before, during, and after migration. FUNDING: UK National Institute for Health Research Imperial Biomedical Research Centre, Imperial College Healthcare Charity, the Wellcome Trust, and UK National Institute for Health Research Health Protection Research Unit in Healthcare-associated Infections and Antimictobial Resistance at Imperial College London

Software for the frontiers of quantum chemistry:An overview of developments in the Q-Chem 5 package

This article summarizes technical advances contained in the fifth major release of the Q-Chem quantum chemistry program package, covering developments since 2015. A comprehensive library of exchange–correlation functionals, along with a suite of correlated many-body methods, continues to be a hallmark of the Q-Chem software. The many-body methods include novel variants of both coupled-cluster and configuration-interaction approaches along with methods based on the algebraic diagrammatic construction and variational reduced density-matrix methods. Methods highlighted in Q-Chem 5 include a suite of tools for modeling core-level spectroscopy, methods for describing metastable resonances, methods for computing vibronic spectra, the nuclear–electronic orbital method, and several different energy decomposition analysis techniques. High-performance capabilities including multithreaded parallelism and support for calculations on graphics processing units are described. Q-Chem boasts a community of well over 100 active academic developers, and the continuing evolution of the software is supported by an “open teamware” model and an increasingly modular design

Surgical site infection after gastrointestinal surgery in high-income, middle-income, and low-income countries: a prospective, international, multicentre cohort study

Background: Surgical site infection (SSI) is one of the most common infections associated with health care, but its importance as a global health priority is not fully understood. We quantified the burden of SSI after gastrointestinal surgery in countries in all parts of the world. Methods: This international, prospective, multicentre cohort study included consecutive patients undergoing elective or emergency gastrointestinal resection within 2-week time periods at any health-care facility in any country. Countries with participating centres were stratified into high-income, middle-income, and low-income groups according to the UN's Human Development Index (HDI). Data variables from the GlobalSurg 1 study and other studies that have been found to affect the likelihood of SSI were entered into risk adjustment models. The primary outcome measure was the 30-day SSI incidence (defined by US Centers for Disease Control and Prevention criteria for superficial and deep incisional SSI). Relationships with explanatory variables were examined using Bayesian multilevel logistic regression models. This trial is registered with ClinicalTrials.gov, number NCT02662231. Findings: Between Jan 4, 2016, and July 31, 2016, 13 265 records were submitted for analysis. 12 539 patients from 343 hospitals in 66 countries were included. 7339 (58·5%) patient were from high-HDI countries (193 hospitals in 30 countries), 3918 (31·2%) patients were from middle-HDI countries (82 hospitals in 18 countries), and 1282 (10·2%) patients were from low-HDI countries (68 hospitals in 18 countries). In total, 1538 (12·3%) patients had SSI within 30 days of surgery. The incidence of SSI varied between countries with high (691 [9·4%] of 7339 patients), middle (549 [14·0%] of 3918 patients), and low (298 [23·2%] of 1282) HDI (p < 0·001). The highest SSI incidence in each HDI group was after dirty surgery (102 [17·8%] of 574 patients in high-HDI countries; 74 [31·4%] of 236 patients in middle-HDI countries; 72 [39·8%] of 181 patients in low-HDI countries). Following risk factor adjustment, patients in low-HDI countries were at greatest risk of SSI (adjusted odds ratio 1·60, 95% credible interval 1·05–2·37; p=0·030). 132 (21·6%) of 610 patients with an SSI and a microbiology culture result had an infection that was resistant to the prophylactic antibiotic used. Resistant infections were detected in 49 (16·6%) of 295 patients in high-HDI countries, in 37 (19·8%) of 187 patients in middle-HDI countries, and in 46 (35·9%) of 128 patients in low-HDI countries (p < 0·001). Interpretation: Countries with a low HDI carry a disproportionately greater burden of SSI than countries with a middle or high HDI and might have higher rates of antibiotic resistance. In view of WHO recommendations on SSI prevention that highlight the absence of high-quality interventional research, urgent, pragmatic, randomised trials based in LMICs are needed to assess measures aiming to reduce this preventable complication