Site-directed M2 proton channel inhibitors enable synergistic combination therapy for rimantadine-resistant pandemic influenza.

Pandemic influenza A virus (IAV) remains a significant threat to global health. Preparedness relies primarily upon a single class of neuraminidase (NA) targeted antivirals, against which resistance is steadily growing. The M2 proton channel is an alternative clinically proven antiviral target, yet a near-ubiquitous S31N polymorphism in M2 evokes resistance to licensed adamantane drugs. Hence, inhibitors capable of targeting N31 containing M2 (M2-N31) are highly desirable. Rational in silico design and in vitro screens delineated compounds favouring either lumenal or peripheral M2 binding, yielding effective M2-N31 inhibitors in both cases. Hits included adamantanes as well as novel compounds, with some showing low micromolar potency versus pandemic "swine" H1N1 influenza (Eng195) in culture. Interestingly, a published adamantane-based M2-N31 inhibitor rapidly selected a resistant V27A polymorphism (M2-A27/N31), whereas this was not the case for non-adamantane compounds. Nevertheless, combinations of adamantanes and novel compounds achieved synergistic antiviral effects, and the latter synergised with the neuraminidase inhibitor (NAi), Zanamivir. Thus, site-directed drug combinations show potential to rejuvenate M2 as an antiviral target whilst reducing the risk of drug resistance.

Aerobic glycolysis is important for zebrafish larval wound closure and tail regeneration.

The underlying mechanisms of appendage regeneration remain largely unknown and uncovering these mechanisms in capable organisms has far-reaching implications for potential treatments in humans. Recent studies implicate a requirement for metabolic reprogramming reminiscent of the Warburg effect during successful appendage and organ regeneration. As changes are thus predicted to be highly dynamic, methods permitting direct, real-time visualisation of metabolites at the tissue and organismal level would offer a significant advance in defining the influence of metabolism on regeneration and healing. We sought to examine whether glycolytic activity was altered during larval fin regeneration, utilising the genetically encoded biosensor, Laconic, enabling the spatiotemporal assessment of lactate levels in living zebrafish. We present evidence for a rapid increase in lactate levels within min following injury, with a role of aerobic glycolysis in actomyosin contraction and wound closure. We also find a second wave of lactate production, associated with overall larval tail regeneration. Chemical inhibition of glycolysis attenuates both the contraction of the wound and regrowth of tissue following tail amputation, suggesting aerobic glycolysis is necessary at two distinct stages of regeneration.

Enhancing Behavior Change Technique Coding Methods: Identifying Behavioral Targets and Delivery Styles in Smoking Cessation Trials.

BACKGROUND: The behavior change technique (BCT) taxonomy v1 is often used in systematic reviews for identifying active components of interventions. Its utility could be enhanced by linking BCTs to specific target behaviors and qualifying BCT delivery style. PURPOSE: To determine whether behavioral targets and delivery styles of BCTs can be coded reliably and to determine the utility of coding these characteristics. METHODS: As part of a large systematic review of 142 smoking cessation trials, two researchers independently coded publicly and privately held intervention and comparator group materials, specifying the behavioral target (quitting, abstinence, medication adherence, or treatment engagement) and delivery style (tailored vs. not tailored; active participation vs. passive receipt) of each BCT. RESULTS: Researchers coded 3,843 BCTs, which were reliably attributed to behavioral targets (AC1 = 0.92, PABAK = 0.91). Tailoring (AC1 = 0.80, PABAK = 0.74) and participation (AC1 = 0.71, PABAK = 0.64) were also coded reliably. There was considerable variability between groups in quitting and abstinence BCTs (ranges: 0-41; 0-18) and in tailoring and participation (ranges: 0-20; 0-32), but less variability for medication adherence and treatment engagement (ranges: 0-6; 0-7). CONCLUSIONS: Behavioral targets and delivery styles of BCTs can be reliably identified and occur with sufficient frequency in smoking cessation trials for inclusion in quantitative syntheses (e.g., meta-regression analyses). Systematic reviewers could consider adopting these methods to evaluate the impact of intervention components targeting different behaviors, as well as the benefits of different BCT delivery styles.

Loss-of-function mutations in CAST cause peeling skin, leukonychia, acral punctate keratoses, cheilitis, and knuckle pads.

Calpastatin is an endogenous specific inhibitor of calpain, a calcium-dependent cysteine protease. Here we show that loss-of-function mutations in calpastatin (CAST) are the genetic causes of an autosomal-recessive condition characterized by generalized peeling skin, leukonychia, acral punctate keratoses, cheilitis, and knuckle pads, which we propose to be given the acronym PLACK syndrome. In affected individuals with PLACK syndrome from three families of different ethnicities, we identified homozygous mutations (c.607dup, c.424A>T, and c.1750delG) in CAST, all of which were predicted to encode truncated proteins (p.Ile203Asnfs∗8, p.Lys142∗, and p.Val584Trpfs∗37). Immunohistochemistry shows that staining of calpastatin is reduced in skin from affected individuals. Transmission electron microscopy revealed widening of intercellular spaces with chromatin condensation and margination in the upper stratum spinosum in lesional skin, suggesting impaired intercellular adhesion as well as keratinocyte apoptosis. A significant increase of apoptotic keratinocytes was also observed in TUNEL assays. In vitro studies utilizing siRNA-mediated CAST knockdown revealed a role for calpastatin in keratinocyte adhesion. In summary, we describe PLACK syndrome, as a clinical entity of defective epidermal adhesion, caused by loss-of-function mutations in CAST.

Pharmacist services for non-hospitalised patients.

BACKGROUND: This review focuses on non-dispensing services from pharmacists, i.e. pharmacists in community, primary or ambulatory-care settings, to non-hospitalised patients, and is an update of a previously-published Cochrane Review. OBJECTIVES: To examine the effect of pharmacists' non-dispensing services on non-hospitalised patient outcomes. SEARCH METHODS: We searched CENTRAL, MEDLINE, Embase, two other databases and two trial registers in March 2015, together with reference checking and contact with study authors to identify additional studies. We included non-English language publications. We ran top-up searches in January 2018 and have added potentially eligible studies to 'Studies awaiting classification'. SELECTION CRITERIA: Randomised trials of pharmacist services compared with the delivery of usual care or equivalent/similar services with the same objective delivered by other health professionals. DATA COLLECTION AND ANALYSIS: We used standard methodological procedures of Cochrane and the Effective Practice and Organisation of Care Group. Two review authors independently checked studies for inclusion, extracted data and assessed risks of bias. We evaluated the overall certainty of evidence using GRADE. MAIN RESULTS: We included 116 trials comprising 111 trials (39,729 participants) comparing pharmacist interventions with usual care and five trials (2122 participants) comparing pharmacist services with services from other healthcare professionals. Of the 116 trials, 76 were included in meta-analyses. The 40 remaining trials were not included in the meta-analyses because they each reported unique outcome measures which could not be combined. Most trials targeted chronic conditions and were conducted in a range of settings, mostly community pharmacies and hospital outpatient clinics, and were mainly but not exclusively conducted in high-income countries. Most trials had a low risk of reporting bias and about 25%-30% were at high risk of bias for performance, detection, and attrition. Selection bias was unclear for about half of the included studies.Compared with usual care, we are uncertain whether pharmacist services reduce the percentage of patients outside the glycated haemoglobin target range (5 trials, N = 558, odds ratio (OR) 0.29, 95% confidence interval (CI) 0.04 to 2.22; very low-certainty evidence). Pharmacist services may reduce the percentage of patients whose blood pressure is outside the target range (18 trials, N = 4107, OR 0.40, 95% CI 0.29 to 0.55; low-certainty evidence) and probably lead to little or no difference in hospital attendance or admissions (14 trials, N = 3631, OR 0.85, 95% CI 0.65 to 1.11; moderate-certainty evidence). Pharmacist services may make little or no difference to adverse drug effects (3 trials, N = 590, OR 1.65, 95% CI 0.84 to 3.24) and may slightly improve physical functioning (7 trials, N = 1329, mean difference (MD) 5.84, 95% CI 1.21 to 10.48; low-certainty evidence). Pharmacist services may make little or no difference to mortality (9 trials, N = 1980, OR 0.79, 95% CI 0.56 to 1.12, low-certaintly evidence).Of the five studies that compared services delivered by pharmacists with other health professionals, no studies evaluated the impact of the intervention on the percentage of patients outside blood pressure or glycated haemoglobin target range, hospital attendance and admission, adverse drug effects, or physical functioning. AUTHORS' CONCLUSIONS: The results demonstrate that pharmacist services have varying effects on patient outcomes compared with usual care. We found no studies comparing services delivered by pharmacists with other healthcare professionals that evaluated the impact of the intervention on the six main outcome measures. The results need to be interpreted cautiously because there was major heterogeneity in study populations, types of interventions delivered and reported outcomes.There was considerable heterogeneity within many of the meta-analyses, as well as considerable variation in the risks of bias.

Genomic Landscape of Esophageal Squamous Cell Carcinoma in a Japanese Population.

BACKGROUND & AIMS: Esophageal squamous cell carcinoma (ESCC) is the predominant form of esophageal cancer in Japan. Smoking and drinking alcohol are environmental risk factors for ESCC, whereas single nucleotide polymorphisms in ADH1B and ALDH2, which increase harmful intermediates produced by drinking alcohol, are genetic risk factors. We conducted a large-scale genomic analysis of ESCCs from patients in Japan to determine the mutational landscape of this cancer. METHODS: We performed whole-exome sequence analysis of tumor and nontumor esophageal tissues collected from 144 patients with ESCC who underwent surgery at 5 hospitals in Japan. We also performed single-nucleotide polymorphism array-based copy number profile and germline genotype analyses of polymorphisms in ADH1B and ALDH2. Polymorphisms in CYP2A6, which increase harmful effects of smoking, were analyzed. Functions of TET2 mutants were evaluated in KYSE410 and HEK293FT cells. RESULTS: A high proportion of mutations in the 144 tumor samples were C to T substitution in CpG dinucleotides (called the CpG signature) and C to G/T substitutions with a flanking 5' thymine (called the APOBEC signature). Based on mutational signatures, patients were assigned to 3 groups, which associated with environmental (drinking and smoking) and genetic (polymorphisms in ALDH2 and CYP2A6) factors. Many tumors contained mutations in genes that regulate the cell cycle (TP53, CCND1, CDKN2A, FBXW7); epigenetic processes (MLL2, EP300, CREBBP, TET2); and the NOTCH (NOTCH1, NOTCH3), WNT (FAT1, YAP1, AJUBA) and receptor-tyrosine kinase-phosphoinositide 3-kinase signaling pathways (PIK3CA, EGFR, ERBB2). Mutations in EP300 and TET2 correlated with shorter survival times, and mutations in ZNF750 associated with an increased number of mutations of the APOBEC signature. Expression of mutant forms of TET2 did not increase cellular levels of 5-hydroxymethylcytosine in HEK293FT cells, whereas knockdown of TET2 increased the invasive activity of KYSE410 ESCC cells. Computational analyses associated the mutations in NFE2L2 we identified with transcriptional activation of its target genes. CONCLUSIONS: We associated environmental and genetic factors with base substitution patterns of somatic mutations and provide a registry of genes and pathways that are disrupted in ESCCs. These findings might be used to design specific treatments for patients with esophageal squamous cancers.

Ion Channel Function and Cross-Species Determinants in Viral Assembly of Nonprimate Hepacivirus p7.

UNLABELLED: Nonprimate hepacivirus (NPHV), the closest homolog of hepatitis C virus (HCV) described to date, has recently been discovered in horses. Even though the two viruses share a similar genomic organization, conservation of the encoded hepaciviral proteins remains undetermined. The HCV p7 protein is localized within endoplasmic reticulum (ER) membranes and is important for the production of infectious particles. In this study, we analyzed the structural and functional features of NPHV p7 in addition to its role during virus assembly. Three-dimensional homology models for NPHV p7 using various nuclear magnetic resonance spectroscopy (NMR) structures were generated, highlighting the conserved residues important for ion channel function. By applying a liposome permeability assay, we observed that NPHV p7 exhibited liposome permeability features similar to those of HCV p7, indicative of similar ion channel activity. Next, we characterized the viral protein using a p7-based trans-complementation approach. A similar subcellular localization pattern at the ER membrane was observed, although production of infectious particles was likely hindered by genetic incompatibilities with HCV proteins. To further characterize these cross-species constraints, chimeric viruses were constructed by substituting different regions of HCV p7 with NPHV p7. The N terminus and transmembrane domains were nonexchangeable and therefore constitute a cross-species barrier in hepaciviral assembly. In contrast, the basic loop and the C terminus of NPHV p7 were readily exchangeable, allowing production of infectious trans-complemented viral particles. In conclusion, comparison of NPHV and HCV p7 revealed structural and functional homology of these proteins, including liposome permeability, and broadly acting determinants that modulate hepaciviral virion assembly and contribute to the host-species barrier were identified. IMPORTANCE: The recent discovery of new relatives of hepatitis C virus (HCV) enables for the first time the study of cross-species determinants shaping hepaciviral pathogenesis. Nonprimate hepacivirus (NPHV) was described to infect horses and represents so far the closest homolog of HCV. Both viruses encode the same viral proteins; however, NPHV protein functions remain poorly understood. In this study, we aimed to dissect NPHV p7 on a structural and functional level. By using various NMR structures of HCV p7 as templates, three-dimensional homology models for NPHV p7 were generated, highlighting conserved residues that are important for ion channel function. A p7-based trans-complementation approach and the construction of NPHV/HCV p7 chimeric viruses showed that the N terminus and transmembrane domains were nonexchangeable. In contrast, the basic loop and the C terminus of NPHV p7 were readily exchangeable, allowing production of infectious viral particles. These results identify species-specific constraints as well as exchangeable determinants in hepaciviral assembly.

Interventions to improve antibiotic prescribing practices for hospital inpatients.

BACKGROUND: Antibiotic resistance is a major public health problem. Infections caused by multidrug-resistant bacteria are associated with prolonged hospital stay and death compared with infections caused by susceptible bacteria. Appropriate antibiotic use in hospitals should ensure effective treatment of patients with infection and reduce unnecessary prescriptions. We updated this systematic review to evaluate the impact of interventions to improve antibiotic prescribing to hospital inpatients. OBJECTIVES: To estimate the effectiveness and safety of interventions to improve antibiotic prescribing to hospital inpatients and to investigate the effect of two intervention functions: restriction and enablement. SEARCH METHODS: We searched the Cochrane Central Register of Controlled Trials (CENTRAL) (the Cochrane Library), MEDLINE, and Embase. We searched for additional studies using the bibliographies of included articles and personal files. The last search from which records were evaluated and any studies identified incorporated into the review was January 2015. SELECTION CRITERIA: We included randomised controlled trials (RCTs) and non-randomised studies (NRS). We included three non-randomised study designs to measure behavioural and clinical outcomes and analyse variation in the effects: non- randomised trials (NRT), controlled before-after (CBA) studies and interrupted time series (ITS) studies. For this update we also included three additional NRS designs (case control, cohort, and qualitative studies) to identify unintended consequences. Interventions included any professional or structural interventions as defined by the Cochrane Effective Practice and Organisation of Care Group. We defined restriction as 'using rules to reduce the opportunity to engage in the target behaviour (or increase the target behaviour by reducing the opportunity to engage in competing behaviours)'. We defined enablement as 'increasing means/reducing barriers to increase capability or opportunity'. The main comparison was between intervention and no intervention. DATA COLLECTION AND ANALYSIS: Two review authors extracted data and assessed study risk of bias. We performed meta-analysis and meta-regression of RCTs and meta-regression of ITS studies. We classified behaviour change functions for all interventions in the review, including those studies in the previously published versions. We analysed dichotomous data with a risk difference (RD). We assessed certainty of evidence with GRADE criteria. MAIN RESULTS: This review includes 221 studies (58 RCTs, and 163 NRS). Most studies were from North America (96) or Europe (87). The remaining studies were from Asia (19), South America (8), Australia (8), and the East Asia (3). Although 62% of RCTs were at a high risk of bias, the results for the main review outcomes were similar when we restricted the analysis to studies at low risk of bias.More hospital inpatients were treated according to antibiotic prescribing policy with the intervention compared with no intervention based on 29 RCTs of predominantly enablement interventions (RD 15%, 95% confidence interval (CI) 14% to 16%; 23,394 participants; high-certainty evidence). This represents an increase from 43% to 58% .There were high levels of heterogeneity of effect size but the direction consistently favoured intervention.The duration of antibiotic treatment decreased by 1.95 days (95% CI 2.22 to 1.67; 14 RCTs; 3318 participants; high-certainty evidence) from 11.0 days. Information from non-randomised studies showed interventions to be associated with improvement in prescribing according to antibiotic policy in routine clinical practice, with 70% of interventions being hospital-wide compared with 31% for RCTs. The risk of death was similar between intervention and control groups (11% in both arms), indicating that antibiotic use can likely be reduced without adversely affecting mortality (RD 0%, 95% CI -1% to 0%; 28 RCTs; 15,827 participants; moderate-certainty evidence). Antibiotic stewardship interventions probably reduce length of stay by 1.12 days (95% CI 0.7 to 1.54 days; 15 RCTs; 3834 participants; moderate-certainty evidence). One RCT and six NRS raised concerns that restrictive interventions may lead to delay in treatment and negative professional culture because of breakdown in communication and trust between infection specialists and clinical teams (low-certainty evidence).Both enablement and restriction were independently associated with increased compliance with antibiotic policies, and enablement enhanced the effect of restrictive interventions (high-certainty evidence). Enabling interventions that included feedback were probably more effective than those that did not (moderate-certainty evidence).There was very low-certainty evidence about the effect of the interventions on reducing Clostridium difficile infections (median -48.6%, interquartile range -80.7% to -19.2%; 7 studies). This was also the case for resistant gram-negative bacteria (median -12.9%, interquartile range -35.3% to 25.2%; 11 studies) and resistant gram-positive bacteria (median -19.3%, interquartile range -50.1% to +23.1%; 9 studies). There was too much variance in microbial outcomes to reliably assess the effect of change in antibiotic use. Heterogeneity of intervention effect on prescribing outcomesWe analysed effect modifiers in 29 RCTs and 91 ITS studies. Enablement and restriction were independently associated with a larger effect size (high-certainty evidence). Feedback was included in 4 (17%) of 23 RCTs and 20 (47%) of 43 ITS studies of enabling interventions and was associated with greater intervention effect. Enablement was included in 13 (45%) of 29 ITS studies with restrictive interventions and enhanced intervention effect. AUTHORS' CONCLUSIONS: We found high-certainty evidence that interventions are effective in increasing compliance with antibiotic policy and reducing duration of antibiotic treatment. Lower use of antibiotics probably does not increase mortality and likely reduces length of stay. Additional trials comparing antibiotic stewardship with no intervention are unlikely to change our conclusions. Enablement consistently increased the effect of interventions, including those with a restrictive component. Although feedback further increased intervention effect, it was used in only a minority of enabling interventions. Interventions were successful in safely reducing unnecessary antibiotic use in hospitals, despite the fact that the majority did not use the most effective behaviour change techniques. Consequently, effective dissemination of our findings could have considerable health service and policy impact. Future research should instead focus on targeting treatment and assessing other measures of patient safety, assess different stewardship interventions, and explore the barriers and facilitators to implementation. More research is required on unintended consequences of restrictive interventions.

mTOR signalling, embryogenesis and the control of lung development.

The existence of a nutrient sensitive "autocatakinetic" regulator of embryonic tissue growth has been hypothesised since the early 20th century, beginning with pioneering work on the determinants of foetal size by the Australian physiologist, Thorburn Brailsford-Robertson. We now know that the mammalian target of rapamycin complexes (mTORC1 and 2) perform this essential function in all eukaryotic tissues by balancing nutrient and energy supply during the first stages of embryonic cleavage, the formation of embryonic stem cell layers and niches, the highly specified programmes of tissue growth during organogenesis and, at birth, paving the way for the first few breaths of life. This review provides a synopsis of the role of the mTOR complexes in each of these events, culminating in an analysis of lung branching morphogenesis as a way of demonstrating the central role mTOR in defining organ structural complexity. We conclude that the mTOR complexes satisfy the key requirements of a nutrient sensitive growth controller and can therefore be considered as Brailsford-Robertson's autocatakinetic centre that drives tissue growth programmes during foetal development.

Birt-Hogg-Dube syndrome is a novel ciliopathy.

Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant disorder where patients are predisposed to kidney cancer, lung and kidney cysts and benign skin tumors. BHD is caused by heterozygous mutations affecting folliculin (FLCN), a conserved protein that is considered a tumor suppressor. Previous research has uncovered multiple roles for FLCN in cellular physiology, yet it remains unclear how these translate to BHD lesions. Since BHD manifests hallmark characteristics of ciliopathies, we speculated that FLCN might also have a ciliary role. Our data indicate that FLCN localizes to motile and non-motile cilia, centrosomes and the mitotic spindle. Alteration of FLCN levels can cause changes to the onset of ciliogenesis, without abrogating it. In three-dimensional culture, abnormal expression of FLCN disrupts polarized growth of kidney cells and deregulates canonical Wnt signalling. Our findings further suggest that BHD-causing FLCN mutants may retain partial functionality. Thus, several BHD symptoms may be due to abnormal levels of FLCN rather than its complete loss and accordingly, we show expression of mutant FLCN in a BHD-associated renal carcinoma. We propose that BHD is a novel ciliopathy, its symptoms at least partly due to abnormal ciliogenesis and canonical Wnt signalling.

Viroporins: structure, function and potential as antiviral targets.

The channel-forming activity of a family of small, hydrophobic integral membrane proteins termed 'viroporins' is essential to the life cycles of an increasingly diverse range of RNA and DNA viruses, generating significant interest in targeting these proteins for antiviral development. Viroporins vary greatly in terms of their atomic structure and can perform multiple functions during the virus life cycle, including those distinct from their role as oligomeric membrane channels. Recent progress has seen an explosion in both the identification and understanding of many such proteins encoded by highly significant pathogens, yet the prototypic M2 proton channel of influenza A virus remains the only example of a viroporin with provenance as an antiviral drug target. This review attempts to summarize our current understanding of the channel-forming functions for key members of this growing family, including recent progress in structural studies and drug discovery research, as well as novel insights into the life cycles of many viruses revealed by a requirement for viroporin activity. Ultimately, given the successes of drugs targeting ion channels in other areas of medicine, unlocking the therapeutic potential of viroporins represents a valuable goal for many of the most significant viral challenges to human and animal health.

High-Specificity Imaging Mass Spectrometry.

Imaging mass spectrometry (IMS) enables highly multiplexed, untargeted tissue mapping for a broad range of molecular classes, facilitating in situ biological discovery. Yet, challenges persist in molecular specificity, which is the ability to discern one molecule from another, and spatial specificity, which is the ability to link untargeted imaging data to specific tissue features. Instrumental developments have dramatically improved IMS spatial resolution, allowing molecular observations to be more readily associated with distinct tissue features across spatial scales, ranging from larger anatomical regions to single cells. High-performance mass analyzers and systems integrating ion mobility technologies are also becoming more prevalent, further improving molecular coverage and the ability to discern chemical identity. This review provides an overview of recent advancements in high-specificity IMS that are providing critical biological context to untargeted molecular imaging, enabling integrated analyses, and addressing advanced biomedical research applications.

Experiences of children's self-wetting (including urinary incontinence) in Cox's Bazar's Rohingya refugee camps, Bangladesh.

Self-wetting is the leakage of urine, either due to the medical condition of urinary incontinence (UI), or because a person does not want to, or cannot, access a toileting facility in time. This study explored the attitudes towards self-wetting and experiences of children (aged five to 11), their caregivers, community leaders and humanitarian practitioners in the Rohingya refugee camps in Cox's Bazar, Bangladesh. We particularly focused on how water, sanitation and hygiene (WASH) and protection interventions might assist in improving these experiences. We purposively selected participants from two camps where our partner organisation works. We conducted Key Informant Interviews (KIIs) with community leaders and camp officials, Story Book (SB) sessions with Rohingya children and in-depth Interviews (IDIs) with caregivers of children who participated in the SB sessions, as well as surveying communal toilets. Self-wetting by children was common and resulted in them feeling embarrassed, upset and uncomfortable, and frightened to use the toilet at night; many children also indicated that they would be punished by their caregivers for self-wetting. Key informants indicated that caregivers have difficulty handling children's self-wetting due to a limited amount of clothing, pillows, and blankets, and difficulty cleaning these items. It was evident that the available toilets are often not appropriate and/or accessible for children. Children in the Rohingya camps appear to self-wet due to both the medical condition of UI and because the sanitation facilities are inappropriate. They are teased by their peers and punished by their caregivers. Although WASH and protection practitioners are unable to drastically alter camp conditions or treat UI, the lives of children who self-wet in these camps could likely be improved by increasing awareness on self-wetting to decrease stigma and ease the concerns of caregivers, increasing the number of child-friendly toilets and increasing the provision of continence management materials.

Connexins in epidermal homeostasis and skin disease.

The expression of multiple connexin (Cx) types in the epidermis, their differential expression during wound closure and the association of skin pathology with specific Cx gene mutations, are indicative of important functions for Cxs in the skin. In this review, we focus on the role of Cx proteins in the epidermis and during wound healing and discuss mutations in Cx genes which cause skin disease. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.

Harlequin ichthyosis: ABCA12 mutations underlie defective lipid transport, reduced protease regulation and skin-barrier dysfunction.

Harlequin ichthyosis (HI) is a devastating autosomal recessive congenital skin disease. It has been vital to elucidate the biological importance of the protein ABCA12 in skin-barrier permeability, following the discovery that ABCA12 gene mutations can result in this rare disease. ATP-binding cassette transporter A12 (ABCA12) is a member of the subfamily of ATP-binding cassette transporters and functions to transport lipid glucosylceramides (GlcCer) to the extracellular space through lamellar granules (LGs). GlcCer are hydrolysed into hydroxyceramides extracellularly and constitute a portion of the extracellular lamellar membrane, lipid envelope and lamellar granules. In HI skin, loss of function of ABCA12 due to null mutations results in impaired lipid lamellar membrane formation in the cornified layer, leading to defective permeability of the skin barrier. In addition, abnormal lamellar granule formation (distorted shape, reduced in number or absent) could further cause aberrant production of LG-associated desquamation enzymes, which are likely to contribute to the impaired skin barrier in HI. This article reviews current opinions on the patho-mechanisms of ABCA12 action in HI and potential therapeutic interventions based on targeted molecular therapy and gene therapy strategies.

Neprilysin Inhibitors in Heart Failure: The Science, Mechanism of Action, Clinical Studies, and Unanswered Questions.

This article provides a contemporary review and a new perspective on the role of neprilysin inhibition in heart failure (HF) in the context of recent clinical trials and addresses potential mechanisms and unanswered questions in certain HF patient populations. Neprilysin is an endopeptidase that cleaves a variety of peptides such as natriuretic peptides, bradykinin, adrenomedullin, substance P, angiotensin I and II, and endothelin. It has a broad role in cardiovascular, renal, pulmonary, gastrointestinal, endocrine, and neurologic functions. The combined angiotensin receptor and neprilysin inhibitor (ARNi) has been developed with an intent to increase vasodilatory natriuretic peptides and prevent counterregulatory activation of the angiotensin system. ARNi therapy is very effective in reducing the risks of death and hospitalization for HF in patients with HF and New York Heart Association functional class II to III symptoms, but studies failed to show any benefits with ARNi when compared with angiotensin-converting enzyme inhibitors or angiotensin receptor blocker in patients with advanced HF with reduced ejection fraction or in patients following myocardial infarction with left ventricular dysfunction but without HF. These raise the questions about whether the enzymatic breakdown of natriuretic peptides may not be a very effective solution in advanced HF patients when there is downstream blunting of the response to natriuretic peptides or among post-myocardial infarction patients in the absence of HF when there may not be a need for increased natriuretic peptide availability. Furthermore, there is a need for additional studies to determine the long-term effects of ARNi on albuminuria, obesity, glycemic control and lipid profile, blood pressure, and cognitive function in patients with HF.

Gradients of glucose metabolism regulate morphogen signalling required for specifying tonotopic organisation in the chicken cochlea.

In vertebrates with elongated auditory organs, mechanosensory hair cells (HCs) are organised such that complex sounds are broken down into their component frequencies along a proximal-to-distal long (tonotopic) axis. Acquisition of unique morphologies at the appropriate position along the chick cochlea, the basilar papilla, requires that nascent HCs determine their tonotopic positions during development. The complex signalling within the auditory organ between a developing HC and its local niche along the cochlea is poorly understood. Using a combination of live imaging and NAD(P)H fluorescence lifetime imaging microscopy, we reveal that there is a gradient in the cellular balance between glycolysis and the pentose phosphate pathway in developing HCs along the tonotopic axis. Perturbing this balance by inhibiting different branches of cytosolic glucose catabolism disrupts developmental morphogen signalling and abolishes the normal tonotopic gradient in HC morphology. These findings highlight a causal link between graded morphogen signalling and metabolic reprogramming in specifying the tonotopic identity of developing HCs.

Understanding children's experiences of self-wetting in humanitarian contexts: An evaluation of the Story Book methodology.

Little is known about how children in humanitarian contexts experience self-wetting. Children can wet themselves due to having the medical condition of urinary incontinence (the involuntary leakage of urine), or due to them not wanting to or not being able to use the toilet facilities available (social or functional incontinence). Self-wetting is a global public health challenge: the physical health of children can suffer; they can miss out on educational and social opportunities; they may face increased protection risks; and the emotional effect on daily life can be significantly negative. The Story Book methodology was developed to facilitate conversations with children aged five to eleven in humanitarian contexts (specifically refugee settlements in Adjumani District, Uganda; and refugee camps in Cox's Bazar, Bangladesh) about self-wetting to understand how humanitarian professionals can best meet the needs of children that wet themselves. This paper has evaluated how far the Story Book methodology meets the specific requirements of conducting research a) in a humanitarian context; b) with young children; and c) on a personal and highly sensitive topic. Data has been used from Story Book sessions held with children in Adjumani District and Cox's Bazar, and from semi-structured interviews held with adults known to have participated in the planning and/or facilitation of the sessions. The evaluation found that although the Story Book methodology provided deep insights into how children in humanitarian contexts experience self-wetting, it was not always implemented as designed; it is not practical to implement in humanitarian settings; and it was not acceptable to all participants and facilitators as a research tool. Changes have been recommended to improve the methodology as a research tool to better understand how children experience personal health issues, but even with such changes the methodology will remain better suited to non-humanitarian contexts.

Arylstibonic acids are potent and isoform-selective inhibitors of Cdc25a and Cdc25b phosphatases.

Arylstibonates structurally resemble phosphotyrosine side chains in proteins and here we addressed the ability of such compounds to act as inhibitors of a panel of mammalian tyrosine and dual-specificity phosphatases. Two arylstibonates both possessing a carboxylate side chain were identified as potent inhibitors of the protein tyrosine phosphatase PTP-ß. In addition, they inhibited the dual-specificity, cell cycle regulatory phosphatases Cdc25a and Cdc25b with sub-micromolar potency. However, the Cdc25c phosphatase was not affected demonstrating that arylstibonates may be viable leads from which to develop isoform specific Cdc25 inhibitors.

Key functions for gap junctions in skin and hearing.

Cx (connexin) proteins are components of gap junctions which are aqueous pores that allow intercellular exchange of ions and small molecules. Mutations in Cx genes are linked to a range of human disorders. In the present review we discuss mutations in ß-Cx genes encoding Cx26, Cx30, Cx30.3 and Cx31 which lead to skin disease and deafness. Functional studies with Cx proteins have given insights into disease-associated mechanisms and non-gap junctional roles for Cx proteins.