A Small Conductance Calcium-Activated K⁺ Channel in C. elegans, KCNL-2, Plays a Role in the Regulation of the Rate of Egg-Laying

In the nervous system of mice, small conductance calcium-activated potassium (SK) channels function to regulate neuronal excitability through the generation of a component of the medium afterhyperpolarization that follows action potentials. In humans, irregular action potential firing frequency underlies diseases such as ataxia, epilepsy, schizophrenia and Parkinson's disease. Due to the complexity of studying protein function in the mammalian nervous system, we sought to characterize an SK channel homologue, KCNL-2, in C. elegans, a genetically tractable system in which the lineage of individual neurons was mapped from their early developmental stages. Sequence analysis of the KCNL-2 protein reveals that the six transmembrane domains, the potassium-selective pore and the calmodulin binding domain are highly conserved with the mammalian homologues. We used widefield and confocal fluorescent imaging to show that a fusion construct of KCNL-2 with GFP in transgenic lines is expressed in the nervous system of C. elegans. We also show that a KCNL-2 null strain, kcnl-2(tm1885), demonstrates a mild egg-laying defective phenotype, a phenotype that is rescued in a KCNL-2-dependent manner. Conversely, we show that transgenic lines that overexpress KCNL-2 demonstrate a hyperactive egg-laying phenotype. In this study, we show that the vulva of transgenic hermaphrodites is highly innervated by neuronal processes and by the VC4 and VC5 neurons that express GFP-tagged KCNL-2. We propose that KCNL-2 functions in the nervous system of C. elegans to regulate the rate of egg-laying. © 2013 Chotoo et al

What influences people's responses to public health messages for managing risks and preventing infectious diseases? A rapid systematic review of the evidence and recommendations

BACKGROUND: Individual behaviour changes, such as hand hygiene and physical distancing, are required on a population scale to reduce transmission of infectious diseases such as COVID-19. However, little is known about effective methods of communicating risk reducing information, and how populations might respond. OBJECTIVE: To synthesise evidence relating to what (1) characterises effective public health messages for managing risk and preventing infectious disease and (2) influences people's responses to messages. DESIGN: A rapid systematic review was conducted. Protocol is published on Prospero CRD42020188704. DATA SOURCES: Electronic databases were searched: Ovid Medline, Ovid PsycINFO and Healthevidence.org, and grey literature (PsyarXiv, OSF Preprints) up to May 2020. STUDY SELECTION: All study designs that (1) evaluated public health messaging interventions targeted at adults and (2) concerned a communicable disease spread via primary route of transmission of respiratory and/or touch were included. Outcomes included preventative behaviours, perceptions/awareness and intentions. Non-English language papers were excluded. SYNTHESIS: Due to high heterogeneity studies were synthesised narratively focusing on determinants of intentions in the absence of measured adherence/preventative behaviours. Themes were developed independently by two researchers and discussed within team to reach consensus. Recommendations were translated from narrative synthesis to provide evidence-based methods in providing effective messaging. RESULTS: Sixty-eight eligible papers were identified. Characteristics of effective messaging include delivery by credible sources, community engagement, increasing awareness/knowledge, mapping to stage of epidemic/pandemic. To influence intent effectively, public health messages need to be acceptable, increase understanding/perceptions of health threat and perceived susceptibility. DISCUSSION: There are four key recommendations: (1) engage communities in development of messaging, (2) address uncertainty immediately and with transparency, (3) focus on unifying messages from sources and (4) frame messages aimed at increasing understanding, social responsibility and personal control. Embedding principles of behavioural science into public health messaging is an important step towards more effective health-risk communication during epidemics/pandemics

A pilot study evaluating GSK1070806 inhibition of interleukin-18 in renal transplant delayed graft function.

INTRODUCTION: Delayed graft function (DGF) following renal transplantation is a manifestation of acute kidney injury (AKI) leading to poor long-term outcome. Current treatments have limited effectiveness in preventing DGF. Interleukin-18 (IL18), a biomarker of AKI, induces interferon-γ expression and immune activation. GSK1070806, an anti-IL18 monoclonal antibody, neutralizes activated (mature) IL18 released from damaged cells following inflammasome activation. This phase IIa, single-arm trial assessed the effect of a single dose of GSK1070806 on DGF occurrence post donation after circulatory death (DCD) kidney transplantation. METHODS: The 3 mg/kg intravenous dose was selected based on prior studies and physiologically based pharmacokinetic (PBPK) modeling, indicating the high likelihood of a rapid and high level of IL18 target engagement when administered prior to kidney allograft reperfusion. Utilization of a Bayesian sequential design with a background standard-of-care DGF rate of 50% based on literature, and confirmed via extensive registry data analyses, enabled a statistical efficacy assessment with a minimal sample size. The primary endpoint was DGF frequency, defined as dialysis requirement ≤7 days post transplantation (except for hyperkalemia). Secondary endpoints included safety, pharmacokinetics and pharmacodynamic biomarkers. RESULTS: GSK1070806 administration was associated with IL18-GSK1070806 complex detection and increased total serum IL18 levels due to IL18 half-life prolongation induced by GSK1070806 binding. Interferon-γ-induced chemokine levels declined or remained unchanged in most patients. Although the study was concluded prior to the Bayesian-defined stopping point, 4/7 enrolled patients (57%) had DGF, exceeding the 50% standard-of-care rate, and an additional two patients, although not reaching the protocol-defined DGF definition, demonstrated poor graft function. Six of seven patients experienced serious adverse events (SAEs), including two treatment-related SAEs. CONCLUSION: Overall, using a Bayesian design and extensive PBPK dose modeling with only a small sample size, it was deemed unlikely that GSK1070806 would be efficacious in preventing DGF in the enrolled DCD transplant population. TRIAL REGISTRATION: NCT02723786

Increased Basal Activity Is a Key Determinant in the Severity of Human Skeletal Dysplasia Caused by TRPV4 Mutations

TRPV4 is a mechanically activated Ca2+-passing channel implicated in the sensing of forces, including those acting on bones. To date, 33 mutations are known to affect human bone development to different extents. The spectrum of these skeletal dysplasias (SD) ranges from dominantly inherited mild brachylomia (BO) to neonatal lethal forms of metatropic dysplasia (MD). Complexities of the results from fluorescence and electrophysiological studies have led to questions on whether channel activity is a good predictor of disease severity. Here we report on a systematic examination of 14 TRPV4 mutant alleles covering the entire SD spectrum. Expressed in Xenopus oocyte and without any stimulation, the wild-type channel had a ∼1% open probability (Po) while those of most of the lethal MD channels approached 100%. All mutant channels had higher basal open probabilities, which limited their further increase by agonist or hypotonicity. The magnitude of this limitation revealed a clear correlation between the degree of over-activity (the molecular phenotype) and the severity of the disease over the entire spectrum (the biological phenotype). Thus, while other factors are at play, our results are consistent with the increased TRPV4 basal activity being a critical determinant of the severity of skeletal dysplasia. We discuss how the channel over-activity may lead to the “gain-of-function” phenotype and speculate that the function of wild-type TRPV4 may be secondary in normal bone development but crucial in an acute process such as fracture repair in the adult

Blood lactate levels in 31 female dogs with pyometra

<p>Abstract</p> <p>Background</p> <p>Canine pyometra is a life-threatening disease common in countries where spaying of dogs is not routinely performed. The disease is associated with endotoxemia, sepsis, systemic inflammatory response syndrome (SIRS) and a 3–4% mortality rate. Blood lactate analysis is clinically valuable in predicting prognosis and survival, evaluating tissue perfusion and treatment response in human and veterinary critical care settings. The aims of the present study were to investigate 1) the blood lactate levels of female dogs with pyometra by a hand-held analyser and 2) if these levels are related with the clinical status or other biochemical or hematological disorders.</p> <p>Methods</p> <p>In total 31 female dogs with pyometra admitted for surgical ovariohysterectomy and 16 healthy female control dogs were included in the present study. A complete physical examination including SIRS-status determination was performed. Blood samples for lactate concentrations, hematological and biochemical parameters, acid-base and blood gas analysis and other laboratory parameters were collected and subsequently analysed. The diagnosis pyometra was verified with histopathological examination of the uterus and ovaries. Increased hospitalisation length and presence of SIRS were used as indicators of outcome.</p> <p>Results</p> <p>In the pyometra group the median blood lactate level was 1,6 mmol l^-1(range <0.8–2.7 mmol l^-1). In the control group the median lactate level was 1,2 mmol l^-1(range <0.8–2.1 mmol l^-1). Of the 31 bitches 19 (61%) fulfilled 2 or more criteria for SIRS at inclusion, 10 bitches (32%) fulfilled 3 of the SIRS criteria whereas none accomplished more than 3 criteria. Lactate levels did not differ significantly between the pyometra and control group, or between the SIRS positive and SIRS negative dogs with pyometra. Increased lactate concentration (>2.5 mmol l^-1) was demonstrated in one female dog with pyometra (3%), and was not associated with longer hospitalisation or presence of SIRS. Lactate measurement was not indicative of peritonitis. None of the bitches died during or within two months of the hospital stay. The measurements of temperature, heart rate, respiratory rate, percentage bandforms of neutrophilic granulocytes, α₂-globulins, creatinin, pvCO₂, TCO₂and base excess showed significant differences between the SIRS positive and the SIRS negative pyometra cases.</p> <p>Conclusion</p> <p>Increased blood lactate concentrations were demonstrated in 3% (1/31), and SIRS was present in 61% (19/31) of the female dogs with pyometra. Preoperative lactate levels were not related with presence of SIRS or prolonged hospitalisation. Lactate measurement was not indicative of peritonitis. The value of a single and repeated lactate analysis in more severely affected cases remains to be determined.</p

Emerging role of the calcium-activated, small conductance, SK3 K ⁺ channel in distal tubule function: Regulation by TRPV4

The Ca2+-activated, maxi-K (BK) K+ channel, with low Ca2+-binding affinity, is expressed in the distal tubule of the nephron and contributes to flow-dependent K+ secretion. In the present study we demonstrate that the Ca2+-activated, SK3 (KCa2.3) K + channel, with high Ca2+-binding affinity, is also expressed in the mouse kidney (RT-PCR, immunoblots). Immunohistochemical evaluations using tubule specific markers demonstrate significant expression of SK3 in the distal tubule and the entire collecting duct system, including the connecting tubule (CNT) and cortical collecting duct (CCD). In CNT and CCD, main sites for K+ secretion, the highest levels of expression were along the apical (luminal) cell membranes, including for both principal cells (PCs) and intercalated cells (ICs), posturing the channel for Ca2+- dependent K+ secretion. Fluorescent assessment of cell membrane potential in native, split-opened CCD, demonstrated that selective activation of the Ca2+-permeable TRPV4 channel, thereby inducing Ca2+ influx and elevating intracellular Ca2+ levels, activated both the SK3 channel and the BK channel leading to hyperpolarization of the cell membrane. The hyperpolarization response was decreased to a similar extent by either inhibition of SK3 channel with the selective SK antagonist, apamin, or by inhibition of the BK channel with the selective antagonist, iberiotoxin (IbTX). Addition of both inhibitors produced a further depolarization, indicating cooperative effects of the two channels on Vm. It is concluded that SK3 is functionally expressed in the distal nephron and collecting ducts where induction of TRPV4-mediated Ca2+ influx, leading to elevated intracellular Ca2+ levels, activates this high Ca2+- affinity K+ channel. Further, with sites of expression localized to the apical cell membrane, especially in the CNT and CCD, SK3 is poised to be a key pathway for Ca2+-dependent regulation of membrane potential and K+ secretion. © 2014 Berrout et al

Signal transduction underlying the control of urinary bladder smooth muscle tone by muscarinic receptors and β-adrenoceptors

The normal physiological contraction of the urinary bladder, which is required for voiding, is predominantly mediated by muscarinic receptors, primarily the M3 subtype, with the M2 subtype providing a secondary backup role. Bladder relaxation, which is required for urine storage, is mediated by β-adrenoceptors, in most species involving a strong β3-component. An excessive stimulation of contraction or a reduced relaxation of the detrusor smooth muscle during the storage phase of the micturition cycle may contribute to bladder dysfunction known as the overactive bladder. Therefore, interference with the signal transduction of these receptors may be a viable approach to develop drugs for the treatment of overactive bladder. The prototypical signaling pathway of M3 receptors is activation of phospholipase C (PLC), and this pathway is also activated in the bladder. Nevertheless, PLC apparently contributes only in a very minor way to bladder contraction. Rather, muscarinic-receptor-mediated bladder contraction involves voltage-operated Ca2+ channels and Rho kinase. The prototypical signaling pathway of β-adrenoceptors is an activation of adenylyl cyclase with the subsequent formation of cAMP. Nevertheless, cAMP apparently contributes in a minor way only to β-adrenoceptor-mediated bladder relaxation. BKCa channels may play a greater role in β-adrenoceptor-mediated bladder relaxation. We conclude that apart from muscarinic receptor antagonists and β-adrenoceptor agonists, inhibitors of Rho kinase and activators of BKCa channels may have potential to treat an overactive bladder

Greenhouse Gases and other Airborne Pollutants from Household Stoves in China: a Database for Emission Factors

Emissions from household stoves, especially those using solid fuels, can contribute significantly to greenhouse gas (GHG) inventories and have adverse health impacts. Few data are available on emissions from the numerous types of cookstoves used in developing countries. We have systematically measured emissions from 56 fuel/stove combinations in India and China, a large fraction of the combinations in use world-wide. A database was generated containing emission factors of direct and indirect GHGs and other airborne pollutants such as CO2, CO, CH4, TNMHC, N2O, SO2, NOx, TSP, etc. In this paper, we report on the 28 fuel/stove combinations tested in China. Since fuel and stove parameters were measured simultaneously along with the emissions, the database allows construction of complete carbon balances and analyses of the trade-off of emissions per unit fuel mass and emissions per delivered energy. Results from the analyses show that the total emissions per unit delivered energy were substantially greater from burning the solid fuels than from burning the liquid or gaseous fuels, due to lower thermal and combustion efficiencies for solid-fuel/stove combinations. For a given biomass fuel type, increasing overall stove efficiency tends to increase emissions of products of incomplete combustion. Biomass fuels are typically burned with substantial production of non-CO2 GHGs with greater radiative forcing, indicating that biomass fuels have the potential to produce net global warming commitments even when grown renewably