Homo- and hetero-dimeric subunit interactions set affinity and efficacy in metabotropic glutamate receptors.

Metabotropic glutamate receptors (mGluRs) are dimeric class C G-protein-coupled receptors that operate in glia and neurons. Glutamate affinity and efficacy vary greatly between the eight mGluRs. The molecular basis of this diversity is not understood. We used single-molecule fluorescence energy transfer to monitor the structural rearrangements of activation in the mGluR ligand binding domain (LBD). In saturating glutamate, group II homodimers fully occupy the activated LBD conformation (full efficacy) but homodimers of group III mGluRs do not. Strikingly, the reduced efficacy of Group III homodimers does not arise from differences in the glutamate binding pocket but, instead, from interactions within the extracellular dimerization interface that impede active state occupancy. By contrast, the functionally boosted mGluR II/III heterodimers lack these interface 'brakes' to activation and heterodimer asymmetry in the flexibility of a disulfide loop connecting LBDs greatly favors occupancy of the activated conformation. Our results suggest that dimerization interface interactions generate substantial functional diversity by differentially stabilizing the activated conformation. This diversity may optimize mGluR responsiveness for the distinct spatio-temporal profiles of synaptic versus extrasynaptic glutamate.

Effects of temperature on the pharmacokinetics, optimal dosage, tissue residue, and withdrawal time of florfenicol in asian seabass (lates calcarifer).

Drug behavior in the bodies of fish is largely influenced by the water temperature. Antimicrobial drugs are needed for the control of bacterial outbreaks in farmed fish including Asian seabass (Lates calcarifer). However, little is known about the temperature effect on appropriate drug uses in this species. The purpose of this study was to investigate the differences in pharmacokinetics (PK), optimal dosages, tissue depletion, and withdrawal time (WDT) of florfenicol (FF) in Asian seabass reared at 25 and 30 °C. In the PK study, the fish were administered with a single oral dose of 10 mg/kg FF. The optimal dosing regimen was determined by the pharmacokinetic-pharmacodynamic (PK-PD) approach. In the tissue depletion and WDT study, FF was administered at the optimal dosages once daily for 5 days and the WDT was determined by linear regression analysis based on the sum of FF and its metabolite florfenicol amine (FFA) in the muscle/skin. When the temperature was increased from 25 to 30 °C, the elimination half-life of FF was significantly decreased from 11.0 to 7.2 h. While the other PK parameters were not changed significantly, the calculated optimal dosages for the target minimum inhibitory concentration (MIC) of 2 µg/mL were 10.9 and 22.0 mg/kg/day, respectively for 25 and 30 °C. The sum of FF + FFA is a preferable marker residue for WDT determination because differential FF metabolism was observed at different temperatures. The depletion half-life of the muscle/skin was shortened from 41.1 to 32.4 h by the 5 °C temperature increase. Despite different absolute amounts of FF given between the two temperature levels, the WDTs were very similar at 6-7 days. Thus, it appears that a single temperature-independent WDT can potentially be assigned when the drug was applied at the optimal dosage.

Effects of Different Shading Treatments on the Biomass and Transcriptome Profiles of Tea Leaves (Camellia sinensis L.) and the Regulatory Effect on Phytohormone Biosynthesis.

Our previous study showed that colored net shading treatments had comparable effects on the reduction of bitter and astringent compounds such as flavonol glycosides in tea leaves, compared with black net shading treatment, whereas the effects on the biomass and phytohormones are still unclear. In this study, we investigated the phytohormone and transcriptome profiles of tea leaves under different shading treatments, using black, blue, and red nets with the same shade percentages. The bud density, fresh weight of 100 buds, and yield under blue net shading treatments were greatly elevated by 2.00-fold, 1.24-fold, and 2.48-fold, compared with black net shading treatment, while their effects on flavonoid composition were comparable with black net shading treatment. The transcriptome profiles of different shade net-treated samples were well resolved and discriminated from control. The KEGG result indicated that the pathways of phenylpropanoid biosynthesis, MAPK signaling pathways, and plant hormone signal transduction were differentially regulated by different shading treatments. The co-expression analysis showed that the contents of salicylic acid and melatonin were closely correlated with certain light signal perception and signaling genes (p < 0.05), and UVR8, PHYE, CRY1, PHYB, PHOT2, and HY5 had more close interactions with phytohormone biosynthetic genes (p < 0.05). Our results suggest that different shading treatments can mediate the growth of tea plants, which could be attributed to the regulatory effect on phytohormones levels, providing an instruction for the production of summer/autumn tea and matcha.

Selective Photoswitchable Allosteric Agonist of a G Protein-Coupled Receptor.

G protein-coupled receptors (GPCRs) are the most common targets of drug discovery. However, the similarity between related GPCRs combined with the complex spatiotemporal dynamics of receptor activation in vivo has hindered drug development. Photopharmacology offers the possibility of using light to control the location and timing of drug action by incorporating a photoisomerizable azobenzene into a GPCR ligand, enabling rapid and reversible switching between an inactive and active configuration. Recent advances in this area include (i) photoagonists and photoantagonists that directly control receptor activity but are nonselective because they bind conserved sites, and (ii) photoallosteric modulators that bind selectively to nonconserved sites but indirectly control receptor activity by modulating the response to endogenous ligand. In this study, we designed a photoswitchable allosteric agonist that targets a nonconserved allosteric site for selectivity and activates the receptor on its own to provide direct control. This work culminated in the development of aBINA, a photoswitchable allosteric agonist that selectively activates the Gi/o-coupled metabotropic glutamate receptor 2 (mGluR2). aBINA is the first example of a new class of precision drugs for GPCRs and other clinically important signaling proteins.

Does the new-type urbanization construction improve the efficiency of agricultural green water utilization in the Yangtze River Economic Belt?

It is very important to control agricultural water pollution and promote agricultural water saving, for high-quality development of Yangtze River Economic Belt (YREB). The efficiency of agricultural green water utilization (EAGWU) needs financial and technical support from the new-type urbanization, which also change agricultural production mode and resource utilization level. This paper introduces non-point source water pollution into the output, adopts the super efficiency-slack model (SE-SBM) to measure the EAGWU, and uses difference generalized method of moments (DIF-GMM) to examine how new-type urbanization affects EAGWU from its four core characteristics. The results of EAGWU show that the overall efficiency value has been increasing rapidly in the research period, while the eastern provinces performed better and the central provinces performed worse. On the other hand, the overall difference in EAGWU first diverged and then shrunk, while economically developed provinces has been converging all the time. The results of driving factor estimation show that population urbanization has a significantly positive effect on EAGWU, with the rural labor force transfer and agricultural land circulation. Economic urbanization and urban-rural integration have negative effects, with the widening gap of absolute income and the compressed space of agricultural development. The EAGWU lag phase has a positive effect, because of the ratchet or cumulative effect, while equilibrium-urbanization has an insignificant effect. The conclusions will provide preferable recommendations for decision-making of green and water-saving development in agriculture.

Size-modulated optical property of gold nanorods for sensitive and colorimetric detection of thiourea in fruit juice.

As an important sulfur compound, thiourea (TU) has caused great concern because of its wide application as well as its serious toxicity and hazard to the environment. Thus, it is necessary to develop a sensitive and selective method for TU analysis. In this work, gold nanorods (AuNRs) acted as an optical probe to realize the sensitive and colorimetric detection of TU. In HCl medium, Fe3+ at low concentration was difficult to oxide Au0 to form Au+ because of the high redox potential or the positive Gibbs free energy change. However, this process was possible when TU was present since the association constant between Au+ and TU is great enough to bind with TU to form a stable complex to further promote the etching of AuNRs, resulting in the lower aspect ratio of AuNRs with the blue shift and intensity decrease in extinction spectra, accompanied by the divisive colors of AuNRs solution or colorful dark-field light scattering imaging of single AuNR. The blue-shift of AuNRs longitudinal plasmon resonance absorption (LPRA) band was proportional to the concentration of TU in the range of 1-250 nM and the limit of detection (3σ/k) was as low as 0.4 nM. In addition, the colorimetric method was proven with high selectivity in the presence of potential interfering compounds, which was successfully applied to the detection of TU in fruit juice samples. This proposed colorimetric method provides a simple, sensitive yet selective measurement tool for TU sensing, which may offer new opportunities in the development of colorimetric sensors for food safety in the future.

Study on the Spatial Differentiation of Public Health Service Capabilities of European Union under the Background of the COVID-19 Crisis.

Access to public health services is a cause that benefits the people and concerns the vital interests of the people. Everyone has access to basic health care services. The continuous improvement in people's health is an important indicator of the improvement in people's quality of life. This paper selects data from the European Union (EU) on aspects of public health expenditure, medical care resources, and government emergency coordination capacity from the period 2008 to 2017. Principal component analysis and factor analysis are used to measure their public health service capacity scores and conduct a comparative analysis. On this basis, the TOBIT model is adopted to explore the driving factors that lead to the spatial differentiation of public health service capabilities, and to combine it with the data of the COVID-19 epidemic as of 8 August 2020 from the official announcements of the World Health Organization and governments for further thinking. The results indicate that the public health service capacity of countries in the EU is showing a gradual increase. The capacity in Western Europe is, in turn, higher than that of Northern Europe, Southern Europe and Eastern Europe. In addition, the overall capacity in Western Europe is relatively high, but it is not balanced and stable, while Northern Europe has remained stable and balanced at a high level. Population density, degree of opening up, education level, economic development level, technological innovation level, and degree of aging have a positive effect on public health service capabilities. The level of urbanization has a negative effect on it. However, in countries with strong public health service capabilities, the epidemic of COVID-19 is more severe. The emergence of this paradox may be related to the detection capabilities of countries, the high probability of spreading thCOVID-19 epidemic, the inefficient implementation of government policy, the integrated system of the EU and the adverse selection of youth. This paper aims to improve the ability of the EU to respond to public health emergencies, improve the utilization of medical and health resources, and better protect people's health from the perspective of public health service capacity.

Conformational pathway provides unique sensitivity to a synaptic mGluR.

Metabotropic glutamate receptors (mGluRs) are dimeric G-protein-coupled receptors that operate at synapses. Macroscopic and single molecule FRET to monitor structural rearrangements in the ligand binding domain (LBD) of the mGluR7/7 homodimer revealed it to have an apparent affinity ~4000-fold lower than other mGluRs and a maximal activation of only ~10%, seemingly too low for activation at synapses. However, mGluR7 heterodimerizes, and we find it to associate with mGluR2 in the hippocampus. Strikingly, the mGluR2/7 heterodimer has high affinity and efficacy. mGluR2/7 shows cooperativity in which an unliganded subunit greatly enhances activation by agonist bound to its heteromeric partner, and a unique conformational pathway to activation, in which mGluR2/7 partially activates in the Apo state, even when its LBDs are held open by antagonist. High sensitivity and an unusually broad dynamic range should enable mGluR2/7 to respond to both glutamate transients from nearby release and spillover from distant synapses.

Genetically Targeted Optical Control of an Endogenous G Protein-Coupled Receptor.

G protein-coupled receptors (GPCRs) are membrane proteins that play important roles in biology. However, our understanding of their function in complex living systems is limited because we lack tools that can target individual receptors with sufficient precision. State-of-the-art approaches, including DREADDs, optoXRs, and PORTL gated-receptors, control GPCR signaling with molecular, cell type, and temporal specificity. Nonetheless, these tools are based on engineered non-native proteins that may (i) express at nonphysiological levels, (ii) localize and turnover incorrectly, and/or (iii) fail to interact with endogenous partners. Alternatively, membrane-anchored ligands (t-toxins, DARTs) target endogenous receptors with molecular and cell type specificity but cannot be turned on and off. In this study, we used a combination of chemistry, biology, and light to control endogenous metabotropic glutamate receptor 2 (mGluR2), a Family C GPCR, in primary cortical neurons. mGluR2 was rapidly, reversibly, and selectively activated with photoswitchable glutamate tethered to a genetically targeted-plasma membrane anchor (membrane anchored Photoswitchable Orthogonal Remotely Tethered Ligand; maPORTL). Photoactivation was tuned by adjusting the length of the PORTL as well as the expression level and geometry of the membrane anchor. Our findings provide a template for controlling endogenous GPCRs with cell type specificity and high spatiotemporal precision.

Anti inflammatory effect of asiaticoside on human umbilical vein endothelial cells induced by ox-LDL.

Early diagnosis and changes associated with atherosclerosis are crucial in clinical medicine. However, atherosclerosis is a multifactorial disease. Asiaticoside (AA), a triterpenoid derived from Centella asiatica, has anti-inflammatory activity. Endothelium-derived nitric oxide is important in modulating vascular tone in a distinct vessel size-dependent manner; it plays a dominant role in conduit arteries and endothelium-dependent hyperpolarisation in resistance vessels. This study evaluated the effects of AA administration on human umbilical endothelial cells with oxidised low-density lipoprotein-induced inflammation. We measured the levels of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Our results indicated that 10-30 µM AA modulated endothelial hyper permeability, adenosine triphosphate levels, ICAM-1 expression, VCAM-1 expression, E-selectin levels, and PECAM-1 expression to 90% (p < 0.005), 80% (p < 0.05), 105% (p < 0.01), 65% (p < 0.005), 70% (p < 0.05), and 105% (p < 0.01), respectively. Taken together, our data suggest that AA inhibits the augmentation of endothelial permeability, thus preventing the early events of atherosclerosis.

Vitamins supplementation affects the onset of preeclampsia.

Preeclampsia may affect between 2-8% of all pregnancies. It seriously affects maternal health after pregnancy. This meta-analysis was performed to define the efficacy of vitamins supplementation on the risk of preeclampsia. Potential articles were systematically searched on the databases of Pubmed, Embase and Web of Science up to May 2016. Relative risk (RR) and 95% confidence intervals (95%CIs) were used to analyze the relationship of vitamins supplementation with risk of preeclampsia. Cochran Q test was used to test inter-study heterogeneity. Begg's funnel plot was adopted to assess the potential publication bias. 28 eligible studies were selected. Pooled results indicated that vitamins supplementation could reduce the risk of preeclampsia (RR = 0.74, 95%CI = 0.64-0.86). The studies with non-randomized controlled trial (RCT) analysis also suggested the significant relationship of vitamins supplementation with risk of preeclampsia (RR = 0.60, 95%CI = 0.42-0.85). However, negative results were observed in studies with RCT analysis. Subgroup analysis by vitamin type was performed among the studies with RCT analysis. The results indicated that vitamin D supplementation could significantly reduce the risk of preeclampsia (RR = 0.41, 95%CI = 0.22-0.78). Similar results were observed in the studies with multivitamins supplementation (RR = 0.69, 95%CI = 0.51-0.93). Vitamins supplementation could reduce the onset of preeclampsia.

Intravenous administration of tirofiban versus loading dose of oral clopidogrel for preventing thromboembolism in stent-assisted coiling of intracranial aneurysms.

Background Thromboembolic complications after stenting of intracranial aneurysms may be affected by antiplatelet administration. Aims This retrospective study aimed to assess the safety of intravenous tirofiban versus loading dose of oral clopidogrel for preventing thromboembolism in stent-assisted coiling of intracranial aneurysms. Methods From January 2006 to December 2013, 281 patients with cerebral aneurysms were treated with stent-coiling using two antiplatelet strategies in comparison: the initial strategy (a loading dose of ≥300 mg clopidogrel followed by dual antiplatelet, clopidogrel group) and the modified strategy (intravenous administration of tirofiban 8 µg/kg over 3 min followed by a maintenance dose of 0.1 µg/kg/min for 24 h, tirofiban group). The end points were rates of perioperative thromboembolic events and intracranial hemorrhages. Results Thromboembolic events were observed more often in the clopidogrel group (13/120 aneurysms, 10.83%) than the tirofiban group (6/178 aneurysms, 3.37%; P = 0.010), with no increase in the rate of intracranial hemorrhages ( P = 0.164). In the ruptured subgroups, thromboembolic events were significantly fewer in the tirofiban subgroup (5/128, 3.91%) compared with the clopidogrel subgroup (7/53, 13.21%; P = 0.043) with no increase in the rate of hemorrhage ( P = 0.360). Conclusions Intravenous administration of tirofiban is safe in intracranial aneurysms treated with stent-assisted coiling.

Sequential Steps of CRAC Channel Activation.

Interaction between the endoplasmic reticulum protein STIM1 and the plasma membrane channel ORAI1 generates calcium signals that are central for diverse cellular functions. How STIM1 binds and activates ORAI1 remains poorly understood. Using electrophysiological, optical, and biochemical techniques, we examined the effects of mutations in the STIM1-ORAI1 activating region (SOAR) of STIM1. We find that SOAR mutants that are deficient in binding to resting ORAI1 channels are able to bind to and boost activation of partially activated ORAI1 channels. We further show that the STIM1 binding regions on ORAI1 undergo structural rearrangement during channel activation. The results suggest that activation of ORAI1 by SOAR occurs in multiple steps. In the first step, SOAR binds to ORAI1, partially activates the channel, and induces a rearrangement in the SOAR-binding site of ORAI1. That rearrangement of ORAI1 then permits sequential steps of SOAR binding, via distinct molecular interactions, to fully activate the channel.

The Development of the Chinese Intermittent Exotropia Questionnaire.

PURPOSE: The Intermittent Exotropia Questionnaire (IXTQ) is a child, proxy, and parent report of health-related quality of life specific to children with intermittent exotropia (IXT). The present study aimed to develop a Chinese-language version of the IXTQ (CIXTQ) and evaluate its validity and reliability when used in Chinese IXT children and their parents. METHODS: The IXTQ was translated into Chinese. One hundred seventy-five IXT children (2 to 17 years old) and 151 orthotropic control children (2 to 17 years old) along with one of their parents were recruited. Children 5 to 17 years old completed the 5- to 7-year-old or the 8- to 17-year-old child questionnaire of the CIXTQ according to their age. Parents of all children (2 to 17 years old) completed the proxy and parent questionnaires of the CIXTQ. Psychometric properties of the CIXTQ were examined for floor and ceiling effects, construct validity, item-internal consistency, discriminative validity, Cronbach α coefficient and test-retest reliability. RESULTS: No items were found to have strong floor or ceiling effects. Principal component analysis identified that the CIXTQ had a similar structure to the original English version. The median scores of each questionnaire in the CIXTQ among children with IXT and their parents were significantly lower than those among control subjects (P < .001). Cronbach α coefficients ranged from 0.869 to 0.931, and test-retest reliabilities ranged from 0.898 to 0.981, for each questionnaire in the CIXTQ. CONCLUSIONS: The CIXTQ is a useful tool to evaluate the influence of IXT on health-related quality of life among Chinese IXT children and their parents.

Mechanism of Assembly and Cooperativity of Homomeric and Heteromeric Metabotropic Glutamate Receptors.

G protein-coupled receptors (GPCRs) mediate cellular responses to a wide variety of extracellular stimuli. GPCR dimerization may expand signaling diversity and tune functionality, but little is known about the mechanisms of subunit assembly and interaction or the signaling properties of heteromers. Using single-molecule subunit counting on class C metabotropic glutamate receptors (mGluRs), we map dimerization determinants and define a heterodimerization profile. Intersubunit fluorescence resonance energy transfer measurements reveal that interactions between ligand-binding domains control the conformational rearrangements underlying receptor activation. Selective liganding with photoswitchable tethered agonists conjugated to one or both subunits of covalently linked mGluR2 homodimers reveals that receptor activation is highly cooperative. Strikingly, this cooperativity is asymmetric in mGluR2/mGluR3 heterodimers. Our results lead to a model of cooperative activation of mGluRs that provides a framework for understanding how class C GPCRs couple extracellular binding to dimer reorganization and G protein activation.

Design Principles of Inert Substrates for Exploiting Gold Clusters' Intrinsic Catalytic Reactivity.

Ultralow stability of gold clusters prohibits the understanding of their intrinsic reactivity (that is vital for revealing the origin of gold's catalytic properties). Using density functional theory including many-body dispersion method, we aim to ascertain effective ways in exploiting gold clusters' intrinsic reactivity on carbon nanotubes (CNTs). We find that the many body van der Waals interactions are essential for gold clusters' reactivity on CNTs and even for O2 activation on these supported clusters. Furthermore, curvature and dopant of CNTs are found to qualitatively change the balance between physisorption and chemisorption for gold clusters on CNTs, determining the clusters' morphology, charge states, stability, and reactivity, which rationalize the experimental findings. Remarkably, N doped small curvature CNTs, which effectively stabilize gold clusters and retain their inherent geometric/electronic structures, can be promising candidates for exploiting gold clusters' intrinsic reactivity.

Paraoxonase 2 serves a proapopotic function in mouse and human cells in response to the Pseudomonas aeruginosa quorum-sensing molecule N-(3-Oxododecanoyl)-homoserine lactone.

Pseudomonas aeruginosa use quorum-sensing molecules, including N-(3-oxododecanoyl)-homoserine lactone (C12), for intercellular communication. C12 activated apoptosis in mouse embryo fibroblasts (MEF) from both wild type (WT) and Bax/Bak double knock-out mice (WT MEF and DKO MEF that were responsive to C12, DKOR MEF): nuclei fragmented; mitochondrial membrane potential (Δψmito) depolarized; Ca(2+) was released from the endoplasmic reticulum (ER), increasing cytosolic [Ca(2+)] (Cacyto); and caspase 3/7 was activated. DKOR MEF had been isolated from a nonclonal pool of DKO MEF that were non-responsive to C12 (DKONR MEF). RNAseq analysis, quantitative PCR, and Western blots showed that WT and DKOR MEF both expressed genes associated with cancer, including paraoxonase 2 (PON2), whereas DKONR MEF expressed little PON2. Adenovirus-mediated expression of human PON2 in DKONR MEF rendered them responsive to C12: Δψmito depolarized, Cacyto increased, and caspase 3/7 activated. Human embryonic kidney 293T (HEK293T) cells expressed low levels of endogenous PON2, and these cells were also less responsive to C12. Overexpression of PON2, but not PON2-H114Q (no lactonase activity) in HEK293T cells caused them to become sensitive to C12. Because [C12] may reach high levels in biofilms in lungs of cystic fibrosis (CF) patients, PON2 lactonase activity may control Δψmito, Ca(2+) release from the ER, and apoptosis in CF airway epithelia. Coupled with previous data, these results also indicate that PON2 uses its lactonase activity to prevent Bax- and Bak-dependent apoptosis in response to common proapoptotic drugs like doxorubicin and staurosporine, but activates Bax- and Bak-independent apoptosis in response to C12.

Pseudomonas aeruginosa quorum-sensing molecule homoserine lactone modulates inflammatory signaling through PERK and eI-F2α.

Pseudomonas aeruginosa secrete N-(3-oxododecanoyl)-homoserine lactone (HSL-C12) as a quorum-sensing molecule to regulate bacterial gene expression. Because HSL-C12 is membrane permeant, multiple cell types in P. aeruginosa-infected airways may be exposed to HSL-C12, especially adjacent to biofilms where local (HSL-C12) may be high. Previous reports showed that HSL-C12 causes both pro- and anti-inflammatory effects. To characterize HSL-C12's pro- and anti-inflammatory effects in host cells, we measured protein synthesis, NF-κB activation, and KC (mouse IL-8) and IL-6 mRNA and protein secretion in wild-type mouse embryonic fibroblasts (MEF). To test the role of the endoplasmic reticulum stress inducer, PERK we compared these responses in PERK(-/-) and PERK-corrected PERK(-/-) MEF. During 4-h treatments of wild-type MEF, HSL-C12 potentially activated NF-κB p65 by preventing the resynthesis of IκB and increased transcription of KC and IL-6 genes (quantitative PCR). HSL-C12 also inhibited secretion of KC and/or IL-6 into the media (ELISA) both in control conditions and also during stimulation by TNF-α. HSL-C12 also activated PERK (as shown by increased phosphorylation of eI-F2α) and inhibited protein synthesis (as measured by incorporation of [(35)S]methionine by MEF). Comparisons of PERK(-/-) and PERK-corrected MEF showed that HSL-C12's effects were explained in part by activation of PERK→phosphorylation of eI-F2α→inhibition of protein synthesis→reduced IκBα production→activation of NF-κB→increased transcription of the KC gene but reduced translation and secretion of KC. HSL-C12 may be an important modulator of early (up to 4 h) inflammatory signaling in P. aeruginosa infections.

Stoichiometry and specific assembly of Best ion channels.

Human Bestrophin 1 (hBest1) is a calcium-activated chloride channel that regulates neuronal excitability, synaptic activity, and retinal homeostasis. Mutations in hBest1 cause the autosomal-dominant Best macular dystrophy (BMD). Because hBest1 mutations cause BMD, but a knockout does not, we wondered if hBest1 mutants exert a dominant negative effect through interaction with other calcium-activated chloride channels, such as hBest2, 3, or 4, or transmembrane member 16A (TMEM16A), a member of another channel family. The subunit architecture of Best channels is debated, and their ability to form heteromeric channel assemblies is unclear. Using single-molecule subunit analysis, we find that each of hBest1, 2, 3, and 4 forms a homotetrameric channel. Despite considerable conservation among hBests, hBest1 has little or no interaction with other hBests or mTMEM16A. We identify the domain responsible for assembly specificity. This domain also plays a role in channel function. Our results indicate that Best channels preferentially self-assemble into homotetramers.

Thapsigargin blocks Pseudomonas aeruginosa homoserine lactone-induced apoptosis in airway epithelia.

Pseudomonas aeruginosa secretes N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule to regulate gene expression. Micromolar concentrations are found in the airway surface liquid of infected lungs. Exposure of the airway surface to C12 caused a loss of transepithelial resistance within 1 h that was accompanied by disassembly of tight junctions, as indicated by relocation of the tight junction protein zonula occludens 1 from the apical to the basolateral pole and into the cytosol of polarized human airway epithelial cell cultures (Calu-3 and primary tracheal epithelial cells). These effects were blocked by carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone, a pan-caspase blocker, indicating that tight junction disassembly was an early event in C12-triggered apoptosis. Short-duration (10 min) pretreatment of airway epithelial (Calu-3 and JME) cells with 1 µM thapsigargin (Tg), an inhibitor of Ca(2+) uptake into the endoplasmic reticulum (ER), was found to be protective against the C12-induced airway epithelial barrier breakdown and also against other apoptosis-related effects, including shrinkage and fragmentation of nuclei, activation of caspase 3/7 (the executioner caspase in apoptosis), release of ER-targeted redox-sensitive green fluorescent protein into the cytosol, and depolarization of mitochondrial membrane potential. Pretreatment of Calu-3 airway cell monolayers with BAPTA-AM [to buffer cytosolic Ca(2+) concentration (Cacyto)] or Ca(2+)-free solution + BAPTA-AM reduced C12 activation of apoptotic events, suggesting that C12-triggered apoptosis may involve Ca(2+). Because C12 and Tg reduced Ca(2+) concentration in the ER and increased Cacyto, while Tg increased mitochondrial Ca(2+) concentration (Camito) and C12 reduced Camito, it is proposed that Tg may reduce C12-induced apoptosis in host cells not by raising Cacyto, but by preventing C12-induced decreases in Camito.