TMEM16A Research Articles

Structural and functional investigations of CLCA proteins as potentiators of TMEM16 family members

The calcium-activated chloride channel regulator (CLCA) family of proteins have been suggested as drug targets for a number of diseases including asthma, COPD, cystic fibrosis, Crohn’s disease, infectious diarrhea, and some cancers. However, a detailed understanding of their biological functions has lagged far behind their association with diseases. CLCA proteins were originally presumed to be chloride channels but work by ourselves and others have demonstrated them to be secreted regulators that act on endogenous channels.

Investigating interacting partners of bestrophin channels

The bestrophin family of anion channels consists of four mammalian paralogs, namely bestrophin1-4 (Best1-4), at least two members of which play important functions in the eye. Best1 is predominantly expressed in the retinal pigment epithelium (RPE) and is required for generating a vision-related electrical signal named “light peak”. Mutations in the human BEST1 gene have been genetically linked to a spectrum of untreatable retinal degenerative disorders collectively known as bestrophinopathies. Meanwhile, Best2 is highly expressed in non-pigmented ciliary epithelial (NPE) cells, and Best2-/- mice exhibit a reduction of intra-ocular pressure (IOP), underlying the contribution of Best2 to IOP and the potential of Best2 as a drug target for relieving ocular hypertension.

Mechanical Stress Modulates Calcium-Activated-Chloride Currents in Differentiating Lens Cells.

During accommodation, the lens changes focus by altering its shape following contraction and relaxation of the ciliary muscle. At the cellular level, these changes in shape may be accompanied by fluid flow in and out of individual lens cells. We tested the hypothesis that some of this flow might be directly modulated by pressure-activated channels. In particular, we used the whole cell patch clamp technique to test whether calcium-activated-chloride channels (CaCCs) expressed in differentiating lens cells are activated by mechanical stimulation. Our results show that mechanical stress, produced by focally perfusing the lens cell at a constant rate, caused a significant increase in a chloride current that could be fully reversed by stopping perfusion. The time course of activation and recovery from activation of the flow-induced current occurred rapidly over a time frame similar to that of accommodation. The flow-induced current could be inhibited by the TMEM16A specific CaCC blocker, Ani9, suggesting that the affected current was predominantly due to TMEM16A chloride channels. The mechanism of action of mechanical stress did not appear to involve calcium influx through other mechanosensitive ion channels since removal of calcium from the bath solution failed to block the flow-induced chloride current. In conclusion, our results suggest that CaCCs in the lens can be rapidly and reversibly modulated by mechanical stress, consistent with their participation in regulation of volume in this organ.

Polymodal Control of TMEM16x Channels and Scramblases.

The TMEM16A/anoctamin-1 calcium-activated chloride channel (CaCC) contributes to a range of vital functions, such as the control of vascular tone and epithelial ion transport. The channel is a founding member of a family of 10 proteins (TMEM16x) with varied functions; some members (i.e., TMEM16A and TMEM16B) serve as CaCCs, while others are lipid scramblases, combine channel and scramblase function, or perform additional cellular roles. TMEM16x proteins are typically activated by agonist-induced Ca2+ release evoked by Gq-protein-coupled receptor (GqPCR) activation; thus, TMEM16x proteins link Ca2+-signalling with cell electrical activity and/or lipid transport. Recent studies demonstrate that a range of other cellular factors—including plasmalemmal lipids, pH, hypoxia, ATP and auxiliary proteins—also control the activity of the TMEM16A channel and its paralogues, suggesting that the TMEM16x proteins are effectively polymodal sensors of cellular homeostasis. Here, we review the molecular pathophysiology, structural biology, and mechanisms of regulation of TMEM16x proteins by multiple cellular factors.

Identification of Biomarker in Brain-specific Gene Regulatory Network Using Structural Controllability Analysis.

Brain tumor research has been stapled for human health while brain network research is crucial for us to understand brain activity. Here the structural controllability theory is applied to study three human brain-specific gene regulatory networks, including forebrain gene regulatory network, hindbrain gene regulatory network and neuron associated cells cancer related gene regulatory network, whose nodes are neural genes and the edges represent the gene expression regulation among the genes. The nodes are classified into two classes: critical nodes and ordinary nodes, based on the change of the number of driver nodes upon its removal. Eight topological properties (out-degree DO, in-degree DI, degree D, betweenness B, closeness CA, in-closeness CI, out-closeness CO and clustering coefficient CC) are calculated in this paper and the results prove that the critical genes have higher score of topological properties than the ordinary genes. Then two bioinformatic analysis are used to explore the biologic significance of the critical genes. On the one hand, the enrichment scores in several kinds of gene databases are calculated and reveal that the critical nodes are richer in essential genes, cancer genes and the neuron related disease genes than the ordinary nodes, which indicates that the critical nodes may be the biomarker in brain-specific gene regulatory network. On the other hand, GO analysis and KEGG pathway analysis are applied on them and the results show that the critical genes mainly take part in 14 KEGG pathways that are transcriptional misregulation in cancer, pathways in cancer and so on, which indicates that the critical genes are related to the brain tumor. Finally, by deleting the edges or routines in the network, the robustness analysis of node classification is realized, and the robustness of node classification is proved. The comparison of neuron associated cells cancer related GRN (Gene Regulatory Network) and normal brain-specific GRNs (including forebrain and hindbrain GRN) shows that the neuron-related cell cancer-related gene regulatory network is more robust than other types.

English

BACKGROUND Gastrointestinal stromal tumour (GIST) was first named in 1983. Gastrointestinal stromal tumours (GISTs) are a special kind of tumours which are derived from mesenchymal tissues of gastrointestinal tract and arises from the interstitial cells of Cajal, the pacemaker cells of the gastrointestinal (GI) tract responsible for the contractions of smooth muscles.1 Determination of the type of mutations in GIST plays a major role in assessing the risk of progression of the disease and also allows determination of the clinical management and treatment. More accurate GIST diagnosis is possible by using simultaneously various types of antibodies to immunohistochemistry methods in routine procedures.2 METHODS In this descriptive cross sectional study expression of DOG1, CD117 & PDGFRA was analysed in 70 patients with histopathologically diagnosed specimens of gastrointestinal stromal tumour, received in the Department of Pathology, Government Medical College, Thiruvananthapuram, using the immunohistochemical method. RESULTS On evaluating the CD117, DOG1 & PDGFRA expression in GIST by immunohistochemistry showed 71.4%, 84.3% and 55.7% positivity respectively. Most of the patients fall in the age group of 60 – 70 years with a slight male predominance. Most common location of GIST is stomach with tumour size of 5 – 10cm. On microscopic evaluation spindle type GIST was predominant histopathological type. Considering the risk groups, histological type, mitotic count and tumour size, PDGFRA expression is more in low-risk groups. PDGFRA expression has insignificant relation with clinicopathologic features including age, sex, site of lesion, risk groups, histologic type, mitotic count and tumour size. Relationship between positive expression by CD117 & DOG1 with risk group & site of lesion are not statistically significant. When compared to the similar studies in literature, the obtained results are concordant. CONCLUSIONS Our study concluded that, 71.4% positive immunoreactions for CD117, 84.3% positive immunoreactions for DOG1 & 55.7% positive immunoreactions for PDGFRA. PDGFRA expression has insignificant relation with clinicopathologic features including age, sex, site of lesion, risk groups, histologic type, mitotic count and tumour size. Relationship between positive expression by CD117 & DOG1 with risk group & site of lesion are not statistically significant. The importance of this study is that PDGFRA expression in tumours can be considered for treatment by using tyrosine kinase inhibitors and avapritinib. So PDGFRA testing in GIST show a new path in the targeted therapy. KEY WORDS GIST (Gastrointestinal Stromal Tumour), Discovered on GIST 1(DOG1), Cluster of Differentiation (CD117), Platelet Derived Growth Factor Receptor A(PDGFRA), Anoctamin 1(ANO1), Succinate Dehydrogenase (SDH), Risk Group, Mitotic Count

Twelve-year follow up of a case of autosomal recessive bestrophinopathy with transient resolution of retinal edema in one eye.

To report 12-year follow-up of a patient with ARB. A 25-year-old man presented with blurred vision in his both eyes (OU). Best-corrected visual acuity (BCVA) was 20/63 Snellen equivalent in the right eye (OD) and 20/32 Snellen equivalent in the left eye. The intraocular pressures and anterior segment examination were unrevealing in OU. Posterior segment examination revealed multiple yellowish flecks and dots in the posterior pole in OU. Optical coherence tomography (OCT) showed subretinal fluid (SRF), intraretinal hyporeflective spaces, elongated and shaggy photoreceptors and outer retinal defects. Fundus autofluorescence demonstrated mottling hyperautofluorescence and hypoautofluorescence in the posterior pole in OU. Fluorescein angiography illustrated hyperfluorescence in the posterior pole and surrounding the arcades in OU. Multifocal electroretinography objectified mild to markedly abnormal responses in all ring areas in OU. Molecular genetic testing confirmed two heterozygous sequence variations in the BEST1 gene. At 4 years of follow-up, OCT revealed a complete resolution of SRF and a partial resolution of intraretinal hyporeflective spaces in the OD with corresponding improvement in the BCVA to 20/23 Snellen equivalent in the OD, even though outer retinal defects persisted. Our patient denied recent changes in his alimentary habits and medical history at that time. Posteriorly, SRF and intraretinal hyporeflective spaces reappeared in the OD. To the best of our knowledge, this is the first case report of ARB with a transient resolution of retinal edema in one eye without medical treatment and dietary therapy.

Systems Approaches to Unravel Molecular Function: High-content siRNA Screen Identifies TMEM16A Traffic Regulators as Potential Drug Targets for Cystic Fibrosis

An attractive approach to treat people with Cystic Fibrosis (CF), a life-shortening disease caused by mutant CFTR, is to compensate for the absence of this chloride/bicarbonate channel by activating alternative (non-CFTR) chloride channels. One obvious target for such “mutation-agnostic” therapeutic approach is TMEM16A (anoctamin-1/ANO1), a calcium-activated chloride channel (CaCC) which is also expressed in the airways of people with CF, albeit at low levels. To find novel TMEM16A regulators of both traffic and function, with the main goal of identifying candidate CF drug targets, we performed a fluorescence cell-based high-throughput siRNA microscopy screen for TMEM16A trafficking using a double-tagged construct expressed in human airway cells. About 700 genes were screened (2 siRNAs per gene) of which 262 were identified as candidate TMEM16A modulators (179 siRNAs enhanced and 83 decreased TMEM16A traffic), being G-protein coupled receptors (GPCRs) enriched on the primary hit list. Among the 179 TMEM16A traffic enhancer siRNAs subjected to secondary screening 20 were functionally validated. Further hit validation revealed that siRNAs targeting two GPCRs – ADRA2C and CXCR3 – increased TMEM16A-mediated chloride secretion in human airway cells, while their overexpression strongly diminished calcium-activated chloride currents in the same cell model. The knockdown, and likely also the inhibition, of these two TMEM16A modulators is therefore an attractive potential therapeutic strategy to increase chloride secretion in CF.

Function and Regulation of the Calcium-Activated Chloride Channel Anoctamin 1 (TMEM16A).

Various human tissues express the calcium-activated chloride channel Anoctamin 1 (ANO1), also known as TMEM16A. ANO1 allows the passive chloride flux that controls different physiological functions ranging from muscle contraction, fluid and hormone secretion, gastrointestinal motility, and electrical excitability. Overexpression of ANO1 is associated with pathological conditions such as hypertension and cancer. The molecular cloning of ANO1 has led to a surge in structural, functional, and physiological studies of the channel in several tissues. ANO1 is a homodimer channel harboring two pores - one in each monomer - that work independently. Each pore is activated by voltage-dependent binding of two intracellular calcium ions to a high-affinity-binding site. In addition, the binding of phosphatidylinositol 4,5-bisphosphate to sites scattered throughout the cytosolic side of the protein aids the calcium activation process. Furthermore, many pharmacological studies have established ANO1 as a target of promising compounds that could treat several illnesses. This chapter describes our current understanding of the physiological roles of ANO1 and its regulation under physiological conditions as well as new pharmacological compounds with potential therapeutic applications.

UPDATED UNDERSTANDING OF THE MOLECULAR TARGETS OF RADIOIODINE IN DIFFERENTIATED THYROID CANCER.

Radioactive iodine (RAI) therapy is a mainstay adjuvant treatment for thyroid cancer. Administration of RAI therapy after total or near-total thyroidectomy has shown a survival advantage in numerous properly selected patients. However, the role of RAI therapy after reoperation for persistent or recurrent differentiated thyroid carcinomas (DTCs) is unclear. One reason may be the possible downregulation of the I- transport system after primary surgery. RAI is transported by the sodium iodide symporter (NIS), PENDRIN, anoctamin 1 (ANO1) and cystic fibrosis transmembrane conductance regulator (CFTR) and emits β particles that destroy follicular cells. The identification of pathways of iodide (I-) transport has allowed use of the transport system to render tumours susceptible to RAI treatment via gene therapy. This review focuses on the effect of RAI therapy in follicular cell-derived thyroid cancers and offers potential novel targets that enable improved radioiodine uptake and thus an improved prognosis of thyroid cancer.

Paneth Cell Secretion invivo Requires Expression ofTmem16a and Tmem16f.

Paneth cells play a central role in intestinal innate immune response. These cells are localized at the base of small intestinal crypts of Lieberkuhn. The calcium-activated chloride channel TMEM16A and the phospholipid scramblase TMEM16F control intracellular Ca2+ signaling and exocytosis. We analyzed the role of TMEM16A and TMEM16F for Paneth cells secretion. Mice with intestinal epithelial knockout of Tmem16a (Tmem16a-/-) and Tmem16f (Tmem16f-/-) were generated. Tissue structures and Paneth cells were analyzed, and Paneth cell exocytosis was examined in small intestinal organoids invitro. Intracellular Ca2+ signals were measured and were compared between wild-type and Tmem16 knockout mice. Bacterial colonization and intestinal apoptosis were analyzed. Paneth cells in the crypts of Lieberkuhn from Tmem16a-/- and Tmem16f-/- mice demonstrated accumulation of lysozyme. Tmem16a and Tmem16f were localized in wild-type Paneth cells but were absent in cells from knockout animals. Paneth cell number and size were enhanced in the crypt base and mucus accumulated in intestinal goblet cells of knockout animals. Granule fusion and exocytosis on cholinergic and purinergic stimulation were examined online. Both were strongly compromised in the absence of Tmem16a or Tmem16f and were also blocked by inhibition of Tmem16a/f. Purinergic Ca2+ signaling was largely inhibited in Tmem16a knockout mice. Jejunal bacterial content was enhanced in knockout mice, whereas cellular apoptosis was inhibited. The present data demonstrate the role of Tmem16 for exocytosis in Paneth cells. Inhibition or activation of Tmem16a/f is likely to affect microbial content and immune functions present in the small intestine.

Case Report of Adult-Onset Vitelliform Macular Dystrophy

Adult-onset vitelliform macular dystrophy is a bilateral dystrophy characterized by round subretinal yellow macular lesions. The dystrophy manifests between the third and fifth decades of life and has a variable genetic inheritance. A case of adult-onset vitelliform macular dystrophy is presented with the use of electrophysiological testing, fluorescein angiography, fundus autofluorescence and optical coherence tomography. CE Notification: This article is available as a COPE accredited CE course. You may take this course for 1-hour credit. Read the article and take the qualifying test to earn your credit. Click here to Enroll (https://www.crojournal.com/case-report-of-adult-onset-vitelliform-macular-dystrophy)

Drought effects on the mineral composition of the leaves of Actinidia species

Against the background of global climate change, drought stress has become one of the environmental limiting factors that can significantly influence the growth and development of crop plants. Drought stress conditions also cause changes in plant physiological and metabolic processes. The influence of soil drought on the mineral composition of the leaves of two Actinidia species with С3-type photosynthesis, namely, Actinidia argutа (Siebold & Zucc.) Planch. ex Miq. cultivar ‘Taezhny Dar’ and Actinidia kolomikta (Maxim. & Rupr.) Maxim. cultivar ‘Narodnaya’, was studied through energy dispersive spectrometry. The investigations were carried out during 2020 to 2021 at the Department of Genofonde and Bioresources of Plants, Federal Scientific Center for Horticulture, Moscow. The present research revealed that actinidia leaves contained the following major elements: K (11.19 mass% to 13.84 mass%), Ca (7.83% to 12.08 mass%), Cl (6.20 mass% to 7.33 mass%), and Mg (2.98 mass% to 3.44 mass%). Low values were recorded for Mo (1.19 mass% to 4.49 mass%) and P (0.83 mass% to 1.25 mass%). In both species, the mineral elements K and Ca were present at high levels. A positive correlation was observed between Mg–P, K–Mn, Mn–Se, Cu-Se, P–Si, Na–Mo, and Si–Mn in the leaves of A. argutа and between Cl–Ca, Mo; P–Si, Mo; and K–Ca in the leaves of A. kolomikta. Under stress conditions, the ratios of K/Ca and K/P declined to 0.9 and 6.3, respectively, whereas those of K/Cl, K/Mg, and K/Mo increased to 3.8, 4.4, and 2.7, respectively. The present studies confirmed that actinidia leaves contained high concentrations of minerals, especially K, Ca, P, and Mg, and that the accumulation of mineral elements in actinidia plant leaves under drought conditions varied depending on the species.

Biochemical and cytological features of onion bulbs and leaves collected from various ecogeographical origins

Against the background of global climate change, drought stress has become one of the environmental limiting factors that can significantly influence the growth and development of crop plants. Drought stress conditions also cause changes in plant physiological and metabolic processes. The influence of soil drought on the mineral composition of the leaves of two Actinidia species with С3-type photosynthesis, namely, Actinidia argutа (Siebold & Zucc.) Planch. ex Miq. cultivar ‘Taezhny Dar’ and Actinidia kolomikta (Maxim. & Rupr.) Maxim. cultivar ‘Narodnaya’, was studied through energy dispersive spectrometry. The investigations were carried out during 2020 to 2021 at the Department of Genofonde and Bioresources of Plants, Federal Scientific Center for Horticulture, Moscow. The present research revealed that actinidia leaves contained the following major elements: K (11.19 mass% to 13.84 mass%), Ca (7.83% to 12.08 mass%), Cl (6.20 mass% to 7.33 mass%), and Mg (2.98 mass% to 3.44 mass%). Low values were recorded for Mo (1.19 mass% to 4.49 mass%) and P (0.83 mass% to 1.25 mass%). In both species, the mineral elements K and Ca were present at high levels. A positive correlation was observed between Mg–P, K–Mn, Mn–Se, Cu-Se, P–Si, Na–Mo, and Si–Mn in the leaves of A. argutа and between Cl–Ca, Mo; P–Si, Mo; and K–Ca in the leaves of A. kolomikta. Under stress conditions, the ratios of K/Ca and K/P declined to 0.9 and 6.3, respectively, whereas those of K/Cl, K/Mg, and K/Mo increased to 3.8, 4.4, and 2.7, respectively. The present studies confirmed that actinidia leaves contained high concentrations of minerals, especially K, Ca, P, and Mg, and that the accumulation of mineral elements in actinidia plant leaves under drought conditions varied depending on the species.

Genome-wide analysis identifies gallstone-susceptibility loci including genes regulating gastrointestinal motility.

Genome-wide association studies (GWAS) have identified several risk loci for gallstone disease. As with most polygenic traits, it is likely that many genetic determinants are undiscovered. The aim of this study was to identify genetic variants that represent new targets for gallstone research and treatment. We performed a GWAS of 28,627 gallstone cases and 348,373 controls in the UK Biobank, replicated findings in a Scottish cohort (1089 cases, 5228 controls), and conducted a GWA meta-analysis (43,639 cases, 506,798 controls) with the FinnGen cohort. We assessed pathway enrichment using gene-based then gene-set analysis and tissue expression of identified genes in Genotype-Tissue Expression project data. We constructed a polygenic risk score (PRS) and evaluated phenotypic traits associated with the score. Seventy-five risk loci were identified (p<5 ×10-8 ), of which 46 were new. Pathway enrichment revealed associations with lipid homeostasis, glucuronidation, phospholipid metabolism, and gastrointestinal motility. Anoctamin 1 (ANO1) and transmembrane Protein 147 (TMEM147), both in novel, replicated loci, are expressed in the gallbladder and gastrointestinal tract. Both regulate gastrointestinal motility. The gallstone risk allele rs7599-A leads to suppression of hepatic TMEM147 expression, suggesting that the protein protects against gallstone formation. The highest decile of the PRS demonstrated a 6-fold increased odds of gallstones compared with the lowest decile. The PRS was strongly associated with increased body mass index, serum liver enzymes, and C-reactive protein concentrations, and decreased lipoprotein cholesterol concentrations. This GWAS demonstrates the polygenic nature of gallstone risk and identifies 46 novel susceptibility loci. We implicate genes influencing gastrointestinal motility in the pathogenesis of gallstones.

Positive modulation of the TMEM16B mediated currents by TRPV4 antagonist

Calcium-activated chloride channels (CaCCs) play important roles in many physiological processes and their malfunction is implicated in diverse pathologies such as cancer, asthma, and hypertension. TMEM16A and TMEM16B proteins are the structural components of the CaCCs. Recent studies in cell cultures and animal models have demonstrated that pharmacological inhibition of CaCCs could be helpful in the treatment of some diseases, however, there are few specific modulators of these channels. CaCCs and Transient Receptor Potential Vanilloid-4 (TRPV4) channels are co-expressed in some tissues where they functionally interact. TRPV4 is activated by different stimuli and forms a calcium permeable channel that is activated by GSK1016790A and antagonized by GSK2193874. Here we report that GSK2193874 enhances the chloride currents mediated by TMEM16B expressed in HEK cells at nanomolar concentrations and that GSK1016790A enhances native CaCCs of Xenopus oocytes. Thus, these compounds may be used as a tool for the study of CaCCs, TRPV4 and their interactions.

Anoctamin 5 Knockout Mouse Model Recapitulates LGMD2L Muscle Pathology and Offers Insight Into in vivo Functional Deficits

Mutations in the Anoctamin 5 (Ano5) gene that result in the lack of expression or function of ANO5 protein, cause Limb Girdle Muscular Dystrophy (LGMD) 2L/R12, and Miyoshi Muscular Dystrophy (MMD3). However, the dystrophic phenotype observed in patient muscles is not uniformly recapitulated by ANO5 knockout in animal models of LGMD2L. Here we describe the generation of a mouse model of LGMD2L generated by targeted out-of-frame deletion of the Ano5 gene. This model shows progressive muscle loss, increased muscle weakness, and persistent bouts of myofiber regeneration without chronic muscle inflammation, which recapitulates the mild to moderate skeletal muscle dystrophy reported in the LGMD2L patients. We show that these features of ANO5 deficient muscle are not associated with a change in the calcium-activated sarcolemmal chloride channel activity or compromised in vivo regenerative myogenesis. Use of this mouse model allows conducting in vivo investigations into the functional role of ANO5 in muscle health and for preclinical therapeutic development for LGMD2L.

Identification of Piezo1 channels in perivascular adipose tissue (PVAT) and their potential role in vascular function

The vasculature constantly experiences distension/pressure exerted by blood flow and responds to maintain homeostasis. We hypothesized that activation of the stretch sensitive, non-selective cation channel Piezo1 would directly increase vascular contraction in a way that might be modified by perivascular adipose tissue (PVAT). The presence and function of Piezo1 was investigated by RT-PCR, immunohistochemistry, and isolated tissue bath contractility. Superior and mesenteric resistance arteries, aortae, and their PVATs from male Sprague Dawley rats were used. Piezo1 mRNA was detected in aortic vessels, aortic PVAT, mesenteric vessels, and mesenteric PVAT. Both adipocytes and stromal vascular fraction of mesenteric PVAT expressed Piezo1 mRNA. In PVAT, expression of Piezo1 mRNA was greater in magnitude than that of Piezo2, transient receptor potential cation channel, subfamily V, member 4 (TRPV4), anoctamin 1, calcium activated chloride channel (TMEM16), and Pannexin1 (Panx1). Piezo1 protein was present in endothelium and PVAT of rat aortic and in PVAT of mesenteric artery. The Piezo1 agonists Yoda1 and Jedi2 (1 nM - 10 µM) did not stimulate aortic contraction [max < 10% phenylephrine (PE) 10 µM contraction] or relaxation in tissues + or -PVAT. Depolarizing the aorta by modestly elevated extracellular K+ did not unmask aortic contraction to Yoda1 (max <10% PE 10 µM contraction). Finally, the Piezo1 antagonist Dooku1 did not modify PE-induced aorta contraction + or -PVAT. Surprisingly, Dooku1 directly caused aortic contraction in the absence (Dooku1 =26 ± 11; Vehicle = 11 ± 11%PE contraction) but not in the presence of PVAT (Dooku1 = 2 ± 1; Vehicle = 8 ± 5% PE contraction). Thus, Piezo1 is present and functional in the isolated rat aorta but does not serve direct vascular contraction with or without PVAT. We reaffirmed the isolated mouse aorta relaxation to Yoda1, indicating a species difference in Piezo1 activity between mouse and rat.

Intravitreal bevacizumab treatment for exudative choroidal neovascularisation in best vitelliform macular dystrophy.

To describe results of intravitreal bevacizumab treatment of the secondary choroidal neovascularisation in Best vitelliform macular dystrophy in an adult and paediatric patient, and present the management of three asymptomatic patients with confirmed BEST1 gene mutation. Five patients from the same family with the Best vitelliform macular dystrophy are presented. In two patients (aged 63 and 4 years) secondary choroidal neovascularisation caused a rapid decline in visual acuity. In the adult patient with advanced Best vitelliform macular dystrophy, visual acuity did not improve despite eight intravitreal bevacizumab injections to the right eye. The formation of a central scar and rapid reoccurrence of choroidal neovascularisation three months after completing the initial treatment affected the outcome. As for the paediatric patient with bilateral choroidal neovascularisation in the vitelliform stage of Best vitelliform macular dystrophy, a complete recovery of visual acuity was observed after two (left eye) and three (right eye) bevacizumab injections, with adjunctive amblyopia treatment. The other three patients with an abnormal electrooculogram reported no visual problems during more than 10 years of follow-up. Minimal changes were seen on optical coherence tomography in the youngest patient. Intravitreal bevacizumab seems to be an effective treatment for exudative choroidal neovascularisation in the vitelliform stage of Best vitelliform macular dystrophy; however, it may not be beneficial in the advanced stages of Best vitelliform macular dystrophy. It is important to regularly screen all family members with an abnormal electrooculogram and confirmed mutation for vitelliform changes and choroidal neovascularisation from an early age. The decision for anti-vascular endothelial growth factor treatment should be made on a case-to-case basis as complications may arise.

Cinobufagin Exerts Anticancer Activity in Oral Squamous Cell Carcinoma Cells through Downregulation of ANO1.

Anoctamin1 (ANO1), a calcium-activated chloride channel, is frequently overexpressed in several cancers, including oral squamous cell carcinoma (OSCC). OSCC is a highly aggressive cancer and the most common oral malignancy. ANO1 has been proposed as a potential candidate for targeted anticancer therapy. In this study, we performed a cell-based screening to identify novel regulators leading to the downregulation of ANO1, and discovered cinobufagin, which downregulated ANO1 expression in oral squamous cell carcinoma CAL-27 cells. ANO1 protein levels were significantly reduced by cinobufagin in a dose-dependent manner with an IC50 value of ~26 nM. Unlike previous ANO1 inhibitors, short-term (≤10 min) exposure to cinobufagin did not alter ANO1 chloride channel activity and ANO1-dependent intestinal smooth muscle contraction, whereas long-term (24 h) exposure to cinobufagin significantly reduced phosphorylation of STAT3 and mRNA expression of ANO1 in CAL-27 cells. Notably, cinobufagin inhibited cell proliferation of CAL-27 cells expressing high levels of ANO1 more potently than that of ANO1 knockout CAL-27 cells. In addition, cinobufagin significantly reduced cell migration and induced caspase-3 activation and PARP cleavage in CAL-27 cells. These results suggest that downregulation of ANO1 by cinobufagin is a potential mechanism for the anticancer effect of cinobufagin in OSCC.