TMEM16A Research Articles

New Evidence on BEST1 Genetic Variant Identified in a Patient with Best Vitelliform Macular Dystrophy Phenotype

Best Disease (BD), also known as Best Vitelliform Macular Dystrophy (BVMD), represents an inherited autosomal dominant macular dystrophy with a juvenile age of onset [1]. It is a phenotypically heterogeneous, bilateral condition that affects the retina and Retinal Pigment Epithelium (RPE) caused by pathogenic variants in the BEST1 gene located on chromosome 11q12-13 [2,3]. Typical fundus findings in BD are egg yolk-like, round or oval, lesions seen in the macula, and affected eyes may demonstrate various clinical stages, ranging from the previtelliform stage to Choroidal Neovascularization (CNV) [4]. The macular appearance in all stages is deceptive, as most patients maintain relatively good visual acuity throughout the course of the disease. Patients commonly experience visual compromise in early adulthood, although the age of onset can range from childhood to late adulthood [3] and most patients with BD maintain good vision in at least one eye. The presence of subretinal fluid or CNV has been associated with a poorer visual prognosis [4]. In this case report, we describe a patient with clinical features suggestive of Best disease. We discuss the differential diagnosis and we present the multimodal imaging of the retina used for both the diagnosis and follow up. We also report a genetic study that demonstrates more evidence on a novel genetic variant in the BEST1 gene. The same genetic mutation has been recently reported as a novel variant in a single patient with BVMD [5].

DOG1 is commonly expressed in pancreatic adenocarcinoma but unrelated to cancer aggressiveness.

BackgroundDOG1 (ANO1; TMEM16A) is a voltage-gated calcium-activated chloride and bicarbonate channel. DOG1 is physiologically expressed in Cajal cells, where it plays an important role in regulating intestinal motility and its expression is a diagnostic hallmark of gastrointestinal stromal tumors (GIST). Data on a possible role of DOG1 in pancreatic cancer are rare and controversial. The aim of our study was to clarify the prevalence of DOG1 expression in pancreatic cancer and to study its association with parameters of cancer aggressiveness.MethodsDOG1 expression was analyzed by immunohistochemistry in 599 pancreatic cancers in a tissue microarray format and in 12 cases of pancreatitis on large tissue sections.ResultsDOG1 expression was always absent in normal pancreas but a focal weak expression was seen in four of 12 cases of pancreatitis. DOG1 expression was, however, common in pancreatic cancer. Membranous and cytoplasmic DOG1 expression in tumor cells was highest in pancreatic ductal adenocarcinomas (61% of 444 interpretable cases), followed by cancers of the ampulla Vateri (43% of 51 interpretable cases), and absent in 6 acinus cell carcinomas. DOG1 expression in tumor associated stroma cells was seen in 76 of 444 (17%) pancreatic ductal adenocarcinomas and in seven of 51 (14%) cancers of the ampulla Vateri. Both tumoral and stromal DOG1 expression were unrelated to tumor stage, grade, lymph node and distant metastasis, mismatch repair protein deficiency and the density of CD8 positive cytotoxic T-lymphocytes in the subgroups of ductal adenocarcinomas and cancers of ampulla Vateri. Overall, the results of our study indicate that DOG1 may represent a potential biomarker for pancreatic cancer diagnosis and a putative therapeutic target in pancreatic cancer. However, DOG1 expression is unrelated to pancreatic cancer aggressiveness.

Vendian Hydrogeochemistry of the Siberian Platform

Abstract —The Vendian deposits of the Siberian Platform are represented by four regional horizons (from bottom to top): Nepa, Vilyuchan, Tira, and Danilovka. The TDS of brines in the Nepa and Vilyuchan horizons varies from 170.3 to 470.1 g/L, and the TDS in the overlying Tira and Danilovka horizons, from 84.6 to 583.1 g/L. Brines of Cl Na, Cl Na–Ca, Cl Ca–Na, Cl Ca, and Cl Ca–Mg types have been identified. The pH values of the brines vary from 3.0 to 7.0; acidic and weakly acidic brines (pH = 3.0–5.5) prevail. The redox potential Eh varies from –440 mV (highly reducing conditions) to +130 mV (oxidizing conditions), averaging –176 mV. Areal regional hydrogeochemical zoning is controlled by the outer (on the periphery of the Siberian Platform, where infiltration of meteoric waters took place) and inner feeding areas. The outer feeding areas bear infiltrogenic brines of different ages (group I), mostly of Cl Na composition. They are characterized by TDS ≤ 280 g/L, rNa/rCl ≤ 1.55, Cl/Br ≤ 2512, and average Ca/Cl = 0.3. The average values of the integrated brine metamorphization index S (according to S.L. Shvartsev) range from 50 to 200. In the intraplatform feeding areas, there are several phases of brines that underwent gravitational sinking into the Vendian horizons in the periods when Cambrian salt-bearing basins existed on the platform. These brines were metamorphosed during filtration along the fracture and fault zones. The intrusion of traps in the late Permian–early Triassic was accompanied by hydrothermal activity. The waters of the enclosing Cambrian dolomite horizons were heated to 800–1000 °C; their subsequent interaction with carbonate rocks and evaporites (in particular, chlorides) resulted in extrastrong saturated brines. These brines were also subjected to gravitational sinking into the Vendian reservoirs, which was the main cause of the inverse vertical hydrogeochemical zoning in the hydrogeologic subsalt formation. The brine metamorphism processes and temperature changes in the rock horizons during the intrusion of traps and their subsequent cooling led to a significant alteration of the enclosing rocks under interaction with brines and to salinization of the reservoirs, an increase in the total salinity of the brines, and catagenetic changes in their composition. The brines closest to the maximum saturation with halite were found in reservoir zones with the highest salinization and near faults and boundaries of intrusive bodies. During the pre-Vilyuchan, pre-Nepa, pre-Tira, and pre-Danilovka sedimentation breaks, infiltrogenic waters penetrated into the Vendian sediments. Geochemically, the sedimentogenic, infiltrogenic, and metamorphic strong brines (group II) have a predominantly Cl Ca or Cl Ca–Mg composition with TDS > 350 g/L. They are characterized by low rNa/rCl ratios (on average, 0.3), Cl/Br ≤ 100, high Ca/Cl values (on average, 0.4), and S > 250.

Disease expression caused by different variants in the BEST1 gene: genotype and phenotype findings in bestrophinopathies.

Purpose:To analyse the spectrum of clinical features and molecular genetic data in a series of patients carrying likely disease-associated variants in the BEST1 gene.Methods:Retrospective observational analysis of clinical data extracted from the medical records of visual function, multimodal imaging and electrophysiology of 62 eyes of 31 patients. Molecular genetic analysis was performed by means of panel-based NGS or Sanger sequencing.Results:The spectrum of variants in the BEST1 gene comprised 19 different variants and three of which are novel. Fundus photographs and OCT images allowed categorization of 52 eyes as Best vitelliform macular dystrophy (BVMD) with stages 1 to 5 and 10 eyes with autosomal recessive bestrophinopathy (ARB), with more severe phenotype. One patient was shown to be heterozygous for a variant, which has so far been described only in ARB, but this patient had the BVMD phenotype. There was no significant progression of the visual acuity during the follow-up period of 5 years both in BVMD and ARB. The most prevalent pattern of fundus autofluorescence (FAF) in BVMD was ‘patchy’. There were diverse visual field defects in static automated perimetry (SAP) depending on the stage. The Arden ratio was significantly lower in ARB patients and in eyes with stage 5 of BVMD.Conclusions:The genotype does not always predict the phenotype in patients with BVMD and ARB; however, having two mutations in the BEST1 gene causes a more severephenotype. FAFhelped to distinguish ARB from BVMD. Most of the observed eyesdidnotprogressfunctionallyduringthefollow-up.ARBandtheatrophicstageof BVMD as the disease end-stage had the worst visual functions and EOG results.

Theaflavin binds to a druggable pocket of TMEM16A channel and inhibits lung adenocarcinoma cell viability

As a calcium-activated chloride channel regulated by the intracellular Ca2+ concentration and membrane potential, TMEM16A has attracted considerable attention and has been proposed as a novel anticancer drug target. We have previously reported that the pocket above the ion conductance pore could be a nonselective inhibitor-binding pocket. However, whether this pocket is druggable remains unexplored. In this study, we performed virtual screening to target the presumed inhibitor-binding pocket and identified a highly effective TMEM16A inhibitor, theaflavin (TF: a tea polyphenol in black tea). Molecular dynamics simulations revealed that theaflavin adopts a “wedge insertion mode” to block the ion conduction pore and induces pore closure. Moreover, the binding mode showed that the TF pedestal plays an important role in pore blockade, and R515, R535, T539, K603, E623, and E633 were determined to be most likely to interact directly with the pedestal. Mutagenesis experiment results corroborated the mechanism through which TF binds to this pocket. Combined with the quantitative calculation results, our data indicated that the three hydroxyl groups on the pedestal may be the most crucial pharmacophores for TMEM16A inhibition by TF. Finally, antitumor experiments revealed that TF could target TMEM16A to inhibit the proliferation and migration of LA795 cells, indicating the potential therapeutic effect of TF on the growth of lung adenocarcinoma with high TMEM16A expression. The successful application of drug screening strategies based on this binding pocket highlights new directions for discovering superior modulators and contributes to the development of novel therapeutics for lung adenocarcinoma.

Multimodal imaging and genetic analysis of adult-onset best vitelliform macular dystrophy in Chinese patients.

Compared to juvenile-onset best vitelliform macular dystrophy (BVMD), adult-onset BVMD is not well characterized and lacks strict diagnostic criteria. The present study aimed to evaluate the clinical and genetic characteristics of four advanced-age Chinese patients with adult-onset BVMD by combining multimodal imaging and genetic analysis. The four patients (all older than 50 years) were diagnosed with adult-onset BVMD at Zhongshan Ophthalmic Center (Guangzhou, China). Comprehensive ophthalmic examinations were performed, including analyses of best-corrected visual acuity, intraocular pressure, slit-lamp examination, fundus photography, optical coherence tomography, fundus fluorescein angiography and electrooculography. Genomic DNA was extracted from leukocytes isolated from peripheral blood obtained from these patients, their family members and 200 unrelated subjects from the same population. A total of 11 exons of the bestrophin-1 (BEST1) gene were amplified using PCR and sequenced. All of the four patients presented with lesions in the macular area. The patients were diagnosed with adult-onset BVMD based on multimodal imaging and genetic analysis. A total of four recurrent mutations, namely c.763C>T (p.Arg255Trp, p.R255W) in exon 7, c.584C>T (p.Ala195Val, p.A195V) in exon 5, c.910_912del GAT (p.304delAsp, p.D304del) in exon 8 and c.310G>C (p.Asp104His, p.D104H) in exon 4 of BEST1, were identified. Sorting intolerant from tolerant predicted that the amino acid substitutions p.R255W, p.A195V and p.D104H in the BEST1 protein were causing the damage. Combining multimodal imaging and genetic analysis was helpful in confirming the diagnosis of patients with adult-onset BVMD. These results maybe valuable for clinical and genetic counseling and for the development of therapeutic interventions for patients with BVMD.

Targeting TMEM16A-encoded Ca2+-activated Cl− channels: a new paradigm for antihypertensive therapy?

Hypertension is the leading risk factor for the development of heart diseases and stroke. Many hypertensive patients experience undesirable side effects to conventional antihypertensive pharmacotherapy. Cil etal. documented the antihypertensive profile of a novel molecule, TMinh-23 (2-bromodifluoroacetylamino-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylic acid o-tolylamide), in the spontaneously hypertensive rat model of systemic hypertension. They showed that this agent reduces blood pressure by inhibiting transmembrane member 16A-encoded calcium-activated chloride channels in vascular myocytes from resistance arteries. If validated, TMinh-23 could become a useful clinical tool.

Autosomal Dominant ANO5-Related Disorder Associated With Myopathy and Gnathodiaphyseal Dysplasia.

ObjectiveTo investigate the molecular basis of muscle disease and gnathodiaphyseal dysplasia (GDD) in a large kindred with 11 (6 women and 5 men) affected family members.MethodsWe performed clinical assessment of 3 patients and collected detailed clinical and family history data on 8 additional patients. We conducted molecular genetic analyses on 5 patients using comprehensive neuromuscular disorder panels, exome sequencing (ES), and targeted testing for specific genetic variants. We analyzed the segregation of the muscle and bone phenotypes with the underlying molecular cause.ResultsThe unique clinical presentation of recurrent episodes of rhabdomyolysis associated with muscle cramps, hyperCKemia, muscle hypertrophy, with absent or mild muscle weakness, as well as cemento-osseous lesions of the mandible, with or without bone fractures and other skeletal abnormalities, prompted us to look for the underlying molecular cause of the disorder in this kindred. Molecular testing revealed a missense variant in anoctamin 5 (ANO5) designated as c.1538C>T; p.Thr513Ile, which was previously described in a large kindred with GDD. In silico analysis, searching publicly available databases, segregation analysis, as well as functional studies performed by another group provide strong evidence for pathogenicity of the variant. ES data in the proband excluded the contribution of additional genetic factors.ConclusionsThis report described the coexistence of muscle and bone phenotypes in the same patients with ANO5-related disorder. Our data challenge recent results that suggested complete dichotomy of these phenotypes and the proposed loss-of-function and gain-of-function mechanisms for the skeletal and muscle phenotypes, respectively.

Emerging Modulators of TMEM16A and Their Therapeutic Potential.

Calcium-activated chloride channels (CaCCs) are widespread chloride channels which rely on calcium activation to perform their functions. In 2008, TMEM16A (also known as anoctamin1, ANO1) was identified as the molecular basis of the CaCCs, which provided the possibility to study the physiological function of CaCCs. TMEM16A is widely expressed in various cells and controls basic physiological functions, including neuronal and cardiac excitability, nerve transduction, smooth muscle contraction, epithelial Cl- secretion and fertilization. However, the abnormal function of TMEM16A may cause a variety of diseases, including asthma, gastrointestinal motility disorder and various cancers. Therefore, TMEM16A is a putative drug target for many diseases, and it is important to determine specific and efficient modulators of TMEM16A channel. In recent years, we and others have screened several natural modulators of TMEM16A against cancers and gastrointestinal motility dysfunction. This article reviews the screening methods, efficacy of TMEM16A modulators and pharmacological effects of TMEM16A modulators on different diseases. GRAPHIC ABSTACT.

Muscle involvement assessed by quantitative magnetic resonance imaging in patients with anoctamin 5 deficiency.

Using magnetic resonance imaging (MRI) and stationary dynamometry, the aim was to investigate the muscle affection in paraspinal muscles and lower extremities and compare the muscle affection in men and women with anoctamin 5 (ANO5) deficiency. Seventeen patients (seven women) with pathogenic ANO5-mutations were included. Quantitative muscle fat fraction of back and leg muscles were assessed by Dixon MRI. Muscle strength was assessed by stationary dynamometer. Results were compared with 11matched, healthy controls. Muscle involvement pattern in men with ANO5-deficiency is characterized by a severe fat replacement of hamstrings, adductor and gastrocnemius muscles, while paraspinal muscles are only mildly affected, while preserved gracilis and sartorius muscles were hypertrophied. Women with ANO5-myopathy, of the same age as male patients, were very mildly affected, showing muscle affection and strength resembling that found in healthy persons, with the exception of the gluteus minimus and medius and gastrocnemii muscles that were significantly replaced by fat. Although individual muscles showed clear asymmetric involvement in a few muscle groups, the overall muscle involvement was symmetric. Patients with ANO5-deficiency have relatively preserved paraspinal muscles on imaging and only mild reduction of trunk extension strength in men only. Our study quantifies the large difference in muscle affection in lower extremity between women and men with ANO5-deficiency. The clinical notion is that affection may be very asymmetric in ANO5-deficiency, but the present study shows that while this may be true for a few muscles, the general impression is that muscle affection is very symmetric.

Anoctamin 5 promotes osteosarcoma development by increasing degradation of Nel-like proteins 1 and 2.

Anoctamin 5 (ANO5) is a member of the Anoctamin (ANO) family of calcium-activated chloride channels. Although ANO5 expression is upregulated in various cancers, its role in osteosarcoma remains largely unknown. In this study, bioinformatics analysis, western blot, and immunohistochemical staining revealed that ANO5 was upregulated in osteosarcoma cell lines and osteosarcoma tissues, and ANO5 expression was positively associated with tumor size, tumor grade, and metastasis. Functional experiments demonstrated that inhibition of ANO5 decreased, while ANO5 overexpression increased, osteosarcoma cell proliferation and mobility in vitro. Immunoprecipitation, western blot, and confocal microscopy experiments showed that ANO5 bound to and promoted the degradation of Nel-like proteins 1 (NELL1) and 2 (NELL2). Moreover, a subcutaneous tumor transplantation model revealed that ANO5 knockdown reduced osteosarcoma cell proliferation and increased NELL1 and NELL2 expression in vivo. Finally, rescue experiments showed that knockdown of NELL1 or NELL2 reversed the inhibitory effects of ANO5 knockdown on osteosarcoma cell proliferation and migration. These results demonstrated that upregulation of ANO5 promoted osteosarcoma development by decreasing the stability of the NELL1 and NELL2 proteins and that ANO5 may be an effective target for the treatment of osteosarcoma.

Calcium-activated chloride channel is involved in the onset of diarrhea triggered by EGFR tyrosine kinase inhibitor treatment in rats

EGFR tyrosine kinase inhibitors (TKIs) are mainly used to treat non-small cell lung cancer; however, adverse effects such as severe diarrhea represent a major obstacle towards the continuation of EGFR-TKIs therapy. Chloride channels, which control the fluid flow in the intestinal lumen, are proposed as an important target to remediate EGFR-TKIs-induced diarrhea, but the mechanism remains unclear. The aim of this study was to clarify the mechanism underlying EGFR-TKIs-induced diarrhea with a particular focus on the role of intestinal chloride channels. Here, we show that osimertinib-treated rats exhibit diarrhea and an increase in fecal water content without showing any severe histopathological changes. This diarrhea was attenuated by intraperitoneal treatment with the calcium-activated chloride channel (CaCC) inhibitor CaCCinh-A01. These findings were confirmed in afatinib-treated rats with diarrhea. Moreover, treatment with the Japanese traditional herbal medicine, hangeshashinto (HST), decreased fecal water content and improved fecal appearance in rats treated with EGFR-TKIs. HST inhibited the ionomycin-induced CaCC activation in HEK293 cells in patch-clamp current experiments and its active ingredients were identified. In conclusion, secretory diarrhea induced by treatment with EGFR-TKIs might be partially mediated by the activation of CaCC. Therefore, blocking the CaCC could be a potential new treatment for EGFR-TKI-induced diarrhea.

Diethylstilbestrol, a Novel ANO1 Inhibitor, Exerts an Anticancer Effect on Non-Small Cell Lung Cancer via Inhibition of ANO1.

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality; thus, therapeutic targets continue to be developed. Anoctamin1 (ANO1), a novel drug target considered for the treatment of NSCLC, is a Ca2+-activated chloride channel (CaCC) overexpressed in various carcinomas. It plays an important role in the development of cancer; however, the role of ANO1 in NSCLC is unclear. In this study, diethylstilbestrol (DES) was identified as a selective ANO1 inhibitor using high-throughput screening. We found that DES inhibited yellow fluorescent protein (YFP) fluorescence reduction caused by ANO1 activation but did not inhibit cystic fibrosis transmembrane conductance regulator channel activity or P2Y activation-related cytosolic Ca2+ levels. Additionally, electrophysiological analyses showed that DES significantly reduced ANO1 channel activity, but it more potently reduced ANO1 protein levels. DES also inhibited the viability and migration of PC9 cells via the reduction in ANO1, phospho-ERK1/2, and phospho-EGFR levels. Moreover, DES induced apoptosis by increasing caspase-3 activity and PARP-1 cleavage in PC9 cells, but it did not affect the viability of hepatocytes. These results suggest that ANO1 is a crucial target in the treatment of NSCLC, and DES may be developed as a potential anti-NSCLC therapeutic agent.

Anoctamin1 Induces Hyperproliferation of HaCaT Keratinocytes and Triggers Imiquimod-Induced Psoriasis-Like Skin Injury in Mice.

Psoriasis, a long-lasting and multifactorial skin disease, is related to comorbidities such as metabolic disease, depression, and psoriatic arthritis. Psoriasis occurs due to a variety of factors including keratinocyte hyperproliferation, inflammation, and abnormal differentiation. Proinflammatory cytokines upregulated by increased activation of keratinocytes and immune cells in the skin trigger progression of psoriasis. This study aimed to investigate the effects of anoctamin1 (ANO1) on psoriasis development in vitro and in vivo. We analyzed the proliferation of HaCaT keratinocytes and ANO1-related ERK and AKT signaling pathways after ANO1 inhibitor (T16Ainh-A01 and Ani9) treatment and knock-down of ANO1. Furthermore, after applying imiquimod (IMQ) cream or coapplying IMQ cream and T16Ainh-A01 on mouse ears, we not only observed psoriatic symptoms, including ear thickening, but also quantified the effects of treatment on ERK and AKT signaling-involved proteins and proinflammatory cytokines. Inhibition of ANO1 attenuated the proliferation of HaCaT cells and induced reduction of pERK1/2. Coapplication of IMQ and T16Ainh-A01 on ears of mice reduced not only symptoms of IMQ-induced psoriasis such as thickening and erythema, but also expression of ANO1 and pERK1/2 compared to that of application of IMQ alone. In addition, the expression levels of IL-17A, IL-17F, IL-22, IL-23, IL-6, IL-1β, and TNF-α increased after applying IMQ and were significantly reduced by coapplying IMQ and T16Ainh-A01. These results aid in understanding the underlying mechanisms of ANO1 in epidermal layer keratinocyte hyperproliferation and suggest the potential of ANO1 as a target to treat psoriasis.

Best Vitelliform Dystrophy; Pathophysiology, Findings, Diagnosis, and Treatment

Best vitelliform dystrophy is the second most common hereditary macular dystrophy. It is an early-onset, progressive disease with autosomal dominant inheritance. The BEST1 gene is located on the long arm of the 11th chromosome and is responsible for the production of a transmembrane protein called bestrophin-1. The disease takes its name from the classic egg yolk-like macular lesion. Its histopathology shows increased lipofuscin in retinal pigment epithelium (RPE), photoreceptor loss, sub RPE deposits, and cell and material accumulation in the subretinal area. As long as complications such as choroidal neovascularization, macular scar, and geographical atrophy do not develop, the disease has a relatively good prognosis. The aim of this review is to summarize the possible etiopathogenesis, examination findings, imaging and electrophysiology characteristics, and treatment approaches of Best vitelliform dystrophy in the light of current literature.

DOG1 expression is in common human tumours: a tissue microarray study on more than 15,000 tissue samples

DOG1 (Discovered on GIST1) is a voltage-gated calcium-activated chloride and bicarbonate channel that is highly expressed in interstitial cells of Cajal and in gastrointestinal stromal tumours (GIST) derived from Cajal cells. To systematically determine in what tumour entities and normal tissue types DOG1 may be further expressed, a tissue microarray (TMA) containing 15,965 samples from 121 different tumour types and subtypes as well as 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. DOG1 immunostaining was found in 67 tumour types including GIST (95.7%), oesophageal squamous cell carcinoma (31.9%), pancreatic ductal adenocarcinoma (33.6%), adenocarcinoma of the papilla Vateri (20%), squamous cell carcinoma of the vulva (15.8%) and the oral cavity (15.3%), mucinous ovarian cancer (15.3%), oesophageal adenocarcinoma (12.5%), endometrioid endometrial cancer (12.1%), neuroendocrine carcinoma of the colon (11.1%) and diffuse gastric adenocarcinoma (11%). Low level-DOG1 immunostaining was seen in 17 additional tumour entities. In conclusion, our data identify several tumour entities that can show DOG1 expression levels at similar levels as in GIST. Although DOG1 is tightly linked to a diagnosis of GIST in spindle cell tumours, the differential diagnosis is much broader in DOG1 positive epithelioid neoplasms.

Expression of the CLCA4 Gene in Esophageal Carcinoma and Its Impact on the Biologic Function of Esophageal Carcinoma Cells.

Background Esophageal carcinoma (ESCA) is one of the malignant tumors with a high mortality rate worldwide, which seriously affects people's health. Calcium-activated chloride channel 4 (CLCA4) was reported to be a tumor inhibitor in hepatocellular carcinoma. Nevertheless, the role of CLCA4 in ESCA is still unclear. Methods RT-qPCR and western blot assay were used to test the expression pattern of CLCA4 in ESCA tissues and cells. CCK-8 assay was performed to detect the effect of CLCA4 overexpression on cell proliferation in ESCA cells. Transwell assay was used to measure the effect of CLCA4 upregulation on migration and invasion abilities of ESCA cells. Animal experiments were conducted to investigate the role of CLCA4 upregulation in tumor growth in vivo. Results CLCA4 was significantly reduced in ESCA tissues and correlated with T stage, differentiation, and lymph node metastasis. CLCA4 overexpression was found to inhibit cell proliferation, migration, invasion, and EMT progression in ESCA cells. Moreover, CLCA4 overexpression suppressed tumor growth in vivo. Conclusion CLCA4 was suggested to act as a tumor inhibitor in ESCA and might be a therapeutic target gene for the treatment of patients with ESCA.

Integrated statistical and hydro-geochemical approach to identify the origin and process of saline contamination of Remila plain groundwater (Khenchela, Algeria)

ABSTRACTGroundwater is widely used in the semi-arid region of Remila plain (Khenchela, Algeria) for urban and agricultural supplies. An integrated statistical and hydro-geochemical approach was performed with 70 water samples in order to identify the main processes and the origin of water salinisation. The results have suggested the dominance of three chemical facies: Sulphato cloruro calcic (SO4–Cl–Ca) in the northeastern part, Sulphato cloruro calci magnisian (SO–4Cl–Ca–Mg) in most of the waters andalkali-earth bicarbonate (HCO3–Ca–Mg) in the southeastern part. Although based on principal component analysis and hierarchical clustering analysis, the statistical approach identified three water groups: (1) saline water (17 %; total dissolved solids >1000 mg l−1 with the dominance of Sulphate (SO42−)); (2) moderately saline water (17 %) with a dominance of bicarbonate (HCO3−); and (3) moderately saline water (66 %) with mixed facies. The binary diagrams confirmed the predominance of three processes: evaporite dissolution and/or precipitation, combined by ionic exchange. In the northeastern part of the area, however, another process was detected – the saline intrusion of Sabkha water, favoured by extensive groundwater use.

Isotopic tracers of sources of water for springs from the Edwards Aquifer, Central Texas, USA

Abstract The Edwards Aquifer (EA) in Central Texas provides water supply for over two million people and contains springs that are hydrologically and ecologically important to the region. The residence time of groundwater in the EA ranges from a few days to many thousands of years, since water in the aquifer is contained and transported within both matrix porosity and large conduits. In this study, stable isotopes of water from five springs are investigated for tracing the origin of water and hydrological processes in the EA system during 2017–2019. There is a quick response of the isotopic signals measured at these springs to changes in the isotopic compositions of precipitation. By utilizing an isotope mixing model, we have identified sources of water for these springs with a bi-modal distribution of groundwater supply in the EA: water supplied from deep groundwater with a longer residence time (an average of 67%) and supplemental epikarst interflow with a shorter residence time (an average of 33%). The evolution of hydrochemical water types from HCO3–Ca to HCO3·Cl–Ca·Mg along the EA flowpaths indicates that inputs from epikarst interflow are greater in springs within the artesian zone than the springs within the contributing zone.

Coping with the calcium overload caused by cell injury: ER to the rescue.

Cells maintain their cytosolic calcium (Ca2+) in nanomolar range and use controlled increase in Ca2+ for intracellular signaling. With the extracellular Ca2+ in the millimolar range, there is a steep Ca2+ gradient across the plasma membrane (PM). Thus, injury that damages PM, leads to a cytosolic Ca2+ overload, which helps activate PM repair (PMR) response. However, in order to survive, the cells must cope with the Ca2+ overload. In a recent study (Chandra et al. J Cell Biol, doi: 10.1083/jcb.202006035) we have examined how cells cope with injury-induced cytosolic Ca2+ overload. By monitoring Ca2+ dynamics in the cytosol and endoplasmic reticulum (ER), we found that PM injury-triggered increase in cytosolic Ca2+ is taken up by the ER. Pharmacological inhibition of ER Ca2+ uptake interferes with this process and compromises the repair ability of the injured cells. Muscle cells from patients and mouse model for the muscular dystrophy showed that lack of Anoctamin 5 (ANO5)/Transmembrane protein 16E (TMEM16E), an ER-resident putative Ca2+-activated chloride channel (CaCC), are poor at coping with cytosolic Ca2+ overload. Pharmacological inhibition of CaCC and lack of ANO5, both prevent Ca2+ uptake into ER. These studies identify a requirement of Cl- uptake by the ER in sequestering injury-triggered cytosolic Ca2+ increase in the ER. Further, these studies show that ER helps injured cells cope with Ca2+ overload during PMR, lack of which contributes to muscular dystrophy due to mutations in the ANO5 protein.