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Inflammatory airway diseases like cystic fibrosis, asthma and COVID-19 are characterized by high levels of pulmonary cytokines. Two well-established antiparasitic drugs, niclosamide and ivermectin, are intensively discussed for the treatment of viral inflammatory airway infections. Here, we examined these repurposed drugs with respect to their anti-inflammatory effects in airways in vivo and in vitro. Niclosamide reduced mucus content, eosinophilic infiltration and cell death in asthmatic mouse lungs in vivo and inhibited release of interleukins in the two differentiated airway epithelial cell lines CFBE and BCi-NS1.1 in vitro. Cytokine release was also inhibited by the knockdown of the Ca2+-activated Cl− channel anoctamin 1 (ANO1, TMEM16A) and the phospholipid scramblase anoctamin 6 (ANO6, TMEM16F), which have previously been shown to affect intracellular Ca2+ levels near the plasma membrane and to facilitate exocytosis. At concentrations around 200 nM, niclosamide inhibited inflammation, lowered intracellular Ca2+, acidified cytosolic pH and blocked activation of ANO1 and ANO6. It is suggested that niclosamide brings about its anti-inflammatory effects at least in part by inhibiting ANO1 and ANO6, and by lowering intracellular Ca2+ levels. In contrast to niclosamide, 1 µM ivermectin did not exert any of the effects described for niclosamide. The present data suggest niclosamide as an effective anti-inflammatory treatment in CF, asthma, and COVID-19, in addition to its previously reported antiviral effects. It has an advantageous concentration–response relationship and is known to be well tolerated. [ABSTRACT FROM AUTHOR]

The Ca2+ activated Cl− channel TMEM16A (anoctamin 1; ANO1) is expressed in secretory epithelial cells of airways and intestine. Previous studies provided evidence for a role of ANO1 in mucus secretion. In the present study we investigated the effects of the two ANO1-inhibitors niclosamide (Niclo) and benzbromarone (Benz) in vitro and in vivo in mouse models for cystic fibrosis (CF) and asthma. In human CF airway epithelial cells (CFBE), Ca2+ increase and activation of ANO1 by adenosine triphosphate (ATP) or ionomycin was strongly inhibited by 200 nM Niclo and 1 µM Benz. In asthmatic mice airway mucus secretion was inhibited by intratracheal instillation of Niclo or Benz. In homozygous F508del-cftr mice, intestinal mucus secretion and infiltration by CD45-positive cells was inhibited by intraperitoneal injection of Niclo (13 mg/kg/day for 7 days). In homozygous F508del-cftr rats intestinal mucus secretion was inhibited by oral application of Benz (5 mg/kg/day for 60 days). Taken together, well tolerated therapeutic concentrations of niclosamide and benzbromarone corresponding to plasma levels of treated patients, inhibit ANO1 and intracellular Ca2+ signals and may therefore be useful in inhibiting mucus hypersecretion and mucus obstruction in airways and intestine of patients suffering from asthma and CF, respectively. [ABSTRACT FROM AUTHOR]

The TMEM16A (ANO1) Cl- channel is activated by Ca2+ in a voltage-dependent manner. It is broadly expressed and was shown to be also present in renal proximal tubule (RPT). KCNQ1 is an entirely different K+ selective channel that forms the cardiac IKS potassium channel together with its ß-subunit KCNE1. Surprisingly, KCNE1 has been claimed to interact with TMEM16A, and to be required for activation of TMEM16A in mouse RPT. Interaction with KCNE1 was reported to switch TMEM16A from a Ca22+-dependent to a voltage-dependent ion channel. Here we demonstrate that KCNE1 is not expressed in mouse RPT. TMEM16A expressed in RPT is activated by angiotensin II and ATP in a KCNE1-independent manner. Coexpression of KCNE1 does not change TMEM16A to a voltage gated Cl- channel and Ca2+-dependent regulation of TMEM16A is fully maintained in the presence of KCNE1. While overexpressed KCNE1 slightly affects Ca2+-dependent regulation of TMEM16A, the data provide no evidence for KCNE1 being an auxiliary functional subunit for TMEM16A. [ABSTRACT FROM AUTHOR]

The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel and the epithelial Na+ channel (ENaC) play essential roles in transepithelial ion and fluid transport in numerous epithelial tissues. Inhibitors of both channels have been important tools for defining their physiological role in vitro. However, two commonly used CFTR inhibitors, CFTRinh-172 and GlyH-101, also inhibit non-CFTR anion channels, indicating they are not CFTR specific. However, the potential off-target effects of these inhibitors on epithelial cation channels has to date not been addressed. Here, we show that both CFTR blockers, at concentrations routinely employed by many researchers, caused a significant inhibition of store-operated calcium entry (SOCE) that was time-dependent, poorly reversible and independent of CFTR. Patch clamp experiments showed that both CFTRinh-172 and GlyH-101 caused a significant block of Orai1-mediated whole cell currents, establishing that they likely reduce SOCE via modulation of this Ca2+ release-activated Ca2+ (CRAC) channel. In addition to off-target effects on calcium channels, both inhibitors significantly reduced human αβγ-ENaC-mediated currents after heterologous expression in Xenopus oocytes, but had differential effects on δβγ-ENaC function. Molecular docking identified two putative binding sites in the extracellular domain of ENaC for both CFTR blockers. Together, our results indicate that caution is needed when using these two CFTR inhibitors to dissect the role of CFTR, and potentially ENaC, in physiological processes. [ABSTRACT FROM AUTHOR]

Objectives: This in vitro study aimed to investigate the optical attenuation of light at 405, 660 and 780 nm sent through sound and carious human enamel and dentin, including respective individual caries zones, as well as microscopically sound-appearing tissue close to a carious lesion. Materials and methods: Collimated light transmission through sections of 1000–125-µm thickness was measured and used to calculate the attenuation coefficient (AC). The data were statistically analysed with a MANOVA and Tukey's HSD. Precise definition of measurement points enabled separate analysis within the microstructure of lesions: the outer and inner halves of enamel (D1, D2), the translucent zone (TZ) within dentin lesions and its adjacent layers, the enamel side of the translucent zone (ESTZ) and the pulpal side of the translucent zone (PSTZ). Results: The TZ could be distinguished from its adjacent layers and from caries-free dentin at 125 µm. Sound-appearing dentin close to caries lesions significantly differed from caries-free dentin at 125 µm. While sound and carious enamel exhibited a significant difference (p < 0.05), this result was not found for D1 and D2 enamel lesions (p > 0.05). At 405 nm, no difference was found between sound and carious dentin (p > 0.05). Conclusions: Light optical means enable the distinction between sound and carious tissue and to identify the microstructure of dentin caries partially as well as the presence of tertiary dentin formation. Information on sample thickness is indispensable when interpreting the AC. Clinical relevance: Non-ionising light sources may be suitable to detect lesion progression and tertiary dentin. [ABSTRACT FROM AUTHOR]

Objectives: This in vitro study analysed potential of early proximal caries detection using 3D range data of teeth consisting of near-infrared reflection images at 850 nm (NIRR). Materials and methods: Two hundred fifty healthy and carious permanent human teeth were arranged pairwise, examined with bitewing radiography (BWR) and NIRR and validated with micro-computed tomography. NIRR findings were evaluated from buccal, lingual and occlusal (trilateral) views according to yes/no decisions about presence of caries. Reliability assessments included kappa statistics and revealed high agreement for both methods. Statistical analysis included cross tabulation and calculation of sensitivity, specificity and AUC. Results: Underestimation of caries was 24.8% for NIRR and 26.4% for BWR. Overestimation was 10.4% for occlusal NIRR and 0% for BWR. Trilateral NIRR had overall accuracy of 64.8%, overestimation of 15.6% and underestimation of 19.6%. NIRR and BWR showed high specificity and low sensitivity for proximal caries detection. Conclusions: NIRR achieved diagnostic results comparable to BWR. Trilateral NIRR assessments overestimated presence of proximal caries, revealing stronger sensitivity for initial caries detection than BWR. Clinical relevance: NIRR provided valid complement to BWR as diagnostic instrument. Investigation from multiple angles did not substantially improve proximal caries detection with NIRR. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) is a life-threatening autosomal recessive disease caused by more than 2100 mutations in the CF transmembrane conductance regulator (CFTR) gene, generating variability in disease severity among individuals with CF sharing the same CFTR genotype. Systems biology can assist in the collection and visualization of CF data to extract additional biological significance and find novel therapeutic targets. Here, we present the CyFi-MAP—a disease map repository of CFTR molecular mechanisms and pathways involved in CF. Specifically, we represented the wild-type (wt-CFTR) and the F508del associated processes (F508del-CFTR) in separate submaps, with pathways related to protein biosynthesis, endoplasmic reticulum retention, export, activation/inactivation of channel function, and recycling/degradation after endocytosis. CyFi-MAP is an open-access resource with specific, curated and continuously updated information on CFTR-related pathways available online at https://cysticfibrosismap.github.io/. This tool was developed as a reference CF pathway data repository to be continuously updated and used worldwide in CF research. [ABSTRACT FROM AUTHOR]

Objectives: This study evaluated the adhesion and whitening effects of a combination of P11-4 self-assembling peptide and hydroxyapatite (peptide-HAP) on bovine enamel. Methods: Forty-six caries-free bovine teeth were selected, and 40 teeth were randomly allocated to one of five groups (n = 8). First, the effects of application frequency, exposure time, and storage in saliva on the whitening effects of an experimental low-concentrated peptide-HAP suspension (0.5 wt% HAP; Curodont, Credentis) were evaluated and compared with a commercial bleaching agent (VivaStyle Paint on Plus, VS, Ivoclar Vivadent). Tooth color was measured using a spectrophotometer (Gretag MacBeth), and color changes ΔE were statistically analyzed. Second, the effects of peptide-HAP concentration (low versus high: 6.25% HAP; Curodont Protect), and its interactions with saliva and postapplication restaining, were investigated. Third, enamel surfaces (n = 2) were treated with low concentration peptide-HAP and high-concentration peptide-HAP in polymeric and monomeric forms (Curodont Protect & Curodont Repair, Credentis) and analyzed by SEM. Results: The ΔE of the low-concentration peptide-HAP suspension did not differ from that of VS. Application frequency, exposure time, and storage in saliva did not have any significant impact on whitening efficacy of the peptide-HAP suspension. Increasing the concentration of the suspension did not promote overall ΔE. SEM observations confirmed the presence of the newly generated peptide and HAP on the enamel surface. Conclusions: The peptide-HAP suspension is a mild tooth whitener, and the adhesion of peptide-HAP to enamel is concentration dependent. Clinical relevance: This peptide-HAP suspension is effective in offsetting discoloration caused by restaining after treatment. [ABSTRACT FROM AUTHOR]

Introduction: The aim of this study was to experimentally investigate the potential of different light wavelengths to distinguish between healthy and carious tissue using a two-circle goniometer. Materials and methods: Tooth slices were prepared from extracted human teeth that were caries free (n = 15) or had occlusal caries lesions (n = 10). The tooth slices were irradiated with diode laser modules of different wavelengths (532, 650, 780 nm). The transmitted and scattered laser light was spatially measured with a detector rotating on a two-circle goniometer. The anisotropy factor and attenuation coefficients were calculated. Results: Enamel was more transparent than dentin and showed wavelength-dependent attenuation. Healthy dentin showed strong light scattering at all wavelengths, independent of the tested wavelength. The calculated attenuation coefficients of carious and healthy tooth tissue differed significantly (p < 0.05; t test). In contrast to healthy enamel, carious enamel showed lower light transmission and an increase in scattering. Differences in the light attenuation of carious versus healthy dentin were less pronounced than those for enamel. Carious dentin was slightly more transparent than healthy dentin. The light of longer wavelengths showed a better penetration of all tooth structures compared with shorter wavelengths. Conclusion: Healthy and carious dentin and enamel exhibited distinct optical properties using laser light at different wavelengths. In dentin, changes in the optical properties caused by caries are significantly less pronounced. Clinical relevance: The clear distinction between healthy and carious enamel makes optical caries diagnostic systems ideal tools for early caries detection. [ABSTRACT FROM AUTHOR]

Objectives: Sufficient depth of cure allows bulk-fill composites to be placed with a 4-mm thickness. This study investigated bulk versus incremental application methods by visualizing shrinkage vectors in flowable bulk-fill and conventional composites. Materials and methods: Cylindrical cavities (diameter = 6 mm, depth = 4 mm) were prepared in 24 teeth and then etched and bonded with OptiBond FL (Kerr, Italy). The composites were mixed with 2 wt% radiolucent glass beads. In one group, smart dentin replacement (SDR, Dentsply) was applied in bulk "SDR-bulk" (n = 8). In two groups, SDR and Tetric EvoFlow (Ivoclar Vivadent) were applied in two 2-mm-thick increments: "SDR-incremental" and "EvoFlow-incremental." Each material application was scanned with a micro-CT before and after light-curing (40 s, 1100 mW/cm2), and the shrinkage vectors were computed via image segmentation. Thereafter, linear polymerization shrinkage, shrinkage stress and gelation time were measured (n = 10). Results: The greatest shrinkage vectors were found in "SDR-bulk" and "SDR-increment2," and the smallest were found in "SDR-increment1-covered" and "EvoFlow-increment1-covered." Shrinkage away from and toward the cavity floor was greatest in "SDR-bulk" and "EvoFlow-increment2," respectively. The mean values of the shrinkage vectors were significantly different between groups (one-way ANOVA, Tamhane's T2 test, p < 0.05). The linear polymerization shrinkage and shrinkage stress were greatest in Tetric EvoFlow, and the gelation time was greatest in "SDR-bulk." Conclusions: The bulk application method had greater values of shrinkage vectors and a higher debonding tendency at the cavity floor. Clinical relevance: Incremental application remains the gold standard of composite insertion. [ABSTRACT FROM AUTHOR]

The aim of this study was to assess near-infrared transillumination with high dynamic range imaging (NIRT-HDRI) for occlusal caries detection in vitro and to compare it with visual inspection using the International Caries Detection and Assessment System II (ICDAS) and digital bitewing radiography (BWR). Sixty-one extracted permanent molars with sound or occlusal carious surfaces without severe cavitation were visually assembled. Two examiners assessed twice these surfaces independently using ICDAS, BWR and NIRT-HDRI. The latter was performed with a prototype consisting of two laser sources (780 nm), a CCD sensor and subsequent processing with image analysis software. Thresholds for carious surfaces, enamel and dentin lesions were defined for all methods. Micro-computed tomography served as the reference standard. Linear weighted Kappa analysis of the methods versus the reference at the threshold carious surface, enamel and dentin lesion revealed 0.59/0.08/0.12 for ICDAS, 0.37/− 0.06/0.58 for BWR and 0.33/− 0.01/0.51 for NIRT-HDRI. Sensitivity values at the three thresholds were 0.85/0.78/0.13 for ICDAS, 0.59/0.00/0.69 for BWR and 0.98/0.33/0.78 for NIRT. Specificity values at the three thresholds were 0.70/0.40/1.00 for ICDAS, 0.9./0.96/0.90 for BWR and 0.30/0.65/0.72 for NIRT-HDRI. Reliability analysis revealed substantial agreement for BWR and NIRT and almost perfect agreement for ICDAS. NIRT exhibited a strong ability to identify occlusal dental decay in general; however, it revealed a tendency towards overestimation. Its strength was the detection of dentin caries lesions compared with ICDAS and BWR. NIRT-HDRI seems to be a suitable method to detect hidden dentin caries as a supplement to visual examination. [ABSTRACT FROM AUTHOR]

Previous analysis of CFTR-knockout (CFTR−/−) in piglets has provided important insights into the pathology of cystic fibrosis. However, controversies exist as to the true contribution of CFTR to the pH balance in airways and intestine. We therefore compared ion transport properties in newborn wild-type (CFTR+/+) and CFTR-knockout (CFTR−/− piglets). Tracheas of CFTR−/− piglets demonstrated typical cartilage malformations and muscle cell bundles. CFTR−/− airway epithelial cells showed enhanced lipid peroxidation, suggesting inflammation early in life. CFTR was mainly expressed in airway submucosal glands and was absent in lungs of CFTR−/− piglets, while expression of TMEM16A was uncompromised. mRNA levels for TMEM16A, TMEM16F, and αβγENaC were unchanged in CFTR−/− airways, while mRNA for SLC26A9 appeared reduced. CFTR was undetectable in epithelial cells of CFTR−/− airways and intestine. Small intestinal epithelium of CFTR−/− piglets showed mucus accumulation. Secretion of both electrolytes and mucus was activated by stimulation with prostaglandin E2 and ATP in the intestine of CFTR+/+, but not of CFTR−/− animals. pH was measured inside small bronchi using a pH microelectrode and revealed no difference between CFTR+/+ and CFTR−/− piglets. Intracellular pH in porcine airway epithelial cells revealed only a small contribution of CFTR to bicarbonate secretion, which was absent in cells from CFTR−/− piglets. In contrast to earlier reports, our data suggest a minor impact of CFTR on ASL pH. In contrast, enhanced amiloride-sensitive Na+ absorption may contribute to lung pathology in CFTR−/− piglets, along with a compromised CFTR- and TMEM16A-dependent Cl− transport. [ABSTRACT FROM AUTHOR]

Objectives: The corona disease (COVID-19) is developing into one of the greatest challenges for healthcare professionals around the world. In this article, we report the detailed actions taken in the Department of Conservative Dentistry and Periodontology, University Hospital, Ludwig-Maximilians-University (LMU), Munich, Germany, during the early phase of the COVID-19 pandemic. Material and methods: After a joint on-site inspection of the dental clinic with the Department of Clinical Microbiology and Hospital Hygiene, existing clinical and hygiene protocols were adapted for COVID-19 patients. Results: A comprehensive summary of the preparation of the facilities as well as pre- treatment, treatment and posttreatment protocols are described and arising problems are being discussed. Conclusions: The importance of rigorous hygiene and treatment protocols as well as a sufficient supply of PPE for dental offices and hospitals is highlighted. The measures reported may be subject to change due to the dynamics of the pandemic. Clinical relevance: The modes of transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (e.g., droplets, aerosols, and fomites) can pose a risk for dental healthcare professionals and patients alike. The presented measures may guide dental faculties and dental practices during the early stage of the COVID-19 crisis. [ABSTRACT FROM AUTHOR]

Polycystic kidney disease (PKD) leads to continuous decline of renal function by growth of renal cysts. Enhanced proliferation and transepithelial chloride secretion through cystic fibrosis transmembrane conductance regulator (CFTR) and Ca2+-activated TMEM16A Cl− channels is thought to cause an increase in cyst volume. Recent work shows the pro-proliferative role of the Ca2+ activated Cl− channel TMEM16A (anoctamin 1), and demonstrates the essential contribution of TMEM16A to CFTR-dependent Cl− secretion. The present data demonstrate an increase in intracellular Ca2+ ([Ca2+]i) signals and Cl− secretion by TMEM16A, in renal collecting duct principle cells from dog (MDCK) and mouse (M1) as well as primary tubular epithelial cells from PKD1−/− knockout mice. M1 organoids proliferated, increased expression of TMEM16A, and secreted Cl− upon knockdown of endogenous polycystin 1 or 2 (PKD1,2), by retroviral transfection with shPKD1 and shPKD2, respectively. Knockdown of PKD1 or PKD2 increased basal intracellular Ca2+ levels and enhanced purinergic Ca2+ release from endoplasmic reticulum. In contrast, ryanodine receptors were found not to be expressed in mouse renal epithelial cells and caffeine had no effects on [Ca2+]i. Ca2+ signals, proliferation, and Cl− secretion were largely reduced by knockdown or blockade of TMEM16A. TMEM16A may be therefore important for enhanced Ca2+ release from IP3-sensitive Ca2+ stores in polycystic kidney disease. Key messages: • ADPKD leads to continuous decline of renal function by growth of renal cysts. • Knockdown of PKD1 or PKD2 increases TMEM16A expression. • TMEM16A enhanced intracellular Ca2+ signals, Cl− secretion, and proliferation. • TMEM16A contributes to cyst growth in ADPKD. [ABSTRACT FROM AUTHOR]

Objectives: The aim of the present in vitro study was to determine the ability of Diagnocam to detect caries at an early stage and to compare it with digital radiography. Materials and methods: One hundred twenty proximal surfaces composed equally of sound and decayed surfaces were evaluated by assessing images captured with Diagnocam (Kavo, Biberach, Germany) and digital radiography (DR). All images were assessed twice by two calibrated dentists with a minimum interval of 2 weeks between examinations. The results were compared with μCT scans. Results: Inter-rater reliability showed nearly perfect agreement; a high intra-rater reliability was calculated. Spearman's rank correlation coefficients showed a strong correlation of Diagnocam and μCT (0.82). DR and μCT achieved a slightly lower correlation (0.73). The surfaces were categorized into sound surfaces, enamel lesions, and dentin lesions to determine intraclass correlation coefficients (ICCs), sensitivity and specificity. ICCs between μCT and DR ranged from 0.20 to 0.63. ICCs between Diagnocam and DR were higher in all categories and ranged from 0.56 to 0.83. Sensitivity, specificity, and accuracy of Diagnocam achieved mostly higher values than DR. In detecting enamel lesions, sensitivity was 0.36 for DR and 0.59 for the DC. The areas under the receiver operating characteristic (ROC) curves of Diagnocam were larger than those of DR in all categories. Conclusions: Diagnocam is more capable of detecting initial proximal lesions than DR and also has a higher sensitivity for dentin lesions. However, caries progression in dentin cannot be reliably determined with Diagnocam. Clinical relevance: Diagnocam may be suitable as a supplement to X-ray diagnostics in clinical use. [ABSTRACT FROM AUTHOR]

The cystic fibrosis transmembrane conductance regulator (CFTR) is the secretory chloride channel in epithelial tissues that has a central role in cystic fibrosis (CF) lung and gastrointestinal disease. A recent publication demonstrates a close association between CFTR and TMEM16A, the calcium-activated chloride channel. Thus, no CFTR chloride currents could be detected in airways and large intestine from mice lacking epithelial expression of TMEM16A. Here, we demonstrate that another plasma membrane–localized TMEM16 paralogue, TMEM16F, can compensate for the lack of TMEM16A. Using TMEM16 knockout mice, human lymphocytes, and a number of human cell lines with endogenous protein expression or heterologous expression, we demonstrate that CFTR can only function in the presence of either TMEM16A or TMEM16F. Double knockout of intestinal epithelial TMEM16A/F expression did not produce offsprings, suggesting a lethal phenotype in utero. Plasma membrane–localized TMEM16A or TMEM16F is required for exocytosis and expression of CFTR in the plasma membrane. TMEM16A/F proteins may therefore have an impact on disease severity in CF. Key messages: • Cystic fibrosis is caused by the defective Cl− channel cystic fibrosis transmembrane conductance regulator (CFTR). • A close relationship exists between CFTR and the calcium-activated chloride channels TMEM16A/TMEM16F. • In conditional airway and intestinal knockout mice, lymphocytes from Scott disease patients and in overexpressing cells, CFTR is not functional in the absence of TMEM16A and TMEM16F. • TMEM16A and TMEM16F support membrane exocytosis and are essential for plasma membrane insertion of CFTR. [ABSTRACT FROM AUTHOR]

Objectives: Bond strength to enamel and dentin depends on the bonding approach or condition. This study investigated the effects of the boundary conditions, in terms of the bonding substrate and the bonding condition, on the shrinkage vectors of a flowable composite. Materials and methods: An experimental cylindrical cavity (diameter = 6 mm, depth = 3 mm) consisting of the enamel floor and the surrounding dentin cavity walls was prepared for the "enamel-floor" group. Cylindrical cavities of the same dimensions were prepared with access from the occlusal enamel into dentin and served as controls. Each cavity model group was divided and bonded with two bonding conditions (n = 9): a self-etch (Adper Easy Bond, 3M ESPE) and a total-etch approach (OptiBond FL, Kerr). The composite (Tetric EvoFlow, Ivoclar Vivadent) was mixed with glass beads, applied to the cavity, scanned twice by micro-CT (uncured and cured states). The scans were evaluated by rigid registration, sphere segmentation, and registration for computing shrinkage vectors. Results: The free surface of all restorations moved downward. The shrinkage vectors in the experimental cavity model pointed downward towards the enamel cavity floor, and the net axial movement was downward. In the control group, shrinkage vectors additionally moved upward, away from the cavity floor. The effect of the bonding substrate and the bonding condition was investigated for the shrinkage vectors and the axial movement (univariate ANOVA). Conclusion: The bonding substrate, enamel, influenced the shrinkage vectors' direction, while the bonding condition caused only variations in the magnitude. Clinical relevance: Bonding to enamel influences shrinkage vectors' direction, while the bonding condition plays only a minor role. Graphical abstract ᅟ [ABSTRACT FROM AUTHOR]

Objectives: This study aimed to assess the effects of fiber incorporation on the contraction stress (CS), time to gelation (TG), and temperature rise (TR) features of two experimental resin-based composites (RBCs) in comparison to those of bulk-fill RBCs and conventional RBCs in simulated, clinically relevant cavities. Materials and methods: CS over 300 s, TG and TR were assessed by a stress-strain analyzer (SSA T80) in two cavity configurations (2 × 4 × 4 mm3/4 × 4 × 4 mm3/configuration value 1 2 and 1 3 ) for two experimental RBCs, eight bulk-fill RBCs (EverX Posterior, Filtek Bulk Fill, SDR, SonicFill, Tetric EvoCeram Bulk Fill, Venus Bulk Fill, X-tra base, and X-tra Fill) and one RBC (Filtek Supreme XTE) by light curing (1200 mW/cm2) (n = 10). The experimental materials used were based on EverX Posterior with modifications made to the fiber and filler content. Statistical analyses were performed via ANOVA; multiple pair-wise comparisons were performed via Tukey's test, and homogeneous subsets were identified ("multcomp" package, R). Results: CS values ranged from 1.00 to 4.07 MPa (2 × 4 × 4 mm3) and from 0.97 to 2.49 MPa (4 × 4 × 4 mm3); TG values ranged from 1.31 to 4.05 s (2 × 4 × 4 mm3) and from 1.39 to 3.84 s (4 × 4 × 4 mm3). SDR values showed lowest stress values in both cavity configurations. The experimental composite with fibers presented significantly higher stress values than did the experimental composite without fibers. Conclusion: Bulk-fill RBCs showed lower levels of stress than did conventional RBCs. The incorporation of fibers had no positive impact on the CS and TG. Clinical relevance: Appropriate material selection may be essential to clinical success because certain RBCs exhibit higher CS, TG, and TR values. [ABSTRACT FROM AUTHOR]

Objective: The objective of this study was to investigate the effects of cavity configuration on the shrinkage vectors of a flowable resin-based composite (RBC) placed in occlusal cavities.Materials and methods: Twenty-seven human molars were divided into three groups (n = 9) according to cavity configuration: “adhesive,” “diverging,” and “cylindrical.” The “adhesive” cavity represented beveled enamel margins and occlusally converging walls, the “diverging” cavity had occlusally diverging walls, and the “cylindrical” cavity had parallel walls (diameter = 6 mm); all cavities were 3 mm deep. Each prepared cavity was treated with a self-etch adhesive (Adper Easy Bond, 3 M ESPE) and filled with a flowable RBC (Tetric EvoFlow, Ivoclar Vivadent) to which had been added 2 wt% traceable glass beads. Two micro-CT scans were performed on each sample (uncured and cured). The scans were then subjected to medical image registration for shrinkage vector calculation. Shrinkage vectors were evaluated three-dimensionally (3D) and in the axial direction.Results: The “adhesive” group had the greatest mean 3D shrinkage vector lengths and upward movement (31.1 ± 10.9 μm; − 13.7 ± 12.1 μm), followed by the “diverging” (27.4 ± 12.1 μm; − 5.7 ± 17.2 μm) and “cylindrical” groups (23.3 ± 11.1 μm; − 3.7 ± 13.6 μm); all groups differed significantly (p < 0.001 for each comparison, one-way ANOVA, Tamhane’s T2).Conclusion: The values and direction of the shrinkage vectors as well as interfacial debonding varied according to the cavity configuration.Clinical relevance: Cavity configuration in terms of wall orientation and beveling of enamel margin influences the shrinkage pattern of composites. [ABSTRACT FROM AUTHOR]

Cystic fibrosis transmembrane conductance regulator (CFTR) is the essential chloride and bicarbonate channel in the apical membrane of epithelial cells. CFTR was also proposed earlier to conduct glutathione (GSH) out of airway epithelial cells to be enriched in the apical airway surface liquid to neutralize reactive oxygen species (ROS). Although earlier studies suggested that release of GSH by wild type (wt) CFTR may lead to an increase in cytosolic ROS, we did not detect different ROS levels in cells expressing wt-CFTR and mutant F508del-CFTR, independent of CFTR-activation or exposure to the ROS donor tert-butyl hydroperoxide. The Ca-activated phospholipid scramblase and ion channel TMEM16F (anoctamin 6, ANO6) is also expressed in airway cells. ANO6 produced outwardly rectifying Cl currents (ORCC) and scrambled plasma membrane phospholipids when activated by increase in cytosolic ROS and consecutive peroxidation of plasma membrane lipids. ANO6 activity is enhanced by CFTR, probably through translocation of signaling proteins to the plasma membrane. The present data suggest that enhanced cell death in CFTR-expressing cells is due to upregulation of ANO6-activity. In ANO6 knockout mice, the number of apoptotic cells in the intestinal epithelium was strongly reduced, supporting the role of ANO6 for cell death. Thus, ANO6 and CFTR act cooperatively on ROS-mediated cell death, which is not further augmented by cAMP-dependent stimulation. We propose that ANO6 supports cell death correlated with expression of CFTR, possibly by inducing ferroptosis. [ABSTRACT FROM AUTHOR]

Activated receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain like (MLKL) are essential components of the necroptotic pathway. Phosphorylated MLKL (pMLKL) is thought to induce membrane leakage, leading to cell swelling and disintegration of the cell membrane. However, the molecular identity of the necroptotic membrane pore remains unclear, and the role of pMLKL for membrane permeabilization is currently disputed. We observed earlier that the phospholipid scramblase and ion channel TMEM16F/anoctamin 6 cause large membrane currents, cell swelling, and cell death when activated by a strong increase in intracellular Ca. We, therefore, asked whether TMEM16F is also central to necroptotic cell death and other cellular events during necroptosis. Necroptosis was induced by TNFα, smac mimetic, and Z-VAD (TSZ) in NIH3T3 fibroblasts and the four additional cell lines HT, 16HBE, H441, and L929. Time-dependent changes in intracellular Ca, cell morphology, and membrane currents were recorded. TSZ induced a small and only transient oscillatory rise in intracellular Ca, which was paralleled by the activation of outwardly rectifying Cl currents, which were typical for TMEM16F/ANO6. Ca oscillations were due to Ca release from endoplasmic reticulum, and were independent of extracellular Ca. The initial TSZ-induced cell swelling was followed by cell shrinkage. Using typical channel blockers and siRNA-knockdown, the Cl currents were shown to be due to the activation of ANO6. However, the knockdown of ANO6 or inhibitors of ANO6 did not inhibit necroptotic cell death. The present data demonstrate the activation of ANO6 during necroptosis, which, however, is not essential for cell death. [ABSTRACT FROM AUTHOR]

Polycystic kidney diseases are characterized by numerous renal cysts that continuously enlarge resulting in compression of intact nephrons and tissue hypoxia. Recently, we have shown that hypoxia-inducible factor (HIF)-1α promotes secretion-dependent cyst expansion, presumably by transcriptional regulation of proteins that are involved in calcium-activated chloride secretion. Here, we report that HIF-1α directly activates expression of the purinergic receptor P2Y2R in human primary renal tubular cells. In addition, we found that P2Y2R is highly expressed in cyst-lining cells of human ADPKD kidneys as well as PKD1 orthologous mouse kidneys. Knockdown of P2Y2R in renal collecting duct cells inhibited calcium-dependent chloride secretion in Ussing chamber analyses. In line with these findings, knockdown of P2Y2R retarded cyst expansion in vitro and prevented ATP- and HIF-1α-dependent cyst growth. In conclusion, P2Y2R mediates ATP-dependent cyst growth and is transcriptionally regulated by HIF-1α. These findings provide further mechanistic evidence on how hypoxia promotes cyst growth. [ABSTRACT FROM AUTHOR]

The anoctamin (ANO, TMEM16) family of Ca-activated Cl channels consists of ten members with different cellular functions (ANO1-10). ANO1 is a Ca-activated Cl channel in secretory epithelial cells of exocrine pancreas, salivary glands, or enterocytes. Expression of ANO1 also promotes cell proliferation and migration of tumor cells. ANO6 is essential for Ca-dependent scrambling of membrane phospholipids in platelets, red blood cells, and lymphocytes. ANO10 modulates Ca signals in macrophages and has a role in cerebellar ataxia and other neurological disorders. All three anoctamins have been proposed to control intracellular Ca signals. Anoctamins may also form the basolateral Ca-activated Cl channel in the retinal pigment epithelium (RPE). We show that native human, bovine, porcine, and mouse RPEs express ANO1, ANO6, and ANO10. Growth arrested and confluent RPR cells expressed ANO1 in the plasma membrane, whereas ANO6 and ANO10 were found in the primary cilium. Ussing chamber experiments showed that the application of ATP to the apical (retinal) side of porcine RPE induced a Ca-activated Cl secretion. Activation was inhibited by basolateral (choroidal) administration of the ANO inhibitors AO1, niflumic acid (NFA), and 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS). The results suggest that ANO1 is responsible for basolateral Ca-dependent Cl secretion in RPE, whereas ANO6 and ANO10 may have different functions, such as modulating Ca signals. [ABSTRACT FROM AUTHOR]

A remarkable feature of apoptosis is the initial massive cell shrinkage, which requires opening of ion channels to allow release of K, Cl, and organic osmolytes to drive osmotic water movement and cell shrinkage. This article focuses on the role of the Cl channels LRRC8, TMEM16/anoctamin, and cystic fibrosis transmembrane conductance regulator (CFTR) in cellular apoptosis. LRRC8A-E has been identified as a volume-regulated anion channel expressed in many cell types. It was shown to be required for regulatory and apoptotic volume decrease (RVD, AVD) in cultured cell lines. Its presence also determines sensitivity towards cytostatic drugs such as cisplatin. Recent data point to a molecular and functional relationship of LRRC8A and anoctamins (ANOs). ANO6, 9, and 10 (TMEM16F, J, and K) augment apoptotic Cl currents and AVD, but it remains unclear whether these anoctamins operate as Cl channels or as regulators of other apoptotic Cl channels, such as LRRC8. CFTR has been known for its proapoptotic effects for some time, and this effect may be based on glutathione release from the cell and increase in cytosolic reactive oxygen species (ROS). Although we find that CFTR is activated by cell swelling, it is possible that CFTR serves RVD/AVD through accumulation of ROS and activation of independent membrane channels such as ANO6. Thus activation of ANO6 will support cell shrinkage and induce additional apoptotic events, such as membrane phospholipid scrambling. [ABSTRACT FROM AUTHOR]

TMEM16A/anoctamin 1/ANO1 and VRAC/LRRC8 are independent chloride channels activated either by increase in intracellular Ca or cell swelling, respectively. In previous studies, we observed overlapping properties for both types of channels. (i) TMEM16A/ANO1 and LRRC8 are inhibited by identical compounds, (ii) the volume-regulated anion channel VRAC requires compartmentalized Ca increase to be fully activated, (iii) anoctamins are activated by cell swelling, (iv) both channels have a role for apoptotic cell death, (v) both channels are possibly located in lipid rafts/caveolae like structures, and (vi) VRAC and anoctamin 1 currents are not additive when each are fully activated. In the present study, we demonstrate in different cell types that loss of LRRC8A expression not only inhibited VRAC, but also attenuated Ca activated Cl currents. Moreover, expression of LRRC8A enhanced Ca activated Cl currents, and both LRRC8A and ANO1 could be coimmunoprecipitated. We found that LRRC8A becomes accessible to biotinylation upon exposure to hypotonic bath solution, while membrane capacitance was not enhanced. When intracellular Ca was increased in ANO1-expressing cells, the membrane capacitance was enhanced and increased binding of FM4-64 to the membrane was observed. As this was not seen in cells lacking ANO1 expression, a role of ANO1 for exocytosis was suggested. We propose that ANO1 and LRRC8A are activated in parallel. Thus, ionomycin or purinergic stimulation will not only activate ANO1 but also LRRC8 currents. Cell swelling will not only activate LRRC8/VRAC, but also stimulate ANO1 currents by enhancing compartmentalized Ca increase and/or through swelling induced autocrine release of ATP. [ABSTRACT FROM AUTHOR]

Activation of ion channels and pores are essential steps during regulated cell death. Channels and pores participate in execution of apoptosis, necroptosis and other forms of caspase-independent cell death. Within the program of regulated cell death, these channels are strategically located. Ion channels can shrink cells and drive them towards apoptosis, resulting in silent, i.e. immunologically unrecognized cell death. Alternatively, activation of channels can induce cell swelling, disintegration of the cell membrane, and highly immunogenic necrotic cell death. The underlying cell death pathways are not strictly separated as identical stimuli may induce cell shrinkage and apoptosis when applied at low strength, but may also cause cell swelling at pronounced stimulation, resulting in regulated necrosis. Nevertheless, the precise role of ion channels during regulated cell death is far from being understood, as identical channels may support regulated death in some cell types, but may cause cell proliferation, cancer development, and metastasis in others. Along this line, the phospholipid scramblase and Cl/nonselective channel anoctamin 6 (ANO6) shows interesting features, as it participates in apoptotic cell death during lower levels of activation, thereby inducing cell shrinkage. At strong activation, e.g. by stimulation of purinergic P2Y receptors, it participates in pore formation, causes massive membrane blebbing, cell swelling, and membrane disintegration. The LRRC8 proteins deserve much attention as they were found to have a major role in volume regulation, apoptotic cell shrinkage and resistance towards anticancer drugs. [ABSTRACT FROM AUTHOR]

Volume regulation is an essential property of any living cell and needs to be tightly controlled. While different types of K channels have been found to participate in the regulation of cell volume, the newly identified volume-regulated anion channel (VRAC) LRRC8 has been claimed to be essential for volume regulation. In unbiased genome-wide small interfering RNA (siRNA) screens, two independent studies identified LRRC8A/Swell1 as an essential component of VRAC, thus being indispensable for cellular volume regulation. We reanalyzed the role of LRRC8A for VRAC and regulatory volume decrease (RVD) in several cell types and under various conditions. While the role of LRRC8A for VRAC and its contribution to RVD is confirmed, we find that it is not essential for swelling-activated anion currents or cellular volume regulation, or apoptotic cell shrinkage. The contribution of LRRC8A is variable and largely depending on the cell type. [ABSTRACT FROM AUTHOR]

Since the discovery of TMEM16A (anoctamin 1, ANO1) as Ca-activated Cl channel, the protein was found to serve different physiological functions, depending on the type of tissue. Subsequent reports on other members of the anoctamin family demonstrated a broad range of yet poorly understood properties. Compromised anoctamin function is causing a wide range of diseases, such as hearing loss (ANO2), bleeding disorder (ANO6), ataxia and dystonia (ANO3, 10), persistent borrelia and mycobacteria infection (ANO10), skeletal syndromes like gnathodiaphyseal dysplasia and limb girdle muscle dystrophy (ANO5), and cancer (ANO1, 6, 7). Animal models demonstrate CF-like airway disease, asthma, and intestinal hyposecretion (ANO1). Although present data indicate that ANO1 is a Ca-activated Cl channel, it remains unclear whether all anoctamins form plasma membrane-localized or intracellular chloride channels. We find Ca-activated Cl currents appearing by expression of most anoctamin paralogs, including the Nectria haematococca homologue nhTMEM16 and the yeast homologue Ist2. As recent studies show a role of anoctamins, Ist2, and the related transmembrane channel-like (TMC) proteins for intracellular Ca signaling, we will discuss the role of these proteins in generating compartmentalized Ca signals, which may give a hint as to the broad range of cellular functions of anoctamins. [ABSTRACT FROM AUTHOR]

During cell swelling, Cl channels are activated to lower intracellular Cl concentrations and to reduce cell volume, a process termed regulatory volume decrease (RVD). We show that anoctamin 6 (ANO6; TMEM16F) produces volume-regulated anion currents and controls cell volume in four unrelated cell types. Volume regulation is compromised in freshly isolated intestinal epithelial cells from Ano6−/− mice and also in lymphocytes from a patient lacking expression of ANO6. Ca influx is activated and thus ANO6 is stimulated during cell swelling by local Ca increase probably in functional nanodomains near the plasma membrane. This leads to stimulation of phospholipase A (PLA) and generation of plasma membrane lysophospholipids, which activates ANO6. Direct application of lysophospholipids also activates an anion current that is inhibited by typical ANO6 blocker. An increase in intracellular Ca supports activation of ANO6, but is not required when PLA is fully activated, while re-addition of arachidonic acid completely blocked ANO6. Moreover, ANO6 is activated by low intracellular Cl concentrations and may therefore operate as a cellular osmosensor. High intracellular Cl concentration inhibits ANO6 and activation by PLA. Taken together, ANO6 supports volume regulation and volume activation of anion currents by action as a Cl channel or by scrambling membrane phospholipids. Thereby, it may support the function of LRRC8 proteins. [ABSTRACT FROM AUTHOR]

Polycystic kidney diseases are characterized by the development of numerous bilateral renal cysts that continuously enlarge resulting in a decline of kidney function due to compression of intact nephrons. Cyst growth is driven by transepithelial chloride secretion which depends on both intracellular cAMP and calcium. Mechanisms that are involved in the regulation of the underlying secretory pathways remain incompletely understood. Here we show that glucose concentration has a strong impact on cyst growth of renal tubular cells within a collagen matrix as well as in embryonic kidneys deficient or competent for Pkd1. Glucose-dependent cyst growth correlates with the transcriptional induction of the calcium-activated chloride channel anoctamin 1 (ANO1) and its increased expression in the apical membrane of cyst-forming cells. Inhibition of ANO1 with the specific inhibitor CaCCinh-AO1 significantly decreases glucose-dependent cyst growth in both models. Ussing chamber analyses revealed increased apical chloride secretion of renal tubular cells upon exposure to high glucose medium which can also be inhibited by the use of CaCCinh-AO1. These data suggest that glycemic control may help to reduce renal cyst growth in patients with polycystic kidney disease. Key message: [ABSTRACT FROM AUTHOR]

Objectives: The aim of this study was to assess the effect of two sealants and two varnishes on the prevention of enamel demineralization, as well as the effect of inattentive surplus enamel-etching by a self-etching primer (SEP). Materials and methods: The sealants ProSeal and Clinpro and the varnishes Protecto and FluorProtector were investigated. For inattentive surplus enamel-etching, Transbond Plus SEP was used. The teeth ( N = 75) underwent a pH-cycling for 4 weeks and were examined by weekly consecutive μCT scans ( t1- t4) to determine mineral loss (Δ Z) and lesion depth (Ld). At t4, we also assessed the fluorescence change (Δ F). Results: For ProSeal, no lesions could be detected. In contrast, we found isolated lesions in the area treated with Clinpro. Teeth with inattentive surplus enamel-etching showed always a higher Δ Z. However, this was not statistically significantly different compared to the teeth treated with the varnishes. The adjacent untreated enamel (except the SEP-treated teeth) always showed significantly more demineralization than any of the treated areas. The Δ F partially confirmed these results. Conclusion: No lesions were shown in the area of application of ProSeal. The other materials did not sufficiently protect the enamel; however, a protective effect of all materials was obvious when comparing the bracket-periphery with the adjacent untreated enamel. Additionally, the area of SEP application showed almost always a significantly less demineralization in comparison to that found on the adjacent untreated enamel. Clinical relevance: The bracket-periphery was not always sufficiently protected. The adjacent untreated enamel did not benefit from the bracket-periphery treatment. [ABSTRACT FROM AUTHOR]

Intestinal epithelial electrolyte secretion is activated by increase in intracellular cAMP or Ca and opening of apical Cl channels. In infants and young animals, but not in adults, Ca-activated chloride channels may cause secretory diarrhea during rotavirus infection. While detailed knowledge exists concerning the contribution of cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) channels, analysis of the role of Ca-dependent Cl channels became possible through identification of the anoctamin (TMEM16) family of proteins. We demonstrate expression of several anoctamin paralogues in mouse small and large intestines. Using intestinal-specific mouse knockout models for anoctamin 1 (Ano1) and anoctamin 10 (Ano10) and a conventional knockout model for anoctamin 6 (Ano6), we demonstrate the role of anoctamins for Ca-dependent Cl secretion induced by the muscarinic agonist carbachol (CCH). Ano1 is preferentially expressed in the ileum and large intestine, where it supports Ca-activated Cl secretion. In contrast, Ano10 is essential for Ca-dependent Cl secretion in jejunum, where expression of Ano1 was not detected. Although broadly expressed, Ano6 has no role in intestinal cholinergic Cl secretion. Ano1 is located in a basolateral compartment/membrane rather than in the apical membrane, where it supports CCH-induced Ca increase, while the essential and possibly only apical Cl channel is CFTR. These results define a new role of Ano1 for intestinal Ca-dependent Cl secretion and demonstrate for the first time a contribution of Ano10 to intestinal transport. [ABSTRACT FROM AUTHOR]

Objectives: The aim of this in vitro study was to consecutively determine the effect of three bonding agents on the prevention of enamel demineralisation at the bracket-periphery and to compare the suitability of micro-computed tomography (μCT) scans and quantitative light-induced fluorescence (QLF) to detect changes within subsurface lesions. Materials and methods: The effect of a resin-modified glass ionomer cement (RMGI) (Fuji Ortho LC), a compomer (Assure) and a composite (Transbond XT) on the prevention of enamel demineralisation at the bracket-periphery was examined. After 7, 14, 21 and 28 days of pH cycling, the teeth ( N = 45) were examined by consecutive μCT scans and by using a customised QLF set-up. Results: Particularly for the RMGI and for the compomer, the QLF and μCT scans showed that the formation and the body of the lesion were not precisely located at the enamel next to the bracket margin. There was an area that was almost protected. The progression of demineralisation was decreased for the RMGI and the compomer-treated teeth. Conclusion: For bonding orthodontic brackets, the RMGI and compomer were comparably able to decrease the progression of white spot lesions (WSL), although the RMGI showed marginally superior protection. Both methods (QLF and μCT scans) were suitable for investigating the longitudinal fluoride effects on WSL, though these effects were more accurately described by mineral (fluorescence) loss or volume changes than by lesion depth. Clinical relevance: The progression of WSL at the bracket-periphery could be altered by using fluoride-releasing bonding agents for bracket application. This approach represents a minimally invasive preventive measure. [ABSTRACT FROM AUTHOR]

Anoctamin 6 (Ano6; TMEM16F gene) is a ubiquitous protein; the expression of which is defective in patients with Scott syndrome, an inherited bleeding disorder based on defective scrambling of plasma membrane phospholipids. For Ano6, quite diverse functions have been described: (1) it can form an outwardly rectifying, Ca-dependent and a volume-regulated Cl channel; (2) it was claimed to be a Ca-regulated nonselective cation channel permeable for Ca; (3) it was shown to be essential for Ca-mediated scrambling of membrane phospholipids; and (4) it can regulate cell blebbing and microparticle shedding. Deficiency of Ano6 in blood cells from Scott patients or Ano6 null mice appears to affect all of these cell responses. Furthermore, Ano6 deficiency in mice impairs the mineralization of osteoblasts, resulting in reduced skeletal development. These diverse results have been obtained under different experimental conditions, which may explain some of the contradictions. This review therefore aims to summarize the currently available information on the diverse roles of Ano6 and tries to clear up some of the existing controversies. [ABSTRACT FROM AUTHOR]

Objective: The aim of this in vitro study was to compare fluorescence-aided caries excavation with conventional excavation based on the Martens and Vickers hardness of dentin at the cavity floor after caries removal. Materials and methods: In total, 20 extracted human teeth with dentin caries were bisected through the lesion center into two halves, which were assigned to either the fluorescence-aided caries excavation group or the conventional excavation group. After the treatment, embedding, mounting, and polishing, a line of indentations from the dental pulp across the sound dentin to the cavity floor was made on each sample. The data were compared with Student's t and Mann-Whitney U tests. Results: The calculated Vickers hardness of the sound dentin was 57 ± 10 kg/mm in the fluorescence-aided caries excavation group and 59 ± 8 kg/mm in the conventional excavation group, which is consistent with the previous studies. The absolute and relative Martens hardness measurements of the cavity floor were 224 ± 93 N/mm and 46 ± 17 %, respectively, in the fluorescence-aided caries excavation group and 412 ± 75 N/mm and 81 ± 14 %, respectively, in the conventional excavation group. Based on either the Martens or Vickers hardness, both the absolute and relative microhardness measurements of the cavity floor after fluorescence-aided caries excavation were significantly lower than the values obtained by conventional excavation. Conclusion: Fluorescence-aided caries excavation showed the tissue-preserving property and was more conservative than the conventional excavation in this in vitro study. [ABSTRACT FROM AUTHOR]

SLC26A11 (human)/Slc26a11 (mouse), also known as kidney brain anion transporter (KBAT), is a member of the SLC26 anion transporter family and shows abundant mRNA expression in the brain. However, its exact cellular distribution and subcellular localization in the brain and its functional identity and possible physiological roles remain unknown. Expression and immunostaining studies demonstrated that Slc26a11 is abundantly expressed in the cerebellum, with a predominant expression in Purkinje cells. Lower expression levels were detected in hippocampus, olfactory bulb, cerebral cortex, and subcortical structures. Patch clamp studies in HEK293 cells transfected with mouse cDNA demonstrated that Slc26a11 can function as a chloride channel that is active under basal conditions and is not regulated by calcium, forskolin, or co-expression with cystic fibrosis transmembrane regulator. Single and double immunofluorescent labeling studies demonstrated the localization of vacuolar (V) H +-ATPase and Slc26a11 (KBAT) in the plasma membrane in Purkinje cells. Functional studies in HEK293 cells indicated that transfection with Slc26a11 stimulated acid transport via endogenous V H +-ATPase. We conclude that Slc26a11 (KBAT) is prominently distributed in output neurons of various subcortical and cortical structures in the central nervous system, with specific expression in Purkinje cells and that it may operate as a chloride channel regulating acid translocation by H +-ATPase across the plasma membrane and in intracellular compartments. [ABSTRACT FROM AUTHOR]

To gain more insight into the consequences of curing contraction within the tooth cavity, we assessed the margin behavior of 12 contemporary restorative systems in class V restorations with margins located on enamel and dentin after mechanical loading and water storage. Mixed class V cavities were prepared on extracted human molars and restored using five etch and rinse and seven self-etch adhesive systems with their corresponding composites. Marginal adaptation was evaluated by using a computer-assisted quantitative marginal analysis in a scanning electron microscope (SEM) on epoxy replicas before, after thermal and mechanical stressing and after 1 year of water storage. The interactions of 'testing conditions', 'adhesive-composite combination' and 'tooth substrate' with 'marginal adaptation' were evaluated by two-way ANOVA. Fatigue, stress and storage conditions had significant effects on the marginal adaptation. Only two groups (Optibond FL and G Bond) presented equal percentages of marginal adaptation on enamel and dentin; in the other groups, the rate of degradation was product dependent. All materials tested showed a distinct behavior on enamel and dentin. In addition to mechanical resistance and long-term stability, differences within materials also exist in their ability to simultaneously bond to enamel and dentin. [ABSTRACT FROM AUTHOR]

Cystic fibrosis lung disease is caused by reduced Cl secretion along with enhanced Na absorption, leading to reduced airway surface liquid and compromised mucociliary clearance. Therapeutic strategies have been developed to activate cystic fibrosis transmembrane conductance regulator (CFTR) or to overcome enhanced Na absorption by the epithelial Na channel (ENaC). In a split-ubiquitin-based two-hybrid screening, we identified stress-associated ER protein 1 (SERP1)/ribosome-associated membrane protein 4 as a novel interacting partner for the ENaC β-subunit. SERP1 is induced during cell stress and interacts with the molecular chaperone calnexin, thus controlling early biogenesis of membrane proteins. ENaC activity was measured in the human airway epithelial cell lines H441 and A549 and in voltage clamp experiments with ENaC-overexpressing Xenopus oocytes. We found that expression of SERP1 strongly inhibits amiloride-sensitive Na transport. SERP1 coimmunoprecipitated and colocalized with βENaC in the endoplasmic reticulum, together with the chaperone calnexin. In contrast to the inhibitory effects on ENaC, SERP1 appears to promote expression of CFTR. Taken together, SERP1 is a novel cochaperone and regulator of ENaC expression. [ABSTRACT FROM AUTHOR]

The cystic fibrosis transmembrane conductance regulator (CFTR) produces a cyclic adenosine monophosphate (cAMP)-dependent Cl conductance of distinct properties that is essential for electrolyte secretion in human epithelial tissues. However, the functional consequences of CFTR expression are multifaceted, encompassing much more than simply supplying a cellular cAMP-regulated Cl conductance. When we expressed CFTR in Xenopus oocytes, we found that extracellular acidic pH activates a Ca-dependent outwardly rectifying Cl conductance that does not reflect CFTR activity. The proton-activated Cl conductance showed biophysical and pharmacological features of a Ca-dependent Cl conductance, most likely mediated by Xenopus TMEM16A. In contrast to the effects of extracellular acidification, intracellular acidification did not activate an endogenous Cl conductance. Proton/CFTR-mediated activation of human TMEM16A was also detected in HEK293 cells. The gating mutant G551D-CFTR conferred proton sensitivity, while deltaF508-CFTR enabled proton activation of TMEM16A only in Xenopus oocytes, which, unlike HEK293 cells, allow deltaF508-CFTR to be trafficked to the cell membrane. Activation of TMEM16A by lysophosphatidic acid was enhanced in the presence of CFTR but was additive with activation by extracellular protons. Because expression of CFTR-E1474X did not confer proton sensitivity, we propose that CFTR translocates a proton receptor to the plasma membrane via its PDZ-binding domain. [ABSTRACT FROM AUTHOR]

Endogenous Ca-activated Cl channels (CaCC) demonstrate biophysical and pharmacological properties that are well represented in cells overexpressing anoctamin 1 (Ano 1, TMEM16A), a protein that has been identified recently as CaCC. Proteins of the anoctamin family (anoctamin 1-10, TMEM16A-K) are widely expressed. The number of reports demonstrating their physiological and clinical relevance is quickly rising. Anoctamins gain additional interest through their potential role in cell volume regulation and malignancy. Available data suggest that Ano 1 forms stable dimers and probably liaise with accessory proteins such as calmodulin or other anoctamins. In order to understand how anoctamins produce Ca-activated Cl currents, it will be necessary to obtain better insight into their molecular structure, interactions with partner proteins, and mode of activation. [ABSTRACT FROM AUTHOR]

Rotavirus infection is the most frequent cause for severe diarrhea in infants, killing more than 600,000 every year. The nonstructural protein NSP4 acts as a rotavirus enterotoxin, inducing secretory diarrhea without any structural organ damage. Electrolyte transport was assessed in the colonic epithelium from pups and adult mice using Ussing chamber recordings. Western blots and immunocytochemistry was performed in intestinal tissues from wild-type and TMEM16A knockout mice. Ion channel currents were recorded using patch clamp techniques. We show that the synthetic NSP4 peptide uses multiple pro-secretory pathways to induce diarrhea, by activating the recently identified Ca-activated Cl channel TMEM16A, and by inhibiting Na absorption by the epithelial Na channel ENaC and the Na/glucose cotransporter SGLT1. Activation of secretion and inhibition of Na absorption by NSP4, respectively, could be potently suppressed by wheat germ agglutinin which probably competes with NSP4 for binding to an unknown glycolipid receptor. The present paper gives a clue as to mechanisms of rotavirus-induced diarrhea and suggests wheat germ agglutinin as a simple and effective therapy. [ABSTRACT FROM AUTHOR]

Bestrophins form Ca2+-activated Cl− channels and regulate intracellular Ca2+ signaling. We demonstrate that bestrophin 1 is localized in the endoplasmic reticulum (ER), where it interacts with stromal interacting molecule 1, the ER-Ca2+ sensor. Intracellular Ca2+ transients elicited by stimulation of purinergic P2Y2 receptors in HEK293 cells were augmented by hBest1. The p21-activated protein kinase Pak2 was found to phosphorylate hBest1, thereby enhancing Ca2+ signaling and activation of Ca2+-dependent Cl− (TMEM16A) and K+ (SK4) channels. Lack of bestrophin 1 expression in respiratory epithelial cells of mBest1 knockout mice caused expansion of ER cisterns and induced Ca2+ deposits. hBest1 is, therefore, important for Ca2+ handling of the ER store and may resemble the long-suspected counterion channel to balance transient membrane potentials occurring through inositol triphosphate (IP3)-induced Ca2+ release and store refill. Thus, bestrophin 1 regulates compartmentalized Ca2+ signaling that plays an essential role in Best macular dystrophy, inflammatory diseases such as cystic fibrosis, as well as proliferation. [ABSTRACT FROM AUTHOR]

The metabolic sensor adenosine-monophosphate-activated kinase (AMPK) detects the cellular energy status and adjusts metabolic activity according to the cytosolic AMP to ATP ratio. Na+ absorption by epithelial Na+ channels (ENaC) is a highly energy-consuming process that is inhibited by AMPK. We show that the catalytic subunit α1 of AMPK inhibits ENaC in epithelial tissues from airways, kidney, and colon and that AMPK regulation of ENaC is absent in AMPKα1−/− mice. These mice demonstrate enhanced electrogenic Na+ absorption that leads to subtle changes in intestinal and renal function and may also affect Na+ absorption and mucociliary clearance in the airways. We demonstrate that AMPK uses the ubiquitin ligase Nedd4-2 to inhibit ENaC by increasing ubiquitination and endocytosis of ENaC. Thus, enhanced expression of epithelial Na+ channels was detected in colon, airways, and kidney of AMPKα1−/− mice. Therefore, AMPKα1 is a physiologically important regulator of electrogenic Na+ absorption and may provide a novel pharmacological target for controlling epithelial Na+ transport. [ABSTRACT FROM AUTHOR]

Proteins of the bestrophin family produce Ca2+-activated Cl− currents and regulate voltage-gated Ca2+ channels. Bestrophin 1 was first identified in the retinal pigment epithelium. Four human paralogs (hBest1–hBest4) exist, and for some bestrophins, dimeric and heterotetrameric structures have been proposed. Here, we demonstrate that hBest1–hBest4 induce Cl− conductances of different amplitudes when expressed in HEK293 cells and when activated through purinergic stimulation. hBest1 mutants that are known to cause autosomal dominant macular dystrophy (Best disease) did not produce a Cl− current. Bestrophins were colocalized and showed molecular and functional interaction in HEK293 cells, overexpressing hBest1 and hBest2 or hBest4. Interaction was confirmed in airway epithelial cells coexpressing endogenous bestrophins. A fraction of hBest2 and hBest4 was expressed in the membrane, while most of hBest1 was found in the endoplasmic reticulum. Nevertheless, hBest1 has a clear role for the adenosine triphosphate (ATP; or uridine triphosphate)-induced Cl− current in both HEK293 and Calu-3 cells. Since native epithelial tissues typically express several bestrophin paralogs, these proteins may exist as heterooligomeric structures. [ABSTRACT FROM AUTHOR]

It has been reported that the cystic fibrosis transmembrane conductance regulator (CFTR) can be activated through cAMP- and protein kinase A-independent pathways involving GTP-binding proteins and an unknown kinase. In this study, we further examined how G protein-coupled pathways regulate CFTR. We demonstrate that stimulation of purinergic P2Y2 receptors in CFTR-expressing oocytes and in airway epithelial cells activates CFTR Cl− currents. Activation of CFTR Cl− currents via P2Y2 was inhibited by CFTRinh-172 and was independent of intracellular Ca2+, protein kinase C, or calmodulin-dependent kinase (CAMK). However, activation of CFTR was suppressed by inhibition of phospholipase C and by the nonselective protein kinase inhibitor staurosporine. Activation of CFTR through P2Y2 receptors was enhanced when Gi proteins were inhibited by pertussis toxin. Inhibition of protein kinase A and of protein kinases downstream of P2Y2 receptors such as mitogen-activated protein kinases, tyrosine kinase, or c- src kinase did not interfere with activation of CFTR. The present results demonstrate an antagonistic regulation of CFTR by P2Y2 receptors: CFTR is inhibited by stimulation of Gi proteins and is activated by stimulation of Gq/11/PLC and an unknown downstream protein kinase. [ABSTRACT FROM AUTHOR]

Previous in vitro studies suggested that Cl− currents produced by the cystic fibrosis transmembrane conductance regulator (CFTR; ABCC7) are inhibited by the α1 isoform of the adenosine monophosphate (AMP)-stimulated kinase (AMPK). AMPK is a serine/threonine kinase that is activated during metabolic stress. It has been proposed as a potential mediator for transport–metabolism coupling in epithelial tissues. All previous studies have been performed in vitro and thus little is known about the regulation of Cl− secretion by AMPK in vivo. Using AMPKα1−/− mice and wild-type littermates, we demonstrate that phenformin, an activator of AMPK, strongly inhibits cAMP-activated Cl− secretion in mouse airways and colon, when examined in ex vivo in Ussing chamber recordings. However, phenformin was equally effective in AMPKα1−/− and wild-type animals, suggesting additional AMPK-independent action of phenformin. Phenformin inhibited CFTR Cl− conductance in basolaterally permeabilized colonic epithelium from AMPKα1+/+ but not AMPKα1−/− mice. The inhibitor of AMPK compound C enhanced CFTR-mediated Cl− secretion in epithelial tissues of AMPKα1−/− mice, but not in wild-type littermates. There was no effect on Ca2+-mediated Cl− secretion, activated by adenosine triphosphate or carbachol. Moreover CFTR-dependent Cl− secretion was enhanced in the colon of AMPKα1−/− mice, as indicated in Ussing chamber ex vivo and rectal PD measurements in vivo. Taken together, these data suggest that epithelial Cl− secretion mediated by CFTR is controlled by AMPK in vivo. [ABSTRACT FROM AUTHOR]

Adenosine is formed from extracellular purines by ecto-5′-nucleotidase (CD73) and is an essential player in allergic airway inflammation. The contribution of adenosine and other purines to electrolyte transport and mucociliary clearance was studied in airways of allergen challenged mice. No signs for allergen-induced inflammation were found in CD73−/− mice, and adenosine monophosphate (AMP) was unable to elicit airway Cl− secretion in these animals. Tracheas of ovalbumin (OVA)-treated BALB/c and CD73+/+ mice were hyperresponsive towards methacholine when assessed by Penh and direct optical measurement of contraction. In addition Cl− secretion activated by ATP and ADP was enhanced. These changes were not observed in CD73−/− mice. Expression of CFTR or CLCA was unchanged upon OVA treatment of CD73 mice, suggesting enhanced Cl− secretion due to upregulated purinergic pathways. Mucociliary clearance was determined by measuring particle transport in excised mouse tracheas and was strongly enhanced in OVA-challenged CD73+/+ mice, but remained unchanged in CD73−/− mice. While mucociliary clearance is activated by allergen exposure independent of functional ecto-5′-nucleotidase, airway inflammation is largely dependent on CD73. Thus, ecto-5′-nucleotidase may provide a novel target for therapeutic intervention, probably by local application of ecto-5′-nucleotidase inhibitors through inhalation. [ABSTRACT FROM AUTHOR]