Distribution and Excretion of BisGMA in Guinea Pigs

Bisphenol-A-glycidyldimethacrylate (BisGMA) is used in many resin-based dental materials. It was shown in vitro that BisGMA was released into the adjacent biophase from such materials during the first days after placement. In this study, the uptake, distribution, and excretion of [14C]BisGMA applied via gastric and intravenous administration (at dose levels well above those encountered in dental care) were examined in vivo in guinea pigs to test the hypothesis that BisGMA reaches cytotoxic levels in mammalian tissues. [14C]BisGMA was taken up rapidly from the stomach and intestine after gastric administration and was widely distributed in the body following administration by each route. Most [14C] was excreted within one day as 14CO2. The peak equivalent BisGMA levels in guinea pig tissues examined were at least 1000-fold less than known toxic levels. The peak urine level in guinea pigs that received well in excess of the body-weightadjusted dose expected in humans was also below known toxic levels. The study therefore did not support the hypothesis

Transillumination and HDR Imaging for Proximal Caries Detection

The purpose of this study was to develop an in vitro model for the validation of near-infrared transillumination (NIRT) for proximal caries detection, to enhance NIRT with high-dynamic-range imaging (HDRI), and to compare both methods, using micro-computed tomography (mu CT) as a reference standard. Both proximal surfaces of 53 healthy or decayed permanent human teeth were examined using the Diagnocam (DC) (KaVo) and NIRT with HDRI (NIRT-HDRI). NIRT was combined with HDRI to improve the diagnostic performance by reducing under- and overexposed image areas. For NIRT-HDRI, an exposure series was captured and merged into a single HDR image. A classification was applied according to lesion depth. All surfaces were assessed twice by 2 trained examiners, and additionally with mu CT for validation. The Kappa statistic was used to calculate inter-rater reliability and agreement between DC and NIRT-HDRI. Inter-rater reliability (weighted Kappa, w) showed very good agreement for the DC (0.90) and NIRT-HDRI (0.96). The overall agreement (w) was almost perfect (0.85). In the individual categories (0 to 4), the agreement (simple Kappa) ranged from almost perfect (category 4) to moderate (1 and 2) to substantial (categories 0 and 3). Sensitivity and specificity of sound surfaces, enamel, and dentin caries ranged from 0.57 to 0.99 and were similar for both methods in the different categories. NIRT-HDRI had a higher sensitivity for sound surfaces and enamel caries, as well as a higher specificity for dentin caries. Regarding the obtained images, HDRI allowed for the detection of caries within a greater range of luminance levels, resulting in a more detailed visualization of structures without under- or overexposure. However, HDRI this did not improve the diagnostics significantly. Distinguishing between a processed demineralized enamel and dentin lesions appears to be a problem specific to NIRT and cannot be balanced using HDRI

Distribution and Excretion of TEGDMA in Guinea Pigs and Mice

The monomer triethyleneglycoldimethacrylate (TEGDMA) is used as a diluent in many resin-based dental materials. It was previously shown in vitro that TEGDMA was released into the adjacent biophase from such materials during the first days after placement. In this study, the uptake, distribution, and excretion of 14C-TEGDMA applied via gastric, intradermal, and intravenous administration at dose levels well above those encountered in dental care were examined in vivo in guinea pigs and mice as a test of the hypothesis that TEGDMA reaches cytotoxic levels in mammalian tissues. 14C-TEGDMA was taken up rapidly from the stomach and small intestine after gastric administration in both species and was widely distributed in the body following administration by each route. Most 14C was excreted within one day as 14 CO2. The peak equivalent TEGDMA levels in all mouse and guinea pig tissues examined were at least 1000-fold less than known toxic levels. The study therefore did not support the hypothesis

Pharmacological Inhibition and Activation of the Ca2+ Activated Cl− Channel TMEM16A

TMEM16A is a Ca2+ activated Cl- channel with important functions in airways, intestine, and other epithelial organs. Activation of TMEM16A is proposed as a therapy in cystic fibrosis (CF) to reinstall airway Cl- secretion and to enhance airway surface liquid (ASL). This CFTR-agnostic approach is thought to improve mucociliary clearance and lung function in CF. This could indeed improve ASL, however, mucus release and airway contraction may also be induced by activators of TMEM16A, particularly in inflamed airways of patients with asthma, COPD, or CF. Currently, both activators and inhibitors of TMEM16A are developed and examined in different types of tissues. Here we compare activation and inhibition of endogenous and overexpressed TMEM16A and analyze potential off-target effects. The three well-known blockers benzbromarone, niclosamide, and Ani9 inhibited both TMEM16A and ATP-induced Ca2+ increase by variable degrees, depending on the cell type. Niclosamide, while blocking Ca2+ activated TMEM16A, also induced a subtle but significant Ca2+ store release and inhibited store-operated Ca2+ influx. Niclosamide, benzbromarone and Ani9 also affected TMEM16F whole cell currents, indicating limited specificity for these inhibitors. The compounds Eact, cinnamaldehyde, and melittin, as well as the phosphatidylinositol diC8-PIP2 are the reported activators of TMEM16A. However, the compounds were unable to activate endogenous TMEM16A in HT29 colonic epithelial cells. In contrast, TMEM16A overexpressed in HEK293 cells was potently stimulated by these activators. We speculate that overexpressed TMEM16A might have a better accessibility to intracellular Ca2+, which causes spontaneous activity even at basal intracellular Ca2+ concentrations. Small molecules may therefore potentiate pre-stimulated TMEM16A currents, but may otherwise fail to activate silent endogenous TMEM16A

Regulation and Function of TMEM16F in Renal Podocytes

The Ca2+-activated phospholipid scramblase and ion channel TMEM16F is expressed in podocytes of renal glomeruli. Podocytes are specialized cells that form interdigitating foot processes as an essential component of the glomerular filter. These cells, which participate in generation of the primary urine, are often affected during primary glomerular diseases, such as glomerulonephritis and secondary hypertensive or diabetic nephropathy, which always leads to proteinuria. Because the function of podocytes is known to be controlled by intracellular Ca2+ signaling, it is important to know about the role of Ca2+-activated TMEM16F in these cells. To that end, we generated an inducible TMEM16F knockdown in the podocyte cell line AB8, and produced a conditional mouse model with knockout of TMEM16F in podocytes and renal epithelial cells of the nephron. We found that knockdown of TMEM16F did not produce proteinuria or any obvious phenotypic changes. Knockdown of TMEM16F affected cell death of tubular epithelial cells but not of glomerular podocytes when analyzed in TUNEL assays. Surprisingly, and in contrast to other cell types, TMEM16F did not control intracellular Ca2+ signaling and was not responsible for Ca2+-activated whole cell currents in podocytes. TMEM16F levels in podocytes were enhanced after inhibition of the endolysosomal pathway and after treatment with angiotensin II. Renal knockout of TMEM16F did not compromise renal morphology and serum electrolytes. Taken together, in contrast to other cell types, such as platelets, bone cells, and immune cells, TMEM16F shows little effect on basal properties of podocytes and does not appear to be essential for renal function

Bestrophin 1 is indispensable for volume regulation in human retinal pigment epithelium cells

In response to cell swelling, volume-regulated anion channels (VRACs) participate in a process known as regulatory volume decrease (RVD). Only recently, first insight into the molecular identity of mammalian VRACs was obtained by the discovery of the leucine-rich repeats containing 8A (LRRC8A) gene. Here, we show that bestrophin 1 (BEST1) but not LRRC8A is crucial for volume regulation in human retinal pigment epithelium (RPE) cells. Whole-cell patch-clamp recordings in RPE derived from human-induced pluripotent stem cells (hiPSC) exhibit an outwardly rectifying chloride current with characteristic functional properties of VRACs. This current is severely reduced in hiPSC-RPE cells derived from macular dystrophy patients with pathologic BEST1 mutations. Disruption of the orthologous mouse gene (Best1−/−) does not result in obvious retinal pathology but leads to a severe subfertility phenotype in agreement with minor endogenous expression of Best1 in murine RPE but highly abundant expression in mouse testis. Sperm from Best1−/− mice showed reduced motility and abnormal sperm morphology, indicating an inability in RVD. Together, our data suggest that the molecular identity of VRACs is more complex—that is, instead of a single ubiquitous channel, VRACs could be formed by cell type- or tissue-specific subunit composition. Our findings provide the basis to further examine VRAC diversity in normal and diseased cell physiology, which is key to exploring novel therapeutic approaches in VRAC-associated pathologies