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 inhibitors of the cystic fibrosis transmembrane conductance regulator exert off-target effects on epithelial cation channels

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

Nanostructured amorphous gallium phosphide on silica for nonlinear and ultrafast nanophotonics

Nanophotonics based on high refractive index dielectrics relies on appreciable contrast between the indices of designed nanostructures and their immediate surrounding, which can be achieved by the growth of thin films on low-index substrates. Here we propose the use of high index amorphous gallium phosphide (a-GaP), fabricated by radio-frequency sputter deposition, on top of a low refractive index glass substrate and thoroughly examine its nanophotonic properties. Spectral ellipsometry of the amorphous material demonstrates the optical properties to be considerably close to crystalline gallium phosphide (c-GaP), with low-loss transparency for wavelengths longer than 650 nm. When nanostructured into nanopatches, the second harmonic (SH) response of an individual a-GaP patch is characterized to be more than two orders of magnitude larger than the as-deposited unstructured film, with an anapole-like resonant behavior. Numerical simulations are in good agreement with the experimental results over a large spectral and geometrical range. Furthermore, by studying individual a-GaP nanopatches through non-degenerate pump-probe spectroscopy with sub-10 fs pulses, we find a more than 5% ultrafast modulation of the reflectivity that is accompanied by a slower decaying free carrier contribution, caused by absorption. Our investigations reveal a potential for a-GaP as an adequate inexpensive and CMOS-compatible material for nonlinear nanophotonic applications as well as for photocatalysis.Fil: Tilmann, Benjamin. Ludwig Maximilians Universitat; AlemaniaFil: Grinblat, Gustavo Sergio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Berté, Rodrigo. Ludwig Maximilians Universitat; AlemaniaFil: Özcan, Mehmet. Ludwig Maximilians Universitat; AlemaniaFil: Kunzelmann, Viktoria F.. Technische Universitat München; AlemaniaFil: Nickel, Bert. Ludwig Maximilians Universitat; AlemaniaFil: Sharp, Ian D.. Ludwig Maximilians Universitat; AlemaniaFil: Cortés, Emiliano. Ludwig Maximilians Universitat; AlemaniaFil: Maier, Stefan A.. Ludwig Maximilians Universitat; AlemaniaFil: Li, Yi. Southern University Of Science And Technology; Chin

Platform-directed allostery and quaternary structure dynamics of SAMHD1 catalysis

SAMHD1 regulates cellular nucleotide homeostasis, controlling dNTP levels by catalysing their hydrolysis into 2’-deoxynucleosides and triphosphate. In differentiated CD4+ macrophage and resting T-cells SAMHD1 activity results in the inhibition of HIV-1 infection through a dNTP blockade. In cancer, SAMHD1 desensitizes cells to nucleoside-analogue chemotherapies. Here we employ time-resolved cryogenic-EM imaging and single-particle analysis to visualise assembly, allostery and catalysis by this multi-subunit enzyme. Our observations reveal how dynamic conformational changes in the SAMHD1 quaternary structure drive the catalytic cycle. We capture five states at high-resolution in a live catalytic reaction, revealing how allosteric activators support assembly of a stable SAMHD1 tetrameric core and how catalysis is driven by the opening and closing of active sites through pairwise coupling of active sites and order-disorder transitions in regulatory domains. This direct visualisation of enzyme catalysis dynamics within an allostery-stabilised platform sets a precedent for mechanistic studies into the regulation of multi-subunit enzymes

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

Measurements of CFTR-Mediated Cl- Secretion in Human Rectal Biopsies Constitute a Robust Biomarker for Cystic Fibrosis Diagnosis and Prognosis

BACKGROUND: Cystic Fibrosis (CF) is caused by ∼1,900 mutations in the CF transmembrane conductance regulator (CFTR) gene encoding for a cAMP-regulated chloride (Cl(-)) channel expressed in several epithelia. Clinical features are dominated by respiratory symptoms, but there is variable organ involvement thus causing diagnostic dilemmas, especially for non-classic cases. METHODOLOGY/PRINCIPAL FINDINGS: To further establish measurement of CFTR function as a sensitive and robust biomarker for diagnosis and prognosis of CF, we herein assessed cholinergic and cAMP-CFTR-mediated Cl(-) secretion in 524 freshly excised rectal biopsies from 118 individuals, including patients with confirmed CF clinical diagnosis (n=51), individuals with clinical CF suspicion (n=49) and age-matched non-CF controls (n=18). Conclusive measurements were obtained for 96% of cases. Patients with "Classic CF", presenting earlier onset of symptoms, pancreatic insufficiency, severe lung disease and low Shwachman-Kulczycki scores were found to lack CFTR-mediated Cl(-) secretion (<5%). Individuals with milder CF disease presented residual CFTR-mediated Cl(-) secretion (10-57%) and non-CF controls show CFTR-mediated Cl(-) secretion ≥ 30-35% and data evidenced good correlations with various clinical parameters. Finally, comparison of these values with those in "CF suspicion" individuals allowed to confirm CF in 16/49 individuals (33%) and exclude it in 28/49 (57%). Statistical discriminant analyses showed that colonic measurements of CFTR-mediated Cl(-) secretion are the best discriminator among Classic/Non-Classic CF and non-CF groups. CONCLUSIONS/SIGNIFICANCE: Determination of CFTR-mediated Cl(-) secretion in rectal biopsies is demonstrated here to be a sensitive, reproducible and robust predictive biomarker for the diagnosis and prognosis of CF. The method also has very high potential for (pre-)clinical trials of CFTR-modulator therapies.This work was supported by grants TargetScreen2 (EU/FP6/LSH/2005/037365), PIC/IC/83103/2007; PTDC/MAT/118335/2010; PEstOE/BIA/UI4046/2011 (to BioFIG) and PEstOE/MAT/UI0006/2011 (to CEAUL) from FCT (Portugal); and FAPESP (SPRF, Brazil), CNPq (40.8924/2006/3, Brazil) and Mukoviszidose e.V. S02/10 (Germany). MS and IU are recipients of SFRH/BD/35936/2007 and SFRH/BD/69180/2010 PhD fellowships (FCT, Portugal), respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript