Martin Hardt

Effect of Fluoride Compounds on Enamel Erosion in vitro: A Comparison of Amine, Sodium and Stannous Fluoride

The aim of the study was to evaluate the relevance of cations in different fluoride compounds for their effectiveness as anti-erosive agents. Human enamel samples underwent a de- and re-mineralisation procedure for 10 days. Erosive demineralisation was performed with 0.05 Mcitric acid (pH 2.3) 6 × 2 min daily followed by immersion in the test solution 6 × 2 min each. Test solutions were: SnCl2 (815 ppm Sn; pH 2.6), NaF (250 ppm F; pH 3.5), SnF2 (250 ppm F, 809 ppm Sn; pH 3.5), amine fluoride (AmF, 250 ppm F; pH 3.5), AmF/NaF (250 ppm F; pH 4.3), and AmF/SnF2 (250 ppm F, 390 ppm Sn; pH 4.2). In the control group no fluoridation was performed. Mineral content was monitored by longitudinal microradiography. Finally, scanning electron microscopy was performed. The highest erosive mineral loss was found in the control group (48.0 ± 17.1 µm). Mineral loss was nearly completely inhibited by AmF/SnF2 (5.7 ± 25.1 µm; p ≤ 0.001) and SnF2 (–3.8 ± 14.4 µm; p ≤ 0.001) treatments. Groups treated with SnCl2 (17.6 ± 19.5 µm; p ≤ 0.001) and NaF (13.2 ± 21.7 µm; p ≤ 0.001) showed a decrease in erosive mineral loss, AmF (41.6 ± 16.0 µm) and AmF/NaF (27.7 ± 28.4 µm) had no significant effect on erosion progression. The results indicate considerable differences between the fluoride compounds tested. Treatment with solutions containing SnF2 was most effective.

Tin-containing fluoride solutions as anti-erosive agents in enamel: an in vitro tin-uptake, tissue-loss, and scanning electron micrograph study

Tin-containing fluoride solutions can reduce erosive tissue loss, but the effects of the reaction between tin and enamel are still not clear. During a 10-d period, enamel specimens were cyclically demineralized (0.05 M citric acid, pH 2.3, 6 x 5 min d(-1)) and remineralized (between the demineralization cycles and overnight). In the negative-control group, no further treatment was performed. Three groups were treated (2 x 2 min d(-1)) with tin-containing fluoride solutions (400, 1,400 or 2,100 ppm Sn2+, all 1,500 ppm F-, pH 4.5). Three additional groups were treated with test solutions twice daily, but without demineralization. Tissue loss was determined profilometrically. Energy-dispersive X-ray spectroscopy was used to measure the tin content on and within three layers (10 mum each) beneath the surface. In addition, scanning electron microscopy was conducted. All test preparations significantly reduced tissue loss. Deposition of tin on surfaces was higher without erosion than with erosion, but no incorporation of tin into enamel was found without demineralization. Under erosive conditions, both highly concentrated solutions led to the incorporation of tin up to a depth of 20 mum; the less-concentrated solution led to small amounts of tin in the outer 10 mum. The efficacy of tin-containing solutions seems to depend mainly on the incorporation of tin into enamel.

Effects of toothbrushing force on the mineral content and demineralized organic matrix of eroded dentine

Eroded dentine has a complex histological structure, and its organic fraction becomes increasingly exposed as a result of the continual action of acids. The present study sought to investigate the effects of brushing forces up to 4 N on mineralized and organic dentine fractions. The study was a cyclic demineralization and remineralization experiment (carried out over 9 d). Erosion was performed with HCl (6 x 2 min d-1), pH 1.6. Samples exposed to erosion alone served as controls; test samples were eroded and brushed with a powered toothbrush (2 x 15 s d-1), applying forces of 2, 3, or 4 N. Samples were analysed (using profilometry and longitudinal microradiography) before and after the removal of superficial organic material with collagenase. Randomly selected samples were subjected to scanning electron microscopy. Demineralized organic material was present on all samples regardless of brushing force. Loss values (determined using profilometry) after erosion only, and after brushing with forces of 2, 3, and 4 N, were 11.7 +/- 5.1, 13.6 +/- 11.2, 30.7 +/- 19.0, and 25.5 +/- 20.3, respectively, before treatment with collagenase, and 111.7 +/- 11.6, 122.0 +/- 11.8, 121.9 +/- 15.7, and 123.0 +/- 12.0, respectively, after treatment with collagenase. Microradiography confirmed the results. Significant effects of brushing force were only found on the demineralized organic fraction, and mineral loss was unaffected. The notion that eroded dentine is particularly prone to abrasion should be reconsidered.

Hepatitis B virus infection is dependent on cholesterol in the viral envelope

The viral and cellular determinants leading to binding and entry of hepatitis B virus (HBV) are still not fully understood. We found that HBV infection of primary hepatocyte cultures is dependent on the presence of cholesterol in the viral envelope. Extraction of cholesterol from HBV purified from plasma of HBV‐infected patients with methyl‐beta‐cyclodextrin (MβCD) leads to a strongly reduced level of infection. The cholesterol‐depleted virions showed higher buoyant density (1.23 versus 1.17 g ml−1), a smaller diameter (39 versus 48 nm), but maintained particle integrity, antigenicity and ability to bind to hepatocytes. Although addition of exogenous cholesterol and cholesterol analogues restored the physical appearance of cholesterol‐depleted virions, infectivity was only regained by cholesterol add‐back. Infectivity of HBV produced from cell culture in the presence of inhibitors of cholesterol‐synthesis is severely impaired. Interestingly, cholesterol extraction from cellular membranes, incubation with filipin and the protein tyrosine kinase inhibitor genistein showed no effect on HBV infection, excluding a role of lipid rafts for the infection process of HBV. In summary, presence of cholesterol within the viral envelope is not important for viral binding, but indispensable for the entry process of HBV and might be important for a later step in viral uptake, e.g. fusion in a yet unknown compartment.

Aphid gel saliva: sheath structure, protein composition and secretory dependence on stylet-tip milieu.

In order to separate and analyze saliva types secreted during stylet propagation and feeding, aphids were fed on artificial diets. Gel saliva was deposited as chains of droplets onto Parafilm membranes covering the diets into which watery saliva was secreted. Saliva compounds collected from the diet fluid were separated by SDS-PAGE, while non-soluble gel saliva deposits were processed in a novel manner prior to protein separation by SDS-PAGE. Soluble (watery saliva) and non-soluble (gel saliva) protein fractions were significantly different. To test the effect of the stylet milieu on saliva secretion, aphids were fed on various diets. Hardening of gel saliva is strongly oxygen-dependent, probably owing to formation of sulfide bridges by oxidation of sulphydryl groups. Surface texture of gel saliva deposits is less pronounced under low-oxygen conditions and disappears in dithiothreitol containing diet. Using diets mimicking sieve-element sap and cell-wall fluid respectively showed that the soluble protein fraction was almost exclusively secreted in sieve elements while non-soluble fraction was preferentially secreted at cell wall conditions. This indicates that aphids are able to adapt salivary secretion in dependence of the stylet milieu.

Mechanism of action of tin-containing fluoride solutions as anti-erosive agents in dentine - an in vitro tin-uptake, tissue loss, and scanning electron microscopy study.

Solutions containing tin and fluoride exhibit remarkable anti-erosive properties with tin ions as a major agent. To elucidate its mechanism of action in dentine, the tin uptake on and in the tissue was investigated and related to histological findings and substance loss. Samples were treated twice daily, each treatment lasting for 2 min, with fluoride solutions [pH 4.5; 1,500 parts per million (p.p.m.) F] containing 2,100, 1,400, or 400 p.p.m. Sn as SnCl(2). In experiments 1 and 2, samples were eroded with citric acid (pH 2.3) six times each day, each treatment lasting for 5 min; in experiment 2, the demineralized organic matrix was continuously digested by collagenase; in experiment 3, no erosive challenges were performed. Sample surfaces and cross-sections were investigated using energy dispersive X-ray spectroscopy, scanning electron microscopy, and profilometry. Surface retention of tin was found in almost all treatment groups and was highest in experiment 2. On cross-sections, tin was retained within the organic matrix; in mineralized areas, tin was found mainly within a depth of 10 mum. Test solutions inhibited substance loss significantly; in experiment 2, the effect was dose-dependent. Erosion inhibition seemed to depend mainly on the incorporation of tin in the mineralized dentine when the organic portion was preserved, but on surface precipitation when the organic portion was continuously digested.

Comparison of calcium analysis, longitudinal microradiography and profilometry for the quantitative assessment of erosion in dentine.

Erosion of dentine causes mineral dissolution, while the organic compounds remain at the surface. Therefore, a determination of tissue loss is complicated. Established quantitative methods for the evaluation of enamel have also been used for dentine, but the suitability of these techniques in this field has not been systematically determined. Therefore, this study aimed to compare longitudinal microradiography (LMR), contacting (cPM) and non-contacting profilometry (ncPM), and analysis of dissolved calcium (Ca analysis) in the erosion solution. Results are discussed in the light of the histology of dentine erosion. Erosion was performed with 0.05 M citric acid (pH 2.5) for 30, 60, 90 or 120 min, and erosive loss was determined by each method. LMR, cPM and ncPM were performed before and after collagenase digestion of the demineralised organic surface layer, with an emphasis on moisture control. Scanning electron microscopy was performed on randomly selected specimens. All measurements were converted into micrometres. Profilometry was not suitable to adequately quantify mineral loss prior to collagenase digestion. After 120 min of erosion, values of 5.4 ± 1.9 μm (ncPM) and 27.8 ± 4.6 μm (cPM) were determined. Ca analysis revealed a mineral loss of 55.4 ± 11.5 μm. The values for profilometry after matrix digestion were 43.0 ± 5.5 μm (ncPM) and 46.9 ± 6.2 (cPM). Relative and proportional biases were detected for all method comparisons. The mineral loss values were below the detection limit for LMR. The study revealed gross differences between methods, particularly when demineralised organic surface tissue was present. These results indicate that the choice of method is critical and depends on the parameter under study.

Influence of the digestive enzymes trypsin and pepsin in vitro on the progression of erosion in dentine

OBJECTIVES In patients with eating disorders, gastric and pancreatic enzymes could possibly reach the oral cavity during vomiting and could perhaps degrade the organic matrix of eroded dentine. This in vitro study sought to investigate whether pepsin, trypsin or the combination of both, have an influence on erosive mineral loss in dentine and whether they are able to degrade the organic matrix. METHODS Sixty-four human dentine specimens were prepared and randomly divided into four groups. Specimens were cyclically de- and remineralised for six days. Demineralisation was performed with an HCl-solution (6x 5min daily, pH 1.6) in groups 1 and 3; in groups 2 and 4 the demineralisation solution additionally contained pepsin (750 microg/ml). After demineralisation, specimens of groups 3 and 4 were treated with a trypsin solution (6x 10min daily, 2000 BAEE/ml). After each day, mineral content (mum) was determined microradiographically, and the matrix degradation was determined by hydroxyproline analysis. RESULTS After six days, treatment with pepsin (group 2) or trypsin (group 3) had no significant influence on mineral loss. The combined impact of pepsin and trypsin led to significantly higher mineral loss (group 4: 202.5+/-37.4) compared to all other groups (group 1: 139.1+/-29.5, p<or=0.001; group 2: 108.8+/-34.7, p<or=0.001; group 3: 157.8+/-37.2, p<or=0.05). Hydroxyproline was found in all pepsin-solutions but in no trypsin- or HCl-solutions. CONCLUSION The combined impact of pepsin and trypsin intensified dentine erosion progression in vitro. This could be one reason for the fast proceeding of dental erosion in patients with chronic vomiting.

Structural and optical characterization of RF reactively sputtered CuInS2 thin films

The ternary compound semiconductor CuInS 2 is one of the most attractive materials for high efficiency solar cells due to its bandgap of 1.55 eV which is well matched to the solar spectrum. We deposit CuInS 2 films on float glass substrates by a reactive RF sputter process using a Cu-In alloy target and H 2 S gas. By optimizing the sputter parameters, such as the sputter power, temperature of the substrate, and the flow of H 2 S, high quality films were obtained. The surface morphology, phase structure and composition of the layers were analyzed by atomic force microscopy (AFM), X-ray diffraction (XRD), energy-dispersive X-ray analysis (EDX) and Rutherford backscattering spectroscopy (RBS), respectively. The electrical properties of the films were characterized by Hall effect measurements, photoluminescence (PL) and transmission measurements were performed to examine the optical properties of the films, and the absorption coefficients and the direct hand gap of the films were evaluated by transmission measurements.

Influenza Virus-Induced Caspase-Dependent Enlargement of Nuclear Pores Promotes Nuclear Export of Viral Ribonucleoprotein Complexes

ABSTRACT Influenza A viruses (IAV) replicate their segmented RNA genome in the nucleus of infected cells and utilize caspase-dependent nucleocytoplasmic export mechanisms to transport newly formed ribonucleoprotein complexes (RNPs) to the site of infectious virion release at the plasma membrane. In this study, we obtained evidence that apoptotic caspase activation in IAV-infected cells is associated with the degradation of the nucleoporin Nup153, an integral subunit of the nuclear pore complex. Transmission electron microscopy studies revealed a distinct enlargement of nuclear pores in IAV-infected cells. Transient expression and subcellular accumulation studies of multimeric marker proteins in virus-infected cells provided additional evidence for increased nuclear pore diameters facilitating the translocation of large protein complexes across the nuclear membrane. Furthermore, caspase 3/7 inhibition data obtained in this study suggest that active, Crm1-dependent IAV RNP export mechanisms are increasingly complemented by passive, caspase-induced export mechanisms at later stages of infection. IMPORTANCE In contrast to the process seen with most other RNA viruses, influenza virus genome replication occurs in the nucleus (rather than the cytoplasm) of infected cells. Therefore, completion of the viral replication cycle critically depends on intracellular transport mechanisms that ensure the translocation of viral ribonucleoprotein (RNP) complexes across the nuclear membrane. Here, we demonstrate that virus-induced cellular caspase activities cause a widening of nuclear pores, thereby facilitating nucleocytoplasmic translocation processes and, possibly, promoting nuclear export of newly synthesized RNPs. These passive transport mechanisms are suggested to complement Crm1-dependent RNP export mechanisms known to occur at early stages of the replication cycle and may contribute to highly efficient production of infectious virus progeny at late stages of the viral replication cycle. The report provides an intriguing example of how influenza virus exploits cellular structures and regulatory pathways, including intracellular transport mechanisms, to complete its replication cycle and maximize the production of infectious virus progeny.