A novel Nrf2-miR-29-desmocollin-2 axis regulates desmosome function in keratinocytes.

The Nrf2 transcription factor controls the expression of genes involved in the antioxidant defense system. Here, we identified Nrf2 as a novel regulator of desmosomes in the epidermis through the regulation of microRNAs. On Nrf2 activation, expression of miR-29a and miR-29b increases in cultured human keratinocytes and in mouse epidermis. Chromatin immunoprecipitation identified the Mir29ab1 and Mir29b2c genes as direct Nrf2 targets in keratinocytes. While binding of Nrf2 to the Mir29ab1 gene activates expression of miR-29a and -b, the Mir29b2c gene is silenced by DNA methylation. We identified desmocollin-2 (Dsc2) as a major target of Nrf2-induced miR-29s. This is functionally important, since Nrf2 activation in keratinocytes of transgenic mice causes structural alterations of epidermal desmosomes. Furthermore, the overexpression of miR-29a/b or knockdown of Dsc2 impairs the formation of hyper-adhesive desmosomes in keratinocytes, whereas Dsc2 overexpression has the opposite effect. These results demonstrate that a novel Nrf2-miR-29-Dsc2 axis controls desmosome function and cutaneous homeostasis.

A 3 years retrospective study of survival for zirconia-based single crowns fabricated from intraoral digital impressions.

OBJECTIVE: To evaluate the clinical performance of glass-ceramic/zirconia crowns fabricated using intraoral digital impressions - a retrospective study with a three-year follow-up. METHODS: 70 consecutive patients with a total of 86 glass-ceramic/zirconia crowns were treated by a single clinician using standardized clinical and laboratory protocols. A complete digital workflow was adopted for the purpose except for the veneering procedure for the glass-ceramic crowns. Occlusal adjustments were made before the ceramic glazing procedure. Before cementation, all abutments where carefully cleaned with a 70% alcoholic solution and air dried. Cementation was performed using dual-curing, self-adhesive resin cement. Patients were re-examined after 12, 24 and 36 months, to assess crown chipping/fractures. RESULTS: After the three-year follow-up, none of the zirconia-based restoration was lost ("apparent" survival rate 100%) otherwise, the chipping rate of the veneering material increased from 9.3% after 12 months, to 14% after 24 months to 30.2% after 36 months. As a consequence, the "real" success rate after 3 years was 69.8%. CONCLUSIONS: After 3 years the success rate of zirconia-based crowns was 69.8%, while the incidence of the chipping was 30.2%. Assuming an exponential increase in chipping rate between 12 and 36 months it can be argued that, among others, the fatigue-mechanism could be advocated as the main factor for the failure of glass-ceramic veneered zirconia especially after 24 months.

Correlation between microtensile bond strength data and clinical outcome of Class V restorations.

OBJECTIVE: To determine if the results of resin-dentin microtensile bond strength (µTBS) is correlated with the outcome parameters of clinical studies on non-retentive Class V restorations. METHODS: Resin-dentin µTBS data were obtained from one test center; the in vitro tests were all performed by the same operator. The µTBS testing was performed 8h after bonding and after 6 months of storing the specimens in water. Pre-test failures (PTFs) of specimens were included in the analysis, attributing them a value of 1MPa. Prospective clinical studies on cervical restorations (Class V) with an observation period of at least 18 months were searched in the literature. The clinical outcome variables were retention loss, marginal discoloration and marginal integrity. Furthermore, an index was formulated to be better able to compare the laboratory and clinical results. Estimates of adhesive effects in a linear mixed model were used to summarize the clinical performance of each adhesive between 12 and 36 months. Spearman correlations between these clinical performances and the µTBS values were calculated subsequently. RESULTS: Thirty-six clinical studies with 15 adhesive/restorative systems for which µTBS data were also available were included in the statistical analysis. In general 3-step and 2-step etch-and-rinse systems showed higher bond strength values than the 2-step/3-step self-etching systems, which, however, produced higher values than the 1-step self-etching and the resin modified glass ionomer systems. Prolonged water storage of specimens resulted in a significant decrease of the mean bond strength values in 5 adhesive systems (Wilcoxon, p<0.05). There was a significant correlation between µTBS values both after 8h and 6 months of storage and marginal discoloration (r=0.54 and r=0.67, respectively). However, the same correlation was not found between µTBS values and the retention rate, clinical index or marginal integrity. SIGNIFICANCE: As µTBS data of adhesive systems, especially after water storage for 6 months, showed a good correlation with marginal discoloration in short-term clinical Class V restorations, longitudinal clinical trials should explore whether early marginal staining is predictive for future retention loss in non-carious cervical restorations.

Schwann cell strip for peripheral nerve repair.

Many strategies have been investigated to provide an ideal substitute to treat a nerve gap injury. Initially, silicone conduits were used and more recently conduits fabricated from natural materials such as poly-3-hydroxybutyrate (PHB) showed good results but still have their limitations. Surgically, a new concept optimising harvested autologous nerve graft has been introduced as the single fascicle method. It has been shown that a single fascicle repair of nerve grafting is successful. We investigated a new approach using a PHB strip seeded with Schwann cells to mimic a small nerve fascicle. Schwann cells were attached to the PHB strip using diluted fibrin glue and used to bridge a 10-mm sciatic nerve gap in rats. Comparison was made with a group using conventional PHB conduit tubes filled with Schwann cells and fibrin glue. After 2 weeks, the nerve samples were harvested and investigated for axonal and Schwann cell markers. PGP9.5 immunohistochemistry showed a superior nerve regeneration distance in the PHB strip group versus the PHB tube group (> 10 mm, crossed versus 3.17+/- 0.32 mm respectively, P<0.05) as well as superior Schwann cell intrusion (S100 staining) from proximal (> 10 mm, crossed versus 3.40+/- 0.36 mm, P<0.01) and distal (> 10 mm, crossed versus 2.91+/- 0.31 mm, P<0.001) ends. These findings suggest a significant advantage of a strip in rapidly connecting a nerve gap lesion and imply that single fascicle nerve grafting is advantageous for nerve repair in rats.

A study of the effects of PVAC on works of art on paper and wood: pH and colour change

This paper presents the preliminary findings of pH and colour measurements carried out on artworks on paperand on wood that had been treated with a poly(vinyl acetate) (PVAC) based adhesive in the 1980s. In both cases, areas treated with PVAC proved to be less acidic than untreated areas. Contrary to expectations, the conservation treatments have not, as yet, increased acidity levels in the objects under study. Colour measurements of the works on paper showed that those that had been backed with a cotton fabric using a mixture of methylcellulose and PVAC were less yellow than those from the same print run that had not been backed. This finding suggests that the backing somehow prevented the natural degradation of the support. In view of these preliminary results, further research is clearly needed. This study forms part of a broader ongoing project to assess the role of PVAC in the conservation of a range of cultural assets.

Impairment of the Bacterial Biofilm Stability by Triclosan

The accumulation of the widely-used antibacterial and antifungal compound triclosan (TCS) in freshwaters raises concerns about the impact of this harmful chemical on the biofilms that are the dominant life style of microorganisms in aquatic systems. However, investigations to-date rarely go beyond effects at the cellular, physiological or morphological level. The present paper focuses on bacterial biofilms addressing the possible chemical impairment of their functionality, while also examining their substratum stabilization potential as one example of an important ecosystem service. The development of a bacterial assemblage of natural composition – isolated from sediments of the Eden Estuary (Scotland, UK) – on non-cohesive glass beads (,63 mm) and exposed to a range of triclosan concentrations (control, 2 – 100 mg L21) was monitored over time by Magnetic Particle Induction (MagPI). In parallel, bacterial cell numbers, division rate, community composition (DGGE) and EPS (extracellular polymeric substances: carbohydrates and proteins) secretion were determined. While the triclosan exposure did not prevent bacterial settlement, biofilm development was increasingly inhibited by increasing TCS levels. The surface binding capacity (MagPI) of the assemblages was positively correlated to the microbial secreted EPS matrix. The EPS concentrations and composition (quantity and quality) were closely linked to bacterial growth, which was affected by enhanced TCS exposure. Furthermore, TCS induced significant changes in bacterial community composition as well as a significant decrease in bacterial diversity. The impairment of the stabilization potential of bacterial biofilm under even low, environmentally relevant TCS levels is of concern since the resistance of sediments to erosive forces has large implications for the dynamics of sediments and associated pollutant dispersal. In addition, the surface adhesive capacity of the biofilm acts as a sensitive measure of ecosystem effects

Role of Staphylococcus aureus surface proteins in experimental endocarditis

Résumé Le staphylocoque doré est un pathogène responsable d'une grande variété de maladies chez l'être humain. Il est extrêmement bien équipé de facteurs de virulence, dont les adhésines. Jusqu'à présent, 21 protéines liant des composants de tissus de l'hôte ("microbial surface components reacting with adherence matrix molecules, MSCRAMM") ont été identifiées, par exemple le "clumping factor" A (CIfA) ou la "fibronectin-binding protein" A (FnBPA). Néanmoins, pour la plupart de ces protéines, leur rôle dans la pathogénie des infections à staphylocoque doré reste à être élucidé. Le but de cette thèse est de contribuer à ce processus. Premièrement, les "MSCRAMM" CIfA, CIfB, FnBPA, FnBPB, Cna, SpA, Pls, SdrC, SdrD, SdrE, SasD, SasE, SasF, SasG, Sasl, SasJ et SasK ont été exprimés dans une bactérie substitut, Lactococcus lactis, et testés pour leurs propriétés adhésives et leur pathogénicité dans un modèle d'endocardite expérimentale (voir chapitre 1). Cette technique a préalablement été utilisée avec succès et a l'avantage d'éviter le contexte complexe des redondances et systèmes de régulations propres au staphylocoque doré. Les résultats montrent que, de tous les facteurs de virulence testés, seuls CIfA et FnBPA sont d'importance primordiale dans le développement d'endocardite expérimentale. En ce qui concerne l'internalisation dans les cellules endothéliales, seulement FnPBA et FnBPB en sont capables. En outre, l'adhérence à chacun des ligands testés (fibrinogène, fibronectine, kératine, élastine, collagène, et les caillots de fibrine et plaquettes) est très spécifique et est médiée par une ou plusieures adhésines provenant du staphylocoque doré. Par conséquence, ces protéines pourraient représenter des cibles potentielles pour de futures thérapies anti-adhésives contre le staphylocoque doré. Deuxièmement, l'expression des facteurs de virulence décrits dans le chapitre 1 par les souches recombinantes de lactocoques a été vérifiée par une nouvelle méthode utilisant la spectrométrie de masse (voir chapitre 2). L'expression de toutes ces protéines par les souches recombinantes a pu être confirmée. Cette méthode pourrait être de grande valeur dans la vérification de la présence de protéines quelconques dans toutes sortes d'applications. Troisièmement, deux facteurs de virulence du staphylocoque, CIfA et une forme tronquée de FnBPA, ont été exprimés de façon simultanée dans une souche recombinante de lactocoque (voir chapitre 3}. Contrairement à une souche exprimant la FnBPA entière, une souche exprimant la forme tronquée de FnPBA, qui ne contient plus le domaine capable de lier le fibrinogène, perd complètement sa capacité d'infecter dans le modèle d'endocardite expérimentale. Par contre, il est montré que, en cas de complémentation de la forme tronquée de FnPBA avec le domaine de liaison au fibrinogène de CIfA dans la souche double recombinante, le phénotype intégral de FnBPA est récupéré. En conséquence, les facteurs de virulence sont capables de coopérer dans le but de la pathogénie des infections à staphylocoque doré. Summary Staphylococcus aureus is a human pathogen causing a wide variety of disease. It is extremely well equipped with both secreted and surface-attached virulence factors, which can act as adhesins to host tissues. In total, twenty-one microbial surface components reacting with adherence matrix molecules (MSCRAMMs) have been identified, so far. These include well-characterized adhesins such as clumping factor A (CIfA) or fibronectin-binding protein A (FnBPA). However, for most of them their potential role in the pathogenesis of staphylococcal infections remains to be elucidated. This has been attempted in this thesis work. Firstly, the staphylococcal MSCRAMMs CIfA, CIfB, FnBPA, FnBPB, Cna, SpA, Pls, SdrC, SdrD, SdrE, SasD, SasE, SasF, SasG, Sasl, SasJ, and SasK have been expressed in a surrogate bacterium, Lactococcus lactis, and tested for their in vitro adherence properties and their pathogenicity in the rat model of experimental endocarditis (see chapter 1). This model has successfully been used previously, and has the advantage of bypassing the complex S. aureus background of redundancies and differential regulation. Here, it is shown that of the seventeen tested potential virulence factors, only CIfA and FnBPA are critical for the pathogenesis of experimental endocarditis in rats, while internalization into bovine endothelial cells is mediated exclusively by FnBPA and FnBPB. In addition, the adherence to specific host ligands (fibrinogen, fibronectin, keratin, elastin, collagen, and fibrin-platelet clots) is highly specific and mediated by one or few staphylococcal adhesins, respectively. Thus, these surface proteins may represent potential targets for an anti-adhesive strategy against S. aureus infections. Secondly, the expression of the staphylococcal proteins by L. lactis recombinants described in chapter 1 was tested by a novel method using mass spectrometry (see chapter 2). The expression of all the staphylococcal proteins by the respective recombinant lactococcal strain could be confirmed. This method may prove to be of great value in the confirmation of the presence of any given protein in various experimental settings. Thirdly, two staphylococcal virulence factors, CIfA and a truncated form of FnBPA, were expressed simultaneously in one recombinant lactococcal strain (see chapter 3). In contrast to a recombinant strain expressing full-length FnPBA, a recombinant strain expressing a truncated FnPBA, lacking the domain capable of binding fibrinogen, completely lost infectivity in experimental endocarditis. However, it is shown that the complementation of the truncated form of FnBPA with the fibrinogenbinding domain of CIfA in a double recombinant strain results in the recovery of the complete phenotype of full-length FnBPA. Thus, virulence factors can cooperate in the pathogenesis of staphylococcal infections.

Biological monitoring of chemical exposure : toxicokinetic differences due to age and sex

Human biomonitoring is a widely used method in the assessment of occupational exposure to chemical substances and recommended biological limits are published periodically for interpretation and decision-making. However, it is increasingly recognized that a large variability is associated with biological monitoring, making interpretation less efficient than assumed. In order to improve the applicability of biological monitoring, specific factors responsible for this variability should be identified and their contribution quantified. Among these factors, age and sex are easily identifiable, and present knowledge about pharmaceutical chemicals suggests that they play an important role on the toxicokinetics of occupational chemical agents, and therefore on the biological monitoring results.The aim of the present research project was to assess the influence of age and sex on biological indicators corresponding to organic solvents. This has been done experimentally and by toxicokinetic computer simulation. Another purpose was to explore the effect of selected CYP2E1 polymorphisms on the toxicokinetic profile.Age differences were identified by numerical simulations using a general toxicokinetic model from a previous study which was applied to 14 chemicals, representing 21 specific biological entities, with, among others, toluene, phenol, lead and mercury. These models were runn with the modified parameters, indicating in some cases important differences due to age. The expected changes are mostly of the order of 10-20 %, but differences up to 50 % were observed in some cases. These differences appear to depend on the chemical and on the biological entity considered.Sex differences were quantified by controlled human exposures, which were carried out in a 12 m3 exposure chamber for three organic solvents separately: methyl ethyl ketone, 1-methoxy-2-propanol and 1,1,1-trichloroethane. The human volunteer groups were composed 12 of ten young men and fifteen young women, the latter subdivided into those with and without hormonal contraceptive. They were exposed during six hours at rest and at half of the threshold limit value. The kinetics of the parent compounds (organic volatiles) and their metabolite(s) were followed in blood, urine and expired air over time. Analyses of the solvent and their metabolites were performed by using headspace gas chromatography, CYP2E1 genotypes by using PCR-based RFLP methods. Experimental data were used to calibrate the toxicokinetic models developed for the three solvents. The results obtained for the different biomarkers of exposure mainly showed an effect on the urinary levels of several biomarkers among women due to the use of hormonal contraceptive, with an increase of about 50 % in the metabolism rate. The results also showed a difference due to the genotype CYP2E1*6, when exposed to methyl ethyl ketone, with a tendency to increase CYP2E1 activity when volunteers were carriers of the mutant allele. Simulations showed that it is possible to use simple toxicokinetic tools in order to predict internal exposure when exposed to organic solvents. Our study suggests that not only physiological differences but also exogenous sex hormones could influence CYP2E1 enzyme activity. The variability among the urinary biological indicators levels gives evidence of an interindividual susceptibility, an aspect that should have its place in the approaches for setting limits of occupational exposure.

Fibrinmatrix erhöht Adhärenz von regenerativen peripheren Nervenzellen [Fibrin matrix enhances adherence of peripheral nerve regenerative cells]

Optimal seeding of a nerve conduit with cells is a core problem in tissue engineering of constructing an artificial nerve substitute to gap lesions in the peripheral nerve system. An ideal nerve gap substitute would have to present an equally distributed number of cells that can activate the regrowing axons. This work shows a new in vitro technique of two-step seeding of cells inside a conduit and on layered mats that allows a valuable targeting of the cells and a proven survival in the environment of poly-3-hydroxybutyrate (PHB) conduits. The technique uses two components of diluted fibrin glue Tisseel. Initially, the chosen area on the mat was coated with thrombin followed from the seeding of a fibrinogen-cell compound. Using Sprague Dawley rat cells, we could demonstrate with immunohistochemistry (S100, DAPI) techniques that undifferentiated (uMSC) and Schwann cells (SC) mimicking differentiated mesenchymal stem cells (dMSC) as well as SC can be suspended and targeted significantly better in dissolvable diluted fibrin glue than in growth medium. Analysis showed significantly better values for adherence (p < 0.001) and drop off (p < 0.05) from seeded cells. Using this two-step application allows the seeding of the cells to be more precise and simplifies the handling of cell transplantation.

Feeding and arrestment responses of Diabrotica speciosa to cucurbitacin-content formulations

The objective of this work was to evaluate arrestant and stimulant feeding effects on Diabrotica speciosa (Ger.), using cucurbitacin-content starch-based formulations prepared with varying starch sources, and adding potassium lignate. In a glass slide assay, the wash off resistance of formulations was compared. Potassium lignate did not improve wash off resistance. Lagenaria vulgaris L. powder, in which cucurbitacin B concentration was determined as 0.28%, was added to the most adhesive formulation. The resultant material was used in a two-choice assay in which leaves of common bean, Phaseolus vulgaris L., treated with concentrations of 2.5%, 5%, 10%, 15% and 20% were offered to insects together with untreated control leaves. Greater number of insects and leaves consumed were found on leaves treated with cucurbitacin-content formulation (2.5%, 5% - greatest response -, 10% and 15% concentrations) than on untreated control leaves. The concentration, in which responses were higher, was sprayed in a bean field at 1,000, 1,900 and 3,000 g ha-1. Greater number of beetles was found in plots treated with the highest dosage, 3 and 6 days after spraying. Ten days after spraying, no significant differences were found among dosages, probably due to washoff of the bait.

A randomised controlled pilot study comparing Mepitel(®) and SurfaSoft(®) on paediatric donor sites treated with Recell(®).

This randomized controlled pilot study examined the effects of a silicone net dressing (Mepitel(®)) and a monofilament polyamide woven dressing (SurfaSoft(®)) on the rate of epithelialisation and epidermal maturation, pain, and ease of dressing removal on paediatric donor sites treated with epithelial cell suspension (ReCell(®)). Fifteen children (1-15 years) admitted for acute or reconstructive burns procedures in a tertiary referral hospital in Australia were randomly assigned to the experimental group, Mepitel(®) (n=8) and to the control group, SurfaSoft(®) (n=7). All donor sites were treated with ReCell(®) and covered with the assigned dressing. Measurements of rate of epithelialisation and epidermal maturation, pain, and ease of dressing removal were recorded every two days until the wound was healed. Results showed that there was no difference in the rate of epidermal maturation between the two groups. Less pain and force to remove the dressing was shown in the Mepitel(®) group when compared to SurfaSoft(®). The rate of epithelialisation was found to be an unreliable measure. Although additional research is required to support the results of this study, these results suggest that Mepitel's(®) pliable, self-adhesive and atraumatic properties may improve healing of ReCell(®) treated donor sites with less pain at dressing changes. This pilot study provides a strong base for further research in this area.