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The oral microbiota, which is known to include at least 600 different bacterial species, is found on the teeth and mucosal surfaces as multi-species communities or biofilms. The oral surfaces are covered with a pellicle of proteins absorbed from saliva, and biofilm formation is initiated when primary colonizers, which express surface adhesins that bind to specific salivary components, attach to the oral tissues. Further development then proceeds through co-aggregation of additional species. Over time, the composition of oral biofilms, which varies between different sites throughout the oral cavity, is determined by a combination of environmental factors such as the properties of the underlying surface, nutrient availability and oxygen levels, and bacterial interactions within the community. A complex equilibrium between biofilm communities and the host is responsible for the maintenance of a healthy biofilm phenotype (eubiosis). In the face of sustained environmental perturbation, however, biofilm homeostasis can break down giving rise to dysbiosis, which is associated with the development of oral diseases such as caries and periodontitis.In vitro models have an important part to play in increasing our understanding of the complex processes involved in biofilm development in oral health and disease, and the requirements for experimental system, microbial complexity, and analysis techniques will necessarily vary depending on the question posed. In this chapter we describe some current and well-established methods used in our laboratory for studying oral bacteria in biofilm models which can be adapted to suit the needs of individual users.

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Large volumes of steel slag are produced annually, leading to significant environmental protection and sustainable development issues. An online technology to monitor the solidification process of steel slag can assist in obtaining the right mineralogy to valorize these slags or render them harmless. For this purpose, we investigated the electrical properties and microstructural relationships of a CaO-Al2O3-SiO2-MgO (CASM) slag during cooling using an innovative setup. The electrical impedance was determined over the frequency range of 20 Hz to 300 kHz at two cooling rates, and the solidification behaviour was observed simultaneously by confocal scanning laser microscopy (CSLM). Four zones can be distinguished in the conductivity-temperature curves for the slag cooled at 10 °C/min, whereas only two distinct zones are visible at 100 °C/min. The liquid fraction of the slag has a significant impact on the slag conductivity during cooling. The electrical conductivity is, therefore, an accurate indicator of the solidification degree. Different theoretical and empirical models were evaluated on their ability to relate the bulk conductivity of the slag to the liquid fraction. The empirical Archie's model proved to be the most suitable model for relating the bulk conductivity of the slag to the liquid fraction. In-situ electrical conductivity measurements during cooling can provide an online assessment of the slag solidification process, including indicating the appearance of solid precipitates, monitoring the growth of crystals, indicating complete solidification when no liquid phase remains, and indicating the cooling rate.

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In light of the limited therapeutic options with Carbapenem-Resistant Enterobacterales (CRE) infections, understanding the bacterial risk factors, such as biofilm formation and related gene expression of CRE, is vital. This study investigates the biofilm formation and biofilm-related gene expression of two enteric Enterobacterales with major CR determinants Escherichia coli IMP and Klebsiella pneumoniae NDM-1, which were seen in high prevalence in most common bacterial infections over the past few years. To our knowledge, this is the first study that demonstrated the relationship between biofilm formation and the related gene expression, to understand the potential molecular mechanisms during the biofilm formation in CRE. Biofilms were quantified by tissue culture plate assay at the stages of the biofilm development: initial attachment (6 h), microcolony formation (12 h), maturation (24 h), and dispersion (48 h). In a dispersion, event bacteria detach without any mechanical means and colonise another area. To investigate the influence of different growth conditions on biofilm formation, biofilms were quantified under different growth conditions. In parallel, quantitative real-time PCR (qPCR) assessed the biofilm-related gene expression of a cluster of genes, including biofilm maturation, quorum sensing, stress survival, and antibiotic resistance. Structural changes during biofilm development were assessed via confocal laser scanning microscopy (CLSM). We observed that the biofilm formation of CRE is correlated with the biofilm development stages, with maximum biofilm observed at 24 h at the maturation stage. Our data also showed that biofilm growth, under the condition tested, is the major factor influencing the variability of biofilm gene expression quantification assays. qPCR analyses have demonstrated that the expression of biofilm-related genes is highly correlated with phenotypic biofilm development, and these findings can be further expanded to understand the variation in regulation of such genes in these significant CRE pathogens. Our study demonstrated that both CRE strains, E. coli IMP and K. pneumoniae NDM-1, are high biofilm formers, and genes involved in biofilm development are upregulated during biofilm growth. The characteristic of the increased biofilm formation with the upregulation of antibiotic-resistant and biofilm-related genes indicates the successful pathogenic role of biofilms of these selected CRE and is attributed to their multi-drug resistance ability and successful dissemination of CRE in common bacterial infections.

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The current study aimed to develop pH-responsive cisplatin-loaded liposomes (CDDP@PLs) via the thin film hydration method. Formulations with varied ratios of dioleoyl phosphatidylethanolamine (DOPE) to cholesteryl hemisuccinate (CHEMS) were investigated to obtain the optimal particle size, zeta potential, entrapment efficiency, in vitro release profile, and stability. The particle size of the CDDP@PLs was in the range of 153.2 ± 3.08-206.4 ± 2.26 nm, zeta potential was -17.8 ± 1.26 to -24.6 ± 1.72, and PDI displayed an acceptable size distribution. Transmission electron microscopy revealed a spherical shape with ~200 nm size. Fourier transform infrared spectroscopic analysis showed the physicochemical stability of CDDP@PLs, and differential scanning calorimetry analysis showed the loss of the crystalline nature of cisplatin in liposomes. In vitro release study of CDDP@PLs at pH 7.4 depicted the lower release rate of cisplatin (less than 40%), and at a pH of 6.5, an almost 65% release rate was achieved compared to the release rate at pH 5.5 (more than 80%) showing the tumor-specific drug release. The cytotoxicity study showed the improved cytotoxicity of CDDP@PLs compared to cisplatin solution in MDA-MB-231 and SK-OV-3 cell lines, and fluorescence microscopy also showed enhanced cellular internalization. The acute toxicity study showed the safety and biocompatibility of the developed carrier system for the potential delivery of chemotherapeutic agents. These studies suggest that CDDP@PLs could be utilized as an efficient delivery system for the enhancement of therapeutic efficacy and to minimize the side effects of chemotherapy by releasing cisplatin at the tumor site.

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A- and B-type lamins are type V intermediate filament proteins. Mutations in the genes encoding these lamins cause rare diseases, collectively called laminopathies. A fraction of the cells obtained from laminopathy patients show aberrations in the localization of each lamin subtype, which may represent only the minority of the lamina disorganization. To get a better insight into more delicate and more abundant lamina abnormalities, the lamin network can be studied using super-resolution microscopy. We compared confocal scanning laser microscopy and stimulated emission depletion (STED) microscopy in combination with different fluorescence labeling approaches for the study of the lamin network. We demonstrate the suitability of an immunofluorescence staining approach when using STED microscopy, by determining the lamin layer thickness and the degree of lamin A and B1 colocalization as detected in fixed fibroblasts (co-)stained with lamin antibodies or (co-)transfected with EGFP/YFP lamin constructs. This revealed that immunofluorescence staining of cells does not lead to consequent changes in the detected lamin layer thickness, nor does it influence the degree of colocalization of lamin A and B1, when compared to the transfection approach. Studying laminopathy patient dermal fibroblasts (LMNA c.1130G>T (p.(Arg377Leu)) variant) confirmed the suitability of immunofluorescence protocols in STED microscopy, which circumvents the need for less convenient transfection steps. Furthermore, we found a significant decrease in lamin A/C and B1 colocalization in these patient fibroblasts, compared to normal human dermal fibroblasts. We conclude that super-resolution light microscopy combined with immunofluorescence protocols provides a potential tool to detect structural lamina differences between normal and laminopathy patient fibroblasts.

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BACKGROUND: This study investigated three in vitro models to assist in elucidating possible mode-of-action, which could be adopted to evaluate insecticidal activity of complex, unknown, or multi-constituent formulations. We used a combination of absorbance spectrometry, confocal scanning laser microscopy and microelectrode ion flux estimation (MIFE) to provide insight into potential target sites for insecticides. This study used two insect cell lines and evaluated three pyrethroid insecticides. RESULTS: We observed that the two cell lines produced distinctly different responses. Drosophila melanogaster D.mel-S2 cell line was a useful model to monitor ion flux changes, resulting from insecticides with neural toxicity; however, it was less useful to determine some metabolic pathway indicators of toxic stress. Conversely, the Spodoptera frugiperda Sf9 cell line produced acute reactive oxygen species (ROS) in response to insecticide treatments, but was not highly responsive in electrophysiological experiments. We also showed that the natural, multi-constituent botanical extract of pyrethrum elicited different Na+ , Cl- and Ca2+ ion fluxes than its synthetic, single constituent analogues, α-cypermethrin and esfenvalerate. These two methods used in combination with absorbance spectrometry measuring cell growth inhibition plus cell mortality assays shed some light on cytotoxic responses in differing model cell lines. CONCLUSION: This research highlights the importance of using multiple cell types and interdisciplinary methods to provide a better insight into mode of insecticidal action. This is especially pertinent to novel biopesticide discovery, as the underlying mechanisms for toxicity in initial screening processes are likely to be unknown.

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Real time visualization and tracking of colloidal particles with 3D resolution is essential for probing the local structure and dynamics in complex fluids. Although tracking translational motion of spherical particles is well-known, accessing rotational dynamics of such particles remains a great challenge. Here, we report a novel approach of using fluorescently labeled raspberry-like colloids with an optical anisotropy to concurrently track translational and rotational dynamics in 3 dimensions. The raspberry-like particles are coated by a silica layer of adjustable thickness, which allows tuning the surface roughness. The synthesis and applicability of the proposed method is demonstrated by two types of probes: rough and smoothened. The accuracies of measuring Mean Squared (Angular) Displacements are also demonstrated by using these 2 probes dispersed in 2 different solvents. The presented 3D trackable colloids offer a high potential for wide range of applications and studies, such as probing the dynamics of crystallization, phase transitions, biological interactions and the effect of surface roughness on diffusion.

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PURPOSE: To investigate alterations of corneal layers in eyes treated for acute postoperative endophthalmitis. METHODS: In this retrospective, nonrandomized comparative study, eyes treated with 25 gauge pars plana vitrectomy (PPV) for acute post-cataract endophthalmitis (group A) were compared to eyes receiving uneventful cataract surgery (group B) and uneventful 25 gauge PPV for epiretinal membrane (group C). After a minimum follow-up of 8 months from last surgical procedure, laser scanning in vivo confocal microscopy (IVCM) was performed. RESULTS: Twelve eyes for each group were recruited. Comparing study eyes with control eyes of group B and C, no statistical difference was found in corneal epithelial cell density (p = n.s.), in density of nerve fibers (p = n.s.), mean grade of nerve reflectivity (p = n.s.), mean grade of nerve tortuosity (p = n.s.), mean grade of anterior keratocyte activation (p = n.s.), and corneal endothelium cell density (p = n.s.), whereas a statistically higher mean grade of posterior keratocyte activation was found in group A (p < 0.01). Epithelial and endothelial corneal morphologies were graded as regular in all groups. Langerhans cells and corneal dendritic-shaped hyper-reflective endothelial deposits were found in group A. Both findings were absent in group B and C, and the difference was statistically significant (p < 0.01). CONCLUSIONS: IVCM was a useful tool in the detection of microscopic chronic corneal abnormalities caused by postoperative endophthalmitis. These findings confirmed the presence of a subclinical chronic corneal inflammation localized to the posterior stroma that should be related to the infectious process. Future studies might clarify pathological processes in the acute phase of postoperative endophthalmitis.

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Atypical enteropathogenic Escherichia coli are capable to form biofilm on biotic and abiotic surfaces, regardless of the adherence pattern displayed. Several E. coli mechanisms are regulated by Quorum sensing (QS), including virulence factors and biofilm formation. Quorum sensing is a signaling system that confers bacteria with the ability to respond to chemical molecules known as autoinducers. Suppressor of division inhibitor (SdiA) is a QS receptor present in atypical enteropathogenic E.coli (aEPEC) that detects acyl homoserine lactone (AHL) type autoinducers. However, these bacteria do not encode an AHL synthase, but they are capable of sensing AHL molecules produced by other species, establishing an inter-species bacterial communication. In this study, we performed experiments to evaluate pellicle, ring-like structure and biofilm formation on wild type, sdiA mutants and complemented strains. We also evaluated the transcription of genes involved in different stages of biofilm formation, such as bcsA, csgA, csgD, fliC and fimA. The sdiA mutants were capable of forming thicker biofilm structures and showed increased motility when compared to wild type and complemented strains. Moreover, they also showed denser pellicles and ring-like structures. Quantitative real-time PCR (qRT-PCR) analysis demonstrated increased csgA, csgD and fliC transcription on mutant strains. Biofilm formation, as well as csgD, csgA and fimA transcription decreased on wild type strains by the addition of AHL. These results indicate that SdiA participates on the regulation of these phenotypes in aEPEC and that AHL addition enhances the repressor effect of this receptor on the transcription of biofilm and motility related genes.

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Cryptococcosis is a fungal disease of global significance for which new effective treatments are needed. The conjugation of the synthetic antimicrobial peptide fragment UBI 31-38 to a coumarin derivative showed to be an effective approach for the design of a novel anticryptococcal agent. In addition to antifungal activity, the conjugate exhibited intense fluorescence, which could be valuable for mechanistic investigations of this molecule. In this work, we studied the photophysical properties of the conjugate and confocal scanning laser microscopy was used to inspect the distribution of the peptide-coumarin conjugate in Cryptococcus cell. The synergism of this compound with amphotericin B or fluconazole against C. gattii and C. neoformans strains was also investigated. The results indicated that the fluorescent conjugate alone as well as its combination with amphotericin B are promising tools against cryptococcosis.

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The antioxidant effect of 8PP, a prenylflavonoid from Dalea elegans on Candida albicans biofilms, was investigated. We previously reported that sensitive (SCa) and resistant C. albicans (RCa) biofilms were strongly inhibited by this compound, in a dose-depending manner (50 µM-100 µM), with a prooxidant effect leading to accumulation of endogenous oxidative metabolites and increased antioxidant defenses. In this work, the antifungal activity of high concentrations of 8PP (200-1000 µM), the cellular stress imbalance and the architecture of biofilms were evaluated. Biofilms were studied by crystal violet and confocal scanning laser microscopy (CSLM) with COMSTAT analysis. Superoxide anion radical, the activity of the superoxide dismutase and the total antioxidant capacity were measured. Intracellular ROS were detected by a DCFH-DA and visualized by CSLM; reactive nitrogen intermediates by Griess. An antioxidant effect was detected at 1000 µM and levels of oxidant metabolites remained low, with major changes in the SCa. COMSTAT analysis showed that biofilms treated with higher concentrations exhibited different diffusion distances with altered topographic surface architectures, voids, channels and pores that could change the flow inside the matrix of biofilms. We demonstrate for first time, a concentration-dependent antioxidant action of 8PP, which can alter its antifungal activity on biofilms.

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The formation of biofilms in conjunction with quorum sensing (QS) regulated expression of virulence by opportunistic pathogens contributes significantly to immune evasion and tolerance to a variety of antimicrobial treatments. The present protocol describes methods to determine the in vitro efficacy of potential QS inhibitors (QSIs). Work on Pseudomonas aeruginosa has shown that chemical blockage of QS is a promising new antimicrobial strategy. Several live bacterial reporter systems have been developed to screen extracts and pure compounds for QSI activity. Here we describe the usage of reporter strains consisting of a lasB-gfp or rhlA-gfp fusion in P. aeruginosa for qualitative and quantitative evaluation of the inhibition of two of the major QS pathways, monitored as reduced expression of green fluorescence. By the use of an in vitro flow cell system it is possible to study the QSI activity by monitoring its ability to interfere with the protective functions of bacterial biofilm. For evaluation of the global effects of QSI compounds, we present a protocol for the DNA microarray based transcriptomics. Using these in vitro methods it is possible to evaluate the potential of various QSI compounds.

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Atypical enteropathogenic Escherichia coli are capable to form biofilm on biotic and abiotic surfaces, regardless of the adherence pattern displayed. Several E. coli mechanisms are regulated by Quorum sensing (QS), including virulence factors and biofilm formation. Quorum sensing is a signaling system that confers bacteria with the ability to respond to chemical molecules known as autoinducers. Suppressor of division inhibitor (SdiA) is a QS receptor present in atypical enteropathogenic E. coli (aEPEC) that detects acyl homoserine lactone (AHL) type autoinducers. However, these bacteria do not encode an AHL synthase, but they are capable of sensing AHL molecules produced by other species, establishing an inter-species bacterial communication. In this study, we performed experiments to evaluate pellicle, ring-like structure and biofilm formation on wild type, sdiA mutants and complemented strains. We also evaluated the transcription of genes involved in different stages of biofilm formation, such as bcsA, csgA, csgD, fliC and fimA. The sdiA mutants were capable of forming thicker biofilm structures and showed increased motility when compared to wild type and complemented strains. Moreover, they also showed denser pellicles and ring-like structures. Quantitative real-time PCR (qRT-PCR) analysis demonstrated increased csgA, csgD and fliC transcription on mutant strains. Biofilm formation, as well as csgD, csgA and fimA transcription decreased on wild type strains by the addition of AHL. These results indicate that SdiA participates on the regulation of these phenotypes in aEPEC and that AHL addition enhances the repressor effect of this receptor on the transcription of biofilm and motility related genes.

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Most studies have focused on myoglobin regarding meat colour development, with little focus on the contribution of muscle structure and light scattering. Our aim was to investigate the pH-dependent changes in muscle structure, on the light scattering properties of the meat. Beef longissimus thoracis muscles were segregated into light, medium or dark colour groups (n=18). 'Dark' muscles had a high ultimate pH (pHu), lower lightness (L*), redness (a*), higher myoglobin concentration and deoxymyoglobin content compared to other muscles. Reflectance confocal laser scanning microscopy (rCLSM) revealed 'dark' muscles had decreased global brightness (indicator of light scattering) and increased fibre width. In a secondary experiment, decreasing the homogenisation buffer pH from 6.10 to 5.40 induced a 17% shrinkage in high pHu muscle fibres, which increased light scattering by ~25%. In conclusion, rCLSM demonstrated that muscle structure contributes to the magnitude of light scattering by a pH dependent mechanism.

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OBJECTIVES: To determine the effects of intrinsic wetness on the formation of dentin bonding interfaces of four resin cement systems bonded to dentin under different pulpal pressures. METHODS: Thirty-six freshly extracted third molars were selected and processed for dentin µTBS. The teeth were randomly assigned into 12 experimental groups, according to the adhesive luting system [Adper Single Bond Plus (3M ESPE) combined with two luting agents RelyX ARC (3M ESPE) and heated Filtek Z250 Universal Restorative (3M ESPE), Clearfil CD Bond (Kuraray) combined with Clearfil Esthetic Cement (Kuraray), and RelyX Unicem 2 Automix (3M ESPE)] and pulpal pressure (0, 5, and 20 cm of simulated pulpal pressure). Leucite-reinforced glass-ceramic slabs (IPS Empress CAD, Ivoclar Vivadent) of 3mm thickness were bonded to dentin. The samples were stored in distilled water for 24h and then sectioned in X/Y directions across the adhesive interface to obtain specimens with a cross section of 0.8 ± 0.2mm(2). All sticks were fractured by tension at a crosshead speed of 1.0mm/min and the data were submitted to Kruskal-Wallis and Mann-Whitney Tests (α=0.05). Ultrastructural analysis of the interfaces was performed using Confocal Laser Scanning Microscopy (CLSM) and Scanning Electron Microscopy (SEM). RESULTS: The statistical analyses showed that pulpal pressure decreased µTBS for all groups. Significantly higher µTBS values were obtained in heated Z250 group restored without any pulpal pressure. CLSM showed that the uptake of water through the dentin tubuli and their anastomosis of lateral branches during the adhesive luting procedures prevented adequate formation of the dentin bonding interfaces. SEM showed that the luting film created is material- dependent and all adhesive failure occurred at the resin-dentin interface. CONCLUSION: The constant intrinsic wetness replenishment prevents adequate formation of the hybrid layer. CLINICAL SIGNIFICANCE: Intrinsic moisture during adhesive luting procedures significantly affects the interaction between luting materials and dentin subtract and decreases the quality and bonding strength of the resin-dentin bond.

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Otitis media with effusion (OME) is a highly prevalent disease in children, but the exact pathogenesis and role of bacteria are still not well understood. This study aimed to investigate the presence of otopathogenic bacteria in the middle ear effusion (MEE) and adenoid of children with chronic OME (COME), and to investigate in vivo whether these bacteria, especially Haemophilus influenzae, are organized as a biofilm in the middle ear fluid. MEE and adenoid samples were collected from 21 patients with COME. Extensive bacterial culturing and genotyping was performed on all middle ear and adenoid samples. Fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM) was used to visualize possible biofilm structures for a selection of middle ear effusion samples. 34 MEE samples were collected from 21 patients of which 64.7 % were culture positive for bacteria and 47.0 % were culture positive for Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus and/or Streptococcus pneumoniae. All 21 adenoid samples were culture positive for one or more of these four otopathogens. H. influenzae (35.3 %) and S. pneumoniae (76.2 %) were the most frequently cultured bacteria in the MEE and adenoid samples, respectively. The same bacterial species was found in MEE and adenoid for 84.6 % of the patients and in 81.2 % of the cases where the same species was found in more than one site it involved the same bacterial genotype. FISH and CLSM demonstrated the presence of H. influenzae specific biofilm structures in five of the eight culture positive MEEs that were tested, but in none of the two culture negative MEEs. The findings in this study indicate that the adenoid acts as a reservoir for bacteria in MEE and confirms that biofilms, in at least half of the cases consisting of H. influenzae, are indeed present in the MEE of children with COME. Biofilms may thus play a crucial role in the pathogenesis of COME, which is important in the understanding of this disease and the development of potential future treatment options.

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Liposome-microbubble conjugates are considered as better targeted drug delivery vehicles compared to microbubbles alone. The microbubble in the integrated drug delivery system delivers the drug intracellularly on the target, whereas the liposome component allows loading of high drug dose and extravasation through leaky vasculature. In this work, a new high yielding microbubble production method was used to prepare microbubbles for formulation of the liposome-conjugated drug delivery system. In formulation process, the prepared liposome of 200 nm diameter was attached to the microbubble surface using the avidin-biotin interaction. The analysis of the confocal scanning laser microscope images showed that approximately 8 × 108 microbubbles per millilitre (range: 2-7 µm, mean size 5 ± 0.5 µm) can be efficiently conjugated to the liposomes. The method of conjugation was found to be effective in attaching liposome to microbubbles.

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This investigation provides generic information on the culturable corrosive and the microfouling bacterial community in a firewater distribution system that uses freshwater. Conventional microbiological methods were used for the selective isolation of the major microfouling bacteria. The isolates were characterized by 16S rRNA gene sequencing and the biofilm as well as the corrosion characteristics of the isolates were evaluated. Pseudomonas aeruginosa and Bacillus cereus were predominantly observed in all the samples analysed. Denaturing gradient gel electrophoresis (DGGE) was carried out for the various samples of firewater system (FWS) and the high intensity bands were sequenced to identify the predominant bacteria. Bacterial groups such as Cyanobacteria, Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes were identified. Biofilm thickness was recorded using confocal scanning laser microscopy (CSLM). This was the first study to report Lysinibacillus fusiformis in a firewater system and its role in iron corrosion. Sulphidogenic bacteria Tissierella sp. and Clostridium bifermentans generated sulphides in the range of 400-900 ppm. Significant corrosion rates of carbon steel (CS) coupons were observed up to 4.3 mpy. C. bifermentans induced more localized corrosion in CS with a pit diameter of 50 µm. Overall, the data on the characterization of the fouling bacteria, their biofilm forming potential and subsequent metal deterioration studies supported in designing an effective water treatment program.

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OBJECTIVES: The aim of this study was to evaluate the formation of dentin bonding interfaces using the water-wet and the ethanol-wet techniques under simulated pulpal pressure, and to assess the effect of adhesive solvent and thermomechanical loading. METHODS: Flat dentin surfaces were restored under 20mm-simulated pulpal pressure following two bonding approaches (water-wet and ethanol-wet bonding) in combination with dental adhesives containing ethanol (Single Bond Plus and Scotchbond Multi-Purpose) or acetone (One-Step Plus and All-Bond 2) as solvent. Half of the restorations of each subgroup were subjected to thermocycling followed by cyclic loading (three teeth per group). Bond strength was measured using the microtensile bond strength test and fitted to a Weibull distribution (α=0.05). Ultrastructural analyses of the interface and leakage/nanoleakage evaluation were performed using confocal scanning microscopy (CLSM) and transmission electron microscopy (TEM). RESULTS: Water permeation through dentin tubules during adhesive application prevented adequate penetration of adhesive monomers into the demineralized collagen matrix in both bonding techniques, but more severely for water-wet bonding. Acetone-solvated adhesives showed worse bonding performance and hybridization than ethanol-based systems when applied in the ethanol-wet mode, both before and after thermomechanical challenge. SIGNIFICANCE: The ethanol-wet bonding technique helps to compensate for water permeation from dentin tubules during the bonding procedures to form more stable dentin bonds, especially when used in conjunction to ethanol-solvated systems.

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AIMS: (i) To analyze the in vivo corneal structure and sub-basal plexus nerves in patients with primary Sjögren's syndrome (pSS) and no-SS dry eye by confocal scanning laser microscopy (CSLM) and (ii) to correlate CSLM findings with tear function tests and with patients' subjective dryness. METHODS: Seventeen patients with pSS, 16 no-SS dry eye, and 20 healthy volunteers were included. CSLM parameters taken into consideration included: basal epithelial integrity, corneal thickness, epithelial cellular density, keratocyte activation, and sub-basal plexus morphology. Statistical analysis was carried out using SPSS-13 (Chicago IL, USA). RESULTS: CSLM pachymetric data and the superficial epithelium cell density were significantly lower in pSS versus no-SS dry eye (p < 0.0001); keratocyte activation and sub-basal nerve abnormalities were also more frequent in pSS patients (p < 0.0001). CSLM findings well correlated with both the ocular test results and the patients' perception of ocular dryness at the baseline and over the follow-up. CONCLUSION: CSLM might be a useful novel tool in the assessment of the involvement of the lachrymal functional unit in pSS.

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