Secretory component mediates Candida albicans binding to epithelial cells.

OBJECTIVES: Candida albicans attaches to oral surfaces via a number of mechanisms including adherence mediated by salivary components adsorbed to the C. albicans cell surface. Our goal was to identify the salivary molecules involved. MATERIALS AND METHODS: Biotinylated salivary polypeptides that were bound by C. albicans were detected in extracts from washed, saliva-treated yeast cells by polyacrylamide gel electrophoresis and electroblot or immunoblot transfer analysis and purified by electroelution. Purified material was tested for the ability to promote the adherence of radiolabelled C. albicans yeast cells to cultured epithelial monolayers. RESULTS: Three of the polypeptides bound by C. albicans cells were identified as components of secretory IgA, including secretory component. Using non-denaturing polyacrylamide gel electrophoresis, we demonstrated that secretory component could be detected in its free form in saliva, and was bound by yeast cells. Secretory component which was purified by electroelution from non-denaturing PAGE-separated saliva, without detectable complete IgA, promoted adherence of yeast cells to cultured epithelial monolayers in a dose-dependent fashion. CONCLUSION: These results indicate that despite the inhibitory effect on adherence of IgA specific to C. albicans, IgA components, in particular secretory component, also promote binding to cultured epithelial monolayers.

Detection of Candida albicans ADH1 and ADH2 mRNAs in human archival oral biopsy samples.

OBJECTIVES: The aim of this study was to investigate the relationship between expression of Candida albicans alcohol dehydrogenases (ADH) genes in archival formalin-fixed paraffin-embedded (FFPE) samples from biopsies of leukoplakia. MATERIALS AND METHODS: Archival FFPE samples were obtained from four sample groups: normal oral mucosa, non-dysplastic leukoplakia, chronic hyperplastic candidosis (CHC), and non-CHC dysplastic leukoplakia. The presence of C. albicans was determined by periodic acid Schiff staining and by immunocytochemistry. C. albicans ADH1 and ADH2 mRNAs were detected using reverse transcription PCR. RESULTS: Candida albicans was detected in FFPE samples diagnosed as CHC (the histological diagnoses had been made by specialist oral pathologists, using uniform criteria), but not in any other sample group, including the non-dysplastic leukoplakias. RT-PCR confirmed a significant correlation between the expression of CaADH1 mRNA (P = 0.000), but not for CaADH2 mRNA (P = 0.056) in archival FFPE samples (n = 31) from biopsies of leukoplakia. CONCLUSIONS: Candida albicans was the predominant species in the lesions diagnosed as CHC, and the presence of C. albicans in CHC lesions was associated with a high expression of C. albicans ADH1 mRNA. There was no association between the presence of Candida and malignant transformation in the cases examined; however, the number of cases was limited and further studies are needed to further elucidate the role of C. albicans ADH1 in the pathogenesis of oral squamous cell carcinoma.

Preparedness and competency of New Zealand graduates for general dental practice - perceptions from the workforce.

BACKGROUND: Dental graduates need to demonstrate clinical competency. This mixed-methods study explored the perceptions of clinicians who employ or work with new graduates from the University of Otago, New Zealand, and identified themes reflecting graduates' preparedness for independent practice. METHODS: An online survey using a semantic differential scale and open-ended questions collected opinions and experiences from the workforce. Quantitative data were analysed using SPSS software, and qualitative data were analysed thematically. RESULTS: A representative sample of the workforce was obtained with a response rate of 35% (N = 83). Most clinicians engage new graduates to support the profession and/or rural communities. They perceived that graduates were well prepared in most areas, could translate theory to clinical practice and demonstrate professionalism. Graduates were reportedly stronger in basic dentistry, communication, ethics, and record keeping however were less strong in complex treatment planning, molar endodontics, fixed prosthodontics and exodontia. Clinical exposure during dental training was perceived as more limited, and mentoring and guidance in the transition to practice were deemed to be important. CONCLUSIONS: New Zealand dental graduates appear prepared for independent practice; however, maximising clinical opportunities during training, mentoring and early professional development in advanced areas of practice is essential to enhance competency and confidence.

Fumarate Restrains Alveolar Bone Restoration via Regulating H3K9 Methylation.

Nonresolving inflammation causes irreversible damage to periodontal ligament stem cells (PDLSCs) and impedes alveolar bone restoration. The impaired tissue regeneration ability of stem cells is associated with abnormal mitochondrial metabolism. However, the impact of specific metabolic alterations on the differentiation process of PDLSCs remains to be understood. In this study, we found that inflammation altered the metabolic flux of the tricarboxylic acid cycle and induced the accumulation of fumarate through metabolic testing and metabolic flux analysis. Transcriptome sequencing revealed the potential of fumarate in modulating epigenetics. Specifically, histone methylation typically suppresses the expression of genes related to osteogenesis. Fumarate was found to impede the osteogenic differentiation of PDLSCs that exhibited high levels of H3K9me3. Various techniques, including assay for transposase-accessible chromatin with high-throughput sequencing, chromatin immunoprecipitation sequencing, and RNA sequencing, were used to identify the target genes regulated by H3K9me3. Mechanistically, accumulated fumarate inhibited lysine-specific demethylase 4B (KDM4B) activity and increased H3K9 methylation, thus silencing asporin gene transcription. Preventing fumarate from binding to the histone demethylase KDM4B with α-ketoglutarate effectively restored the impaired osteogenic capacity of PDLSCs and improved alveolar bone recovery. Collectively, our research has revealed the significant impact of accumulated fumarate on the regulation of osteogenesis in stem cells, suggesting that inhibiting fumarate production could be a viable therapeutic approach for treating periodontal diseases.

Storage-related stability and antimicrobial efficacy of bottled, neutral-pH Electrolysed Oxidising Water.

OBJECTIVES: Denture stomatitis is prevalent in older people and poses serious health risks. Ready-to-use (RTU) neutral-pH Electrolysed Oxidizing Water (EOW) is an effective environmental disinfectant used in residential care settings and geriatric wards. However, the influence of storage on stability and effectiveness for denture disinfection has not been established. This research investigated the storage-related stability and antimicrobial activity of RTU EOW, and its efficacy against Candida albicans biofilms formed on denture resin. METHODS: The pH, oxidation/reduction potential (mV), available chlorine content (mg/L) and [HOCl] (mM) of RTU EOW (Envirolyte, New Zealand) solutions (n = 22) were measured from bottle opening to 28 days following storage at 4 °C, room temperature (RT) or 37 °C. Staphylococcus aureus and C. albicans cells were incubated in 80% EOW for contact times (CTs) up to 15 min and colony-forming units (cfu) determined. Minimum inhibitory concentrations (MIC90 EOW-HOCl) after CTs up to five minutes were determined for S. aureus and C. albicans reference strains and clinical isolates. C. albicans-denture resin disc biofilms were assessed after a five-minute CT with undiluted EOW by XTT-metabolic activity assay. RESULTS: [HOCl] remained stable when RTU EOW was stored at 4 °C or RT for five months after manufacture. One-minute CT resulted in log10 cfu reductions of >6 for S. aureus and >5 for C. albicans. Mean MIC90 for five-minute CT was 37 µM (S. aureus) and 54 µM (C. albicans). Undiluted EOW reduced C. albicans biofilm metabolic activity by 86%. CONCLUSIONS: RTU neutral-pH EOW is stable over five-months storage and is an effective denture disinfectant. CLINICAL SIGNIFICANCE: The efficacy of the RTU neutral EOW against C. albicans isolates and biofilms formed on denture resin surfaces supports its use as a denture disinfectant and can inform future research to assess its potential for preventing denture-related oral Candida infections in the older population, especially in resource-limited communities.

Clinically significant micafungin resistance in Candida albicans involves modification of a glucan synthase catalytic subunit GSC1 (FKS1) allele followed by loss of heterozygosity.

OBJECTIVES: To determine the mechanism of intermediate- and high-level echinocandin resistance, resulting from heterozygous and homozygous mutations in GSC1 (FKS1), in both laboratory-generated and clinical isolates of Candida albicans. METHODS: The DNA sequences of the entire open reading frames of GSC1, GSL1 (FKS3) and RHO1, which may contribute to the beta-1,3-glucan synthase of a micafungin-susceptible strain and a resistant clinical isolate, were compared. A spontaneous heterozygous mutant isolated by selection for micafungin resistance, and a panel of laboratory-generated homozygous and heterozygous mutants that possessed combinations of the echinocandin-susceptible and -resistant alleles, or mutants with individual GSC1 alleles deleted, were used to compare levels of echinocandin resistance and inhibition of glucan synthase activity. RESULTS: DNA sequence analysis identified a mutation, S645P, in both alleles of GSC1 from the clinical isolate. GSL1 had two homozygous amino acid changes and five non-synonymous nucleotide polymorphisms due to allelic variation. The predicted amino acid sequence of Rho1p was conserved between strains. Reconstruction of the heterozygous (S645/S645F) and homozygous (S645F/S645F) mutation showed that the homozygous mutation conferred a higher level of micafungin resistance (4 mg/L) than the heterozygous mutation (1 mg/L). Exposure of the heterozygous mutant to micafungin resulted in a loss of heterozygosity. Kinetic analysis of beta-1,3-glucan synthase activity showed that the homozygous and heterozygous mutations gave echinocandin susceptibility profiles that correlated with their MIC values. CONCLUSIONS: A homozygous hot-spot mutation in GSC1, caused by mutation in one allele and then loss of heterozygosity, is required for high-level echinocandin resistance in C. albicans. Both alleles of GSC1 contribute equally and independently to beta-1,3-glucan synthase activity.

Halitosis: prevalence, risk factors, sources, measurement and treatment - a review of the literature.

Halitosis, an offensive breath odour, has multiple sources and negative impacts on people's social interactions and quality of life. It is important for health care professionals, including general physicians and dental professionals, to understand its aetiology and risk factors in order to diagnose and treat patients appropriately. In this study, we have reviewed the current literature on halitosis regarding its prevalence, classification, risk factors, sources, measurement and treatment.

Genetic Polymorphisms in FGFR2 Underlie Skeletal Malocclusion.

Fibroblast growth factor receptor 2 (FGFR2) in craniofacial bones mediates osteoprogenitor proliferation, differentiation, and apoptosis. The distortion of proper craniofacial bone growth may cause class II and class III skeletal malocclusion and result in compromised function and aesthetics. Here, we investigated the association between variations in FGFR2 and skeletal malocclusions. First, 895 subjects were included in a 2-stage case-control study with independent populations (stage 1: n = 138 class I, 111 class II, and 81 class III; stage 2: n = 279 class I, 187 class II, and 99 class III). Eight candidate single-nucleotide polymorphisms (SNPs) in FGFR2 were screened and validated. Five SNPs (rs2162540, rs2981578, rs1078806, rs11200014, and rs10736303) were found to be associated with skeletal malocclusions (all P < 0.05). That is, rs2162540 was significantly associated with skeletal class II malocclusion, while others were associated with skeletal class III malocclusion. Electrophoretic mobility shift assay and chromatin immunoprecipitation analysis showed that the common genotypes of rs2981578 and rs10736303 contained the binding sites of RUNX2 and SMAD4. Compared with the common genotypes, the minor genotypes at these 2 SNPs decreased the binding affinity and enhancer effect of RUNX2 and SMAD4, as well the levels of FGFR2 expression. In addition, FGFR2 expression contributed positively to osteogenic differentiation in vitro. Thus, we identified FGFR2 as a skeletal malocclusion risk gene, and FGFR2 polymorphisms regulated its transcriptional expression and then osteogenic differentiation.

Simultaneous wireless assessment of intra-oral pH and temperature.

OBJECTIVES: Intra-oral pH plays an important role in the pathogenesis of tooth erosion and decay, but there is limited information about its variation in real life settings. The aims of this research were to: 1) develop a wireless device, which can be used to continuously monitor intra-oral pH and temperature in real-time; 2) test and validate the device under controlled laboratory conditions; and 3) collect data in a natural environment in a sample of healthy volunteers. METHODS: A wireless device for measuring pH and temperature simultaneously was developed, calibrated and validated against the gold standard glass electrode pH meter. A smart phone was used as data logger. The wireless device was embedded in an oral appliance and worn by eleven participants (mean age 31.1±6.9years) for 24h, while conducting standardised drinking tasks and regular daily activities. RESULTS: The wireless device could accurately measure pH and temperature both in vitro and in vivo. The recovery time following the swallow of a standard acidic drink varied markedly among individuals (mean=1.3±0.9min). The intra-oral pH and temperature recorded in the natural environment also showed a large inter- and intra-individual variability. The average intra-oral pH when asleep (6.7±0.5) was lower (p<0.001) than when awake (7.2±0.5). The average intra-oral temperature during sleep (35.6±0.5°C) was higher (p<0.001) than when awake (34.5±0.7°C). CONCLUSIONS: Intra-oral pH and temperature can be continuously and wirelessly assessed in real-life settings, and show individual-specific patterns with circadian variations. Intra-oral pH becomes slightly acidic during sleep while intra-oral temperature increases and fluctuates less. CLINICAL SIGNIFICANCE: We propose a wireless device that is capable of measuring intra-oral pH over a 24-h period. We found marked inter-individual variation after acidic stimuli, and day to sleep time variation of both intra-oral temperature and pH. Our approach may provide new insight into the relationship between oral pH, tooth wear and decay.

Learning the ABC of oral fungal drug resistance.

ATP-binding cassette (ABC) proteins are ubiquitous in prokaryotes and eukaryotes. They are involved in energy-dependent transport of molecules across membranes. ABC proteins are often promiscuous transporters that can translocate a variety of substrates. In oral fungi, especially in Candida species, they have been implicated as major contributors to the high-level azole resistance of clinical isolates from infections that do not respond to drug therapy. Although this is predominantly due to efflux of azoles from the cells, ABC proteins can contribute to fungal drug resistance in other ways as well. Cells in biofilms are notoriously resistant to antifungal agents. ABC proteins can contribute to this resistance through the efflux of drugs. Biofilms are complex communities of myriad microorganisms which, to survive in such a milieu, need to communicate with, and respond to, other microorganisms and their products. ABC proteins are involved in the secretion of fungal mating factors and quorum sensing molecules. These molecules affect biofilm structure and behavior that can result in increased drug resistance. Hence, ABC proteins make multiple contributions to oral fungal drug resistance through a variety of responses to environmental signals.

In vitro expression of Candida albicans alcohol dehydrogenase genes involved in acetaldehyde metabolism.

Alcohol consumption is a risk factor for oral cancer, possibly via its conversion to acetaldehyde, a known carcinogen. The oral commensal yeast Candida albicans may be one of the agents responsible for this conversion intra-orally. The alcohol dehydrogenase (Adh) family of enzymes are involved in acetaldehyde metabolism in yeast but, for C. albicans it is not known which family member is responsible for the conversion of ethanol to acetaldehyde. In this study we determined the expression of mRNAs from three C. albicans Adh genes (CaADH1, CaADH2 and CaCDH3) for cells grown in different culture media at different growth phases by Northern blot analysis and quantitative reverse transcription polymerase chain reaction. CaADH1 was constitutively expressed under all growth conditions but there was differential expression of CaADH2. CaADH3 expression was not detected. To investigate whether CaAdh1p or CaAdh2p can contribute to alcohol catabolism in C. albicans, each gene from the reference strain C. albicans SC5314 was expressed in Saccharomyces cerevisiae. Cell extracts from an CaAdh1p-expressing S. cerevisiae recombinant, but not an CaAdh2p-expressing recombinant, or an empty vector control strain, possessed ethanol-utilizing Adh activity above endogenous S. cerevisiae activity. Furthermore, expression of C. albicans Adh1p in a recombinant S. cerevisiae strain in which the endogenous ScADH2 gene (known to convert ethanol to acetaldehyde in this yeast) had been deleted, conferred an NAD-dependent ethanol-utilizing, and so acetaldehyde-producing, Adh activity. We conclude that CaAdh1p is the enzyme responsible for ethanol use under in vitro growth conditions, and may contribute to the intra-oral production of acetaldehyde.

Observation of magnetic coupling between distant metal centers in K3(MnO4)2 by muon spin relaxation measurements.

The site of a probe muon in the double salt K3(MnO4)2, which exhibits an antiferromagnetic phase at low temperature, and the Néel temperature of the salt were determined by zero-field muon spin relaxation. The picture shows the relaxation of the polarization asymmetry after muon insertion and a part of the crystal lattice.

Temperature-related expression of the vacuolar aspartic proteinase (APR1) gene and beta-N-acetylglucosaminidase (HEX1) gene during Candida albicans morphogenesis.

Expression of the Candida albicans vacuolar aspartic proteinase (APR1) and beta-N-acetylglucosaminidase (HEX1) genes was studied when carbon-starved cells of strains ATCC 10261 and A72 were induced to grow as yeast or as germ tube-forming cells. Amounts of APR1 mRNA were similar under yeast or germ tube growth conditions. However, more APR1 mRNA was present in cells grown at 28 degrees C than in cells grown at 37 degrees C. The Apr1 enzyme activity of cell-free extracts was not affected by cellular morphology, culture pH or growth temperature. Amounts of HEX1 mRNA were also higher in N-acetylglucosamine (GlcNAc)-induced cells grown at 28 degrees C than in cells grown at 37 degrees C. There was slightly more HEX1 mRNA in cells grown at pH 4.5 than in cells grown at pH 6.7. The beta-N-acetylglucosaminidase activities of GlcNAc-grown cells correlated with the amounts of HEX1 mRNA and were higher when cells were grown at a lower temperature and at a lower pH. Although a similar temperature- and pH-dependent pattern of HEX1 mRNA expression was seen in cells grown on glucose, the enzyme activities in cell-free extracts were all very low. These data indicate that the APR1 and HEX1 genes play no direct role in the dimorphic transition of C. albicans and that transcription of both genes appears to be temperature regulated when the cells are released from carbon starvation. The expression of HEX1 mRNA is in part under the control of culture pH and translation of HEX1 mRNA seems to be regulated by glucose.

Effect of calcium ion uptake on Candida albicans morphology.

In liquid culture using a synthetic medium, added magnesium but not calcium was required for exponential growth of Candida albicans yeast cells. However, medium without added divalent cations supported 2-3 generations of yeast growth or germ tube induction. The addition of calcium ions (1.0 mM) at any stage during the induction of germ tube formation caused reversion to a yeast mode of growth, in contrast to the effect of zinc and cobalt ions which were toxic to all growth. Inhibition of germ tube formation by calcium was not observed in the presence of either magnesium (10 microM) or manganese (100 microM). The presence of either of these ions caused inhibition of 45Ca uptake in yeast cultures. We conclude that unrestricted calcium uptake resulted in the specific inhibition of C. albicans mycelial growth, indicating a critical role for calcium in the regulation of C. albicans morphogenesis.

Yeast-specific DNA probes and their application for the detection of Candida albicans.

Two DNA fragments cloned from the genome of Candida albicans ATCC 10261 may be useful in the rapid diagnosis of disseminated candidosis. One sequence (probe EOB1) was specific for C. albicans (positive hybridisation with 45 strains tested). The second sequence (probe EOB2) detected C. albicans, as well as five other pathogenic Candida spp. and Saccharomyces cerevisiae, but did not react with human or bacterial DNA. Both probes were repetitive sequences in the genome of C. albicans. Probe EOB1 was used to detect, without DNA amplification, 500 C. albicans yeast cells in 1 ml of human blood.

Loop catheter extraction of ureteral stones: a re-evaluation.

Loop catheter extraction of ureteral stones has been used consistently by our group for over thirty years. Stones of up to 1.5 cm in largest dimension can be removed by this method, and for stones of any size there is less trauma to the ureter than with basket extraction. A representative five-year experience is presented in dealing with 264 ureteral stones, 58 of which were managed by loop extraction.

Saliva promotes Candida albicans adherence to human epithelial cells.

Adhesion of Candida cells to oral surfaces is an initial event in pathogenesis. Since specific immobilized salivary components mediate the binding of Candida albicans to hydroxyapatite, we hypothesized that saliva may also promote adherence to oral epithelia via a similar mechanism. In an in vitro model, C. albicans ATCC 10261 yeast cells adhered in a saturable manner to monolayers of three cultured human epithelial cell lines (A549, HEp-2, and HET-1A). The addition of whole saliva to the assay promoted the binding of C. albicans to all cell lines in a dose-dependent manner, but pre-incubation of the epithelial cells with pooled whole saliva had no effect on subsequent adherence. Pre-incubation of the yeast cells with pooled whole saliva, however, significantly enhanced (by up to 120%, P < 0.05) binding to epithelial cell monolayers, and pooled saliva that had been pre-incubated with C. albicans yeast cells was defective in promoting yeast adherence. There was a negative correlation (r = 0.68, P < 0.005) between specific IgA titers against whole cells of C. albicans and adherence-promoting activities for individual saliva samples. The adhesion-inhibitory effect of specific anti-C. albicans IgA was reversed by depletion of IgA from saliva by affinity chromatography. Factors in whole saliva, therefore, bound to the yeast cells, counter the C. albicans-specific salivary IgA inhibitory effect on adhesion and promote the adherence of C. albicans yeast cells to cultured epithelial cells.

Oral Candida: clearance, colonization, or candidiasis?

Candida albicans is frequently isolated from the human mouth, yet few carriers develop clinical signs of candidiasis. Oral candidiasis presents clinically in many forms. This reflects the ability of the yeast to colonize different oral surfaces and the variety of factors which predispose the host to Candida colonization and subsequent infection. Colonization of the oral cavity appears to be facilitated by several specific adherence interactions between C. albicans and oral surfaces which enable the yeast to resist host clearance mechanisms. Thus, Candida has been shown to adhere to complement receptors, various extracellular matrix proteins, and specific sugar residues displayed on host or bacterial surfaces in the oral cavity. Oral candidiasis results from yeast overgrowth and penetration of the oral tissues when the host's physical and immunological defenses have been undermined. Tissue invasion may be assisted by secreted hydrolytic enzymes, hyphal formation, and contact sensing. While these and other phenotypic characteristics may endow certain Candida species or strains with a competitive advantage in the oral cavity, it is the host's immune competence that ultimately determines whether clearance, colonization, or candidiasis occurs.

Interactions of Actinomyces naeslundii strains T14V and ATCC 12104 with saliva, collagen and fibrinogen.

Approximately similar numbers of actinomyces cells adhered to hydroxylapatite beads coated with saliva, collagen or fibrinogen. Adherence generally was unaffected by the presence of free saliva. Binding of cells to collagen- or fibrinogen-coated beads was reduced in the presence of either free collagen or fibrinogen. Glucan inhibited bacterial adherence only to collagen-coated hydroxylapatite beads. It is suggested that actinomyces bind to saliva-, collagen- or fibrinogen-coated surfaces by different mechanisms, but that these mechanisms involve some common bacterial cell-surface components.

Clinical efficacy of the Abbott Tacrolimus II assay for the IMx.

Monitoring tacrolimus is essential to maintain therapeutic concentrations. Performance of the new Abbott Tacrolimus assay (FK II) was evaluated and compared to the original tacrolimus assay (FK I). 189 trough whole blood samples from transplant cases were included in the study. Samples (n = 117) with FK I concentrations > 5 ng/mL were reanalyzed with the FK II assay. Patient samples (n = 43) that had FK I concentration < 5 ng/mL with apparent mean and range of 3.1 ng/mL and 0.7 to 4.5 ng/mL, respectively, were also reanalyzed with FK II to yield a mean of 5.9 ng/mL with a range of 2.9 to 10.8 ng/mL. Checking for patient compliance, samples (n = 10) with a FK I concentration of 0 ng/mL were re-analyzed. With one exception of a mislabeled cyclosporine sample, all samples (n = 9) showed FK506 levels greater than 2 ng/mL with the FK II assay. The FK II assay was shown to be a clinically efficacious assay, with improved sensitivity and acceptable precision versus the previous FK I assay.