Effect of soft foods on primary human gingival epithelial cell growth and the wound healing process.

OBJECTIVES: Investigate the effect of soft diet foods on gingival epithelial cell growth, migration, and mediator secretion. METHODS: Human gingival epithelial cells were stimulated for various time periods with the following soft diet foods: orange juice, drinkable yogurt, and a nutritional drink. Cell growth was determined by an MTT assay and cell migration was investigated by a scratch assay and F-actin filament staining. Keratin production was analyzed by Western blot and wound healing mediators IL-6 and human ß-defensin 2 were quantified by ELISA. RESULTS: We demonstrate, for the first time, that certain soft diet foods increased the production of keratin 5, 14, and 19 by gingival epithelial cells. These proteins were known to be produced by proliferating cells. The soft foods tested also stimulated gingival epithelial cells to produce IL-6 and human ß-defensin 2. Soft foods are capable of promoting gingival epithelial cell migration by increasing F-actin production, which is part of the wound healing process. Results varied depending on the foods tested. CONCLUSION: Gingival epithelial cells interacted with the soft diet foods under study. This interaction was shown to upregulate keratin expression, as well as IL-6 and human ß-defensin 2 secretions. Furthermore, following cell wound, the soft foods upregulated post-scratch cell migration and F-actin production. Overall data suggest that the choice of foods in soft diets following oral surgery may influence the wound healing process of gingival epithelial cells.

Perioperative Systemic Corticosteroids in Orthognathic Surgery: A Systematic Review and Meta-Analysis.

PURPOSE: Perioperative systemic corticosteroids are broadly used in orthognathic surgery to prevent postoperative complications, but it is unclear whether this practice is beneficial and concerns about potential side effects have been raised. The purpose of this systematic review and meta-analysis was to assess the effects of perioperative systemic corticosteroids on clinically important outcomes in patients undergoing orthognathic surgery. MATERIALS AND METHODS: The authors conducted a systematic review of randomized controlled trials evaluating the effect of systemic corticosteroids in orthognathic surgery compared with placebo or any other intervention. The authors searched Medline, Embase, Cochrane Central, CINAHL, Lilacs, Scopus, and Web of Science and references of included trials. The primary outcome was the incidence of postoperative reintubation during the index hospitalization. The secondary outcomes were hospital length of stay, decreases in facial edema, and adverse events. Data were summarized using Mantel-Haenszel random-effects models. RESULTS: Of the 1,098 trials retrieved, 8 were included (n = 234). No trial evaluated the risk of postoperative reintubation. One trial evaluated the duration of hospital stay and showed no difference associated with the intervention. There was a decrease in facial edema with the use of systemic corticosteroids (n = 80; standardized mean difference, -1.07; 95% confidence interval, -1.99 to -0.16; I2 = 67%). Three trials reported side effects, such as postoperative surgical site bleeding, hypersensitivity, and stomach discomfort with intake of corticosteroids. The 8 trials had an unclear risk of bias. CONCLUSION: The authors observed no evidence of effect of systemic corticosteroids on the risk of reintubation and hospital length of stay in orthognathic surgery. Although facial edema decrease was observed to be improved with the intervention, adverse effects were inconsistently screened and reported. Thus, the use of systemic steroids in orthognathic surgery is not supported by strong evidence.

HYPERBARIC OXYGEN THERAPY FOR HEAD AND NECK IRRADIATED PATIENTS WITH SPECIAL ATTENTION TO ORAL AND MAXILLOFACIAL TREATMENTS.

Although radiation therapy is a common treatment for head and neck cancer, osteoradionecrosis (ORN) represents a major complication during or after treatment. Hyperbaric oxygen is often mentioned as a prophylactic and therapeutic treatment for ORN. In this article, we review the literature on hyperbaric oxygen therapy in head and neck irradiated patients. The widespread use of such therapy for the prevention and treatment of ORN appears to be based mainly on personal beliefs and experience, as no consensus exists in the scientific literature about its efficacy. Randomized controlled trials are, thus, needed to assess the real impact of hyperbaric oxygen therapy in head and neck irradiated patients. More fundamental research is also needed to clarify the pathophysiology of ORN, which in turn would help identify appropriate treatments.

Sch9 kinase integrates hypoxia and CO2 sensing to suppress hyphal morphogenesis in Candida albicans.

The yeast-hypha transition is an important virulence trait of Candida albicans. We report that the AGC kinase Sch9 prevents hypha formation specifically under hypoxia at high CO(2) levels. sch9 mutants showed no major defects in growth and stress resistance but a striking hyperfilamentous phenotype under hypoxia (<10% O(2)), although only in the presence of elevated CO(2) levels (>1%) and at temperatures of <37°C during surface growth. The sch9 hyperfilamentous phenotype was independent of Rim15 kinase and was recreated by inhibition of Tor1 kinase by rapamycin or caffeine in a wild-type strain, suggesting that Sch9 suppression requires Tor1. Caffeine inhibition also revealed that both protein kinase A isoforms, as well as transcription factors Czf1 and Ace2, are required to generate the sch9 mutant phenotype. Transcriptomal analyses showed that Sch9 regulates most genes solely under hypoxia and in the presence of elevated CO(2). In this environment, Sch9 downregulates genes encoding cell wall proteins and nutrient transporters, while under normoxia Sch9 and Tor1 coregulate a minor fraction of Sch9-regulated genes, e.g., by inducing glycolytic genes. Other than in Saccharomyces cerevisiae, both sch9 and rim15 mutants showed decreased chronological aging under normoxia but not under hypoxia, indicating significant rewiring of the Tor1-Sch9-Rim15 pathway in C. albicans. The results stress the importance of environmental conditions on Sch9 function and establish a novel response circuitry to both hypoxia and CO(2) in C. albicans, which suppresses hypha formation but also allows efficient nutrient uptake, metabolism, and virulence.

Expression of TPK1 and TPK2 genes encoding PKA catalytic subunits during growth and morphogenesis in Candida albicans.

The transcript levels of Candida albicans TPK1 and TPK2 genes, encoding PKA catalytic subunits, as well as phosphotransferase activity, were measured in the parental strain CAI4 and in homozygous tpk1Delta and tpk2Delta mutants during vegetative growth and during yeast-to-mycelial transition in N-acetylglucosamine liquid inducing medium at 37 degrees C. We observed two TPK2 transcripts, a major one of 1.8 kb and a minor one of 1.4 kb, and established by 3'-RACE that they originate from the recognition of the three polyadenylation signals present in the 3' untranslated region of the gene. During vegetative growth of CAI4 strain, the expression profiles of TPK1 and TPK2 varied similarly, reaching maximal expression at the late logarithmic phase. TPK1 mRNA levels were lower than those of TPK2 at all stages measured. In the corresponding homozygous tpk mutants, mRNA levels and the expression patterns of TPK1 and TPK2 were similar to those of CAI4, suggesting that the loss of one catalytic isoform is not compensated by overexpression of the other. Changes in PKA specific activity roughly correlated with fluctuations of mRNA expression levels. During yeast-to-mycelial transition, a sharp increase in TPK1 mRNA levels and in PKA-specific activity correlated with the onset of germ-tube formation in strain tpk2Delta. We also showed that tpk1Delta strain exhibited a delayed morphogenetic shift in comparison with CAI4 and tpk2Delta strains in several liquid inducing media, reinforcing the idea that Tpk1p is important for faster germ-tube appearance.

Expression levels and subcellular localization of Bcy1p in Candida albicans mutant strains devoid of one BCY1 allele results in a defective morphogenetic behavior.

We investigated expression, functionality and subcellular localization of C. albicans Bcy1p, the PKA regulatory subunit, in mutant strains having one BCY1 allele fused to a green fluorescent protein (GFP). DE-52 column chromatography of soluble extracts of yeast cells from strains bearing one BCY1 allele (fused or not to GFP) showed co-elution of Bcy1p and Bcy1p-GFP with phosphotransferase activity, suggesting that interaction between regulatory and catalytic subunits was not impaired by the GFP tag. Subcellular localization of Bcy1p-GFP supports our previous hypothesis on the nuclear localization of the regulatory subunit, which can thus tether the PKA catalytic subunit to the nucleus. Protein modeling of CaBcy1p, showed that the fusion of the GFP tag to Bcy1p C-terminus did not significantly disturb its proper folding. Bcy1p levels in mutant strains having one or both BCY1 alleles, led us to establish a direct correlation between the amount of protein and the number of alleles, indicating that deletion of one BCY1 allele is not fully compensated by overexpression of the other. The morphogenetic behavior of several C. albicans mutant strains bearing one or both BCY1 alleles, in a wild-type and in a TPK2 null genetic background, was assessed in N-acetylglucosamine (GlcNAc) liquid medium at 37 degrees C. Strains with one BCY1 allele tagged or not, behaved similarly, displaying pseudohyphae and true hyphae. In contrast, hyphal morphology was almost exclusive in strains having both BCY1 alleles, irrespective of the GFP insertion. It can be inferred that a tight regulation of PKA activity is needed for hyphal growth.

Virulence of the fungal pathogen Candida albicans requires the five isoforms of protein mannosyltransferases.

The PMT gene family in Candida albicans encodes five isoforms of protein mannosyltransferases (Pmt proteins Pmt1p, Pmt2p, Pmt4p, Pmt5p, and Pmt6p) that initiate O mannosylation of secretory proteins. We compared virulence characteristics of pmt mutants in two complex, three-dimensional models of localized candidiasis, using reconstituted human epithelium (RHE) and engineered human oral mucosa (EHOM); in addition, mutants were tested in a mouse model of hematogenously disseminated candidiasis (HDC). All pmt mutants showed attenuated virulence in the HDC model and at least one model of localized candidiasis. The pmt5 mutant, which lacks in vitro growth phenotypes, was less virulent in the EHOM and HDC assays but had no consistent phenotype in the RHE assay. In contrast, the pmt4 and pmt6 mutants were less virulent in the RHE and HDC assays but not in the EHOM assay. The results stress the contribution of all Pmt isoforms to the virulence of C. albicans and suggest that the importance of individual Pmt isoforms may differ in specific host niches. We propose that Pmt proteins may be suitable targets for future novel classes of antifungal agents.

Candida albicans lacking the gene encoding the regulatory subunit of protein kinase A displays a defect in hyphal formation and an altered localization of the catalytic subunit.

The fungal pathogen Candida albicans switches from a yeast-like to a filamentous mode of growth in response to a variety of environmental conditions. We examined the morphogenetic behavior of C. albicans yeast cells lacking the BCY1 gene, which encodes the regulatory subunit of protein kinase A. We cloned the BCY1 gene and generated a bcy1 tpk2 double mutant strain because a homozygous bcy1 mutant in a wild-type genetic background could not be obtained. In the bcy1 tpk2 mutant, protein kinase A activity (due to the presence of the TPK1 gene) was cyclic AMP independent, indicating that the cells harbored an unregulated phosphotransferase activity. This mutant has constitutive protein kinase A activity and displayed a defective germinative phenotype in N-acetylglucosamine and in serum-containing medium. The subcellular localization of a Tpk1-green fluorescent protein (GFP) fusion protein was examined in wild-type, tpk2 null, and bcy1 tpk2 double mutant strains. The fusion protein was observed to be predominantly nuclear in wild-type and tpk2 strains. This was not the case in the bcy1 tpk2 double mutant, where it appeared dispersed throughout the cell. Coimmunoprecipitation of Bcy1p with the Tpk1-GFP fusion protein demonstrated the interaction of these proteins inside the cell. These results suggest that one of the roles of Bcy1p is to tether the protein kinase A catalytic subunit to the nucleus.

cDNA cloning, biochemical and phylogenetic characterization of beta- and beta'-subunits of Candida albicans protein kinase CK2.

We have previously reported that Candida albicans protein kinase CK2 is composed of two distinct catalytic (alpha- and alpha'-) and two distinct regulatory (beta- and beta'-) subunits. We report here the isolation of two cDNAs clones, CaCKB1 and CaCKB2, encoding C. albicans beta- and beta'-subunits, respectively. The predicted beta- and beta'-proteins have calculated molecular masses of 34 kDa and 31 kDa and show all major features conserved in beta-subunits of other organisms, including the N-terminal autophosphorylation site, the internal acidic region and a potential metal-binding motif. The deduced amino acid sequence of C. albicans beta-subunit displays 48% identity with that of Saccharomyces cerevisiae and has an unusually long C-terminal acidic region containing a putative autophosphorylation site. C. albicans beta' shows 54% sequence identity with its S. cerevisiae homologue. Semi-quantitative RT-PCR analyses indicate that the mRNAs corresponding to both subunits are present in similar amounts in the yeast and hyphal forms of the fungus. To evaluate the biochemical properties of C. albicans beta- and beta'-subunits, both proteins were expressed in Escherichia coli and purified. Experiments performed in vitro indicate that both recombinant subunits reconstitute a fully functional holoenzyme when incubated with stoichiometric amounts of human recombinant alpha-subunit, as judged by their ability to abolish basal phosphorylation of calmodulin by human recombinant alpha-subunit and the reversion of the inhibitory effect by polylysine. In addition, both regulatory subunits can be phosphorylated by human recombinant alpha subunit. Phylogenetic analysis of beta- and beta'-proteins of C. albicans and other organisms shows that the CKB gene duplication occurred before the split of the ascomycete and basidiomycete lineages.

The two isoforms of the cAMP-dependent protein kinase catalytic subunit are involved in the control of dimorphism in the human fungal pathogen Candida albicans.

We have cloned the Candida albicans TPK2 gene encoding a cAMP-dependent protein kinase (PKA) catalytic subunit and generated a tpk2 homozygous null mutant to assess its ability to germinate in liquid media. N-acetylglucosamine (GlcNAc)-induced germ-tube formation was attenuated in the tpk2 strain and enhanced by compounds that are known to increase the PKA activity in situ. Germination was completely blocked in the presence of the myristoylated derivative of the heat-stable PKA inhibitor (MyrPKI). These results indicate that TPK1 acts positively in regulating the morphogenetic transition in C. albicans in the absence of the TPK2 gene. We were able to identify an mRNA from this second form of PKA in both wild-type and tpk2 null mutant cells. We found that PKA activity measured in the mutant lacking the TPK2 gene was about 10% of that displayed by the wild-type. The finding that the germinative response of tpk2 null mutant to serum was severely diminished at low serum concentrations indicates that the level of PKA is an important determinant of filamentous growth at low serum concentrations. The extent of germination attained at higher serum concentrations (5%) was similar in the wild-type and in the tpk2 null mutant strains suggesting that under these conditions germination was triggered through a PKA-independent pathway.

Interactions between regulatory and catalytic subunits of the Candida albicans cAMP-dependent protein kinase are modulated by autophosphorylation of the regulatory subunit.

The cAMP-dependent protein kinase (PKA) from Candida albicans is a tetramer composed of two catalytic subunits (C) and two type II regulatory subunits (R). To evaluate the role of a putative autophosphorylation site of the R subunit (Ser(180)) in the interaction with C, this site was mutated to an Ala residue. Recombinant wild-type and mutant forms of the R subunit were expressed in Escherichia coli and purified. The wild-type recombinant R subunit was fully phosphorylated by the purified C subunit, while the mutant form was not, confirming that Ser(180) is the target for the autophosphorylation reaction. Association and dissociation experiments conducted with both recombinant R subunits and purified C subunit showed that intramolecular phosphorylation of the R subunit led to a decreased affinity for C. This diminished affinity was reflected by an 8-fold increase in the concentration of R subunit needed to reach half-maximal inhibition of the kinase activity and in a 5-fold decrease in the cAMP concentration necessary to obtain half-maximal dissociation of the reconstituted holoenzyme. Dissociation of the mutant holoenzyme by cAMP was not affected by the presence of MgATP. Metabolic labeling of yeast cells with [(32)P]orthophosphate indicated that the R subunit exists as a serine phosphorylated protein. The possible involvement of R subunit autophosphorylation in modulating C. albicans PKA activity in vivo is discussed.