Modulation of Candida albicans virulence in in vitro biofilms by oral bacteria.

Candida-associated denture stomatitis presents as erythema of the palatal mucosa and is caused by biofilms containing the fungus Candida albicans that co-reside with oral bacteria on the denture-fitting surface. This study aimed to assess the effect of several frequently encountered oral bacteria on the expression of C. albicans virulence factors in in vitro polymicrobial biofilms. Biofilms containing C. albicans and selected bacterial species were grown on denture acrylic, and analysed by microscopy and by qPCR for expression of putative virulence genes. Candida albicans-only biofilms showed limited hyphal production. Hyphal development was significantly (P < 0·001) increased when biofilms also contained four species of oral bacteria (Streptococcus sanguinis, Streptococcus gordonii, Actinomyces odontolyticus and Actinomyces viscosus), as was the expression of virulence genes (P < 0·05). Importantly, inclusion of Porphyromonas gingivalis in the biofilm consortium resulted in significant (P < 0·05) inhibition of virulence gene expression and production of hyphae. The in vitro expression of C. albicans virulence factors was modulated in polymicrobial biofilms. The complexity of this modulation was highlighted by the reversal of effects following introduction of a single bacterial species into a biofilm community. SIGNIFICANCE AND IMPACT OF THE STUDY: The impact of individual bacterial species on Candida albicans virulence highlights both the complexity of predicting infection mediated by polymicrobial communities and the potential for management through pro- or prebiotic therapy. The possibility to selectively modulate microbial virulence by addition of, or treatment with pro- or prebiotics avoids the use of conventional antimicrobial compounds, thus reducing the contribution to potential drug resistance. Understanding which bacterial species modulate virulence, and the mechanisms by which this occurs, particularly in biofilms, provides excellent foundations for further research questions, and the potential for novel clinical interventions.

Indicators of dental anxiety in children just prior to treatment.

OBJECTIVES: We evaluated the relationship between child dental anxiety and selected child and parental characteristics. STUDY DESIGN: Children and their parents were interviewed at the New York University, College of Dentistry, Pediatric Dentistry Clinic. The Children's Fear Survey Schedule - Dental Subscale (CFSS-DS) evaluated child self-reported anxiety; the Modified Dental Anxiety Scale (MDAS) measured self-reported parental anxiety when the parent received dental treatment. RESULTS: Ninety-three children and their parents completed the questionnaires. Mean CFSS-DS scores were higher for girls than boys (32.5 vs. 26.3, p=0.003) and for children whose accompanying parents had MDAS scores of 11+ vs. ≥ 11 (32.8 vs. 26.6, p=0.001). There was little difference in mean CFSS-DS scores among those aged 6-10 yrs. vs. 11-14 yrs. (30.1 vs. 29.3). Significant correlations were found between CFSS-DS and both gender (Spearman's rho, rs=0.31) and MDAS scores (rs=0.33), but not between CFSS-DS and child age (rs=-0.05). Controlling simultaneously for gender, MDAS score and child age, a high CFSS-DS score (38+ vs. ≥ 38) was positively associated with girls (ORadj=3.76, 95% CI: 1.13-12.54) and an MDAS score of ≤ 15 vs. ≥ 11 (ORadj=2.50, 0.73-8.54), but weakly and inversely associated with age (ORadj=0.80, 0.25-2.52). CONCLUSION: Child gender and parental anxiety are indicators of child dental anxiety.

Occupational exposure to body fluids among health care workers in Georgia.

BACKGROUND: Health care workers (HCWs) are at increased risk of being infected with blood-borne pathogens. AIMS: To evaluate risk of occupational exposure to blood-borne viruses and determine the prevalence of human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV) among HCWs in Georgia. METHODS: The sample included HCWs from seven medical institutions in five cities in Georgia. A self-administered questionnaire was used to collect information on demographic, occupational and personal risk factors for blood-borne viruses. After obtaining informed consent, blood was drawn from the study participants for a seroprevalence study of HBV, HCV and HIV infections. RESULTS: There were 1386 participating HCWs from a number of departments, including surgery (29%), internal medicine (19%) and intensive care (19%). Nosocomial risk events were reported by the majority of HCWs, including accidental needlestick injury (45%), cuts with contaminated instruments (38%) and blood splashes (46%). The most frequent risk for receiving a cut was related to a false move during a procedure, reassembling devices and handing devices to a colleague. The highest proportion of needlestick injuries among physicians (22%) and nurses (39%) was related to recapping of used needles. No HIV-infected HCW was identified. Prevalence of HCV infection was 5%, anti-HBc was present among 29% with 2% being HBsAg carriers. CONCLUSIONS: Data from this study can be utilized in educational programs and implementation of universal safety precautions for HCWs in Georgia to help achieve similar reductions in blood-borne infection transmission to those achieved in developed countries.

Imaging gene delivery in a mouse model of congenital neuronal ceroid lipofuscinosis.

Adeno-associated virus (AAV)-mediated gene replacement for lysosomal disorders have been spurred by the ability of some serotypes to efficiently transduce neurons in the brain and by the ability of lysosomal enzymes to cross-correct among cells. Here, we explored enzyme replacement therapy in a knock-out mouse model of congenital neuronal ceroid lipofuscinosis (NCL), the most severe of the NCLs in humans. The missing protease in this disorder, cathepsin D (CathD) has high levels in the central nervous system. This enzyme has the potential advantage for assessing experimental therapy in that it can be imaged using a near-infrared fluorescence (NIRF) probe activated by CathD. Injections of an AAV2/rh8 vector-encoding mouse CathD (mCathD) into both cerebral ventricles and peritoneum of newborn knock-out mice resulted in a significant increase in lifespan. Successful delivery of active CathD by the AAV2/rh8-mCathD vector was verified by NIRF imaging of mouse embryonic fibroblasts from knock-out mice in culture, as well as by ex vivo NIRF imaging of the brain and liver after gene transfer. These studies support the potential effectiveness and imaging evaluation of enzyme replacement therapy to the brain and other organs in CathD null mice via AAV-mediated gene delivery in neonatal animals.

Suicidal gene therapy in an NF-κB-controlled tumor environment as monitored by a secreted blood reporter.

The nuclear factor-κB (NF-κB) is known to be activated in many cancer types including lung, ovarian, astrocytomas, melanoma, prostate as well as glioblastoma, and has been shown to correlate with disease progression. We have cloned a novel NF-κB-based reporter system (five tandem repeats of NF-κB responsive genomic element (NF; 14 bp each)) to drive the expression cassette for both a fusion between the yeast cytosine deaminase and uracil phosphoribosyltransferase (CU) as a therapeutic gene and the secreted Gaussia luciferase (Gluc) as a blood reporter, separated by an internal ribosomal entry site (NF-CU-IGluc). We showed that malignant tumor cells have high expression of Gluc, which correlates to high activation of NF-κB. When NF-κB was further activated by tumor necrosis factor-α in these cells, we observed up to 10-fold increase in Gluc levels and therefore transgene expression in human glioma cells served to greatly enhance the sensitization of these cells to the prodrug, 5-fluorocytosine both in cultured cells and in vivo subcutaneous tumor xenograft model. This inducible system provides a tool to enhance the expression of imaging and therapeutic genes for cancer therapy.

The biology underlying molecular imaging in oncology: from genome to anatome and back again.

Cancers are complex, evolving, multiscale ecosystems that are characterized by profound spatial and temporal heterogeneity. The interactions in cancer are non-linear in that small changes in one variable can have large changes on another. These multiple interacting phenotypes and spatial scales can best be understood with appropriate mathematical and computational models. Imaging is central to this investigation because it can non-destructively and longitudinally characterize spatial variations in the tumour phenotype and environment so that the system dynamics over time can be captured quantitatively.

Resource partitioning in bumble bees: the role of behavioral factors.

Small Bombus ternarius workers for age most frequently on the distal parts of goldenrod flower clusters when large B. terricola workers are present. This shift results from B. ternarius avoiding B. terricola. In this way B. ternarius can exploit, without conflict, resources whose availability changes constantly because of fluctuating numbers of larger consumers.

Aldehyde Pheromones in Lepidoptera: Evidence for an Acetate Ester Precursor in Choristoneura fumiferana.

Labeling studies of the eastern spruce budworm in vivo indicate that trans-11-tetradecenyl acetate is synthesized specifically in the pheromone-producing gland and is degraded in concert with pheromone release; hence it may be a precursor to the trans-11-tetradecenal pheromone. Radioactivity from exogenously added labeled fatty acids did not appear to be directly incorporated into the ester, suggesting that de novo biosynthesis from acetate is the major route of ester biosynthesis. Conversion of the acetate ester to alcohol and aldehyde functional groups may be the principal method of regulating pheromone specificity between species of Choristoneura.

ggr-Aminobutyric Acid, a Neurotransmitter, Induces Planktonic Abalone Larvae to Settle and Begin Metamorphosis.

gamma-Aminobutyric acid (a simple amino acid and potent neurotransmitter in human brain and other tissues of higher animals) and certain of its congeners rapidly and synchronously induce planktonic larvae of the red abalone, Haliotis rufescens, to settle and commence behavioral and developmental metamorphosis. These naturally occurring inducers of algal origin apparently are responsible, in part, for the substrate-specific recruitment, induction of settling, and the onset of metamorphosis of abalone and other planktonic larvae upon specific algae which provide naturally favorable habitats for the young of these species in coastal waters. These observations provide a convenient experimental model for further analysis of the basic molecular mechanisms by which environmental and endogenous factors control the recruitment and development of planktonic larvae. Halogenated organic pesticides significantly interfere with larval settling, as quantified in a new bioassay based upon these findings.

A nuclear-encoded form II RuBisCO in dinoflagellates.

The chloroplasts of most dinoflagellates are unusual in that they are surrounded by three membranes and contain the carotenoid peridinin. The ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) in dinoflagellate chloroplasts was found here to also be unusual. Unlike other eukaryotes, dinoflagellates containing peridinin use a form of RuBisCO (form II) previously found only in some species of proteobacteria. Furthermore, this RuBisCO is not encoded in the chloroplast DNA, as is the case in other organisms, but is encoded by the nuclear DNA. The unusual nature of this enzyme and location of its gene support the idea that dinoflagellate chloroplasts may have had a distinctive evolutionary origin.

Hydrogen peroxide induces spawning in mollusks, with activation of prostaglandin endoperoxide synthetase.

Addition of hydrogen peroxide to seawater causes synchronous spawning in gravid male and female abalones, and certain other mollusks as well. This effect is blocked by exposure of the animals to aspirin, an inhibitor of the enzyme catalyzing oxidative synthesis of prostaglandin endoperoxide. Hydrogen peroxide activates this enzymatic reaction in cell-free extracts prepared from abalone eggs (a very rich source of the prostaglandin endoperoxide synthetase); this effect appears to reveal a fundamental property of prostaglandin endoperoxide synthesis. Applicability of these findings to both mariculture and medical purposes is suggested.

Long-range structure in ribonuclease P RNA.

Phylogenetic-comparative and mutational analyses were used to elucidate the structure of the catalytically active RNA component of eubacterial ribonuclease P (RNase P). In addition to the refinement and extension of known structural elements, the analyses revealed a long-range interaction that results in a second pseudoknot in the RNA. This feature strongly constrains the three-dimensional structure of RNase P RNA near the active site. Some RNase P RNAs lack this structure but contain a unique, possibly compensating, structural domain. This suggests that different RNA structures located at different positions in the sequence may have equivalent architectural functions in RNase P RNA.

A link between RNA interference and nonsense-mediated decay in Caenorhabditis elegans.

Double-stranded RNA (dsRNA) inhibits expression of homologous genes by a process involving messenger RNA degradation. To gain insight into the mechanism of degradation, we examined how RNA interference is affected by mutations in the smg genes, which are required for nonsense-mediated decay. For three of six smg genes tested, mutations resulted in animals that were initially silenced by dsRNA but then recovered; wild-type animals remained silenced. The levels of target messenger RNAs were restored during recovery, and RNA editing and degradation of the dsRNA were identical to those of the wild type. We suggest that persistence of RNA interference relies on a subset of smg genes.

What is the clock? Translational regulation of circadian bioluminescence.

An oscillation in the cellular level of specific proteins in the unicellular marine alga Gonyaulax is correlated with the prominent circadian rhythm of bioluminescence of living cells and persists under constant conditions. This regulation involves a daily bout of synthesis of a specific protein, which is controlled by the circadian clock at the translational level.

Ex vivo and in vivo evaluation of residual chlorhexidine gluconate on skin following repetitive exposure to saline and wiping with 2% chlorhexidine gluconate/70% isopropyl alcohol pre-operative skin preparations.

BACKGROUND: Skin antisepsis is performed before surgery to minimize the risk of surgical site infections. Chlorhexidine gluconate (CHG) is routinely used in this application, but it may be removed during surgery when prepped areas are exposed to fluid and repeated blotting. AIM: This work evaluated the effect of adding a film-forming acrylate copolymer to a CHG-containing skin preparation on minimizing CHG loss during a simulated surgical irrigation and wiping procedure. The results were compared with those obtained with a commercially available water-soluble CHG preparation. METHODS: Two studies using excised porcine skin and one study on human volunteers were performed. In each study, the CHG preparations were applied and the treated sites were challenged with repetitive saline soaks and gauze dabbing to simulate surgical conditions. Challenged and unchallenged sites were analysed either for CHG content by high-performance liquid chromatography, or for bacterial log recovery after seeding an indicator organism (reflecting remaining CHG activity). FINDINGS: After irrigation and wiping, skin treated with the film-forming CHG preparation had more CHG remaining both on excised pig skin and in the human model. In the pig model, there was a lower recovery of inoculated bacteria with the CHG preparation containing the film-forming copolymer. No skin irritation or adverse events were reported in the human study. CONCLUSIONS: The addition of a film-forming copolymer has the potential to improve the retention of CHG on skin throughout a surgical procedure compared to a water-soluble preparation. This improved retention may lead to better antimicrobial activity.

Hypervariable Domains of Self-Incompatibility RNases Mediate Allele-Specific Pollen Recognition.

Self-incompatibility (SI) in angiosperms is a genetic mechanism that promotes outcrossing through rejection of self-pollen. In the Solanaceae, SI is determined by a multiallelic S locus whose only known product is an S RNase. S RNases show a characteristic pattern of five conserved and two hypervariable regions. These are thought to be involved in the catalytic function and in allelic specificity, respectively. When the Solanum chacoense S12S14 genotype is transformed with an S11 RNase, the styles of plants expressing significant levels of the transgene reject S11 pollen. A previously characterized S RNase, S13, differs from the S11 RNase by only 10 amino acids, four of which are located in the hypervariable regions. When S12S14 plants were transformed with a chimeric S11 gene in which these four residues were substituted with those present in the S13 RNase, the transgenic plants acquired the S13 phenotype. This result demonstrates that the S RNase hypervariable regions control allelic specificity.

Selection and characterization of a glucokinase-deficient mutant of Tetrahymena thermophila.

We have isolated a mutant of Tetrahymena thermophila that is resistant to inhibition of growth by the glucose analog 2-deoxyglucose. The mutant exhibits a deficiency in a cytoplasmic glucokinase. This enzymatic defect and the attendant inability to convert 2-deoxyglucose to toxic phosphorylated derivatives is apparently the sole basis for the mutant phenotype since transport of glucose and 2-deoxyglucose is unimpaired; there is no elevation of glucose-6-phosphatase activity, which could decrease the level of toxic 2-deoxyglucose metabolites. Genetic analyses have shown that the mutant allele is recessive and inherited as a single Mendelian mutation. The glucokinase-deficient strain described here is useful for the selection of other mutants in this organism and for the investigation of various cellular processes initiated or modulated by glucose and its analogs. We have exploited the molecular defect in this strain to investigate the initial steps in the cyclic AMP-mediated repression of galactokinase gene expression which is caused by glucose.

Purification and properties of galactokinase from Tetrahymena thermophila.

Galactokinase (EC 2.7.1.6; ATP: D-galactose-1-phosphototransferase) was purified 152-fold with an 11% yield from Tetrahymena thermophila maximally derepressed for enzyme synthesis in late stationary phase. The purification procedure utilized sequential acid precipitation, batch DEAE-Sephacel chromatography, differential ammonium sulfate precipitation and narrow range electrofocusing. The apparent molecular weight of the holoenzyme as determined by gel filtration on Sephadex G-200 is 50000-55000. The holoenzyme consists of two subunits of approx. 28000 daltons each, as determined by SDS-polyacrylamide gel electrophoresis. The native enzyme appears to be a single species with an isoelectric point at pH 5.1. Optimal activity was obtained at pH 7.8 and 41 degrees C, with no added monovalent salt. D-Galactose, 2-deoxygalactose and galactosamine all are suitable carbohydrate substrates for the stereospecific galactokinase; only substitution at the C-2 position of galactose retains enzyme recognition. The enzyme utilizes ATP, 2'-dATP and 3'-dATP as phosphate donors; ADP and adenosine-5'-[gamma-thio]triphosphate are inhibitory. The Km values for galactose and ATP were determined to be 0.60 mM and 0.15 mM, respectively. The enzyme requires a divalent cation for activity, with effectiveness being in the order: Mg2+ greater than Co2+ greater than Mn2+ greater than Fe2+. Galactokinases from all eucaryotic sources studied thus far seem to be very similar. Based upon the results reported here, the galactokinases from Tetrahymena and yeast appear to be most similar in their biophysical and biochemical properties.

Hemodynamic changes induced by liposomes and liposome-encapsulated hemoglobin in pigs: a model for pseudoallergic cardiopulmonary reactions to liposomes. Role of complement and inhibition by soluble CR1 and anti-C5a antibody.

BACKGROUND: Intravenous administration of some liposomal drugs can trigger immediate hypersensitivity reactions that include symptoms of cardiopulmonary distress. The mechanism underlying the cardiovascular changes has not been clarified. METHODS AND RESULTS: Anesthetized pigs (n=18) were injected intravenously with 5-mg boluses of large multilamellar liposomes, and the ensuing hemodynamic, hematologic, and laboratory changes were recorded. The significant (P<0.01) alterations included 79+/-9% (mean+/-SEM) rise in pulmonary arterial pressure, 30+/-7% decline in cardiac output, 11+/-2% increase in heart rate, 236+/-54% increase in pulmonary vascular resistance, 71+/-27% increase in systemic vascular resistance, and up to a 100-fold increase in plasma thromboxane B2. These changes peaked between 1 and 5 minutes after injection, subsided within 10 to 20 minutes, were lipid dose-dependent (ED50=4. 5+/-1.4 mg), and were quantitatively reproducible in the same animal several times over 7 hours. The liposome-induced rises of pulmonary arterial pressure showed close quantitative and temporal correlation with elevations of plasma thromboxane B2 and were inhibited by an anti-C5a monoclonal antibody (GS1), by sCR1, or by indomethacin. Liposomes caused C5a production in pig serum in vitro through classic pathway activation and bound IgG and IgM natural antibodies. Zymosan- and hemoglobin-containing liposomes and empty liposomes caused essentially identical pulmonary changes. CONCLUSIONS: The intense, nontachyphylactic, highly reproducible, complement-mediated pulmonary hypertensive effect of minute amounts of intravenous liposomes in pigs represents a unique, unexplored phenomenon in circulation physiology. The model provides highly sensitive detection and study of cardiopulmonary side effects of liposomal drugs and many other pharmaceutical products due to "complement activation-related pseudoallergy" (CARPA).

Suppression of loss-of-function mutations in Escherichia coli ribonuclease P RNA (M1 RNA) by a specific base-pair disruption.

A plasmid encoding ribonuclease P RNA of Escherichia coli (M1 RNA) was mutagenized with hydroxylamine in vitro and defective rnpB genes were identified by screening in an in vivo suppression assay. Defective rnpB sequences were mutagenized with a second round of hydroxylamine to restore activity. We report here that conversion of the C32.G48 base-pair of RNase P RNA to either C.A or U.G restored activity to defective rnpB genes bearing a variety of spatially distinct primary mutations. Disruption of this base-pair in an otherwise wild-type rnpB sequence increased the growth rate of the indicator strain E. coli FS101, consistent with the opening of C32.G48 during in vivo assembly of or catalysis by RNase P.