Effect of gallium on growth of Streptococcus mutans NCTC 10449 and dental tissues.

Gallium-doped phosphate-based glasses (Ga-PBG) were assessed for their impact on Streptococcus mutans and dental mineralisation, firstly by disc diffusion assays followed by biofilms grown on nitrocellulose filter membrane (NFM) and constant-depth film fermentor (CDFF). Short-time exposure (10 min) effects of Ga-PBG on S. mutans biofilm were compared with that of 0.2% chlorhexidine. The effects of Ga-PBG on bovine enamel (which was investigated under pH-cycling condition) and dentine were analysed using transverse microradiography (TMR), profilometry and inductively coupled plasma optical-emission spectrometry (ICP-OES). The disc diffusion assays showed inhibition zones of 24.5 ± 0.5 mm for Ga-PBG compared with controls (C-PBG). Ga-PBG showed statistically significant growth inhibition of S. mutans biofilms on NFM (p = 0.001) and CDFF (p < 0.046) compared with hydroxyapatite (HA) and C-PBG. The CDFF assay revealed a maximum of 2.11 log colony-forming unit (CFU) reduction at 48 h, but short-time exposure effects were comparable with that of 0.2% chlorhexidine only on older biofilms (maximum of 0.59 vs. 0.69 log CFU reduction at 120 h). TMR analyses of the enamel revealed non-significant mineral loss (p = 0.37) only in the case of Ga-PBG samples compared with controls including sodium fluoride. ICP-OES analyses indicated transient gallium adsorption into dentine by calcium displacement. The results confirmed that gallium inhibited S. mutans growth and appears to have the potential to protect the enamel surface under conditions representative of the oral environment. Further work is needed to establish whether it has an application in daily oral hygiene procedures to prevent or reduce caries.

Photobleaching of red fluorescence in oral biofilms.

BACKGROUND AND OBJECTIVE: Many species of oral bacteria can be induced to fluoresce due to the presence of endogenous porphyrins, a phenomenon that can be utilized to visualize and quantify dental plaque in the laboratory or clinical setting. However, an inevitable consequence of fluorescence is photobleaching, and the effects of this on longitudinal, quantitative analysis of dental plaque have yet to be ascertained. MATERIAL AND METHODS: Filter membrane biofilms were grown from salivary inocula or single species (Prevotella nigrescens and Prevotella intermedia). The mature biofilms were then examined in a custom-made lighting rig comprising 405 nm light-emitting diodes capable of delivering 220 W/m(2) at the sample, an appropriate filter and a digital camera; a set-up analogous to quantitative light-induced fluorescence digital. Longitudinal sets of images were captured and processed to assess the degradation in red fluorescence over time. RESULTS: Photobleaching was observed in all instances. The highest rates of photobleaching were observed immediately after initiation of illumination, specifically during the first minute. Relative rates of photobleaching during the first minute of exposure were 19.17, 13.72 and 3.43 arbitrary units/min for P. nigrescens biofilms, microcosm biofilm and P. intermedia biofilms, respectively. CONCLUSION: Photobleaching could be problematic when making quantitative measurements of porphyrin fluorescence in situ. Reducing both light levels and exposure time, in combination with increased camera sensitivity, should be the default approach when undertaking analyses by quantitative light-induced fluorescence digital.

A common neonatal image phenotype predicts adverse neurodevelopmental outcome in children born preterm.

Diffuse white matter injury is common in preterm infants and is a candidate substrate for later cognitive impairment. This injury pattern is associated with morphological changes in deep grey nuclei, the localization of which is uncertain. We test the hypotheses that diffuse white matter injury is associated with discrete focal tissue loss, and that this image phenotype is associated with impairment at 2years. We acquired magnetic resonance images from 80 preterm infants at term equivalent (mean gestational age 29(+6)weeks) and 20 control infants (mean GA 39(+2)weeks). Diffuse white matter injury was defined by abnormal apparent diffusion coefficient values in one or more white matter region (frontal, central or posterior white matter at the level of the centrum semiovale), and morphological difference between groups was calculated from 3D images using deformation based morphometry. Neurodevelopmental assessments were obtained from preterm infants at a mean chronological age of 27.5months, and from controls at a mean age of 31.1months. We identified a common image phenotype in 66 of 80 preterm infants at term equivalent comprising: diffuse white matter injury; and tissue volume reduction in the dorsomedial nucleus of the thalamus, the globus pallidus, periventricular white matter, the corona radiata and within the central region of the centrum semiovale (t=4.42 p<0.001 false discovery rate corrected). The abnormal image phenotype is associated with reduced median developmental quotient (DQ) at 2years (DQ=92) compared with control infants (DQ=112), p<0.001. These findings indicate that specific neural systems are susceptible to maldevelopment after preterm birth, and suggest that neonatal image phenotype may serve as a useful biomarker for studying mechanisms of injury and the effect of putative therapeutic interventions.

Structural characterization by x-ray methods of novel antimicrobial gallium-doped phosphate-based glasses.

Antimicrobial gallium-doped phosphate-based glasses of general composition (P(2)O(5))(0.45)(CaO)(0.16)(Na(2)O)(0.39-x)(Ga(2)O(3))(x) (where x=0, 0.01, 0.03, and 0.05) have been studied using the advanced synchrotron-based techniques of Ga K-edge x-ray absorption spectroscopy and high-energy x-ray diffraction to provide a structural insight into their unique properties. The results show that the Ga(3+) ions are octahedrally coordinated. Furthermore, substitution of Na(2)O by Ga(2)O(3) strengthens the phosphate network structure because the presence of GaO(6) octahedra inhibits the migration of the remaining Na(+) ions. The results are discussed in terms of the use of Na(2)O-CaO-P(2)O(5) glasses as controlled-delivery devices for antimicrobial Ga(3+) ions in biomedical applications. We are thereby able to relate the atomic-scale environment of the Ga(3+) ions beneficially to the glass dissolution, and thus to their ability to disrupt bacterial cell activity by usurping the role of iron.

Controlled delivery of antimicrobial gallium ions from phosphate-based glasses.

Gallium-doped phosphate-based glasses (PBGs) have been recently shown to have antibacterial activity. However, the delivery of gallium ions from these glasses can be improved by altering the calcium ion concentration to control the degradation rate of the glasses. In the present study, the effect of increasing calcium content in novel gallium (Ga2O3)-doped PBGs on the susceptibility of Pseudomonas aeruginosa is examined. The lack of new antibiotics in development makes gallium-doped PBG potentially a highly promising new therapeutic agent. The results show that an increase in calcium content (14, 15 and 16 mol.% CaO) cause a decrease in degradation rate (17.6, 13.5 and 7.3 microg mm(-2) h(-1)), gallium ion release and antimicrobial activity against planktonic P. aeruginosa. The most potent glass composition (containing 14 mol.% CaO) was then evaluated for its ability to prevent the growth of biofilms of P. aeruginosa. Gallium release was found to reduce biofilm growth of P. aeruginosa with a maximum effect (0.86 log(10) CFU reduction compared to Ga2O3-free glasses) after 48 h. Analysis of the biofilms by confocal microscopy confirmed the anti-biofilm effect of these glasses as it showed both viable and non-viable bacteria on the glass surface. Results of the solubility and ion release studies show that this glass system is suitable for controlled delivery of Ga3+. 71Ga NMR and Ga K-edge XANES measurements indicate that the gallium is octahedrally coordinated by oxygen atoms in all samples. The results presented here suggest that PBGs may be useful in controlled drug delivery applications, to deliver gallium ions in order to prevent infections due to P. aeruginosa biofilms.

Polyhydroxyalkanoate biosynthesis in Bacillus cereus SPV under varied limiting conditions and an insight into the biosynthetic genes involved.

AIMS: A new strain of Bacillus, Bacillus cereus SPV, was found to be capable of using a wide range of carbon sources for the production of polyhydroxyalkanoates (PHAs) (Valappil et al. 2007b). Limiting nutrient in the culture conditions is crucial for PHA production. In this study, B. cereus SPV was grown in different culture conditions with limitation of potassium, nitrogen, sulphur and phosphorous to establish the impact of nutritional limitation on PHA production. METHODS AND RESULTS: The PHA yields obtained were found to be 13.4, 38, 13.15 and 33.33% dcw for potassium, nitrogen, sulphur and phosphorus limitations, respectively. Gas chromatography-mass spectrometry analysis of the isolated polymers showed the presence of P(3HB) under nitrogen, sulphur and phosphate-limiting conditions and P(3HB-3HV) copolymer under potassium limiting conditions. This ability of B. cereus SPV to accumulate different PHA monomers from structurally unrelated carbon sources led to an interest in the molecular analysis of PHA biosynthesis in this organism. To achieve this, PCR was used to identify the polyhydroxyalkanoate biosynthetic genes in B. cereus SPV. CONCLUSION: Sequence analysis of the PCR products from B. cereus SPV revealed the sequence of the putative biosynthetic genes, and possible regions involved in substrate binding. The nucleotide sequence reported in this paper is in the GenBank nucleotide sequence database under accession number DQ486135. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report comparing the capability of B. cereus SPV to produce PHAs under different culture conditions of potassium, nitrogen, sulfur and phosphate limitations. The results in this study suggest the unique ability of B. cereus SPV to supply both 3HB and 3HV monomers from a structurally unrelated carbon source, glucose.

Large-scale production and efficient recovery of PHB with desirable material properties, from the newly characterised Bacillus cereus SPV.

A newly characterised Bacillus strain, Bacillus cereus SPV was found to produce PHB at a concentration of 38% of its dry cell weight in shaken flask cultures, using glucose as the main carbon source. Polymer production was then scaled up to 20 L batch fermentations where 29% dry cell weight of PHB was obtained within 48 h. Following this, a simple glucose feeding strategy was developed and the cells accumulated 38% dry cell weight of PHB, an increase in the overall volumetric yield by 31% compared to the batch fermentation. Sporulation is the cause of low PHB productivity from the genus Bacillus [Wu, Q., Huang, H., Hu, G.H., Chen, J., Ho, K.P., Chen, G.Q., 2001. Production of poly-3-hydroxybutyrate by Bacillus sp. JMa5 cultivated in molasses media. Antonie van Leeuwenhoek 80, 111-118]. However, in this study, acidic pH conditions (4.5-5.8) completely suppress sporulation, in accordance with Kominek and Halvorson [Kominek, L.A., Halvorson, H.O., 1965. Metabolism of poly-beta-hydroxybutyrate and acetoin in Bacillus cereus. J. Bacteriol. 90, 1251-1259], and result in an increase in the yield of PHB production. This observation emphasises the potential of the use of Bacillus in the commercial production of PHB and other PHAs. The recovery of the PHB produced was optimised and the isolated polymer characterised to identify its material properties. The polymer extracted, was found to have similar molecular weight, polydispersity index and lower crystallinity index than others reported in literature. Also, the extracted polymer was found to have desirable material properties for potential tissue engineering applications.

Effect of ternary phosphate-based glass compositions on osteoblast and osteoblast-like proliferation, differentiation and death in vitro.

There is currently a need to expand the range of graft materials available to orthopaedic surgeons. This study investigated the effect of ternary phosphate-based glass (PBG) compositions on the behaviour of osteoblast and osteoblast-like cells. PBGs of the formula (in mol.%) P(2)O(5)(50)-CaO(50-X)-Na(2)O(X), where X is either 2, 4, 6, 8 or 10, were produced and their influence on the proliferation, differentiation and death in vitro of adult human bone marrow stromal cells (hBMSCs) and human fetal osteoblast 1.19 (HFOB 1.19) cells were assessed. Tissue culture plastic (TCP) and hydroxyapatite (HA) were used as controls. Exposure to PBGs in culture inhibited cell adhesion and proliferation and increased cell death in both cell types studied. There was no significant difference in percentage cell death between the PBGs, which was significantly greater than the controls. However, compared with other PBGs, a greater number of cells were found on the 48mol.% CaO which may have been due to either increased adherence or proliferation, or both. This composition was capable of supporting osteogenic proliferation and early differentiation, and supports the notion that chemical modification of the glass could lead to a more biologically compatible substrate with the potential to support osteogenic grafting. Realisation of this potential should lead to the development of novel grafting strategies for the treatment of problematic bone defects.

Polyhydroxyalkanoate (PHA) biosynthesis from structurally unrelated carbon sources by a newly characterized Bacillus spp.

A newly acquired polyhydroxyalkanoate (PHA) producing Bacillus spp. was identified to be a strain of Bacillus cereus using a range of microbiological and molecular techniques. This strain, named B. cereus SPV, was found to be capable of using a wide range of carbon sources including glucose, fructose, sucrose, various fatty acids and gluconate for the production of PHAs, an advantage for the commercial production of the polymers. The media used for the polymer production was novel in the context of the genus Bacillus. The PHA, once produced, was found to remain at a constant maximal concentration, without any degradation, a great advantage for the commercial production of the PHAs. This particular strain of Bacillus spp. was able to synthesize various PHAs with 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV) and 4-hydroxybutyrate (4HB)-like monomer units from structurally unrelated carbon sources such as fructose, sucrose and gluconate. This is the first report of the incorporation of a 4HB related monomer containing PHA by the genus Bacillus and from structurally unrelated carbon sources. The PHAs isolated had molecular weights ranging between (0.4 and 0.8) x 10(6) and low polydispersity index values (M(W)/M(N)) ranging from 2.6 to 3.4.

Poly(3- hydroxybutyrate)/Bioglass(®) composite films containing carbon nanotubes.

Poly(3hydroxybutyrate) (P(3HB))/Bioglass(®) composites incorporating multiwalled carbon nanotubes (MWCNTs) have been successfully prepared by the solvent casting technique. The microstructure, electrical properties and bioactivity of the composites were characterized using scanning electron microscopy, x-ray diffraction and current-voltage measurements. Different concentrations of MWCNTs were used to determine their effect on the electrical properties of the composites. MWCNTs and Bioglass(®) particles were found to be homogeneously dispersed throughout the P(3HB) matrix. The electrical resistance of the composite samples decreased on increasing the MWCNT concentration, as expected. An in vitro degradation study in simulated body fluid (SBF) was carried out on composite samples. The formation of hydroxyapatite on the surfaces of P(3HB)/Bioglass(®)/MWCNT composite films was confirmed after two months of immersion in SBF. This hydroxyapatite layer was not formed on the neat polymeric films and on composites containing MWCNTs only (without Bioglass(®)). It was found that the presence of MWCNTs did not hinder the bioactivity of the Bioglass(®) particles, as confirmed by SEM and XRD studies on composite samples.

Ovarian metastasis from primary renal cell carcinoma: report of a case and review of literature.

BACKGROUND: Ovarian metastasis from renal cell adenocarcinoma is quite rare. They are often mistaken for primary clear cell tumours of the ovary because of histological similarity and propensity to appear years before or after the renal primary is detected. CASE: A case of ovarian metastasis from renal cell adenocarcinoma is reported. Patient developed bilateral ovarian metastasis 7 years after diagnosis of renal primary. She is alive 2 years after removal of ovarian tumours, with evidence of metastases to pelvic and paraaortic lymph nodes. Review of literature with analysis of 11 such reported cases in English literature is included. CONCLUSION: Although rare, possibility of metastasis from renal cell adenocarcinoma should be considered as a differential diagnosis in cases of clear cell ovarian tumours. Imaging studies are warranted in such cases to rule out renal tumour. It is important to differentiate between the two because of therapeutic and prognostic implications. Careful histological and histochemical analysis may help to differentiate between the two.