Hydrogen/deuterium exchange mass spectrometry for characterizing phosphoenolpyruvate-induced structural transitions in Mycobacterium tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.1)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Phosphate closes the solution structure of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Mycobacterium tuberculosis

The 5-enolpyruvylshikimate-3-phosphate synthase catalyses the sixth step of the shikimate pathway that is responsible for synthesizing aromatic compounds and is absent in mammals, which makes it a potential target for drugs development against microbial diseases. Here, we report the phosphate binding effects at the structure of the 5-enolpyruvyl shikimate-3-phosphate synthase from Mycobacterium tuberculosis. This enzyme is formed by two similar domains that close on each other induced by ligand binding, showing the occurrence of a large conformation change. We have monitored the phosphate binding effects using analytical ultracentrifugation, small angle X-ray scattering and, circular dichroism techniques. The low resolution results showed that the enzyme in the presence of phosphate clearly presented a more compact structure. Thermal-induced unfolding experiments followed by circular dichroism suggested that phosphate rigidified the enzyme. Summarizing, these data suggested that the phosphate itself is able to induce conformational change resulting in the closure movement in the M. tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase. (c) 2006 Elsevier B.V. All rights reserved.

Allosteric water and phosphate effects in Hoplosternum littorale hemoglobins

This paper reports the results obtained using the osmotic stress method applied to the purified cathodic and anodic hemoglobins (Hbs) from the catfish Hoplosternum littorale, a species that displays facultative accessorial air oxygenation. We demonstrate that water potential affects the oxygen affinity of H. littorale Hbs in the presence of an inert solute (sucrose). Oxygen affinity increases when water activity increases, indicating that water molecules stabilize the high-affinity state of the Hb. This effect is the same as that observed in tetrameric vertebrate Hbs. We show that both anodic and cathodic Hbs show conformational substrates similar to other vertebrate Hbs. For both Hbs, addition of anionic effectors, especially chloride, strongly increases the number of water molecules bound, although anodic Hb did not exhibit sensitivity to saturating levels of ATP. Accordingly, for both Hbs, we propose that the deoxy conformations coexist in at least two anion-dependent allosteric states, T-o and T-x, as occurs for human Hb. We found a single phosphate binding site for the cathodic Hb.

Photodynamic inactivation of biofilms formed by Candida spp., Trichosporon mucoides, and Kodamaea ohmeri by cationic nanoemulsion of zinc 2,9,16,23-tetrakis(phenylthio)-29H, 31H-phthalocyanine (ZnPc)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Morphology and bacterial colonisation of tooth/ceramic restoration interface after different cement excess removal techniques

Objectives: To evaluate the influence of different protocols for resin cement removal during cementation on biofilm formation.Methods: Twenty-eight ceramic blocks, which were injected under pressure, were placed over enamel blocks obtained from freshly extracted bovine incisors. The ceramic blocks were cemented to the enamel blocks using a dual-cured resin cement and the excess resin was removed according to the experimental group: TS: Teflon spatula; BR: brush; BR+: brush and polishing; SB+: scalpel blade and polishing. After autoclaving, the samples were colonised by incubation in a sucrose broth suspension standardised with Streptococcus mutans in microaerophilic stove. Specimens were quantitatively analysed for bacterial adherence at the adhesive interface using confocal laser scanning microscopy and counting the colony forming units, and qualitatively analysed using SEM. The roughness (Ra/Rz/RSm) was also analysed. Data were analysed by 1-way ANOVA and Tukey's test (5%).Results: The roughness values ranged from 0.96 to 1.69 mu m for Ra (p > 0.05), from 11.59 to 22.80 mu m for Rz (p = 0.02 < 0.05) and from 293.2 to 534.3 mu m for RSm (p = 0.00). Bacterial adhesion varied between 1,974,000 and 2,814,000 CFU/ml (p = 0.00). Biofilm mean thickness ranged from 0.477 and 0.556 mu m (p > 0.05), whilst the biovolume values were between 0.388 and 0.547 mu m(3)/mu m(2) (p = 0.04). Lower values for roughness, bacterial adhesion, biofilm thickness and biovolume were found with BR, whilst TS presented the highest values for most of the parameters. SEM images confirmed the quantitative values.Conclusions: The restoration margin morphology and interface roughness affects bacterial accumulation. The brush technique promoted less bacterial colonisation at the adhesive interface than did the other removal methods.Clinical significance: The brush technique seems to be a good option for removing the excess resin cement after adhesive cementation in clinical practice, as indicated by its better results with lower bacterial colonisation. (C) 2012 Elsevier Ltd. All rights reserved.

Effect of Cement Type, Relining Procedure, and Length of Cementation on Pull-out Bond Strength of Fiber Posts

Introduction: As opposed to the cementation metal posts, the cementation of fiber posts has several details that can significantly influence the success of post retention. This study evaluated the effect of the relining procedure, the cement type, and the luted length of the post on fiber posts retention. Methods: One hundred eighty bovine incisors were selected to assess post retention; after endodontic treatment, the canals were flared with diamonds burs. Post holes were prepared in lengths of 5, 7.5, and 10 mm; the fiber posts were relined with composite resin and luted with RelyX ARC, RelyX Unicem, or RelyX Luting 2. All cements are manufactured by 3M ESPE (St. Paul, MN). Samples were subjected to a pull-out bond strength test in a universal testing machine; the results (N) were submitted to a three-way analysis of variance and the Tukey post hoc test (alpha = 0.05). Results: The improvement of post retention occurred with the increase of the post length luted into the root canal; the relining procedure improved the pull-out bond strength. RelyX Unicem and RelyX ARC showed similar values of retention, both showing higher values than RelyX Luting 2. Conclusion: Post length, the reining procedure, and the cement type are all important factors for improving the retention of fiber posts. (J Endod 2010;36:1543-1546)

Does the Ultrasonic Cleaning Medium Affect the Adhesion of Resin Cement to Feldspathic Ceramic?

Purpose: To evaluate the effect of different cleaning media on the adhesion of resin cement to feldspathic ceramic after etching.Materials and Methods: The cementation surfaces of ceramic blocks (N = 20, n = 5 per group) were etched with 10% hydrofluoric acid (HF) gel for 20 s and rinsed for 60 s. They were then randomly assigned to 4 groups: G1: air-water spray+drying (control); G2: ultrasonic cleaning in distilled water for 4 min+drying; G3: ultrasonic cleaning in 99.5% acetone for 4 min+drying; G4: ultrasonic cleaning in 70% alcohol for 4 min+drying. The ceramic blocks were silanized and cemented (RelyX ARC) to the composite blocks. Subsequently, the microtensile bond strength test (mu TBS) was performed. In addition, EDS analysis was made to assess the elemental composition of the conditioned and cleaned ceramic surfaces.Results: A significantly higher mean mu TBS was obtained when specimens had been ultrasonically cleaned in distilled water (G2: 18.8 +/- 0.4 MPa) (p < 0.05) compared to other groups (G1: 16.6 +/- 0.5; G3: 16.1 +/- 0.9; G4: 15.8 +/- 1.4) (one-way ANOVA). EDS analysis indicated the presence of F- only in G1. Dissolved precipitates after HF etching were removed by ultrasonic cleaning.Conclusion: Cleaning the HF-etched ceramic surface ultrasonically in distilled water is recommended, instead of rinsing it with air-water spray only.

Effect of Cement Shade and Light-curing Unit on Bond Strength of a Ceramic Cemented to Dentin

Purpose: To evaluate the effect of cement shade, light-curing unit, and water storage on tensile bond strength (a) of a feldspathic ceramic resin bonded to dentin.Materials and Methods: The dentin surface of 40 molars was exposed and etched with 37% phosphoric acid, then an adhesive system was applied. Forty blocks of feldspathic ceramic (Vita VM7) were produced. The ceramic surface was etched with 10% hydrofluoric acid for 60 s, followed by the application of a silane agent and a dual-curing resin cement (Variolink II). Ceramic blocks were cemented to the treated dentin using either A3 or transparent (Tr) shade cement that was activated using either halogen or LED light for 40 s. All blocks were stored in 37 degrees C distilled water for 24 h before cutting to obtain non-trimmed bar-shaped specimens (adhesive area = 1 mm(2) +/- 0.1) for the microtensile bond strength test. The specimens were randomly grouped according to the storage time: no storage or stored for 150 days in 37 degrees C distilled water. Eight experimental groups were obtained (n = 30). The specimens were submitted to the tensile bond strength test using a universal testing machine at a crosshead speed of 1 mm/min. The data were statistically analyzed using ANOVA and Tukey's post-hoc tests (alpha = 0.05).Results: The mean bond strength values were significantly lower for the corresponding water stored groups, except for the specimens using A3 resin cement activated by halogen light. There was no significance difference in mean bond strength values among all groups after water storage.Conclusion: Water storage had a detrimental effect under most experimental conditions. For both cement shades investigated (Tr and A3) under the same storage condition, the light-curing units (QTH and LED) did not affect the mean microtensile bond strengths of resin-cemented ceramic to dentin.

Hardening of a dual-cure resin cement using QTH and LED curing units

Objective: This study evaluated the surface hardness of a resin cement (RelyX ARC) photoactivated through indirect composite resin (Cristobal) disks of different thicknesses using either a light- emitting diode (LED) or quartz tungsten halogen (QTH) light source. Material and Methods: Eighteen resin cement specimens were prepared and divided into 6 groups according to the type of curing unit and the thickness of resin disks interposed between the cement surface and light source. Three indentations (50 g for 15 s) were performed on the top and bottom surface of each specimen and a mean Vickers hardness number (VHN) was calculated for each specimen. The data were analyzed using two-way ANOVA and Tukey-Kramer test was used for post-hoc pairwise comparisons. Results: Increased indirect resin disk thickness resulted in decreased mean VHN values. Mean VHN values for the top surfaces of the resin cement specimens ranged from 23.2 to 46.1 (QTH) and 32.3 to 41.7 (LED). The LED curing light source produced higher hardness values compared to the QTH light source for 2- and 3-mm-thick indirect resin disks. The differences were clinically, but not statistically significant. Increased indirect resin disk thickness also resulted in decreased mean VHN values for the bottom surfaces of the resin cement: 5.8 to 19.1 (QTH) and 7.5 to 32.0 (LED). For the bottom surfaces, a statistically significant interaction was also found between the type of curing light source and the indirect resin disk thickness. Conclusions: Mean surface hardness values of resin cement specimens decreased with the increase of indirect resin disk thickness. The LED curing light source generally produced higher surface hardness values.

Surface degradation of glass ceramics after exposure to acidulated phosphate fluoride

Objective: This study evaluated the surface degradation effect of acidulated phosphate fluoride (APF) gel exposure on the glassy matrix ceramics as a function of time. Material and methods: Disc-shaped ceramic specimens (N = 120, 10/per ceramic material) were prepared in stainless steel molds (inner diameter: 5 mm, height: 2 mm) using 6 dental ceramics: 3 indicated for ceramic-fused-to-metal (Vita Omega 900, Carmen and Vita Titankeramik), 2 for all-ceramic (Vitadur Alpha and Finesse (R) Low Fusing) and 1 for both types of restorations (IPS d. SIGN). The specimens were wet ground finished, ultrasonically cleaned and auto-glazed. All specimens were subjected to calculation of percentage of mass loss, surface roughness analysis and topographical description by scanning electron microscopy (SEM) before (0 min) and after exposure to 1.23 % APF gel for 4 min and 60 min representing short-and long-term etching effect, respectively. The data were analyzed using two-way ANOVA with repeated measures and Tukey` s test (alpha=0.05). Results: Significant effect of the type of the ceramics (p=0.0000, p=0.0031) and exposure time (p=0.0000) was observed in both surface roughness and percentage of mass loss values, respectively. The interaction factor between both parameters was also significant for both parameters (p=0.0904, p=0.0258). Both 4 min (0.44 +/- 0.1-0.81 +/- 0.2 mu m) and 60 min (0.66 +/- 0.1 - 1.04 +/- 0.3 mu m) APF gel exposure created significantly more surface roughness for all groups when compared to the control groups (0.33 +/- 0.2-0.68 +/- 0.2 mu m) (p<0.05). There were no significant differences in percentage of mass loss between the ceramics at 4 min (p>0.05) but at 60 min exposure, IPS d. SIGN showed the highest percentage of mass loss (0.1151 +/- 0.11). The mean surface roughness for Vita Titankeramik (0.84 +/- 0.2 mu m) and Finesse (R) Low Fusing (0.74.+/- 0.2 mu m) was significantly higher than those of the other ceramics (0.59 +/- 0.1 mu m - 0.49 +/- 0.1 mu m) and Vita Titankeramik (p<0.05) regardless of the exposure time. A positive correlation was found between surface roughness and percentage of mass loss for all ceramic materials [(r=0.518 (Vitadur Alpha), r=0.405 (Vita Omega 900), r=0.580 (Carmen), r=0.687 (IPS d. SIGN), r=0.442 (Finesse (R) Low Fusing), r=0.572 (Vita Titankeramik), Pearson's correlation coefficient)]. The qualitative SEM analysis showed evidence of corrosive attack on all of ceramics at varying degrees. Conclusions: The ceramics indicated for either metal-ceramic or all-ceramic restorations were all vulnerable to surface texture changes and mass loss after short-term and long-term APF gel exposure.