Antimicrobial photodynamic action on dentin using a light-emitting diode light source

Objective: The aim of this study was the evaluation of two different photosensitizers activated by red light emitted by light-emitting diodes (LEDs) in the decontamination of carious bovine dentin. Materials and Methods: Fifteen bovine incisors were used to obtain dentin samples which were immersed in brain-heart infusion culture medium supplemented with 1% glucose, 2% sucrose, and 1% young primary culture of Lactobacillus acidophilus 108 CFU/mL and Streptococcus mutans 108 CFU/mL for caries induction. Three different concentrations of the Photogem solution, a hematoporphyrin derivative (1, 2, and 3 mg/mL) and two different concentrations of toluidine blue O (TBO), a basic dye (0.025 and 0.1 mg/mL) were used. To activate the photosensitizers two different light exposure times were used: 60 sec and 120 sec, corresponding respectively to the doses of 24 J/cm(2) and 48 J/cm(2). Results: After counting the numbers of CFU per milligram of carious dentin, we observed that the use of LED energy in association with Photogem or TBO was effective for bacterial reduction in carious dentin, and that the greatest effect on S. mutans and L. acidophilus was obtained with TBO at 0.1 mg/mL and a dose of 48 J/cm(2). It was also observed that the overall toxicity of TBO was higher than that of Photogem, and that the phototoxicity of TBO was higher than that of Photogem. Conclusion: Based on our data we propose a mathematical model for the photodynamic effect when different photosensitizer concentrations and light doses are used.

Crystallization and preliminary X-ray diffraction analysis of recombinant chlorocatechol 1,2-dioxygenase from Pseudomonas putida

Chlorocatechol 1,2-dioxygenase from the Gram-negative bacterium Pseudomonas putida (Pp 1,2-CCD) is considered to be an important biotechnological tool owing to its ability to process a broad spectrum of organic pollutants. In the current work, the crystallization, crystallographic characterization and phasing of the recombinant Pp 1,2-CCD enzyme are described. Reddish-brown crystals were obtained in the presence of polyethylene glycol and magnesium acetate by utilizing the vapour-diffusion technique in sitting drops. Crystal dehydration was the key step in obtaining data sets, which were collected on the D03B-MX2 beamline at the CNPEM/MCT - LNLS using a MAR CCD detector. Pp 1,2-CCD crystals belonged to space group P6(1)22 and the crystallographic structure of Pp 1,2-CCD has been solved by the MR-SAD technique using Fe atoms as scattering centres and the coordinates of 3-chlorocatechol 1,2-dioxygenase from Rhodococcus opacus (PDB entry 2boy) as the search model. The initial model, which contains three molecules in the asymmetric unit, has been refined to 3.4 A resolution.

A Component of the Xanthomonadaceae Type IV Secretion System Combines a VirB7 Motif with a N0 Domain Found in Outer Membrane Transport Proteins

Type IV secretion systems (T4SS) are used by Gram-negative bacteria to translocate protein and DNA substrates across the cell envelope and into target cells. Translocation across the outer membrane is achieved via a ringed tetradecameric outer membrane complex made up of a small VirB7 lipoprotein (normally 30 to 45 residues in the mature form) and the C-terminal domains of the VirB9 and VirB10 subunits. Several species from the genera of Xanthomonas phytopathogens possess an uncharacterized type IV secretion system with some distinguishing features, one of which is an unusually large VirB7 subunit (118 residues in the mature form). Here, we report the NMR and 1.0 angstrom X-ray structures of the VirB7 subunit from Xanthomonas citri subsp. citri (VirB7(XAC2622)) and its interaction with VirB9. NMR solution studies show that residues 27-41 of the disordered flexible N-terminal region of VirB7(XAC2622) interact specifically with the VirB9 C-terminal domain, resulting in a significant reduction in the conformational freedom of both regions. VirB7(XAC2622) has a unique C-terminal domain whose topology is strikingly similar to that of N0 domains found in proteins from different systems involved in transport across the bacterial outer membrane. We show that VirB7(XAC2622) oligomerizes through interactions involving conserved residues in the N0 domain and residues 42-49 within the flexible N-terminal region and that these homotropic interactions can persist in the presence of heterotropic interactions with VirB9. Finally, we propose that VirB(7XAC2622) oligomerization is compatible with the core complex structure in a manner such that the N0 domains form an extra layer on the perimeter of the tetradecameric ring.

Novel Isolates for Biological Detoxification of Lignocellulosic Hydrolysate

In this paper, two new strians, Issatchenkia occidentalis (Lj-3, CCTCC M 2006097) and Issatchenkia orienalis (S-7, CCTCC M 2006098), isolated from different environments on solid media, were used in the detoxification process of the hemicellulosic hydrolysate of sugarcane bagasse. High-pressure liquid chromatography elution curve of UV-absorption compounds represented by acetic acid, furfural, and guaiacol (toxic compounds found in the hemicellulosic hydrolysate) showed that several chromatographic peaks were evidently diminished for the case of detoxified hydrolysate with isolate strains compared to the high peaks resulted for no detoxified hydrolysate. It was clear that these inhibitors were degraded by the two new isolates during their cultivation process. Fermentation results for the biodetoxified hydrolysate showed an increase in xylitol productivity (Q (p)) by 1.97 and 1.95 times (2.03 and 2.01 g l(-1) h(-1)) and in xylitol yield (Y (p)) by 1.72 and 1.65 times (0.93 and 0.89 g xylitol per gram xylose) for hydrolysate treated with S-7 and Lj-3, respectively, in comparison with no detoxified hydrolysate (1.03 g l(-1) h(-1) and 0.54 g xylitol per gram xylose). This present work demonstrated the importance of Issatchenkia yeast in providing an effective biological detoxification approach to remove inhibitors and improve hydrolysate fermentability, leading to a high xylitol productivity and yield.

Effect of fibre morphology on flocculation of fibre-cement suspensions

The objective of the present research was to evaluate the effect of fibre morphology (e.g., length, width, fibrillation, broken ends, content of fines and number of fibres per gram) on flocculation and drainage properties of fibre-cement suspensions and on physical properties of the fibre-cement composites. Mechanical refining was used to change the morphological properties of Eucalyptus and Pinus pulps. Results show that the mechanical refining increased the size of the formed flocs and decreased the concentration of free small particles (with dimensions between 1 and 20 pm) as a consequence of the increased fibrillation and content of fines, which increased the capacity of the fibres to capture the mineral particles. High levels of refining were necessary for Pinus pulp to obtain cement retention values similar to those obtained by unrefined Eucalyptus pulp. This is due to the higher number of fibres per gram in Eucalyptus pulp than in Pinus pulp. Pulp refining improved the packing of the particles and, although decreased the drainage rate. it contributed to a less porous structure, which improved the microstructure of the composite. (C) 2009 Elsevier Ltd. All rights reserved.

Performance evaluation and phylogenetic characterization of anaerobic fluidized bed reactors using ground tire and pet as support materials for biohydrogen production

This study evaluated two different support materials (ground tire and polyethylene terephthalate [PET]) for biohydrogen production in an anaerobic fluidized bed reactor (AFBR) treating synthetic wastewater containing glucose (4000 mg L(-1)). The AFBR, which contained either ground tire (R1) or PET (R2) as support materials, were inoculated with thermally pretreated anaerobic sludge and operated at a temperature of 30 degrees C. The AFBR were operated with a range of hydraulic retention times (HRT) between 1 and 8 h. The reactor R1 operating with a HRT of 2 h showed better performance than reactor R2, reaching a maximum hydrogen yield of 2.25 mol H(2) mol(-1) glucose with 1.3 mg of biomass (as the total volatile solids) attached to each gram of ground tire. Subsequent 16S rRNA gene sequencing and phylogenetic analysis of particle samples revealed that reactor R1 favored the presence of hydrogen-producing bacteria such as Clostridium, Bacillus, and Enterobacter. (C) 2010 Elsevier Ltd. All rights reserved.

Anaerobic degradation of linear alkylbenzene sulfonate (LAS) in fluidized bed reactor by microbial consortia in different support materials

Four anaerobic fluidized bed reactors filled with activated carbon (R1), expanded clay (R2), glass beads (R3) and sand (R4) were tested for anaerobic degradation of LAS. All reactors were inoculated with sludge from a UASB reactor treating swine wastewater and were fed with a synthetic substrate supplemented with approximately 20 mg l(-1) of LAS, on average. To 560 mg l(-1) COD influent, the maximum COD and LAS removal efficiencies were mean values of 97 +/- 2% and 99 +/- 2%, respectively, to all reactors demonstrating the potential applicability of this reactor configuration for treating LAS. The reactors were kept at 30 degrees C and operated with a hydraulic retention time (HRT) of 18 h. The use of glass beads and sand appear attractive because they favor the development of biofilms capable of supporting LAS degradation. Subsequent 16S rRNA gene sequencing and phylogenetic analysis of samples from reactors R3 and R4 revealed that these reactors gave rise to broad microbial diversity, with microorganisms belonging to the phyla Bacteroidetes, Firmicutes, Actinobacteria and Proteobacteria, indicating the role of microbial consortia in degrading the surfactant LAS. (C) 2010 Elsevier Ltd. All rights reserved.

Host status of citrus rootstocks for the population K5 of Pratylenchus jaehni

Host status of citrus rootstocks for the population K 5 of Pratylenchus jaehni Pratylenchus jaehni has been reported as the main nematode in Brazilian citrus orchards due to the economic losses it causes. Considering the reports of differences and similarities in populations from that species, the host reaction of citrus rootstocks to K 5 population of P. jaehni was evaluated and compared with topotypes of the species. Two experiments were carried out with initial population (Pi) of 180 and 200 specimens per plant. In each experiment, the evaluation was carried out in two periods: at 120 and 245 days after inoculation (DAI) in experiment 1 and at 60 and 240 DAI in experiment 2. The variables obtained were the reproduction factor [RF = final population (Pf)/Pi)] and nematodes per gram of roots (N/g). The nematode density was reduced (RF = 0.00 to 0.02) in trifoliata `Limeira`, sour orange, citrange `Carrizo`, `Cleopatra` tangerine, `Sunki` tangerine, `Volkameriano` lemon and citrumelo `Swingle`; but the nematode density increased (RF = 1.89 to 22.28) in Rangpur lime in both periods of experiment 1 and in the second period of experiment 2. Therefore, the host reaction of citrus rootstocks to K 5 was very similar to that reported to topotypes of P. jaehni in the literature.

Resistance of Santa Ines and crossbred ewes to naturally acquired gastrointestinal nematode infections

This trial was carried out in Piracicaba, Sao Paulo State. Brazil. to comparatively evaluate the degree of resistance to naturally acquired gastrointestinal nematode infections in sheep of the following genetic groups purebred Santa Ines (SI), SI crossbred with Dorper (DO x SI), lie de France (IF x SI), Suffolk (SU x SI), and Texel (TE < SI) Fifteen ewes from each group were raised indoors until 12 months of age. At this age, they were moved to pasture that was naturally contaminated by nematode infective larvae and were evaluated from December to May. 2007. Rainfall ranged from 267 mm in January to 37 mm in April Maximum and minimum mean temperatures ranged from 32 5 degrees C to 19 0 degrees C in March and from 25.9 degrees C to 12.8 degrees C in May. There was an increase in the mean number of eggs per gram of feces (EPG) after animals were placed on pasture with significant difference between the SI (80 EPG) and IF x SI (347 EPG) groups in January: and the DO x SI (386 EPG) and TE x SI (258 EPG) groups in May. The highest mean fecal egg count (FEC), 2073 EPG, was recorded for the TE x SI group in February. All groups showed a progressive reduction in body weight throughout the experiment of 12.0% (TE x SI) to 15.9% (SU x SI). In general. the animals with the highest FEC presented the lowest packed cell volumes (PCV): the highest correlation coefficient between FEC x PCV occurred in the SU x SI sheep in January (r = -0.70; P < 0.01). Similarly, there was an inverse relationship between FEC and blood eosinophil Values, with the highest correlation coefficient in the TE x SI sheep in February (r = -0.64; P < 0.05). Immunoglobulin G (IgG) levels against Haemonchus contortus antigens increased in all groups as a result of the exposure to parasites and remained relatively constant until the end of the study, with the exceptions of SU x SI and TE x SI, which showed a rise in IgG levels during the last sampling that coincided with a reduction in mean FEC. In conclusion. crossbreeding Santa Ines sheep with any of the breeds evaluated can result in a production increase and the maintenance of a satisfactory degree of infection resistance, especially against H. contortus and Trichostrongylus colubriformis. the major nematodes detected in this flock. (c) 2009 Elsevier B.V. All rights reserved.

Apparent digestibility coefficients of selected feed ingredients for dourado Salminus brasiliensis

The excellence of its flesh and fast growth makes the dourado, Salminus brasiliensis, a carnivorous fish native to the Prata basin, a potential candidate for intensive fish farming. This study evaluated the apparent digestibility coefficients (ADC) of energy and nutrients of animal and plant protein sources for the carnivorous Characin dourado S. brasiliensis. Fish (19.5 +/- 5.0 g) were stocked in plastic cages (80-L) and fed pelleted test diets containing 30% of the test ingredient [fish meal (FM), poultry by-product meal (PBM), soybean meal (SBM), and corn gluten meal (CGM) plus 70% of a reference diet (481.4 g kg(-1) of crude protein and 18.63 kJ of gross energy per gram]. After the last daily meal, cages were transferred to cylindrical, conical-bottomed aquarium (200-L) where faeces were collected by sedimentation in a refrigerated container. Except for ADC of protein and energy, all other ADC of nutrients showed significant differences (P < 0.01). ADC values were: 94.3%, 91.3%, 93.1%, and 93.5% for crude protein; 91.0%, 90.3%, 87.8%, and 88.8% for gross energy; 92.1%, 84.5%, 80.6%, and 79.3% for ash; 83.9%, 80.3%, 84.3%, and 84.6% for dry matter; 97.4%, 96.7%, 93.3%, and 91.5% for lipid for FM, PBM, SBM, and CGM, respectively. The average amino acid ADC was: 93.6%, 90.0%, 92.1%, and 92.5% of FM, PBM, SBM, and CGM, respectively. All test ingredients were efficiently used and can partially replace FM in diets for carnivorous dourado.

Long-Chain Acyl-Homoserine Lactones from Methylobacterium mesophilicum: Synthesis and Absolute Configuration

The acyl-homoserine lactones (acyl-HSLs) produced by Methylobacterium mesophilicum isolated from orange trees infected with the citrus variegated chlorosis (CVC) disease have been studied, revealing the occurrence of six long-chain acyl-HSLs, i.e., the saturated homologues (S)-N-dodecanoyl (1) and (S)-N-tetradecanoyl-HSL (5), the uncommon odd-chain N-tridecanoyl-HSL (3), the new natural product (S)-N-(2E)-dodecenoyl-HSL (2), and the rare unsaturated homologues (S)-N-(7Z)-tetradecenoyl (4) and (S)-N-(2E,7Z)-tetradecadienyl-HSL (6). The absolute configurations of all HSLs were determined as 3S. Compounds 2 and 6 were synthesized for the first time. Antimicrobial assays with synthetic acyl-HSLs against Gram-positive bacterial endophytes co-isolated with M. mesophilicum from CVC-infected trees revealed low or no antibacterial activity.

Characterisation of an antiviral pediocin-like bacteriocin produced by Enterococcus faecium

The bacteriocin-producing strain Enterococcus faecium ST5Ha was isolated from smoked salmon and identified by biomolecular techniques. Ent. faecium ST5Ha produces a pediocin-like bacteriocin with activity against several lactic acid bacteria, Listeria spp. and some other human and food pathogens, and remarkably against HSV-1 virus. Bacteriocin ST5Ha was produced at high levels in MRS broth at 30 degrees C and 37 degrees C, reaching a maximum production of 1.0 x 10(9) AU/ml, checked against Listeria ivanovii ATCC19119 as target strain and surrogate of pathogenic strain Listeria monocytogenes. The molecular weight of bacteriocin ST5Ha was estimated to be 4.5 kDa according to tricine-SDS-PAGE data. Ent. faecium ST5Ha harbors a 1.044 kb chromosomal DNA fragment fitting in size to that of pediocin PA-1/AcH. In addition, the sequencing of bacteriocin ST5Ha gene indicated 99% of DNA homology to pediocin PA-1/AcH. The combined application of low levels (below MIC) of ciprofloxacin and bacteriocin ST5Ha resulted in a synergetic effect in the inhibition of target strain L ivanovii ATCC19119. Bacteriocin ST5Ha displayed antiviral activity against HSV-1, an important human pathogen, with a selectivity index of 173. To the best of our knowledge, this is the first report on Ent. faecium as a potential producer of pediocin-like bacteriocin with antiviral activity. (C) 2010 Elsevier Ltd. All rights reserved.

Dissemination of bla(IMP-1)-carrying integron In86 among Klebsiella pneumoniae isolates harboring a new trimethoprim resistance gene dfr23

The genetic context of the bla(IMP-1) gene was evaluated in 9 Klebsiella pneumoniae isolates recovered from 2 hospitals in Sao Paulo, Brazil. All isolates harbored a copy of In86 carrying bla(Imp-1), aac(6`)-31, and aadAl. Eight strains from the same hospital also carried another class I integron harboring a new trimethoprim resistance gene (dfr23) that was chromosomally embedded. In86 was likely to be in a 30-kb nontransferable plasmid and was flanked upstream by a sequence identical to one identified in an IMP-1-producing Pseudomonas putida isolate. The bla(IMP-1)-carrying integron In86 was recently reported from nonfermentative bacilli isolated in Sao Paulo. These isolates appear to be the Source of this integron now acquired by K. pneumoniae strains from different hospitals in the same city. Metallo-beta-lactamase production is still rare among Enterobacteriaceae isolates in Brazil, but the acquisition of genetic structures carrying these mobile resistance determinants is worrisome and could lead to an increase in the prevalence of these phenotypes of resistance. (C) 2008 Elsevier Inc. All rights reserved.

Heterologous expression of a thermophilic esterase in Kluyveromyces yeasts

In the present work, a thermophilic esterase from Thermus thermophilus HB27 was cloned into Kluyveromyces marxianus and into Kluyveromyces lactis using two different expression systems, yielding four recombinant strains. K. lactis showed the highest esterase expression levels (294 units per gram dry cell weight, with 65% of cell-bound enzyme) using an episomal system with the PGK promoter and terminator from Saccharomyces cerevisiae combined with the K. lactis k1 secretion signal. K. marxianus showed higher secretion efficiency of the heterologous esterase (56.9 units per gram dry cell weight, with 34% of cell-bound enzyme) than K. lactis. Hydrolytic activities for the heterologous esterases were maximum at pH values between 8.0 and 9.0 for both yeast species and at temperatures of 50 A degrees C and 45 A degrees C for K. marxianus and K. lactis, respectively. When compared to previously published data on this same esterase produced in the original host or in S. cerevisiae, our results indicate that Kluyveromyces yeasts can be considered good hosts for the heterologous secretion of thermophilic esterases, which have a potential application in biodiesel production or in resolving racemates.

Batch purification of high-purity lysozyme from egg white and characterization of the enzyme modified by PEGylation

PEGylation is one of the most promising and extensively studied strategies for improving the pharmacological properties of proteins as well as their physical and thermal stability. Purified lysozyme obtained from hen egg white by batch mode was modified by PEGylation with methoxypolyethyleneglycol succinimidyl succinato (mPEG-SS, MW 5000). The conjugates produced retained full enzyme activity with the substrate glycol chitosan, independent of degree of enzyme modification, although lysozyme activity with the substrate Micrococcus lysodeikticus was altered according to the degree of modification. The conjugate with a low degree of modification by mPEG-SS retained 67% of its enzyme activity with the M. lysodeikticus substrate. The mPEG-SS was also shown to be a highly reactive polymer. The effects of pH and temperature on PEGylated lysozymes indicated that the conjugate was active over a wide pH range and was stable up to 50 degrees C. This conjugate also showed resistance to proteolytic degradation, remained stable in human serum, and displayed greater antimicrobial activity than native lysozyme against Gram-negative bacteria.