Real-time Polymerase Chain Reaction Quantification of Porphyromonas gingivalis and Tannerella forsythia in Primary Endodontic Infections

Introduction: Porphyromonas gingivalis and Tannerella forsythia are anaerobic bacteria commonly involved in root canal infections. Although previous investigations have assessed these species by strictly qualitative approaches, accurate determination of their cell levels by a sensitive quantitative technique may contribute with additional information regarding relevance in pain of endodontic origin. Method: The root canal levels of P gingivalis, T forsythia, and total bacteria were investigated by a quantitative polymerase chain reaction (PCR) assay based on unique copy molecular markers. A total of 32 symptomatic (n = 14) and asymptomatic (n = 18) cases of endodontic infections were analyzed. Root canal samples were collected; genomic DNA was extracted and submitted to SYBR Green I real-time PCR targeting the rgpB (P gingivalis), bspA (T forsythia), and rpoB (total bacteria) single copy genes. Results: Overall, R gingivalis, T forsythia, and the coexistence of both species were encountered in 28%, 66%, and 22% of the subjects, respectively. P gingivalis and T forsythia levels ranged from 5.65 x 10(-6) to 1.20 x 10(-2) and from 5.76 x 10(-6) to 1.35 x 10(-1). T forsythia was highly prevalent and numerous in the study groups, whereas P gingivalis was moderately frequent and less abundant, displaying 19-fold lower average levels than the former. Conclusions: The endodontic levels of P gingivalis and T forsythia, individually or in conjunction, did not display significant associations with the manifestation of pain of endodontic origin. (J Endod 2009,35:1518-1524)

Digestibility of plant protein-based diets by largemouth bass Micropterus salmoides

Commercial farming of carnivorous fish demands the reduction of environmental impact of feeds; that requires minimal use of dietary animal protein. This study investigated the digestibility of diets formulated exclusively out of plant protein, added feed attractants, by the carnivore largemouth bass, Micropterus salmoides. Juvenile largemouth bass (14.0 +/- 1.0 cm) conditioned to accept artificial, dry feed were confined in polypropylene cages and fed ad libitum in three daily meals, seven experimental diets containing varying levels of vegetable and animal protein sources, added of different feed stimulants. After last daily meal, cages were transferred to cylindrical-conical-bottomed, 200-L aquaria, where faeces were collected by sedimentation into refrigerated containers, preserved and later analysed for chemical composition. Soybean meal can be used as partial substitute of animal protein in diets for largemouth bass; the poultry by-product meal shows as a good option as animal protein source in these rations. Control treatment - 50PP : 50AP - yielded best performances; the need for the use of fish meal in the formulation for carnivorous diets is, at least, questionable. Results of the digestibility trials demonstrated the importance of determining the diet digestibility, if precision in the formulation of least-cost feeds for carnivorous fish is the ultimate goal.

Outcrossing rate in sweet passion fruit based on molecular markers

P>Yellow and sweet passion fruit are insect-pollinated species native to the tropics. Fruits are used commercially for human consumption worldwide. The yellow passion fruit is an outcrossing species with self-incompatible flowers. However, the reproductive system of the sweet passion fruit (Passiflora alata) has not been well elucidated. The objective of this work was to characterize aspects of the mating system in the sweet passion fruit using random amplified polymorphic DNA (RAPD) and microsatellite markers, particularly the rate of outcrossing in P. alata progenies. A multilocus outcrossing rate of t(m) = 0.994 was determined from RAPD and t(m) = 0.940 from microsatellites, supporting P. alata as an outcrossing species. The fixation indices of the maternal generation (F(m)) were -0.200 and 0.071 with RAPD and microsatellite loci, respectively, indicating the absence of inbreeding in the maternal generation. The paternity correlation (r(p)) varied from -0.008 with RAPD markers to 0.208 with microsatellite markers, suggesting a low probability of finding full sibs within the progenies. The results demonstrated that all progenies assessed in this study were derived from outcrossing.

A mosaic of beach bean (Canavalia rosea) caused by an isolate of Cowpea aphid-borne mosaic virus (CABMV) in Brazil

Beach bean (Canavalia rosea) plants showing mosaic symptoms were found at Massaguacu beach, Caraguatatuba, Brazil. A potyvirus was found to be responsible for the symptoms, based on transmission assays and electron microscopy. A positive reaction in ELISA was obtained against cowpea aphid-borne mosaic (CABMV) antisera. Viral identity was confirmed by RT-PCR using specific primers to amplify part of the NIb and the entire CP coding region of the genome and the 3`NTR. Comparison of the amplified sequences with that of CABMV showed a nucleotide sequence identity of 97% for the CP coding region. Thus, the potyvirus from beach bean should be considered a CABMV isolate, referred to as CABMV-Cr.

Orchid fleck symptoms may be caused naturally by two different viruses transmitted by Brevipalpus

Brevipalpus-transmitted viruses (BTV) cause chlorotic, necrotic and/or ringspot lesions in leaves and stems of orchids, citrus, coffee and several other plant species. There are two different types of BTVs, the nuclear and the cytoplasmic, based on maturation locale in the cell and particle morphology. The orchid fleck virus (OFV) is a BTV that infects orchids. Its short rodlike particles are 32-40 nm in diameter, 100-150 nm in length. OFV is found in the nucleus and is associated with intranuclear electronlucent viroplasms. In 1999, transmission electron microscopy analysis revealed a distinct type of virus causing orchid fleck symptoms. The bacilliform particles, 70-80 nm in diameter and 110-120 nm in length, induced electron-dense viroplasm inclusions in infected cells and resembled the cytoplasmic type associated with BTV, such as the citrus leprosis virus C. Our objective in the present study was to verify whether the cytoplasmic type virus found in orchids could be amplified using primers for other cytoplasmic BTVs, such as CiLV-C and Solanum violaefolium ringspot virus (SvRSV). Additionally, we aimed to differentiate the two BTVs found in orchids: the nuclear and the cytoplasmic types of OFV using microscopy and molecular and serological tools. This virus was not amplified by the CiLV-C and SvRSV primers, and neither the molecular nor the serological tools available to the OFV diagnosis reacted with it, demonstrating that they are definitely different viruses.

Evaluation of the genetic variability of orchid fleck virus by single-strand conformational polymorphism analysis and nucleotide sequencing of a fragment from the nucleocapsid gene

The variability of a fragment of the nucleocapsid gene of orchid fleck virus (OFV) was investigated by single-strand conformational polymorphism (SSCP) analysis and nucleotide sequencing. Forty-eight samples of 18 genera of orchids were collected from Brazil, Costa Rica and Australia. The SSCP analysis yielded six different band patterns, and phylogenetic analysis based on the nucleotide fragment sequence obtained in this work and six available in GenBank showed two different groups, one with isolates 023Germany and So-Japan, and other with the rest of the isolates. None of the analyses showed geographic correlation among the Brazilian strains. The data obtained in this study showed a low genetic variation in this region of the genome; the d(N)/d(S) ratio of 0.251-0.405 demonstrated a negative selective pressure that maintains the stability of the analyzed fragments.

Phytoplasma closely related to the pigeon pea witches`-broom phytoplasma (16Sr IX) is associated with citrus huanglongbing symptoms in the state of Sao Paulo, Brazil

In February 2007, sweet orange trees with characteristic symptoms of huanglongbing (HLB) were encountered in a region of Sao Paulo state (SPs) hitherto free of HLB. These trees tested negative for the three liberibacter species associated with HLB. A polymerase chain reaction (PCR) product from symptomatic fruit columella DNA amplifications with universal primers fDI/rPI was cloned and sequenced. The corresponding agent was found to have highest 16S rDNA sequence identity (99%) with the Pigeon pea witches`-broom phytoplasma of group 16Sr IX. Sequences of PCR products obtained with phytoplasma 16S rDNA primer pairs fU5/rU3, fU5/P7 confirm these result.,;. With two primers D7f2/D7r2 designed based oil the 16S rDNA Sequence of the cloned DNA fragment, positive amplifications were obtained from more than one hundred samples including symptomatic fruits and blotchy mottle leaves. Samples positive for phytoplasmas were negative for liberibacters, except for four samples, which were positive for both the phytoplasma and `Candidatus Liberibacter asiaticus`. The phytoplasma was detected by electron microscopy in the sieve tubes of midribs from symptomatic leaves. These results Show that a phytoplasma of group IX is associated with citrus HLB symptoms ill northern, central, and Southern SPs. This phytoplasma has very probably been transmitted to citrus from an external Source of inoculum, but the Putative insect vector is not yet known.

Sensory evaluation and physicochemical optimisation of soy-based desserts using response surface methodology

The objective of this study was to develop a dessert that contains soy protein (SP) (1%, 2%, 3%) and guava juice (GJ) (22%, 27%, 32%) using Response Surface Methodology (RSM) as the optimisation technique. Water activity, physical stability, colour, acidity, pH, iron, and carotenoid contents were analysed. Affective tests were performed to determine the degree of liking of colour, creaminess, and acceptability. The results showed that GJ increased the values of redness, hue angle, chromaticity, acidity, and carotenoid content, while SP reduced water activity. Optimisation suggested a dessert containing 32% GJ and 1.17% SP as the best proportion of these components. This sample was considered a source of fibres, ascorbic acid, copper, and iron and garnered scores above the level of `slightly liked` for sensory attributes. Moreover, RSM was shown to be an adequate approach for modelling the physicochemical parameters and the degree of liking of creaminess of desserts. (C) 2010 Elsevier Ltd. All rights reserved.

Isoflavones and Antioxidant Capacity of Commercial Soy-Based Beverages: Effect of Storage

Samples of 11 different brands of commercially available soy-based beverages (n = 65), including products made from soy protein isolate (SPI) and soy milk, mixed with fruit juice and/or flavoring, were analyzed for their isoflavone content and in vitro antioxidant activity. There was a large variation in isoflavone and total phenolics contents ranging from 0.7 to 13 mg of isoflavones/200 mL and from 6 to 155 mg equivalents of catechin/200 mL, respectively. The antioxidant activity also varied significantly among products. Storage of the beverages at room temperature caused a significant decrease of antioxidant capacity, soluble phenolics, and isoflavone contents after 9 months. When soybeans used for beverage production were stored for up to 6 months in silos, the resulting products were not affected. However, a decrease of malonyl and a proportional increase of free glucosidic forms of isoflavones were observed after storage of both the raw material and the beverages.

Enzymatic-spectrophotometric determination of paraquat in urine samples: A method based on its toxic mechanism

Paraquat is a broad-spectrum contact herbicide that has been encountered worldwide in several cases of accidental, homicidal, and suicidal poisonings. The pulmonary toxicity of this compound is related to the depletion of NADPH in the pneumocytes, which is continuously consumed by the reduction/oxidation of paraquat and reductase enzyme systems in the presence of O(2) (redox cycling). Based on this mechanism, an enzymatic-spectrophotometric method was developed for the determination of paraquat in urine samples. The velocity of NADPH consumption was monitored at 340 nm, every 10 s during 15 min. The velocity of NADPH oxidation correlated with the paraquat levels found in samples. The enzymatic-spectrophotometric method showed to be sensitive, making possible the detection of paraquat in urine samples at concentrations as low as 0.05 mg/L.

Novel sequence types (STs) of Staphylococcus aureus isolates causing clinical and subclinical mastitis in flocks of sheep in the northeast of Brazil

Staphylococcus aureus is one of the most important infectious mastitis causative agents in small ruminants. In order to know the distribution of Staph. aureus strains associated with infectious mastitis in flocks of sheep in the northeast of Brazil and establish whether these clones are related to the strains distributed internationally, this study analysed the genetic diversity of Staph. aureus isolates from cases of clinical and subclinical mastitis in ewes by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). In this research, 135 ewes with mastitis from 31 sheep flocks distributed in 15 districts were examined. Staph. aureus was isolated from sheep milk in 9 (29%) out of 31 herds located in 47% of the districts surveyed. MLST analysis allowed the identification of four STs (ST750, ST1728, ST1729 and ST1730). The last three with their respective novel alleles (g/p-220; pta-182 and yqil-180) were recently reported in the Staph. aureus MLST database (http://www.mlst.net). Each novel allele showed only a nucleotide different from those already described. The occurrence of CC133 (ST750 and ST1729) in this study is in agreement with other reports that only a few clones of Staph. aureus seem to be responsible for most cases of mastitis in dairy farms and that some of these clones may have broad geographic distribution. However, the prevalence of CC5 (ST1728 and ST1730)-an important group related to cases of colonization or infection in humans-differs from previous studies by its widespread occurrence and may suggest human contamination followed by selective pressures of the allelic diversifications presented for these STs.

Impedimetric immunosensor for electronegative low density lipoprotein (LDL(-)) based on monoclonal antibody adsorbed on (polyvinyl formal)-gold nanoparticles matrix

Monoclonal antibodies (MAb) have been commonly applied to measure LDL in vivo and to characterize modifications of the lipids and apoprotein of the LDL particles. The electronegative low density lipoprotein (LDL(-)) has an apolipoprotein B-100 modified at oxidized events in vivo. In this work, a novel LDL-electrochemical biosensor was developed by adsorption of anti-LDL(-) MAb on an (polyvinyl formal)-gold nanoparticles (PVF-AuNPs)-modified gold electrode. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to characterize the recognition of LDL-. The interaction between MAb-LDL(-) leads to a blockage in the electron transfer of the [Fe(CN)(6)](4-)/K(4)[Fe(CN)(6)](3-) redox couple, which may could result in high change in the electron transfer resistance (R(CT)) and decrease in the amperometric responses in CV analysis. The compact antibody-antigen complex introduces the insulating layer on the assembled surface, which increases the diameter of the semicircle, resulting in a high R(CT), and the charge transferring rate constant k(0) decreases from 18.2 x 10(-6) m/s to 4.6 x 10(-6) m/s. Our results suggest that the interaction between MAb and lipoprotein can be quantitatively assessed by the modified electrode. The PVF-AuNPs-MAb system exhibited a sensitive response to LDL(-), which could be used as a biosensor to quantify plasmatic levels of LDL(-). (C) 2011 Elsevier B.V. All rights reserved.

Enantioselective Arylations Catalyzed by Carbohydrate-Based Chiral Amino Alcohols

The application of carbohydrate-derived amino alcohols in the asymmetric arylation of aldehydes by using arylboronic acids as the source of transferable aryl groups is described. The best ligand is derived from the readily available sugar D-xylose and it mediates the addition of a range of arylboronic acids to various aromatic aldehydes in excellent yields and high enantiomeric excesses.

3D-Pharmacophore mapping of thymidine-based inhibitors of TMPK as potential antituberculosis agents

Tuberculosis (TB) is the primary cause of mortality among infectious diseases. Mycobacterium tuberculosis monophosphate kinase (TMPKmt) is essential to DNA replication. Thus, this enzyme represents a promising target for developing new drugs against TB. In the present study, the receptor-independent, RI, 4D-QSAR method has been used to develop QSAR models and corresponding 3D-pharmacophores for a set of 81 thymidine analogues, and two corresponding subsets, reported as inhibitors of TMPKmt. The resulting optimized models are not only statistically significant with r (2) ranging from 0.83 to 0.92 and q (2) from 0.78 to 0.88, but also are robustly predictive based on test set predictions. The most and the least potent inhibitors in their respective postulated active conformations, derived from each of the models, were docked in the active site of the TMPKmt crystal structure. There is a solid consistency between the 3D-pharmacophore sites defined by the QSAR models and interactions with binding site residues. Moreover, the QSAR models provide insights regarding a probable mechanism of action of the analogues.

Emulsified systems based on glyceryl monostearate and potassium cetyl phosphate: Scale-up and characterization of physical properties

introducing a pharmaceutical product on the market involves several stages of research. The scale-up stage comprises the integration of previous phases of development and their integration. This phase is extremely important since many process limitations which do not appear on the small scale become significant on the transposition to a large one. Since scientific literature presents only a few reports about the characterization of emulsified systems involving their scaling-up, this research work aimed at evaluating physical properties of non-ionic and anionic emulsions during their manufacturing phases: laboratory stage and scale-up. Prototype non-ionic (glyceryl monostearate) and anionic (potassium cetyl phosphate) emulsified systems had the physical properties by the determination of the droplet size (D[4,3 1, mu m) and rheology profile. Transposition occurred from a batch of 500-50,000 g. Semi-industrial manufacturing involved distinct conditions: intensity of agitation and homogenization. Comparing the non-ionic and anionic systems, it was observed that anionic emulsifiers generated systems with smaller droplet size and higher viscosity in laboratory scale. Besides that, for the concentrations tested, augmentation of the glyceryl monostearate emulsifier content provided formulations with better physical characteristics. For systems with potassium cetyl phosphate, droplet size increased with the elevation of the emulsifier concentration, suggesting inadequate stability. The scale-up provoked more significant alterations on the rheological profile and droplet size on the anionic systems than the non-ionic. (C) 2008 Elsevier B.V. All rights reserved.