Use of Pyrosequencing and Denaturing Gradient Gel Electrophoresis to Examine the Effects of Probiotics and Essential Oil Blends on Digestive Microflora in Broilers Under Mixed Eimeria Infection

A protective digestive microflora helps prevent and reduce broiler infection and colonization by enteropathogens. In the current experiment, broilers fed diets supplemented with probiotics and essential oil (EO) blends were infected with a standard mixed Eimeria spp. to determine effects of performance enhancers on ileal and cecal microbial communities (MCs). Eight treatment groups included four controls (uninfected-unmedicated [UU], unmedicated-infected, the antibiotic BMD plus the ionophore Coban as positive control, and the ionophore as negative control), and four treatments (probiotics BC-30 and Calsporin; and EO, Crina Poultry Plus, and Crina PoultryAF). Day-old broilers were raised to 14 days in floor pens on used litter and then were moved to Petersime batteries and inoculated at 15 days with mixed Eimeria spp. Ileal and cecal samples were collected at 14 days and 7 days postinfection. Digesta DNA was subjected to pyrosequencing for sequencing of individual cecal bacteria and denaturing gradient gel electrophoresis (DGGE) for determination of changes in ileal and cecal MC according to percentage similarity coefficient (%SC). Pyrosequencing is very sensitive detecting shifts in individual bacterial sequences, whereas DGGE is able to detect gross shifts in entire MC. These combined techniques offer versatility toward identifying feed additive and mild Eimeria infection modulation of broiler MC. Pyrosequencing detected 147 bacterial species sequences. Additionally, pyrosequencing revealed the presence of relatively low levels of the potential human enteropathogens Campylobacter sp. and four Shigella spp. as well as the potential poultry pathogen Clostridiun perfringens. Pre- and postinfection changes in ileal (56%SC) and cecal (78.5%SC) DGGE profiles resulted from the coccidia infection and with increased broiler age. Probiotics and EO changed MC from those seen in UU ilea and ceca. Results potentially reflect the performance enhancement above expectations in comparison to broilers not given the probiotics or the specific EO blends as feed supplements.

High-speed video observations of positive ground flashes produced by intracloud lightning

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Evaluation of Paracoccidioides brasiliensis exoantigen in the detection of delayed dermal hypersensitivity in experimental and human paracoccidioidomycosis

The exoantigen of Paracoccidioides brasiliensis standardized by Camargo et al. [1] (AgR) was used to evaluate the in vivo and in vitro cell immune response of experimental animals and of patients with paracoccidioidomycosis (PBM). Fava Netto antigen (AgF) was tested in parallel as a control antigen. The study was conducted with mice and guinea pigs infected with P. brasiliensis or immunized with its fungal antigens, on patients with PBM and on their respective control groups. The cell immune response was analysed by skin tests, and by the macrophage and leucocyte migration inhibition tests (MMIT and LMIT) in the animals and in the patients, respectively. The skin test with AgR as paracoccidioidin was positive in infected or immunized mice and guinea pigs and negative in control animals. The skin tests with AgR (24 h) showed 96.7% positivity in patients with PBM and were negative in control individuals. Histopathological study of the in vivo tests in the different experimental models was consistent with a delayed hypersensitivity response (DHR). Immunohistochemical study of the skin tests of PBM patients demonstrated a predominance of T lymphocytes, confirming the nature of a DHR to the fungal antigens. The in vitro cell immune response showed variable results for the various experimental models, i.e. significant rates of MMIT in immunized mice, a tendency to positivity in infected guinea pigs, and the absence of migration inhibition in PBM patients. Taken together, the data indicate that the AgR is efficient as paracoccidioidin in the evaluation of DHR in PBM, with an optimum time of reading the test of 24 h.

IgG, IgM and IgA antibody response for the diagnosis and follow-up of paracoccidioidomycosis: Comparison of counterimmunoelectrophoresis and complement fixation

IgG, IgM and IgA antibodies to GP43 (glycoprotein fraction of Paracoccidioides brasiliensis) were measured by ELISA in 63 samples from 23 patients with paracoccidioidomycosis before and twice after chemotherapy was started. Antibodies against P. brasiliensis were detected by indirect immunofluorescence (IF) (IgG, IgM and IgA isotypes), counterimmunoelectrophoresis (CIE) and complement fixation. Two control groups composed of 19 healthy individuals and 12 patients with other diseases (six with histoplasmosis, three with tuberculosis and three with other mycoses). The highest efficiency percentages were found with IgG and IgA- ELISA (100%), IgG-IF (96.2%), CIE (94.4%) and the lowest with CF (75.9%). Highest positive and negative predictive values (100%) were observed for IgG and IgA ELISA. IgG and IgM-ELISA antibodies are more often found in patients with acute than chronic disease (P = 0.01). Four to six months after treatment follow-up showed decreased levels of IgG and IgM-ELISA for acute cases and decreased titres of CIE for chronic cases in relation to pretreatment levels. This study suggests that IgG-ELISA anti-GP43 represents a good marker to monitor clinical response to therapy.

Microhabitat and plant structure of Batrachospermum (Batrachospermales, Rhodophyta) populations in four streams of São Paulo State, southeastern Brazil

Microhabitat and plant structure of seven Batrachospermum populations (four of Batrachospermum delicatulum (= Sirodotia delicatula), one of Batrachospermum macrosporum and two of the 'Chantransia' stage), including the influence of physical variables (current velocity, depth, irradiance and substratum), were investigated in four streams of São Paulo State, southeastern Brazil. The populations of B. delicatulum and the 'Chantransia' stage occurred under very diverse microhabitat conditions, which probably contributes to their wide spatial and seasonal distribution in Brazilian streams. Results suggest branch reconfiguration as a probable mechanism of adaptation to current velocity based on the occurrence of: (i) B. macrosporum (a large mucilaginous form with presumably little ability for branch reconfiguration) under lower current velocity than B. delicatulum; (ii) only dense plants in populations with high current velocities (> 60 cm s-1), whereas 53-77% of dense plants were seen in populations exposed to lower currents (< 40 cm s-1); (iii) positive correlations of plant length with internode length in populations under low current velocities and negative correlation in a population with high velocity (132 cm s-1); and (iv) negative correlations of current velocity with plant diameter and internode length in a population under high flow. This study, involving mainly dioecious populations, revealed that B. delicatulum displayed higher fertilization rates than B. macrosporum. A complementary explanation for a dioecious species to increase fertilization success was proposed consisting of outcrossing among intermingled male and female adjacent plants within an algal spot.

Lamé differential equations and electrostatics

The problem of existence and uniqueness of polynomial solutions of the Lamé differential equation A(x)y″ + 2B(x)y′ + C(x)y = 0, where A(x),B(x) and C(x) are polynomials of degree p + 1,p and p - 1, is under discussion. We concentrate on the case when A(x) has only real zeros aj and, in contrast to a classical result of Heine and Stieltjes which concerns the case of positive coefficients rj in the partial fraction decomposition B(x)/A(x) = ∑j p=0 rj/(x - aj), we allow the presence of both positive and negative coefficients rj. The corresponding electrostatic interpretation of the zeros of the solution y(x) as points of equilibrium in an electrostatic field generated by charges rj at aj is given. As an application we prove that the zeros of the Gegenbauer-Laurent polynomials are the points of unique equilibrium in a field generated by two positive and two negative charges. © 2000 American Mathematical Society.

p16INK4A immunohistochemical overexpression in premalignant and malignant oral lesions infected with human papillomavirus

Human papillomavirus (HPV) is believed to promote the oncogenic process, and the correlation between viral oncoproteins and dysfunction of p16 INK4A tumor suppressor protein in oral lesions is controversial. To test the hypothesis that anogenital HPV types participate in disruption of the regulation of p16INK4A suppressor protein in oral lesions, we analyzed 46 oral biopsy specimens for the presence of HPV 6/11 and 16/18 by in situ hybridization (ISH) and for p16INK4A expression by immunohistochemistry (IHC). Eighteen (39%) of the 46 oral lesions were HPV-positive and 28 (61%) were HPV-negative. HPV 6/11 DNA was found in 5 (11%) and HPV 16/18 in 13 (28%) of 46 biopsies. Nine of the 18 HPV-positive oral lesions (50%), assessed by catalyzed signal amplification coupled to ISH (CSA-ISH), gave high-intensity p16INK4A immunostaining. Focal and diffuse patterns were observed in 11/13 (77%) lesions with HPV 16/18, focal immunopositivity in 3/5 (80%) with HPV 6/11, and negative or sporadic p16-labeling in 18/28 (64%) without the presence of HPV DNA. These results showed a strong association between overexpression of p16 protein and malignant oral lesions, mainly those infected by HPV 16/18. We can conclude that high-risk HPV types are associated with p16 overexpression, and p16 may serve as a biomarker in oral cancer related to high-risk HPV infection.

Analysis of susceptibility profile of Pseudomonas spp. and prevalence of bacterial samples from the surfaces of dental consulting-rooms

The aim of this research was to evaluate the susceptibility profile of Pseudomonas spp. and the prevalence of bacterial samples isolated from horizontal surfaces surrounding wash-basins used by dentists in several adjoined consulting-rooms, at points next to and at a distance from the basin, before and after surgical procedures. Our results showed a high percentage of Gram-positive cocci and Gram-negative bacilli; 34.66% were Staphylococcus spp. and 30.12% were non-fermentative Gram-negative bacilli among which Pseudomonas spp. (40.90%) was the commonest genus. Analysis of the susceptibility profile of Pseudomonas spp. isolates by determining the minimal inhibitory concentration (MIC) of 14 antibiotics showed a great variation among the strains and high rates of resistance to cefazolin, ceftazidime and aztreonan. Of the 14 antibiotics tested, 59.03% were found to be active against all the environmental isolates. Strains were resistant to aztreonan (62.82%), while susceptibility to third generation cephalosporins was variable.

Pseudospin and spin symmetries in the relativistic harmonic oscillator

We compute the analytical solutions of the generalized relativistic harmonic oscillator in 1+1 dimensions, including a linear pseudoscalar potential and quadratic scalar and vector potentials which have equal or opposite signs These are the conditions in which pseudospin or spin symmetries can be realized We consider positive and negative quadratic potentials and present their bound-state solutions for fermions and an-tifermions. We relate the spin-type and pseudospin-type spectra through charge conjugation and γ5 chiral transformations. Finally, we establish a relation of the solutions found with single-particle states of nuclei described by relativistic mean-field theories with tensor interactions and discuss the conditions in which one may have both nucleon and antin-ucleon bound states.

In vitro antimicrobial efficiency of a mouthwash containing triclosan/gantrez and sodium bicarbonate

Several antiseptic substances have been used as adjuncts to routine mechanical procedures of oral hygiene, based on their antimicrobial effects. The objective of this study was to assess in vitro the antimicrobial efficiency of 2 mouthwash containing Triclosan/Gantrez and sodium bicarbonate in comparison to both positive and negative controls. Standard strain samples of Escherichia coli, Pseudomonas aeruginosa, Actinomyces viscosus and Bacillus subtilis were used. Samples of Streptococcus mutans and Gram-negative bacilli were collected from 20 volunteers (10 with a clinically healthy periodontium and 10 presenting biofilm-associated gingivitis). Evaluation of the antimicrobial activity was performed by determining the minimal inhibitory concentration (MIC). The results indicated that the test solution inhibited the growth of both Gram-negative and Gram-positive microorganisms from the volunteers' saliva as well as that of the standard strains at the MIC dilution of 1:20, whereas the MIC dilution of 0.12% chlorhexidine against the same bacteria was 1:80. Thus, even though the tested mouthrinse solution presented an in-vitro antimicrobial activity superior to that of a placebo, it was inferior to that of chlorhexidine.

Development and validation of a Xanthomonas axonopodis pv. citri DNA microarray platform (XACarray) generated from the shotgun libraries previously used in the sequencing of this bacterial genome

Background. From shotgun libraries used for the genomic sequencing of the phytopathogenic bacterium Xanthomonas axonopodis pv. citri (XAC), clones that were representative of the largest possible number of coding sequences (CDSs) were selected to create a DNA microarray platform on glass slides (XACarray). The creation of the XACarray allowed for the establishment of a tool that is capable of providing data for the analysis of global genome expression in this organism. Findings. The inserts from the selected clones were amplified by PCR with the universal oligonucleotide primers M13R and M13F. The obtained products were purified and fixed in duplicate on glass slides specific for use in DNA microarrays. The number of spots on the microarray totaled 6,144 and included 768 positive controls and 624 negative controls per slide. Validation of the platform was performed through hybridization of total DNA probes from XAC labeled with different fluorophores, Cy3 and Cy5. In this validation assay, 86% of all PCR products fixed on the glass slides were confirmed to present a hybridization signal greater than twice the standard deviation of the deviation of the global median signal-to-noise ration. Conclusions. Our validation of the XACArray platform using DNA-DNA hybridization revealed that it can be used to evaluate the expression of 2,365 individual CDSs from all major functional categories, which corresponds to 52.7% of the annotated CDSs of the XAC genome. As a proof of concept, we used this platform in a previously work to verify the absence of genomic regions that could not be detected by sequencing in related strains of Xanthomonas. © 2010 Moreira et al; licensee BioMed Central Ltd.

Relationship between psychological factors and symptoms of TMD in university undergraduate students.

Temporomandibular disorders is a collective term used to describe a number of related disorders involving the temporomandibular joints, masticatory muscles and occlusion with common symptoms such as pain, restricted movement, muscle tenderness and intermittent joint sounds. The multifactorial TMD etiology is related to emotional tension, occlusal interferences, tooth loss, postural deviation, masticatory muscular dysfunction, internal and external changes in TMJ structure and the various associations of these factors. The aim of this study was to evaluate the prevalence of the relationship between signs of psychological distress and temporomandibular disorder in university students. A total 150 volunteers participated in this study. They attended different courses in the field of human science at one public university and four private universities. TMD was assessed by the Research Diagnostic Criteria (RDC) questionnaire. Anxiety was measured by means of a self-evaluative questionnaire, Spielberger's Trait-State anxiety inventory, to evaluate students'state and trait anxiety. The results of the two questionnaires were compared to determine the relationship between anxiety levels and severity degrees of chronic TMD pain by means of the chi-square test. The significance level was set at 5%. The statistical analysis showed that the TMD degree has a positive association with state-anxiety (p = 0.008; p < 0.05) and negative with trait-anxiety (p = 0.619; p < 0.05). Moreover a high TMD rate was observed among the students (40%). This study concluded that there is a positive association between TMD and anxiety.

UMP kinase from Mycobacterium tuberculosis: Mode of action and allosteric interactions, and their likely role in pyrimidine metabolism regulation

The pyrH-encoded uridine 5′-monophosphate kinase (UMPK) is involved in both de novo and salvage synthesis of DNA and RNA precursors. Here we describe Mycobacterium tuberculosis UMPK (MtUMPK) cloning and expression in Escherichia coli. N-terminal amino acid sequencing and electrospray ionization mass spectrometry analyses confirmed the identity of homogeneous MtUMPK. MtUMPK catalyzed the phosphorylation of UMP to UDP, using ATP-Mg 2+ as phosphate donor. Size exclusion chromatography showed that the protein is a homotetramer. Kinetic studies revealed that MtUMPK exhibits cooperative kinetics towards ATP and undergoes allosteric regulation. GTP and UTP are, respectively, positive and negative effectors, maintaining the balance of purine versus pyrimidine synthesis. Initial velocity studies and substrate(s) binding measured by isothermal titration calorimetry suggested that catalysis proceeds by a sequential ordered mechanism, in which ATP binds first followed by UMP binding, and release of products is random. As MtUMPK does not resemble its eukaryotic counterparts, specific inhibitors could be designed to be tested as antitubercular agents. © 2010 Elsevier Inc. All rights reserved.

Cellular damages in the Allium cepa test system, caused by BTEX mixture prior and after biodegradation process

Petroleum and derivatives have been considered one of the main environmental contaminants. Among petroleum derivatives, the volatile organic compounds benzene, toluene, ethylbenzene and xylene (BTEX) represent a major concern due to their toxicity and easy accumulation in groundwater. Biodegradation methods seem to be suitable tools for the clean-up of BTEX contaminants from groundwater. Genotoxic and mutagenic potential of BTEX prior and after biodegradation process was evaluated through analyses of chromosomal aberrations and MN test in meristematic and F 1 root cells using the Allium cepa test system. Seeds of A. cepa were germinated into five concentrations of BTEX, non-biodegraded and biodegraded, in ultra-pure water (negative control), in MMS 4×10 -4M (positive control) and in culture medium used in the biodegradation (blank biodegradation control). Results showed a significant frequency of both chromosomal and nuclear aberrations. The micronucleus (MN) frequency in meristematic cells was significant for most of tested samples. However, MN was not present in significant levels in the F 1 cells, suggesting that there was no permanent damage for the meristematic cell. The BTEX effects were significantly reduced in the biodegraded samples when compared to the respective non-biodegraded concentrations. Therefore, in this study, the biodegradation process showed to be a reliable and effective alternative to treat BTEX-contaminated waters. Based on our results and available data, the BTEX toxicity could also be related to a synergistic effect of its compounds. © 2011 Elsevier Ltd.

Reproduction, food dynamics and exploitation level of Oreochromis niloticus (Perciformes: Cichlidae) from artisanal fisheries in Barra Bonita Reservoir, Brazil

Nile tilapia (Oreochromis niloticus), which is exotic to South America, is the most common species caught in artisanal fisheries at the Barra Bonita Reservoir, Southeastern Brazil. This species is of great socioeconomic importance for the region and keeps active a population of about 500 fishers. In the present study we assess reproduction, food dynamics and level of exploitation of O. niloticus, caught by artisanal fisheries in the Barra Bonita Reservoir. Specimens were collected monthly, from July 2004-June 2005, and a total of 1 715 specimens were analyzed. Each specimen was examined to obtain biological and biometric data: standard length (cm), total weight (g), reproductive data (sex and stage of maturation), and stomach contents (empty, partly full, and full). We also estimated the sex ratio (by macroscopic observation of gonads), reproductive period (by ovarian development and seasonal average of gonadosomatic index in females), and feeding habits (by stomach contents). The possible relationship between abiotic factors and the reproductive period was statistically verified using Spearman's Rank Correlation. The FiSAT (ELEFAN I) package was used to assess growth parameters, mortality rates and to infer exploitation rate from standard length frequencies. The O. niloticus population had a sex ratio of 1.3:1 (M:F). Results indicated that ripe females were captured throughout the year, with a higher frequency during the winter-2004 (with a frequency of 59%, at a mean temperature of 20.5°C), and in spring-2004 (with a frequency of 60.5% at a mean temperature of 21.18°C). The GSI mean values obtained by season were: winter-2004: 1.71; spring-2004: 1.72; summer-2005: 0.80, and autumn-2005: 1.19. The Spearman correlation indicated positive values with respect to pH, dissolved oxygen, electric conductivity, transparency and chlorophyll a, and negative values with respect to temperature, accumulated rainfall and altimetric benchmark. The main food items were phytoplankton and periphytic algae, observed in 99.6% of the analyzed stomachs. The estimated growth and mortality parameters were: L∞=33.60cm, k=0.63/year, longevity= 4.76years, Z=2.81/ year, M=1.20/year and F=1.61/year. The weight-length relationship was Ln Wt=-2.8532+2.8835 Ln Lp. The estimated yield per recruit values were as follows: E=0.570, Emax=0.776, E0.1=0.604 and E0.5=0.349. These results indicate that a well established population of O. niloticus is present at Barra Bonita Reservoir; with an active reproduction throughout the year, more intense during winter and spring, and that O. niloticus is a phytoplanktophagus species. There were no indications that this species is being overfished, we therefore recommend that, due to its exotic condition, no restrictions need to be taken on its fishing activities.