Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples

A real-time polymerase chain reaction (PCR) assay was developed for rapid identification of Bacillus anthracis in environmental samples. These samples often harbor Bacillus cereus bacteria closely related to B. anthracis, which may hinder its specific identification by resulting in false positive signals. The assay consists of two duplex real-time PCR: the first PCR allows amplification of a sequence specific of the B. cereus group (B. anthracis, B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus pseudomycoides, and Bacillus mycoides) within the phosphoenolpyruvate/sugar phosphotransferase system I gene and a B. anthracis specific single nucleotide polymorphism within the adenylosuccinate synthetase gene. The second real-time PCR assay targets the lethal factor gene from virulence plasmid pXO1 and the capsule synthesis gene from virulence plasmid pXO2. Specificity of the assay is enhanced by the use of minor groove binding probes and/or locked nucleic acids probes. The assay was validated on 304 bacterial strains including 37 B. anthracis, 67 B. cereus group, 54 strains of non-cereus group Bacillus, and 146 Gram-positive and Gram-negative bacteria strains. The assay was performed on various environmental samples spiked with B. anthracis or B. cereus spores. The assay allowed an accurate identification of B. anthracis in environmental samples. This study provides a rapid and reliable method for improving rapid identification of B. anthracis in field operational conditions.

Biohazard neutralization based on lipid technology

A rapid detection and neutralization method for biowarfare agents would be a great biodefense in war times. With this purpose, liposomes were developed following the lipid film formation, rehydration, and extrusion procedure as the production method. MgOCl2 was encapsulated in the liposomes and it was tested with three different bacterium B. cereus; B. thuringiensis; and B. subtilis. For specificity, the liposomes were modified with a polyclonal antibody against B. cereus and B. subtilis. The liposomes were characterized using a Malvern Zetasizer Instrument, and the study revealed stability of the liposomes stored at 4°C for a period of 15 days. A live/dead assay revealed a significant reduction of bacterium incubated with MgOCl2-liposomes. Smaller reduction percentages, but yet significant, were observed with the MgOCl2-immunoliposomes. A colony growth assay revealed a significant reduction percentage for empty liposomes, MgOCl2-liposomes, and MgOCl2-immunoliposomes incubated with B. thuringiensis.

Parasite-host interaction of the protoplast isolates of Entomophthora egressa with the eastern hemlock looper and the eastern spruce budworm

Hemocytes of the insects Lambdina fiscellaria fiscellaria and Choristoneura fumiferana did not adhere to the protoplasts of ~he fungus EntomoEhthora egressa. Hemocyte reaction for both insect species to test-particles was not suppressed by the protoplasts. The spherule cells of _-L. fiscellaria fiscellaria adhered to the spherical hyphal bodies and hyphae of ~· ~gressa. The granular cells of -c. fumiferana adhered to the hyphae of ~· egress~. Protoplasts exposed to papain were attacked by the granular ·cells of -c. fumiferana. Spent growth medium of both protoplast isolates produced paralysis when injected into -c. fumiferana larvae. Evidence suggests that heat-stable proteins may be involved. Protoplast isolates showed differences in the growth rates and regeneration sequences using coagulated egg yolk medium, a highly modified version of Grace's insect tissue . culture medium (MGM) and modifications of MGM and in the presence of C0₂. The isolates also differed in the changes that they induced in MGM composition during protoplast growth and in the rates of glucose utilization and protein secretion. The serum of c. fumiferana larvae contained protein(s) which we believe adhere to the cell membranes of the protoplasts of E. egressa. Evidence is presented for hemocyteplasn~ interaction in the presence of protoplasts. Components in the larval serum were found to influence protoplast growth patterns. The possibility of antiprotoplast serum activity is presented. Melanin, toxic levels of ninhydrinpositive compounds and antiprotoplast proteins may have been involved in this activity. The granular cells of -L. fiscellaria fiscellaria and Q• fumiferana adhered to the hyphae of ,Rhizopus ~i$rican~. Spores of Absidia repens and the bacteria Escherichia coli and Bacillus cereus adhered to the granular cells of both species of· insects. The granular cells and plasmatocytes of -c. fumiferana were capable of phagocytosing -B. cereus. Adhesion of .A... . repens spores to c. fumiferana granular cells ~ . - was stimulated by N-acetylglucosamine and glucosamine, moderately reduced by D-fucose, D-arabinose, D-mannose, D-galatose and sucrose and mildly reduced by D-glucose, D-fructose and trehalose. There was no evidence of humoral opsonins in larval hemolymph favoring test-particle-hemocyte interaction. Granular cells of c. fumiferana exposed to papain had reduced affinities for A. repens spores.

Synthesis and antimicrobial activity of some 2- Piperidinomethylamino-4-(7-H/substituted coumarin-3-yl)- 6-chlorosubstitutedphenyl pyrimidines

Purpose: To prepare and evaluate some 2-piperidinomethylamino-4-(7-H/substitutedcoumarin-3-yl)-6- chlorosubstitutedphenyl pyrimidines as antimicrobial agents. Methods: Some 2-piperidinomethylamino-4-(7-H/substitutedcoumarin-3-yl)-6-chlorosubstitutedphenyl pyrimidines were prepared by reacting 2-amino-4-(7-H/substitutedcoumarin-3-yl)-6- (chlorosubstitutedphenyl) pyrimidines with piperidine and formaldehyde. The chemical structures of the synthesized compounds were elucidated by Fourier transform infrared (FTIR), 1H-nuclear magnetic resonance (1H-NMR), mass spectrometry and elemental analysis. These compounds were investigated for their antimicrobial activity against ten bacteria and five fungi by serial plate dilution method using standard drugs, namely, ofloxacin and ketoconazole, respectively, and their minimum inhibitory concentrations (MICs) were also determined. Results: A total of eighteen new compounds (1a-18a) were synthesized. Compound 6a (MIC = 50 μg/mL; p < 0.05 or less) displayed the highest activity against S. aureus , E. faecalis , Staphylococcus epidermidis , B. subtilis , and B. cereus . Compound 6a further showed good activity (MIC = 25 μg/mL; p < 0.05 or less) against E. coli ; P. aeruginosa K. pneumonia , B. bronchiseptica , and P. vulgaris . Compounds 6a (MIC = 25 μg/mL; p < 0.0001) and 17a (MIC = 25 μg/mL; p < 0.0001) displayed very good activity against C. albicans , A. niger , A. flavus , M. purpureous , and P. citrinum , respectively. Analysis of structure-activity relationship revealed that the presence of bromo group at 7-postion of the coumarin moiety along with the 4-chlorophenyl group at position-6 of the pyrimidine ring is critical for antimicrobial activity against Gram-positive bacteria, Gram negative bacteria and fungi. Conclusion: The synthesized 2-piperidino derivatives are better antifungal and antibacterial agents than the earlier reported 2-morpholino derivatives, but require further investigations against other microbial strains to ascertain their broad spectrum antimicrobial activity.

Microbially derived bioactive peptides to improve human health

This thesis describes a study of various methods to produce bioactive peptides. Initially, the generation of anti-Cronobacter spp. peptides by fermentation of milk protein is described. Lactobacillus johnsonii DPC6026 was used to generate two previously described antimicrobial peptides. Phenotypic analysis indicated unsatisfactory casein hydrolysis. The genome of the strain was sequenced and annotated. Results showed a number of unique features present, most notably a large symmetrical inversion of approximately 750kb in comparison with the human isolate L. johnsonii NCC 533. The data suggest significant genetic diversity and intra-species genomic rearrangements within the L. johnsonii spp.. Cronobacter spp. have emerged as pathogens of concern to the powdered infant formula industry. Chapters 3 and 4 of this thesis describe novel methods to generate two antimicrobial peptides, Caseicin A and B. In Chapter 3 a bank of Bacillus strains was generated and investigated for caseicin production. Following casein hydrolysis by specific B. cereus and B. thuringiensis strains the peptides of interest were generated. Chapter 4 describes a sterile enzymatic method to generate peptides from casein. Bioinformatic tools were used to predict enzymes capable of liberating caseicin peptides from casein. Hydrolysates were generated using suitable enzymes, examined and some were found to produce peptides with activity against Cronobacter spp.. This study establishes a potential industrial-grade method to generate antimicrobial peptides. Administration of GLP-1 leads to improved glycaemic control in diabetes patients. Generation of a recombinant lactic acid bacteria capable of producing a GLP-1 analogue is described in Chapter 5. In-vivo analysis confirmed insulinotropic activity. The results illustrate a method using bacteriocin producing cellular machinery to generate bioactive peptides. This thesis describes the generation of bioactive peptides by bacterial fermentation, tailored enzymatic hydrolysis and recombinant bacterial methods. The techniques described contribute to bioactive peptide research with regards novel methods of production and industrial scale-up.

An Asp49 Phospholipase A2 From Snake Venom Induces Cyclooxygenase-2 Expression And Prostaglandin E2 Production Via Activation Of Nf-κb, P38mapk, And Pkc In Macrophages.

Phospholipases A2 (PLA2) are key enzymes for production of lipid mediators. We previously demonstrated that a snake venom sPLA2 named MT-III leads to prostaglandin (PG)E2 biosynthesis in macrophages by inducing the expression of cyclooxygenase-2 (COX-2). Herein, we explored the molecular mechanisms and signaling pathways leading to these MT-III-induced effects. Results demonstrated that MT-III induced activation of the transcription factor NF-κB in isolated macrophages. By using NF-κB selective inhibitors, the involvement of this factor in MT-III-induced COX-2 expression and PGE2 production was demonstrated. Moreover, MT-III-induced COX-2 protein expression and PGE2 release were attenuated by pretreatment of macrophages with SB202190, and Ly294002, and H-7-dihydro compounds, indicating the involvement of p38MAPK, PI3K, and PKC pathways, respectively. Consistent with this, MT-III triggered early phosphorylation of p38MAPK, PI3K, and PKC. Furthermore, SB202190, H-7-dihydro, but not Ly294002 treatment, abrogated activation of NF-κB induced by MT-III. Altogether, these results show for the first time that the induction of COX-2 protein expression and PGE2 release, which occur via NF-κB activation induced by the sPLA2-MT-III in macrophages, are modulated by p38MAPK and PKC, but not by PI3K signaling proteins.

Draft Genome Sequence Of Ft9, A Novel Bacillus Cereus Strain Isolated From A Brazilian Thermal Spring.

A Bacillus cereus strain, FT9, isolated from a hot spring in the midwest region of Brazil, had its entire genome sequenced.

Staphylococcus Aureus Enterotoxins A And B Inhibit Human And Mice Eosinophil Chemotaxis And Adhesion In Vitro.

Staphylococcus aureus aggravates the allergic eosinophilic inflammation. We hypothesized that Staphylococcus aureus-derived enterotoxins directly affect eosinophil functions. Therefore, this study investigated the effects of Staphylococcal enterotoxins A and B (SEA and SEB) on human and mice eosinophil chemotaxis and adhesion in vitro, focusing on p38 MAPK phosphorylation and intracellular Ca(2+) mobilization. Eosinophil chemotaxis was evaluated using a microchemotaxis chamber, whereas adhesion was performed in VCAM-1 and ICAM-1-coated plates. Measurement of p38 MAPK phosphorylation and intracellular Ca(2+) levels were monitored by flow cytometry and fluorogenic calcium-binding dye, respectively. Prior incubation (30 to 240 min) of human blood eosinophils with SEA (0.5 to 3 ng/ml) significantly reduced eotaxin-, PAF- and RANTES-induced chemotaxis (P<0.05). Likewise, SEB (1 ng/ml, 30 min) significantly reduced eotaxin-induced human eosinophil chemotaxis (P<0.05). The reduction of eotaxin-induced human eosinophil chemotaxis by SEA and SEB was prevented by anti-MHC monoclonal antibody (1 μg/ml). In addition, SEA and SEB nearly suppressed the eotaxin-induced human eosinophil adhesion in ICAM-1- and VCAM-1-coated plates. SEA and SEB prevented the increases of p38 MAPK phosphorylation and Ca(2+) levels in eotaxin-activated human eosinophils. In separate protocols, we evaluated the effects of SEA on chemotaxis and adhesion of eosinophils obtained from mice bone marrow. SEA (10 ng/ml) significantly reduced the eotaxin-induced chemotaxis along with cell adhesion to both ICAM-1 and VCAM-1-coated plates (P<0.05). In conclusion, the inhibition by SEA and SEB of eosinophil functions (chemotaxis and adhesion) are associated with reductions of p38 MAPK phosphorylation and intracellular Ca(2+) mobilization.

Update On Ultraviolet A And B Radiation Generated By The Sun And Artificial Lamps And Their Effects On Skin.

Solar radiation, especially ultraviolet A (UVA) and ultraviolet B (UVB), can cause damage to the human body, and exposure to the radiation may vary according to the geographical location, time of year and other factors. The effects of UVA and UVB radiation on organisms range from erythema formation, through tanning and reduced synthesis of macromolecules such as collagen and elastin, to carcinogenic DNA mutations. Some studies suggest that, in addition to the radiation emitted by the sun, artificial sources of radiation, such as commercial lamps, can also generate small amounts of UVA and UVB radiation. Depending on the source intensity and on the distance from the source, this radiation can be harmful to photosensitive individuals. In healthy subjects, the evidence on the danger of this radiation is still far from conclusive.

Avaliação do teor e da estabilidade de vitaminas do complexo B e vitamina C em bebidas isotônicas e energéticas

Vitamin C stability and concentration was evaluated in isotonic beverages and B group vitamins (B1, B2, B3, B5 and B6) in power beverages. The amount of vitamins was found to be above of that declared on the labels, even after the shelf life had been exceeded. A small decrease in the amount of B group vitamins was observed during the shelf life of the products. In the case of vitamin C this decrease was slightly higher. The present research shows the need of increased quality control and inspection.

Simultaneous determination of B-Group vitamins in enriched cookies

The objective of this research was to determine the levels of enrichment of vitamins B1, B2, B6 and B3 in different types and brands of enriched cookies. The chromatographic separation was performed in a C18 column with gradient elution and UV detection at 254 and 287 nm. The results show that only 5 of the 24 brands evaluated are in accordance with the Brazilian legislation with respect to the vitamin content declared on the labels. However, consumption of approximately 100-150 g of most of the brands supplies the recommended dietary intake for children and adults of the vitamins evaluated.

Curiosidades sobre a reação aldólica utilizada como etapa chave na síntese Brasileira dos ácidos pterídicos A e B

This work describes an overview of our synthesis of pteridic acids A and B and discloses some interesting results related to the lithium enolate-mediated aldol reaction used as key step to set up the C5-C15 fragment of these natural products. This first example, as far we know, of an aldol reaction between a chiral enolate of a (Z) enone and a chiral aldehyde has driven us to a series of experiments showing the remarkable relation between enolization selectivity and reaction conditions.

Influence of light and temperature on seed germination of Cereus pernambucensis Lemaire (Cactaceae)

The objective of the present study was to evaluate the effects of light and temperature on germination of Cereus pernambucensis seeds, a species of columnar cactus native to Brazil and naturally incident in the restinga. Cereus pernambucensis seeds were incubated under different temperatures, from 5 to 45 °C, with 5 °C intervals, and under alternating temperatures of 15-20 °C, 15-30 °C, 20-25 °C, 20-30 °C, 20-35 °C, 25-30 °C, 25-35 °C, and 30-35 °C, both under continuous white light and dark. The seeds were also incubated in a gradient of phytochrome photoequilibrium at 25 °C. The highest percentage germination in this species was between 25 and 30 °C. The minimum temperature was between 15 and 20 °C and the maximum between 35 and 40 °C. Alternating temperatures did not affect the percentage of seed germination, but it did alter the rate and synchronization indexes. Seeds incubated in the dark did not germinate under any of the conditions tested, indicating that this species when cultivated present light sensitive seeds controlled by phytochrome. The seeds can tolerate a lot of shade conditions, germinating under very low fluence response of phytochrome.

Differentiation of Melipona quadrifasciata L. (Hymenoptera, Apidae, Meliponini) subspecies using cytochrome b PCR-RFLP patterns

Melipona quadrifasciata quadrifasciata and M. quadrifasciata anthidioides are subspecies of M. quadrifasciata, a stingless bee species common in coastal Brazil. These subspecies are discriminated by the yellow stripe pattern of the abdominal tergites. We found Vsp I restriction patterns in the cytochrome b region closely associated to each subspecies in 155 M. quadrifasciata colonies of different geographical origin. This mitochondrial DNA molecular marker facilitates diagnosis of M. quadrifasciata subspecies matrilines and can be used to establish their natural distribution and identify hybrid colonies.

Methodology of a nationwide cross-sectional survey of prevalence and epidemiological patterns of hepatitis A, B and C infection in Brazil

Um inquérito de base populacional foi conduzido na população urbana de todas as capitais e do Distrito Federal no Brasil para fornecer informações sobre a prevalência de hepatites virais e fatores de risco, entre 2005 e 2009. Este artigo descreve o delineamento e a metodologia do estudo que envolveu a população com idade entre 5 e 19 anos para hepatite A e 10 a 69 anos para hepatite B e C. As entrevistas e amostras de sangue foram obtidas através de visitas domiciliares e a amostra selecionada a partir de uma amostragem estratificada em múltiplos estágios (por conglomerado) com igual probabilidade para cada domínio de estudo (região e faixa etária). Nacionalmente, 19.280 residências e ~31.000 indivíduos foram selecionados. O tamanho da amostra foi suficiente para detectar uma prevalência em torno de 0,1% e para avaliar os fatores de risco por região. A metodologia apresentou-se viável para distinguir entre diferentes padrões epidemiológicos da hepatite A, B e C. Estes dados serão de valia para a avaliação das políticas de vacinação e para o desenho de estratégias de controle.