Enterohemorrhagic Escherichia coli O157: H7 from healthy dairy cattle in Mid-West Brazil: occurrence and molecular characterization

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 represents the major Shiga toxin-producing E. coli (STEC) strain related to large outbreaks and severe diseases such as hemorrhagic colitis (HC) and the potentially lethal hemolytic uremic syndrome (HUS). The aim of this study was to report the occurrence and molecular characterization of O157:H7 isolates obtained by rectal swab from 52 healthy dairy cattle belonging to 21 farms in Mid-West of Brazil. Detection of 16SrRNA, stx1, stx2, rfbO157, fliCh7, eae, ehxA, saa, cnf1, chuA, yjaA and TSPE4.C2 genes was performed by PCR. The isolates were further characterized by serotyping. Two hundred and sixty E. coli isolates were obtained, of which 126 were characterized as STEC. Two isolates from the same cow were identified as serotype O157:H7. Both isolates presented the stx2, eae, ehxA, saa and cnf1 virulence factor genes and the chuA gene in the phylogenetic classification (virulent group D), suggesting that they were clones. The prevalence of O157:H7 was found to be 1.92% (1/52 animals), demonstrating that healthy dairy cattle from farms in the Mid-West of Brazil are an important reservoir for highly pathogenic E. coli O157:H7.

Rikosperusteisen erityistarkastuksen laadun osa-alueet

Viimeisenä kymmenenä vuotena valtiovalta on halunnut panostaa harmaan talouden torjuntaan. Tätä kautta panoksia on lisätty myös talousrikollisuuden torjuntaan, mm. lisäämällä tutkiville viranomaisille myönnettyjen resurssien määrää. Poliisi yhdessä syyttäjän kanssa on keskeisessä roolissa talousrikostutkinnassa ja -torjunnassa. Talousrikosten tunnusmerkistön toteen näyttämisessä rikosperusteiselle erityistarkastuksella on erityisen tärkeä rooli. Tutkielmassa on paneuduttu rikosperusteisen erityistarkastuksen laadun määrittelyyn ja laatuun vaikuttaviin tekijöihin. Tutkielman lähdeaineistona käytettiin mm. teoriakirjallisuutta tilintarkastusalalta sekä talousrikollisuutta käsittelevästä kirjallisuudesta. Lisäksi käytännön työn kannalta olennaisia seikkoja on kerätty asiantuntijahaastatteluilla. Tämän tutkielman tuloksena on eritelty tarkkaan rikosperusteisen erityistarkastuksen laatuun vaikuttavia tekijöitä. Kyseiset tekijät on jaoteltu neljän eri pääkriteerin kautta. Tarkastuksen laatukriteereitä taas on tarkasteltu suhteessa tilintarkastusalaan ja tuloksena on luotu kriteereiden jaottelu, joka helpottaa laatuun vaikuttavien tekijöiden hahmottamista.

Sorption of oxadiazon in soils cultivated in the brazilian cerrado

The objective of this study was to evaluate oxadiazon sorption in different soils of the Brazilian Cerrado, highlighting the correlations of lethal doses of this herbicide capable of inhibiting 50% of the dry matter accumulation of the bio-indicator (LD50) among the chemical characteristics of the soil and its direct and indirect effects. The experiment was carried out in a greenhouse in a randomized block design and four repetitions. Each experimental unit consisted of a pot with increasing rates of oxadiazon and oat (Avena sativa), as the bio-indicator species. For sorption evaluation, washed sand and 22 soils (substrates) from Cerrado Brazilian's Alliaceae cultivated areas were used. LD50 and sorption ratio (SR) = [(LD50soil - LD50sand)/LD50sand] to the substrates were determined. Pearson correlation analysis was performed between the chemical characteristics of the substrates and the LD50 of oxadiazon. A path analysis was quantified, to deploy only the significant correlations estimated in direct and indirect effects of the characters on LD50, which is a basic variable. A more pronounced LD50 (528.09 g ha-1) for the Cerrado soil sample resulted in higher SR (> 53.00), while in the washed sand substrate, LD50 corresponded only to 9.74 g ha-1 of the oxadiazon (available in soil). It was concluded that oxadiazon sorption is influenced by the chemical characteristics of the soils, highlighting the correlation with pH (CaCl2), magnesium content, aluminum, organic matter, organic carbon, and aluminum saturation.

Imazapyr herbicide efficacy on floating macrophyte control and ecotoxicology for non-target organisms

the aims of this study were to determine imazapyr efficacy for floating macrophyte control and ecotoxicology for non-target organisms. For the floating macrophyte control efficacy tests were used the doses of 0,5; 1,0; 2,0; 2,5; 3,0; 3,5 and 4,0 L ha-1 and a control with 10 replicates. The acute toxicology for non-target organisms was estimated by lethal concentration 50% (LC50 and EC50). The floating macrophyte control efficacy was over 90%. Imazapyr was classified as moderately toxic for the following biomarkers: L. minor, H. eques, B. rerio, P. caudimaculatus, P. canaliculata, and P. mesopotamicus and lightly toxic for A. caroliniana. Thus, imazapyr herbicide is a tool with great potential to be used on floating macrophyte control (E. crassipes, P. stratiotes e S. molesta) in Brazil and this practice can be evaluated by the use of application biomarkers.

Ecophysiology and respiratory metabolism during the germination of Inga sessilis (Vell.) Mart. (Mimosaceae) seeds subjected to hypoxia and anoxia

This paper presents a study on the respiratory metabolism of germinating seeds of Inga sessilis subjected to normoxia, hypoxia and anoxia. Although it is typical of environments where waterlogging seldom occurs, 40% of its seeds are able to germinate under hypoxia; yet, anoxia periods over 96 h are lethal to the seeds. Ethanol is the main product of the seeds anaerobic metabolism, but the steep increase in lactate after 24 h anoxia or 48 h hypoxia may explain the drop in seed viability.

Quantitative Analysis of Novel Prostate Cancer Markers in Tissue

Prostate cancer is a heterogeneous disease affecting an increasing number of men all over the world, but particularly in the countries with the Western lifestyle. The best biomarker assay currently available for the diagnosis of the disease, the measurement of prostate specific antigen (PSA) levels from blood, lacks specificity, and even when combined with invasive tests such as digital rectal exam and prostate tissue biopsies, these methods can both miss cancers, and lead to overdiagnosis and subsequent overtreatment of cancers. Moreover, they cannot provide an accurate prognosis for the disease. Due to the high prevalence of indolent prostate cancers, the majority of men affected by prostate cancer would be able to live without any medical intervention. Their latent prostate tumors would not cause any clinical symptoms during their lifetime, but few are willing to take the risk, as currently there are no methods or biomarkers to reliably differentiate the indolent cancers from the aggressive, lethal cases that really are in need of immediate medical treatment. This doctoral work concentrated on validating 12 novel candidate genes for use as biomarkers for prostate cancer by measuring their mRNA expression levels in prostate tissue and peripheral blood of men with cancer as well as unaffected individuals. The panel of genes included the most prominent markers in the current literature: PCA3 and the fusion gene TMPRSS2-ERG, in addition to BMP-6, FGF-8b, MSMB, PSCA, SPINK1, and TRPM8; and the kallikrein-related peptidase genes 2, 3, 4, and 15. Truly quantitative reverse-transcription PCR assays were developed for each of the genes for the purpose, time-resolved fluorometry was applied in the real-time detection of the amplification products, and the gene expression data were normalized by using artificial internal RNA standards. Cancer-related, statistically significant differences in gene transcript levels were found for TMPRSS2-ERG, PCA3, and in a more modest scale, for KLK15, PSCA, and SPINK1. PCA3 RNA was found in the blood of men with metastatic prostate cancer, but not in localized cases of cancer, suggesting limitations for using this method for early cancer detection in blood. TMPRSS2-ERG mRNA transcripts were found more frequently in cancerous than in benign prostate tissues, but they were present also in 51% of the histologically benign prostate tissues of men with prostate cancer, while being absent in specimens from men without any signs of prostate cancer. PCA3 was shown to be 5.8 times overexpressed in cancerous tissue, but similarly to the fusion gene mRNA, its levels were upregulated also in the histologically benign regions of the tissue if the corresponding prostate was harboring carcinoma. These results indicate a possibility to utilize these molecular assays to assist in prostate cancer risk evaluation especially in men with initially histologically negative biopsies.

Conflicting genomes, the demic theory & biodiversity

Organismic-centered Darwinism, in order to use direct phenotypes to measure natural selection's effect, necessitates genome's harmony and uniform coherence plus large population sizes. However, modern gene-centered Darwinism has found new interpretations to data that speak of genomic incoherence and disharmony. As a result of these two conflicting positions a conceptual crisis in Biology has arisen. My position is that the presence of small, even pocket-size, demes is instrumental in generating divergence and phenotypic crisis. Moreover, the presence of parasitic genomes as in acanthocephalan worms, which even manipulate suicidal behavior in their hosts; segregation distorters that change meiosis and Mendelian ratios; selfish genes and selfish whole chromosomes, such as the case of B-chromosomes in grasshoppers; P-elements in Drosophila; driving Y-chromosomes that manipulate sex ratios making males more frequent, as in Hamilton's X-linked drive; male strategists and outlaw genes, are eloquent examples of the presence of real conflicting genomes and of a non-uniform phenotypic coherence and genome harmony. Thus, we are proposing that overall incoherence and disharmony generate disorder but also more biodiversity and creativeness. Finally, if genes can manipulate natural selection, they can multiply mutations or undesirable characteristics and even lethal or detrimental ones, hence the accumulation of genetic loads. Outlaw genes can change what is adaptively convenient even in the direction of the trait that is away from the optimum. The optimum can be "negotiated" among the variants, not only because pleiotropic effects demand it, but also, in some cases, because selfish, outlaw, P-elements or extended phenotypic manipulation require it. With organismic Darwinism the genome in the population and in the individual was thought to act harmoniously without conflicts, and genotypes were thought to march towards greater adaptability. Modern Darwinism has a gene-centered vision in which genes, as natural selection's objects can move in dissonance in the direction which benefits their multiplication. Thus, we have greater opportunities for genomes in permanent conflict.

Cardiovascular, respiratory and metabolic responses to temperature and hypoxia of the winter frog Rana catesbeiana

The objective of the present study was to determine the effects of hypoxia and temperature on the cardiovascular and respiratory systems and plasma glucose levels of the winter bullfrog Rana catesbeiana. Body temperature was maintained at 10, 15, 25 and 35oC for measurements of breathing frequency, heart rate, arterial blood pressure, metabolic rate, plasma glucose levels, blood gases and acid-base status. Reducing body temperature from 35 to 10oC decreased (P<0.001) heart rate (bpm) from 64.0 ± 3.1 (N = 5) to 12.5 ± 2.5 (N = 6) and blood pressure (mmHg) (P<0.05) from 41.9 ± 2.1 (N = 5) to 33.1 ± 2.1 (N = 6), whereas no significant changes were observed under hypoxia. Hypoxia-induced changes in breathing frequency and acid-base status were proportional to body temperature, being pronounced at 25oC, less so at 15oC, and absent at 10oC. Hypoxia at 35oC was lethal. Under normoxia, plasma glucose concentration (mg/dl) decreased (P<0.01) from 53.0 ± 3.4 (N = 6) to 35.9 ± 1.7 (N = 6) at body temperatures of 35 and 10oC, respectively. Hypoxia had no significant effect on plasma glucose concentration at 10 and 15oC, but at 25oC there was a significant increase under conditions of 3% inspired O2. The arterial PO2 and pH values were similar to those reported in previous studies on non-estivating Rana catesbeiana, but PaCO2 (37.5 ± 1.9 mmHg, N = 5) was 3-fold higher, indicating increased plasma bicarbonate levels. The estivating bullfrog may be exposed not only to low temperatures but also to hypoxia. These animals show temperature-dependent responses that may be beneficial since during low body temperatures the sensitivity of most physiological systems to hypoxia is reduced

Neutralizing capacity of a new monovalent anti-Bothrops atrox antivenom: comparison with two commercial antivenoms

Three horse-derived antivenoms were tested for their ability to neutralize lethal, hemorrhagic, edema-forming, defibrinating and myotoxic activities induced by the venom of Bothrops atrox from Antioquia and Chocó (Colombia). The following antivenoms were used: a) polyvalent (crotaline) antivenom produced by Instituto Clodomiro Picado (Costa Rica), b) monovalent antibothropic antivenom produced by Instituto Nacional de Salud-INS (Bogotá), and c) a new monovalent anti-B. atrox antivenom produced with the venom of B. atrox from Antioquia and Chocó. The three antivenoms neutralized all toxic activities tested albeit with different potencies. The new monovalent anti-B. atrox antivenom showed the highest neutralizing ability against edema-forming and defibrinating effects of B. atrox venom (41 ± 2 and 100 ± 32 µl antivenom/mg venom, respectively), suggesting that it should be useful in the treatment of B. atrox envenomation in Antioquia and Chocó

Induction of neutralizing antibodies in mice immunized with scorpion toxins detoxified by liposomal entrapment

The possibility of producing neutralizing antibodies against the lethal effects of scorpion toxins was evaluated in the mouse model by immunization with an immunogen devoid of toxicity. A toxic fraction (5 mg) from the venom of the scorpion Tityus serrulatus was entrapped in sphingomyelin-cholesterol liposomes. The liposomes were treated for 1 h at 37oC with a 1% (w/w) trypsin solution in 0.2 M sodium carbonate buffer, pH 8.3. This treatment led to a strong reduction in venom toxicity. Immunization was performed as follows: mice were injected sc with 20 µg of the liposome-entrapped toxic fraction on days 1 and 21 and a final injection (20 µg) was administered ip on day 36. After injection of the immunogen, all mice developed an IgG response which was shown to be specific for the toxic antigen. The antibodies were measured 10 days after the end of the immunization protocol. In an in vitro neutralization assay we observed that pre-incubation of a lethal dose of the toxic fraction with immune serum strongly reduced its toxicity. In vivo protection assays showed that mice with anti-toxin antibodies could resist the challenge with the toxic fraction, which killed, 30 min after injection, all non-immune control mice

Thalidomide protects mice against LPS-induced shock

Thalidomide has been shown to selectively inhibit TNF-a production in vitro by lipopolysaccharide (LPS)-stimulated monocytes. TNF-a has been shown to play a pivotal role in the pathophysiology of endotoxic shock. Using a mouse model of LPS-induced shock, we investigated the effects of thalidomide on the production of TNF-a and other cytokines and on animal survival. After injection of 100-350 µg LPS into mice, cytokines including TNF-a, IL-6, IL-10, IL-1ß, GM-CSF and IFN-g were measured in the serum. Administration of 200 mg/kg thalidomide to mice before LPS challenge modified the profile of LPS-induced cytokine secretion. Serum TNF-a levels were reduced by 93%, in a dose-dependent manner, and TNF-a mRNA expression in the spleens of mice was reduced by 70%. Serum IL-6 levels were also inhibited by 50%. Thalidomide induced a two-fold increase in serum IL-10 levels. Thalidomide treatment did not interfere with the production of GM-CSF, IL-1ß or IFN-g. The LD50 of LPS in this model was increased by thalidomide pre-treatment from 150 µg to 300 µg in 72 h. Thus, at otherwise lethal doses of LPS, thalidomide treatment was found to protect animals from death

Genetic engineering of baker's and wine yeasts using formaldehyde hyperresistance-mediating plasmids

Yeast multi-copy vectors carrying the formaldehyde-resistance marker gene SFA have proved to be a valuable tool for research on industrially used strains of Saccharomyces cerevisiae. The genetics of these strains is often poorly understood, and for various reasons it is not possible to simply subject these strains to protocols of genetic engineering that have been established for laboratory strains of S. cerevisiae. We tested our vectors and protocols using 10 randomly picked baker's and wine yeasts all of which could be transformed by a simple protocol with vectors conferring hyperresistance to formaldehyde. The application of formaldehyde as a selecting agent also offers the advantage of its biodegradation to CO2 during fermentation, i.e., the selecting agent will be consumed and therefore its removal during down-stream processing is not necessary. Thus, this vector provides an expression system which is simple to apply and inexpensive to use

Structure and function of the cystic fibrosis transmembrane conductance regulator

Cystic fibrosis (CF) is a lethal autosomal recessive genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR). Mutations in the CFTR gene may result in a defective processing of its protein and alter the function and regulation of this channel. Mutations are associated with different symptoms, including pancreatic insufficiency, bile duct obstruction, infertility in males, high sweat Cl-, intestinal obstruction, nasal polyp formation, chronic sinusitis, mucus dehydration, and chronic Pseudomonas aeruginosa and Staphylococcus aureus lung infection, responsible for 90% of the mortality of CF patients. The gene responsible for the cellular defect in CF was cloned in 1989 and its protein product CFTR is activated by an increase of intracellular cAMP. The CFTR contains two membrane domains, each with six transmembrane domain segments, two nucleotide-binding domains (NBDs), and a cytoplasmic domain. In this review we discuss the studies that have correlated the role of each CFTR domain in the protein function as a chloride channel and as a regulator of the outwardly rectifying Cl- channels (ORCCs).

Photodynamic DNA damage induced by phycocyanin and its repair in Saccharomyces cerevisiae

In the present study, we analyzed DNA damage induced by phycocyanin (PHY) in the presence of visible light (VL) using a set of repair endonucleases purified from Escherichia coli. We demonstrated that the profile of DNA damage induced by PHY is clearly different from that induced by molecules that exert deleterious effects on DNA involving solely singlet oxygen as reactive species. Most of PHY-induced lesions are single strand breaks and, to a lesser extent, base oxidized sites, which are recognized by Nth, Nfo and Fpg enzymes. High pressure liquid chromatography coupled to electrochemical detection revealed that PHY photosensitization did not induce 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) at detectable levels. DNA repair after PHY photosensitization was also investigated. Plasmid DNA damaged by PHY photosensitization was used to transform a series of Saccharomyces cerevisiae DNA repair mutants. The results revealed that plasmid survival was greatly reduced in rad14 mutants, while the ogg1 mutation did not modify the plasmid survival when compared to that in the wild type. Furthermore, plasmid survival in the ogg1 rad14 double mutant was not different from that in the rad14 single mutant. The results reported here indicate that lethal lesions induced by PHY plus VL are repaired differently by prokaryotic and eukaryotic cells. Morever, nucleotide excision repair seems to play a major role in the recognition and repair of these lesions in Saccharomyces cerevisiae.

Ischemic preconditioning reduces peripheral oxidative damage associated with brain ischemia in rats

Brain ischemia followed by reperfusion causes neuronal death related to oxidative damage. Furthermore, it has been reported that subjects suffering from ischemic cerebrovascular disorders exhibit changes in circulating platelet aggregation, a characteristic that might be important for their clinical outcome. In the present investigation we studied tert-butyl hydroperoxide-initiated plasma chemiluminescence and thiol content as measures of peripheral oxidative damage in naive and preconditioned rats submitted to forebrain ischemia produced by the 4-vessel occlusion method. Rats were submitted to 2 or 10 min of global transient forebrain ischemia followed by 60 min or 1, 2, 5, 10 or 30 days of reperfusion. Preconditioned rats were submitted to a 10-min ischemic episode 1 day after a 2-min ischemic event (2 + 10 min), followed by 60 min or 1 or 2 days of reperfusion. It has been demonstrated that such preconditioning protects against neuronal death in rats and gerbils submitted to a lethal (10 min) ischemic episode. The results show that both 2 and 10 min of ischemia cause an increase of plasma chemiluminescence when compared to control and sham rats. In the 2-min ischemic group, the effect was not present after reperfusion. In the 10-min ischemic group, the increase was present up to 1 day after recirculation and values returned to control levels after 2 days. However, rats preconditioned to ischemia (2 + 10 min) and reperfusion showed no differences in plasma chemiluminescence when compared to controls. We also analyzed plasma thiol content since it has been described that sulfhydryl (SH) groups significantly contribute to the antioxidant capacity of plasma. There was a significant decrease of plasma thiol content after 2, 10 and 2 + 10 min of ischemia followed by reperfusion when compared to controls. We conclude that ischemia may cause, along with brain oxidative damage and cell death, a peripheral oxidative damage that is reduced by the preconditioning phenomenon.