Gold Standard Cholera Diagnostics Are Tarnished by Lytic Bacteriophage and Antibiotics.

A fundamental, clinical, and scientific concern is how lytic bacteriophage, as well as antibiotics, impact diagnostic positivity. Cholera was chosen as a model disease to investigate this important question, because cholera outbreaks enable large enrollment, field methods are well established, and the predatory relationship between lytic bacteriophage and the etiologic agent Vibrio cholerae share commonalities across bacterial taxa. Patients with diarrheal disease were enrolled at two remote hospitals in Bangladesh. Diagnostic performance was assessed as a function of lytic bacteriophage detection and exposure to the first-line antibiotic azithromycin, detected in stool samples by mass spectrometry. Among diarrheal samples positive by nanoliter quantitative PCR (qPCR) for V. cholerae (n = 78/849), the odds that a rapid diagnostic test (RDT) or qPCR was positive was reduced by 89% (odds ratio [OR], 0.108; 95% confidence interval [CI], 0.002 to 0.872) and 87% (OR, 0.130; 95% CI, 0.022 to 0.649), respectively, when lytic bacteriophage were detected. The odds that an RDT or qPCR was positive was reduced by more than 99% (OR, 0.00; 95% CI, 0.00 to 0.28) and 89% (OR, 0.11; 95% CI, 0.03 to 0.44), respectively, when azithromycin was detected. Analysis of additional samples from South Sudan found similar phage effects on RDTs; antibiotics were not assayed. Cholera burden estimates may improve by accommodating for the negative effects of lytic bacteriophage and antibiotic exposure on diagnostic positivity. One accommodation is using bacteriophage detection as a proxy for pathogen detection. These findings have relevance for other diagnostic settings where bacterial pathogens are vulnerable to lytic bacteriophage predation.

The effect of grazing system intensification on the growth and meat quality of beef cattle in the Brazilian Atlantic Forest biome.

This study was carried out to evaluate the effects of four levels of intensification of grazing systems: 1) degraded pasture - DP; 2) irrigated pasture with high stocking rate - IHS; 3) dryland pasture with high stocking rate - DHS; 4) dryland pasture with moderate stocking rate - DMS; on growth, muscle development and meat quality of Nellore steers (271±2.2kg of live body weight - BW; 15months old) during two consecutive periods (17 and 15months). The final BW, the average daily BW gain, the hot carcass weight and the dress percentage were greater (P<0.0001), and the ribeye area tended to be greater (P=0.085), in the intensified systems compared to the degraded system. Animals in all systems presented similar back fat. Muscle development increased with the intensification of the grazing systems and meat quality was not affected.

Tiempo de recanalización y reflujo valvular después de la trombosis venosa profunda de los MI / Recanalization time and venous valve reflux following deep venous trombosis in lower limbs

Evaluar con ultrasonido doppler a color el tiempo de recanalización, los eventos de retrombosis y aparición de reflujo venoso en dos grupos de pacientes de acuerdo a la localización de la trombosis venosa profunda (DVT)...La trombosis venosa profunda y sus secuelas pueden ser documentadas por ultrasonografía doppler color. Las trombosis venosas profundas proximales tienen un curso natural desfavorable no solamente en el tiempo de la recanalización sino en la severidad del reflujo venoso. La heparina podría no ser una eficaz alternativa terapéutica en este subgrupo específico de pacientes (TVP proximal)

Tiempo de recanalización y reflujo valvular después de la trombosis venosa profunda de los MI / Recanalization time and venous valve reflux following deep venous trombosis in lower limbs

Evaluar con ultrasonido doppler a color el tiempo de recanalización, los eventos de retrombosis y aparición de reflujo venoso en dos grupos de pacientes de acuerdo a la localización de la trombosis venosa profunda (DVT)...La trombosis venosa profunda y sus secuelas pueden ser documentadas por ultrasonografía doppler color. Las trombosis venosas profundas proximales tienen un curso natural desfavorable no solamente en el tiempo de la recanalización sino en la severidad del reflujo venoso. La heparina podría no ser una eficaz alternativa terapéutica en este subgrupo específico de pacientes (TVP proximal)

Relationship between soil copper content and copper content of selected crop plants in central Chile.

A survey of copper levels in agricultural soils of central Chile revealed two soil clusters-one with a mean copper level of 162 mg/kg and one with a mean copper level of 751 mg/kg of soil. Samples of soils from both soil clusters were characterized on the basis of physicochemical characteristics, and copper extractability was compared by saturation and CaCl2 extraction as well as an acid-leaching procedure (TCLP). We also measured the copper content of various tissues of tomato (Lycopersicon esculentum) and onion (Allium cepa) crops growing on these soils. Other than copper levels, soils from the two clusters were quite similar, with slightly greater levels of molybdenum and cadmium in the high-copper soils. Within each cluster, extracted copper levels and total soil copper levels were not correlated. However, the three extraction procedures solubilized significantly more copper from the high-Cu soils. Mineralogical characterization of the soil particles and depth profiles of soil metal levels in a subsample of sites suggested that highly insoluble copper ore and mining wastes might account for the high copper levels. Neither total nor extractable copper levels allowed statistical prediction of the levels of copper in plant tissue. The edible tissues of both crops had the same mean copper content, regardless of the copper soil level. However, copper contents of stems and leaves were significantly higher for plants growing on the high-Cu soils. These results show that in these soils, high copper levels are associated with very insoluble copper species and thus low bioavailability of copper to crop plants.

Comparative development of Brugia pahangi and variation in acid hydrolase enzyme titers in.

The development of the filarial nematode Brugia pahangi was monitored and compared in susceptible (BLACK EYE) and refractory (ROCK) strains of Aedes aegypti. Simultaneously, the activities of acid phosphatase, beta-glucuronidase, alpha-glucosidase, and N-acetyl-beta-glucosaminidase were measured. Three- to five-day-old females of both strains were fed on infected and uninfected clawed jirds (Meriones unguiculatus) then dissected or homogenized at 2 h, at 24-h intervals for 5 days, and at 8 and 10 days after treatment. Enzyme activities were assayed by a fluorometric procedure. The susceptible strain maintained an 80% infection and 18.6 larvae/mosquito over the 10-day period. In contrast, the refractory strain was initially 33% infected and had a mean of 4.9 larvae/mosquito and this decreased to 20% by 3 days, and to 3% with a mean of 0.33 larvae/mosquito at 10 days. Significantly higher acid phosphatase and beta-glucuronidase activities were observed in the refractory strain at specific time intervals after infection. Alpha-glucosidase and N-acetyl-beta-glucosaminidase were highly variable among strains and according to infection status. Analysis of the results of this study suggests that certain acid hydrolase enzymes could be involved in the elimination of B. pahangi in refractory strains of Ae. aegypti and could be used to monitor biochemical changes in response to filarial nematode infections in certain mosquito populations.

Reduction in susceptibility to Brugia malayi of F2 progeny of Aedes togoi treated with ethyl methanesulfonate.

The susceptibility to Brugia malayi infection was tested in F2 female progeny derived from male and female Aedes togoi treated with ethyl methanesulfonate (EMS). Three-day-old males and females were treated with 0.025, 0.050, and 0.075, 0.10, 0.15, or 0.20% EMS by allowing them to feed for 5 days on sugar cubes containing EMS and then mated at random. Percentage of susceptibility and mean number of infective larvae (L3) in F2 females were analyzed over a 2-wk period. Reductions in susceptibility were significant in the F2 populations arising from the 3 highest EMS concentrations. F2 infections were reduced by 80%, indicating that EMS-induced mutations affect loci associated with filarial nematode susceptibility.

Ligand-induced conformational transitions in Escherichia coli phosphofructokinase 2: evidence for an allosteric site for MgATP2-.

The binding of ligands to phosphofructokinase 2 (Pfk-2) from Escherichia coli induces changes in the fluorescence emission properties of its single tryptophan residue, Trp88, suggesting that upon binding the protein undergoes a conformational change. This fluorescence probe was used to determine the presence of an allosteric site for MgATP2- in the enzyme. Fructose 6-phosphate (fructose-6-P), the first substrate that binds to the enzyme with an ordered bi-bi mechanism, increases the fluorescence up to 30%. The saturation curve for this compound is hyperbolic with a Kd of 6 microM. The titration of Pfk-2 with MgATP2- causes a quenching of fluorescence of about 30% of its initial value, with a blue shift of 7 nm in the emission maximum. The response is cooperative with a Kd of 80 microM and a Hill coefficient of 2. The interaction of MgATP2- cannot take place at the active site in the absence of fructose-6-P, due to the ordered kinetic mechanism. Addition of compounds that bind to the catalytic site of Pfk-2, such as ATP4- or Mg-AMP-PNP, did not produce significant changes in the fluorescence spectrum of Trp88. However, in the absence of Mg2+, the addition of ATP4- to the enzyme-fructose-6-P complex shows a hyperbolic increase of fluorescence of 8%. Acrylamide steady-state quenching experiments for different enzyme-ligand complexes of Pfk-2, indicate that the tryptophan in the enzyme-MgATP2- complex is exposed to a much smaller extent to the solvent than in the free enzyme or in the enzyme-fructose-6-P complex. The effect of the binding of fructose-6-P or MgATP2- on the polarization fluorescence of the emission of Trp88 in Pfk-2 indicates changes in the local mobility of the Trp88 in both enzyme complexes. The average lifetime for Trp88 in Pfk-2 was found to be unusually large, approximately 7.7 ns, and did not vary significantly with the ligation state of the enzyme, which demonstrates that the quenching or enhancement of fluorescence induced by the ligands is mainly due to the complex formation with Pfk-2. These results demonstrate the presence of an allosteric site for MgATP2- in Pfk-2 from E. coli, responsible for the inhibition of the enzyme activity by this ligand.

Diferentes fontes de cálcio na dieta do escargot gigante africano (Achatina fulica) e seu efeito no crescimento e rendimento de carcaça / Different calcium sources and their effect on the carcass of the snail Achatina fulica

Avaliou-se a eficiência do emprego de diferentes fontes de cálcio e o seu efeito sobre a carcaça do escargot Achatina fulica. Montou-se um ensaio, em delineamento inteiramente casualizado, com 4 tratamentos, com 10 animais/repetiçöes, com tamanho semelhante, aos 45 dias de idade, com duraçäo de 120 dias, utilizando a Dieta Base proposta por CUELLAR et al (1986) como padräo. Os tratamentos aplicados por kg de D.B foram: T1(250g/de calcário calcinado), T2(250g/ de farinha de concha de escargot); T3(250g/de osso calcinado) e T4(D.B. c/ 25 por cento de carbonato de cálcio). Ao término do ensaio, os animais foram submetidos a jejum de 7 dias e em seguida abatidos, e individualmente, pesados, mensurados e a carcaça fracionada em: concha, carne, hepatopâncreas, genitália/epifragma e líquidos (sangue, muco e água).

Variation in acid hydrolase enzyme titers in different developmental stages of Aedes togoi.

Genetic mechanisms of filarial nematode susceptibility were studied in Aedes togoi. Acid hydrolases may play an important role in this process, including humoral or cell-mediated defenses. Levels of acid phosphatase, alpha-glucosidase, beta-glucuronidase, and N-acetyl-beta-glucosaminidase were determined for 1st- and 4th-instar larvae, male and female pupae, and 1- and 7-day-old adults using fluorometric and colorimetric assays. Acid phosphatase activity was highest in 1-day-old adults, moderate in larvae and pupae, and lowest in 7-day-old adults. Female 7-day-old adults had significantly higher levels than males of the same age. Moderate levels of alpha-glucosidase were found in larvae, with progressive increases in activity from pupae to 7-day-old adults. Levels in male pupae and 1-day-old males were higher than in females, but activity was twice as high in 7-day-old females. Activity of beta-glucuronidase was greater in adults, with females showing a 2-fold higher level than males at 7 days. In contrast, N-acetyl-beta-glucosaminidase activity was highest in 1st- and 4th-instar larvae and 1-day-old males and females. Activity also was significantly higher in male pupae, slightly greater in 1-day-old males, but twice as high in 7-day-old females when compared to males of the same age. Results showed significant changes and variation in acid hydrolase enzyme titers in the different life stages of Ae. togoi. These and other specific acid hydrolases could prove effective in monitoring biochemical and genetic changes in mosquito populations.

Protons induce calsequestrin conformational changes.

Calsequestrin, a high-capacity, intermediate-affinity, calcium-binding protein present in the lumen of sarcoplasmic reticulum, undergoes extensive calcium-induced conformational changes at neutral pH that cause distinct intrinsic fluorescence changes. The results reported in this work indicate that pH has a marked effect on these calcium-induced intrinsic fluorescence changes, as well as on calorimetric changes produced by the addition of Ca(2+) to calsequestrin. The addition of Ca(2+) at neutral pH produced a marked and cooperative increase in calsequestrin intrinsic fluorescence. In contrast, at pH 6.0 calsequestrin's intrinsic fluorescence was not affected by the addition of Ca(2+), and the same intrinsic fluorescence as that measured in millimolar calcium at neutral pH was obtained. The magnitude and the cooperativity of the calcium-induced intrinsic fluorescence changes decreased as either [H+] or [K+] increased. The evolution of heat production, determined by microcalorimetry, observed upon increasing the molar ratio of Ca(2+) to calsequestrin in 0.15 M KCl, decreased markedly as the pH decreased from pH 8.0 to pH 6.0, indicating that pH modifies the total heat content changes produced by Ca(2+). We propose that protons bind to calsequestrin and induce protein conformational changes that are responsible for the observed proton-induced intrinsic fluorescence and calorimetric changes.

Calcium and gadolinium ions stimulate the GTPase activity of purified chicken brain tubulin through a conformational change.

Ca2+ and Gd3+ stimulated the GTPase activity of chicken brain tubulin 13- and 26-fold, respectively. Mg2+, Tb3+, and Na+ had no effect. This GTPase activity showed a saturation behavior with Ca2+ and Gd3+ with a maximal activity of 0.26 +/- 0.026 and 1.15 +/- 0.78 nmol min-1 per mg of tubulin and semisaturation constants, expressed as the concentration of the cation needed for 50% of saturation, of 0.32 +/- 0.18 and 0.011 +/- 0.007 mM, respectively. In the presence of Ca2+, the GTPase activity was proportional to tubulin concentration in the range 0.9-31.8 microM. The semisaturation constants for the inhibition of tubulin polymerization and for the depolymerization of microtubules by Ca2+ were 0.71 +/- 0.1 and 0.049 +/- 0.043 mM, respectively. The similarity of the Ca2+ semisaturation constants for inhibition of tubulin assembly and stimulation of the GTPase activity suggests that these processes are correlated. These results support the hypothesis that the GTPase activity is related to but not directly involved in the mechanism of inhibition of Ca2+ -dependent tubulin assembly. This inhibition could be better explained by the formation of a nonfunctional conformational state of tubulin induced by Ca2+ that is responsible for the GTPase activity. Quenching of the intrinsic fluorescence of tryptophan induced by Ca2+ showed an apparent dissociation constant of 0.14 +/- 0.005 mM, in the range of values determined through tubulin polymerization inhibition or through the induction of GTPase activity by Ca2+. Acrylamide-induced quenching of the intrinsic fluorescence showed values of the Stern-Volmer constants of 5.4 +/- 0.12 and 5.0 +/- 0.15 M-1 in the absence and presence of Ca2+, respectively. These results support the hypothesis that the inhibition of tubulin polymerization and the induction of the GTPase activity by Ca2+ is mediated by a conformational change. Ca2+ failed to induce depolymerization of GDP-AIF4-microtubules; this could be explained by a model in which Ca-tubulin is unable to assemble into microtubules and the rate of dissociation of GDP-Pi-tubulin from the microtubule ends is extremely slow compared with the rate of GDP-subunit dissociation, supporting the concept that the GTP- and GDP-Pi-tubulin cap at the ends of microtubules regulates their dynamic instability.

Comparison of the substrate specificities of cAMP-dependent protein kinase from bovine heart and Ascaris suum muscle.

The catalytic subunits of cAMP-dependent protein kinases (protein kinase A) from bovine heart and Ascaris suum muscle exhibit only 48% sequence identity and show quantitative differences in substrate specificity. These differences were not obvious at the level of short synthetic substrate peptides but were distinct for some protein substrates. Phosphofructokinase from Ascaris, a physiological substrate, was a better substrate for the protein kinase from the nematode in comparison to the mammalian protein kinase due to a 10-fold lower Michaelis constant. Selective phosphorylation by the two kinases was also observed with some in vitro substrates. In addition, quantitative differences in the interactions between R- and C-subunits from Ascaris and bovine heart were observed. However, several synthetic peptides whose sequence reflected the phosphorylation site of Ascaris suum phosphofructokinase (AKGRSDS*IV), or variations of it, were phosphorylated with the same efficiency by both protein kinases. Based on the data the following are concluded: (1) In agreement with the conservation of structure in the catalytic cleft, the recognition of substrates by protein kinases from phylogenetically distant organisms exhibits similarity. (2) Non-conserved parts of the surface of the protein kinase molecule may contribute to binding of protein substrates and thus to selective recognition.

The catalytic subunit of cAMP-dependent protein kinase from Ascaris suum. The cloning and structure of a novel subtype of protein kinase A.

A complete cDNA clone encoding the catalytic subunit of cAMP-dependent protein kinase of Ascaris suum was constructed from two overlapping partial clones. The encoded sequence of 337 amino acids is 48% identical with the sequence of mouse C alpha subunit. Approximately the same low similarity was found with the sequence of the C subunit from another nematode, Caenorhabditis elegans. The N-terminal 14 amino acids and the myristoylation site of the mammalian protein are not contained in the enzyme from Ascaris. Two cysteines (Cys33 and Cys319) replace a basic residue in the N-terminal region and an acidic amino acid near the C-terminus which are conserved in all known C subunits from other sources. The substitutions provide the possibility of disulfide bridge formation between the N-terminal and C-terminal parts of the protein. There is strong evidence that a single gene encodes cAMP-dependent protein kinase in Ascaris. Modelling of the sequence into the coordinates of the X-ray structure of the mammalian enzyme suggest a high degree of conservation in the three-dimensional structure. However, structural variations occur at the surface of the protein near the catalytic cleft and are likely to account for the variations in substrate specificity previously observed between the purified protein kinase from Ascaris [Thalhofer, H. P., Daum, G., Harris, B. G. & Hofer, H. W. (1988) J. Biol. Chem. 263, 952-957] and the mammalian enzyme.

Reduction in the susceptibility of Aedes aegypti to Brugia malayi infection after treatment with ethyl methanesulfonate.

The susceptibility to Brugia malayi infections of F2 and F4 progenies of Aedes aegypti (Black Eye strain) treated with ethyl methanesulfonate (EMS) was tested. Both 3-day-old males and females were treated with 0.025, 0.050, 0.075 and 0.10% EMS. Control and treated females were then mated with normal or treated males to recover F1 progeny. F2 offspring were derived from sibling intercrosses, and 3 lines were established by further intercross matings to generate the F4. Susceptibility in the 0.025, 0.050, 0.075 and 0.10% EMS F2s was reduced by 13, 12, 4 and 25%, respectively. The 0.025% and 0.050% EMS F2 females showed a 29 to 39% decrease in mean L3 numbers. At 0.075 and 0.10% EMS, mean L3 numbers decreased by 0.8 and 71%, respectively. The F4 populations gave overall infections of 65, 56 and 22% for the control, 0.25 and 0.10% EMS lines, respectively. Mean L3s were reduced by 24 and 77%, respectively, in the 0.025 and 0.10% F4 EMS-selected populations.

Reduced productivity in adult yellowfever mosquito (Diptera: Culicidae) populations.

Male and female Aedes aegypti (L.) mosquitoes of the laboratory strain ROCK were irradiated with 130 mw of argon 514.5 nm laser microbeams for 0.04, 0.25, 0.4, and 0.5 s, respectively. Egg production, percentage hatch, and productivity (average number of adults surviving after 3 wk) were used to assess mutagenic effects. Mortality was high for males in all laser radiation groups and increased with time of exposure. Except for the group treated for 0.25 s, significant reductions in total F1 progeny also were demonstrated for all other experimentals when male parents were exposed to laser radiation. Females showed a high mortality when subjected to 0.4- and 0.5-s laser radiation. No F1 progeny were produced when parental females were exposed for 0.25, 0.4, and 0.5 s. Numbers of F1 progeny from females exposed to 0.04 s of laser radiation were significantly reduced. A comparison of weekly mean number of progeny showed that the important differences in productivity occurred during the first and second week, respectively, when either male or female adult parents were subjected to laser radiation.