A time-dependent, two-step binding mode of the nitro dye flavianic acid to trypsin in acid media

Synthetic dyes bind to proteins causing selective coprecipitation of the complexes in acid aqueous solution by a process of reversible denaturation that can be used as an alternative method for protein fractionation. The events that occur before precipitation were investigated by equilibrium dialysis using bovine trypsin and flavianic acid as a model able to cause coprecipitation. A two-step mode of interaction was found to be dependent on the incubation periods allowed for binding, with pronounced binding occurring after 42 h of incubation. The first step seems to involve hydration effects and conformational changes induced by binding of the first dye molecule, following rapid denaturation due to the binding of six additional flavianate anions to the macromolecule.

Assessment of a two-step nucleic acid amplification assay for detection of Neisseria meningitidis followed by capsular genogrouping

Immediate prevention of meningococcal disease relies in part on the prompt treatment with antibiotics of household and other close contacts of cases; however intervention with effective vaccination relies on identification of serogroup-causing strains. Parenteral antibiotic for patient with suspected meningococcal disease before hospital admission is currently recommended. Laboratory standard methods are hindered by failure to detect bacteria by this medical approach to improve patient prognosis. We assessed two polymerase chain reaction (PCR) assays to detect (crgA) and define the serogroups (siaD, orf-2, and ctrA) of Neisseria meningitidis in 120 cerebrospinal fluid (CSF) samples from positive cases (culture or antigen detection or direct smear). The PCR sensitivity for the identification of N. meningitidis was 100% (95% confidence interval, CI, 96-100%) compared to a sensitivity of 46% for culture (95% CI 37-55%), 61% for latex agglutination test (95% CI 52-70%), and 68% for Gram stain (95% CI 59-76%); PCR specificity was 97% (95% CI 82-100%). PCR correctly identified the serogroups A, B, C, W135, Y, and X in CSF samples with a sensitivity of 88% (95% CI 80-93%); the primer sets were 100% specific. The introduction of PCR-based assays shall increase laboratory confirmed cases, consequently enhancing surveillance of meningococcal disease.

Temporal variability of soil CO2 emission after conventional and reduced tillage described by an exponential decay in time model

To study Assessing the impact of tillage practices on soil carbon losses dependents it is necessary to describe the temporal variability of soil CO2 emission after tillage. It has been argued that large amounts of CO2 emitted after tillage may serve as an indicator for longer-term changes in soil carbon stocks. Here we present a two-step function model based on soil temperature and soil moisture including an exponential decay in time component that is efficient in fitting intermediate-term emission after disk plow followed by a leveling harrow (conventional), and chisel plow coupled with a roller for clod breaking (reduced) tillage. Emission after reduced tillage was described using a non-linear estimator with determination coefficient (R²) as high as 0.98. Results indicate that when emission after tillage is addressed it is important to consider an exponential decay in time in order to predict the impact of tillage in short-term emissions.

Two point deterministic model for acquisition of in vitro pollen grain androgenetic capacity based on wheat studies

This article discusses, from the standpoint of cellular biology, the deterministic and indeterministic androgenesis theories. The role of the vacuole and of various types of stresses on deviation of the microspore from normal development and the point where androgenetic competence is acquired are examined. Based on extensive literature review and data on wheat studies from our laboratory, a model for androgenetic capacity of pollen grain is proposed. A two point deterministic model for in vitro androgenesis is our proposal for acquisition of androgenetic potential of the pollen grain: the first switch point would be early meiosis and the second switch point the uninucleate pollen stage, because the elimination of cytoplasmatic sporophytic determinants takes place at those two strategic moments. Any abnormality in this process allowing the maintenance of sporophytic informational molecules results in the absence of establishment of a gametophytic program, allowing the reactivation of the embryogenic process

Developing novel one-step processes for obtaining food-grade O/W emulsions from pressurized fluid extracts: processes description, state of the art and perspectives

Abstract In this work, a novel on-line process for production of food-grade emulsions containing oily extracts, i.e. oil-in-water (O/W) emulsions, in only one step is presented. This process has been called ESFE, Emulsions from Supercritical Fluid Extraction. With this process, emulsions containing supercritical fluid extracts can be obtained directly from plant materials. The aim in the conception of this process is to propose a new rapid way to obtain emulsions from supercritical fluid extracts. Nowadays the conventional emulsion formulation method is a two-step procedure, i.e. first supercritical fluid extraction for obtaining an extract; secondly emulsion formulation using another device. Other variation of the process was tested and successfully validated originating a new acronymed process: EPFE (Emulsions from Pressurized Fluid Extractions). Both processes exploit the supercritical CO2-essential oils miscibility, in addition, EPFE process exploits the emulsification properties of saponin-rich pressurized aqueous plant extracts. The feasibility of this latter process was demonstrated using Pfaffia glomerata roots as source of saponin-rich extract, water as extracting solvent and clove essential oil, directly extracted using supercritical CO2, as a model dispersed phase. In addition, examples of pressurized fluid-based coupled processes applied for adding value to food bioactive compounds developed in the past five years are reviewed.

Simulation of growth and development of irrigated cowpea in Piauí State by CROPGRO model

The objective of this work was to adapt the CROPGRO model, which is part of the DSSAT system, for simulating the cowpea (Vigna unguiculata) growth and development under soil and climate conditions of the Baixo Parnaíba region, Piauí State, Brazil. In the CROPGRO, only input parameters that define crop species, cultivars, and ecotype were changed in order to characterize the cowpea crop. Soil and climate files were created for the considered site. Field experiments without water deficit were used to calibrate the model. In these experiments, dry matter (DM), leaf area index (LAI), yield components and grain yield of cowpea (cv. BR 14 Mulato) were evaluated. The results showed good fit for DM and LAI estimates. The medium values of R² and medium absolute error (MAE) were, respectively, 0.95 and 264.9 kg ha-1 for DM, and 0.97 and 0.22 for LAI. The difference between observed and simulated values of plant phenology varied from 0 to 3 days. The model also presented good performance for yield components simulation, excluding 100-grain weight, for which the error ranged from 20.9% to 34.3%. Considering the medium values of crop yield in two years, the model presented an error from 5.6%.

Adsorption of glyphosate in chilean soils and its relationship with unoccupied phosphate binding sites

The objective of this work was to investigate glyphosate adsorption by soils and its relationship with unoccupied binding sites for phosphate adsorption. Soil samples of three Chilean soils series - Valdivia (Andisol), Clarillo (Inceptisol) and Chicureo (Vertisol) - were incubated with different herbicide concentrations. Glyphosate remaining in solution was determined by adjusting a HPLC method with a UV detector. Experimental maximum adsorption capacity were 15,000, 14,300 and 4,700 mg g¹ for Valdivia, Clarillo, and Chicureo soils, respectively. Linear, Freundlich, and Langmuir models were used to describe glyphosate adsorption. Isotherms describing glyphosate adsorption differed among soils. Maximum adjusted adsorption capacity with the Langmuir model was 231,884, 17,874 and 5,670 mg g-1 for Valdivia, Clarillo, and Chicureo soils, respectively. Glyphosate adsorption on the Valdivia soil showed a linear behavior at the range of concentrations used and none of the adjusted models became asymptotic. The high glyphosate adsorption capacity of the Valdivia soil was probably a result of its high exchangeable Al, extractable Fe, and alophan and imogolite clay type. Adsorption was very much related to phosphate dynamics in the Valdivia soil, which showed the larger unoccupied phosphate binding sites. However relationship between unoccupied phosphate binding sites and glyphosate adsorption in the other two soils (Clarillo and Chicureo) was not clear.

Detection and identification of dengue virus isolates from Brazil by a simplified reverse transcription - polymerase chain reaction (RT-PCR) method

We show here a simplified RT-PCR for identification of dengue virus types 1 and 2. Five dengue virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD as a negative control, were used in this study. C6/36 cells were infected and supernatants were collected after 7 days. The RT-PCR, done in a single reaction vessel, was carried out following a 1/10 dilution of virus in distilled water or in a detergent mixture containing Nonidet P40. The 50 µl assay reaction mixture included 50 pmol of specific primers amplifying a 482 base pair sequence for dengue type 1 and 210 base pair sequence for dengue type 2. In other assays, we used dengue virus consensus primers having maximum sequence similarity to the four serotypes, amplifying a 511 base pair sequence. The reaction mixture also contained 0.1 mM of the four deoxynucleoside triphosphates, 7.5 U of reverse transcriptase, 1U of thermostable Taq DNA polymerase. The mixture was incubated for 5 minutes at 37ºC for reverse transcription followed by 30 cycles of two-step PCR amplification (92ºC for 60 seconds, 53ºC for 60 seconds) with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized by UV light after staining with ethidium bromide solution. Low virus titer around 10 3, 6 TCID50/ml was detected by RT-PCR for dengue type 1. Specific DNA amplification was observed with all the Brazilian dengue strains by using dengue virus consensus primers. As compared to other RT-PCRs, this assay is less laborious, done in a shorter time, and has reduced risk of contamination

Detection of codon 12 mutation in the k-ras oncogene in pancreatic tumors

Mutations at codons 12, 13, or 61 of the H-ras, K-ras, and N-ras have been detected in human neoplasias by a variety of techniques. Some of these techniques are very sensitive and can detect K-ras mutation in 90% of the cases of pancreatic adenocarcinomas. We analyzed 11 samples of pancreatic adenocarcinoma, three samples of pancreatic mucinous cystadenoma, and two samples without tumors in formalin-fixed paraffin embedded tissue sections. K-ras mutations at codon 12 were detected by a two-step PCR-enriched technique in all the samples of pancreatic adenocarcinoma, but not in cystadenoma or control samples. This technique may be useful for early detection of pancreatic cancer.

Medium-term protocols for in vivo evaluation of chemical modifiers of carcinogenesis

Cancer development is a long-term multistep process which allows interventional measure before the clincial disease emerges. the detection of natural substances which can block the process of carcinogenesis is a important as the identification of anti-tumoral drugs since they might be used in chemoprevention of cancer in high-risk groups. In vivo rodent models of chemical caecinogenesis have been used to study plant-derived inhibitors of carcinofenesis such as indols, coumarins, isothiocyanates, flavones, phenols and allyl-sulfides. Since the standard in vivo rodent bioassay is prolonged and expensive, shorter reliable protocols are needed. Two in vivo medium-term protocols for evaluation of modifiers of carcinogenesis are presented, one related to liver and the other to bladder cancer. Both protocols use rats, last 8 and 36 weeks and are based on the two-step concept of carcinogenesis: initiation and promotion. The protocols use respectively the development of altered foci of hepatocytes expressing immunochistochemically the placental form of gluthation S-transferase and the appearence of pre-neoplastic urothelium and papillomas as the "end-points". the use of these protocols for detection of plantpderived inhibitors of carcinogenesis appear warranted.

Detection of Rotavirus in Sewage and Creek Water: Efficiency of the Concentration Method

Simian rotavirus SA-11, experimentally seeded, was recovered from raw domestic sewage by a two-step concentration procedure, using filtration through a positively charged microporous filter (Zeta Plus 60 S) followed by ultracentrifugation, effecting an 8,000-fold concentration. By this method, a mean recovery of 81% ± 7.5 of the SA-11 virus, was achieved

A Simple Reverse Transcription-Polymerase Chain Reaction for Dengue Type 2 Virus Identification

We show here a simplified reverse transcription-polymerase chain reaction (RT-PCR) for identification of dengue type 2 virus. Three dengue type 2 virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD, as a negative control, were used in this study. C6/36 cells were infected with the virus, and tissue culture fluids were collected after 7 days of infection period. The RT-PCR, a combination of RT and PCR done after a single addition of reagents in a single reaction vessel was carried out following a digestion of virus with 1% Nonidet P-40. The 50ml assay reaction mixture included 50 pmol of a dengue type 2 specific primer pair amplifying a 210 base pair sequence of the envelope protein gene, 0.1 mM of the four deoxynucleoside triphosphates, 7.5U of reverse transcriptase, and 1U of thermostable Taq DNA polymerase. The reagent mixture was incubated for 15 min at 37oC for RT followed by a variable amount of cycles of two-step PCR amplification (92oC for 60 sec, 53oC for 60 sec) with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized with UV light after gel incubation in ethidium bromide solution. DNA bands were observed after 25 and 30 cycles of PCR. Virus amount as low as 102.8 TCID50/ml was detected by RT-PCR. Specific DNA amplification was observed with the three dengue type 2 strains. This assay has advantages compared to other RT-PCRs: it avoids laborious extraction of virus RNA; the combination of RT and PCR reduces assay time, facilitates the performance and reduces risk of contamination; the two-step PCR cycle produces a clear DNA amplification, saves assay time and simplifies the technique

Identification of Mycobacterium tuberculosis complex based on amplification and sequencing of the oxyR pseudogene from stored Ziehl-Neelsen-stained sputum smears in Brazil

A cross-sectional analysis of stored Ziehl-Neelsen (ZN)-stained sputum smear slides (SSS) obtained from two public tuberculosis referral laboratories located in Juiz de Fora, Minas Gerais, was carried out to distinguish Mycobacterium bovis from other members of the Mycobacterium tuberculosis complex (MTC). A two-step approach was used to distinguish M. bovis from other members of MTC: (i) oxyR pseudogene amplification to detect MTC and, subsequently, (ii) allele-specific sequencing based on the polymorphism at position 285 of this gene. The oxyR pseudogene was successfully amplified in 100 of 177 (56.5%) SSS available from 99 individuals. No molecular profile of M. bovis was found. Multivariate analysis indicated that acid-fast bacilli (AFB) results and the source laboratory were associated (p < 0.05) with oxyR pseudogene amplification. SSS that were AFB++ SSS showed more oxyR pseudogene amplification than those with AFB0, possibly due to the amount of DNA. One of the two source laboratories presented a greater chance of oxyR pseudogene amplification, suggesting that differences in sputum conservation between laboratories could have influenced the preservation of DNA. This study provides evidence that stored ZN-SSS can be used for the molecular detection of MTC.

SOIL PHOSPHORUS THRESHOLDS IN EVALUATING RISK OF ENVIRONMENTAL TRANSFER TO SURFACE WATERS IN SANTA CATARINA, BRAZIL

The State of Santa Catarina, Brazil, has agricultural and livestock activities, such as pig farming, that are responsible for adding large amounts of phosphorus (P) to soils. However, a method is required to evaluate the environmental risk of these high soil P levels. One possible method for evaluating the environmental risk of P fertilization, whether organic or mineral, is to establish threshold levels of soil available P, measured by Mehlich-1 extractions, below which there is not a high risk of P transfer from the soil to surface waters. However, the Mehlich-1 extractant is sensitive to soil clay content, and that factor should be considered when establishing such P-thresholds. The objective of this study was to determine P-thresholds using the Mehlich-1 extractant for soils with different clay contents in the State of Santa Catarina, Brazil. Soil from the B-horizon of an Oxisol with 800 g kg-1 clay was mixed with different amounts of sand to prepare artificial soils with 200, 400, 600, and 800 g kg-1 clay. The artificial soils were incubated for 30 days with moisture content at 80 % of field capacity to stabilize their physicochemical properties, followed by additional incubation for 30 days after liming to raise the pH(H2O) to 6.0. Soil P sorption curves were produced, and the maximum sorption (Pmax) was determined using the Langmuir model for each soil texture evaluated. Based on the Pmax values, seven rates of P were added to four replicates of each soil, and incubated for 20 days more. Following incubation, available P contents (P-Mehlich-1) and P dissolved in the soil solution (P-water) were determined. A change-point value (the P-Mehlich-1 value above which P-water starts increasing sharply) was calculated through the use of segmented equations. The maximum level of P that a soil might safely adsorb (P-threshold) was defined as 80 % of the change-point value to maintain a margin for environmental safety. The P-threshold value, in mg dm-3, was dependent on the soil clay content according to the model P-threshold = 40 + Clay, where the soil clay content is expressed as a percentage. The model was tested in 82 diverse soil samples from the State of Santa Catarina and was able to distinguish samples with high and low environmental risk.

Zinc adsorption in highly weathered soils

The objective of this work was to assess the effects of pH and ionic strength upon zinc adsorption, in three highly weathered variable charge soils. Adsorption isotherms were elaborated from batch adsorption experiments, with increasing Zn concentrations (0-80 mg L-1), and adsorption envelopes were constructed through soil samples reactions with 0.01, 0.1 and 1 mol L-1 Ca(NO3)2 solutions containing 5 mg L-1 of Zn, with an increasing pH value from 3 to 8. Driving force of reaction was quantified by Gibbs free energy and separation factor. Isotherms were C-, H- and L-type and experimental results were fitted to nonlinear Langmuir model. Maximum adsorption ranged from 59-810 mg kg-1, and Zn affinity was greater in subsoil (0.13-0.81 L kg-1) than in the topsoil samples (0.01-0.34 L kg-1). Zinc adsorption was favorable and spontaneous, and showed sharply increase (20-90%) in the 4-6 pH range. No effect of ionic strength was observed at pH values below 5, because specific adsorption mechanisms predominated in the 3-5 pH range. Above pH 5, and in subsoil samples, Zn was adsorbed by electrostatic mechanisms, since ionic strength effect was observed. Despite depth and ionic strength effects, Zn adsorption depends mainly on the pH.