Cheap, rapid and efficient DNA extraction method to perform multilocus microsatellite genotyping on all Schistosoma mansoni stages

Schistosomes are endoparasites causing a serious human disease called schistosomiasis. The quantification of parasite genetic diversity is an essential component to understand the schistosomiasis epidemiology and disease transmission patterns. In this paper, we propose a novel assay for a rapid, low costly and efficient DNA extraction method of egg, larval and adult stages of Schistosoma mansoni. One euro makes possible to perform 60,000 DNA extraction reactions at top speed (only 15 min of incubation and 5 handling steps).

Improving the production of the denatured recombinant N-terminal domain of rhoptry-associated protein 2 from a Plasmodium falciparum target in the pathology of anemia in falciparum malaria

Rhoptry-associated protein 2 (RAP2) is known to be discharged from rhoptry onto the membrane surface of infected and uninfected erythrocytes (UEs) ex vivo and in vitro and this information provides new insights into the understanding of the pathology of severe anemia in falciparum malaria. In this study, a hexahistidine-tagged recombinant protein corresponding to residues 5-190 of the N-terminal of Plasmodium falciparum RAP2 (rN-RAP2) was produced using a new method of solubilization and purification. Expression was induced with D-lactose, a less expensive alternative inducer to the more common isopropyl-²-D-thio-galactopyranosidase. The recombinant protein was purified using two types of commercially-available affinity columns, iminodiacetic and nitrilotriacetic. rN-RAP2 had immunogenic potential, since it induced high titers of anti-RAP2 antibodies in mice. These antibodies recognized full-length RAP2 prepared from Triton X-100 extracts from two strains of P. falciparum. In fact, the antibody recognized a 29-kDa product of RAP2 cleavage as well as 82 and 70-kDa products of RAP1 cleavage. These results indicate that the two antigens share sequence epitopes. Our expressed protein fragment was shown to contain a functional epitope that is also present in rhoptry-derived ring surface protein 2 which attaches to the surface of both infected and UEs and erythroid precursor cells in the bone marrow of malaria patients. Serum from malaria patients who developed anemia during infection recognized rN-RAP2, suggesting that this protein fragment may be important for epidemiological studies investigating whether immune responses to RAP2 exacerbate hemolysis in falciparum malaria patients.

Inheritance of Schistosoma mansoni infection incompatibility in Biomphalaria alexandrina snails

In this study, we looked at the inheritance of susceptibility and resistance to Schistosoma mansoni infection in the first generation of crossbred Biomphalaria alexandrina snails. Our ultimate goal is to use such information to develop a biological method of controlling schistosomiasis. We infected laboratory-bred snails with S. mansoni miracidia and examined cercarial shedding to determine susceptibility and resistance. Five parental groups were used: Group I contained 30 susceptible snails, Group II contained 30 resistant snails, Group III contained 15 susceptible and 15 resistant snails, Group IV contained 27 susceptible and three resistant snails and Group V contained three susceptible and 27 resistant snails. The percentage of resistant snails in the resulting progeny varied according to the ratio of susceptible and resistant parents per group; they are 7%, 100%, 68%, 45% and 97% from Groups I, II, III, IV and V, respectively. On increasing the percentage of resistant parent snails, the percentage of resistant progeny increased, while cercarial production in their susceptible progeny decreased.

The activity of a metronidazole analogue and its β-cyclodextrin complex against Trypanosoma cruzi

In this study we prepared an inclusion complex between an iodide analogue of metronidazole (MTZ-I) and cyclodextrin (CD) to develop a safer and more effective method of treating Trypanosoma cruzi infections. According to our results, MTZ-I and MTZ-I:β-CD were 10 times more active than MTZ, demonstrating that the presence of an iodine atom on the side chain increased the trypanocidal activity while maintaining its cytotoxicity. The selective index shows that MTZ-I was 10 times more active against T. cruzi than it was against mammalian cells. The modification of MTZ side chains provides a promising avenue for the development of new drugs.

Root system distribution of sugar cane as related to nitrogen fertilization, evaluated by two methods: monolith and probes

Few studies on sugar cane have evaluated the root system of the crop, in spite of its importance. This is mainly due to the difficulty of evaluation and high variability of results. The objective of this study was to develop an evaluation method of the cane root system by means of probes so as to evaluate the mass, distribution and metabolically active roots related to N fertilization at planting. For this purpose, an experiment was conducted in an Arenic Kandiustults with medium texture in Jaboticabal/SP, in a randomized block design with four replications and four treatments: control (without N) and 40, 80 and 120 kg ha-1 of N applied in the form of urea in the planting furrow of the cane variety SP81 3250. One week before harvest, a urea-15N solution was applied at the cane stalk base to detect active metabolism in the root system. Trenches of 1.5 m length and 0.6 m depth were opened between two sugar cane rows for root sampling by two methods: monoliths (0.3, 0.2 and 0.15 m wide, deep and long respectively) taken from the trench wall and by probe (internal diameter 0.055 m). For each method, 15 samples per plot were collected. The roots were separated from the soil in a sieve (2 mm mesh), oven-dried (at 65 ºC) and the dry matter was measured. Root sampling by probes resulted in root mass that did not differ from the evaluation in monoliths, indicating that this evaluation method may be used for sugar cane root mass, although neither the root distribution in the soil profile nor the rhizome mass were efficiently evaluated, due to the small sample volume. Nitrogen fertilization at planting did not result in a greater root accumulation in the sugar cane plant, but caused changes in the distribution of the root system in the soil. The absence of N fertilization led to a better root distribution in the soil profile, with 50, 34 and 16 % in the 0-0.2, 0.2-0.4 and 0.4-0.6 m layers, respectively; in the fertilized treatments the roots were concentrated in the surface layer, with on average 70, 17 and 13 % for the same layers. The metabolically active roots were concentrated in the center of the cane stool, amounting to 40 % of the total root mass, regardless of N fertilization (application of 120 kg ha-1 N or without N).

Complexometric titration with potenciometric indicator to determination of calcium and magnesium in soil extracts¹

This study proposes a method of direct and simultaneous determination of the amount of Ca2+ and Mg2+ present in soil extracts using a Calcium Ion-Selective Electrode and by Complexometric Titration (ISE-CT). The results were compared to those obtained by conventional analytical techniques of Complexometric Titration (CT) and Flame Atomic Absorption Spectrometry (FAAS). There were no significant differences in the determination of Ca2+ and Mg2+ in comparison with CT and FAAS, at a 95 % confidence level. Additionally, results of this method were more precise and accurate than of the Interlaboratorial Control (IC).

An improved method for measuring soil microbial activity by gas phase flow injection analysis

The rate of carbon dioxide production is commonly used as a measure of microbial activity in the soil. The traditional method of CO2 determination involves trapping CO2 in an alkali solution and then determining CO2 concentration indirectly by titration of the remaining alkali in the solution. This method is still commonly employed in laboratories throughout the world due to its relative simplicity and the fact that it does not require expensive, specific equipment. However, there are several drawbacks: the method is time-consuming, requires large amounts of chemicals and the consistency of results depends on the operator's skills. With this in mind, an improved method was developed to analyze CO2 captured in alkali traps, which is cheap and relatively simple, with a substantially shorter sample handling time and reproducibility equivalent to the traditional titration method. A comparison of the concentration values determined by gas phase flow injection analysis (GPFIA) and titration showed no significant difference (p > 0.05), but GPFIA has the advantage that only a tenth of the sample volume of the titration method is required. The GPFIA system does not require the purchase of new, costly equipment but the device was constructed from items commonly found in laboratories, with suggestions for alternative configurations for other detection units. Furthermore, GPFIA for CO2 analysis can be equally applied to samples obtained from either the headspace of microcosms or from a sampling chamber that allows CO2 to be released from alkali trapping solutions. The optimised GPFIA method was applied to analyse CO2 released from degrading hydrocarbons from a site contaminated by diesel spillage.

Fertilization response likelihood for the interpretation of leaf analyses

Leaf analysis is the chemical evaluation of the nutritional status where the nutrient concentrations found in the tissue reflect the nutritional status of the plants. Thus, a correct interpretation of the results of leaf analysis is fundamental for an effective use of this tool. The purpose of this study was to propose and compare the method of Fertilization Response Likelihood (FRL) for interpretation of leaf analysis with that of the Diagnosis and Recommendation Integrated System (DRIS). The database consisted of 157 analyses of the N, P, K, Ca, Mg, S, Cu, Fe, Mn, Zn, and B concentrations in coffee leaves, which were divided into two groups: low yield (< 30 bags ha-1) and high yield (> 30 bags ha-1). The DRIS indices were calculated using the method proposed by Jones (1981). The fertilization response likelihood was computed based on the approximation of normal distribution. It was found that the Fertilization Response Likelihood (FRL) allowed an evaluation of the nutritional status of coffee trees, coinciding with the DRIS-based diagnoses in 84.96 % of the crops.

Recommendation of soil fertility levels for willow in the southern highlands of Santa Catarina

The species Salix x rubens is being grown on the Southern Plateau of Santa Catarina since the 1940s, but so far the soil fertility requirements of the crop have not been assessed. This study is the first to evaluate the production profile of willow plantations in this region, based on the modified method of Summer & Farina (1986), for the recommendation of fertility levels for willow. By this method, based on the law of Minimum and of Maximum for willow production for the conditions on the Southern Plateau of Santa Catarina, the following ranges could be recommended: pH: 5.0-6.5; P: 12-89 mg dm-3; Mg: 3.2-7.5 mg; Zn: 5.0-8.3 mg dm-3; Cu: 0.8-4.6 mg dm-3; and Mn; 20-164 mg dm-3. The Ca/Mg ratio should be between 1.2 and 2.9. For K and Ca only the lower (sufficiency level), but not the upper threshold (excess) was established, with respectively 114 mg dm-3 and 5.3 cmol c dm-3. It was also possible to determine the upper threshold for Al and the Al/Ca ratio, i.e., 1.7 cmol c dm-3 and 0.28, respectively. For maximum yields, the clay in the soil surface layer should be below 320 g dm-3.

Replication of an ivg protocol to estimate bioaccessible arsenic in materials from a gold mining area in Brazil

Tests for bioaccessibility are useful in human health risk assessment. No research data with the objective of determining bioaccessible arsenic (As) in areas affected by gold mining and smelting activities have been published so far in Brazil. Samples were collected from four areas: a private natural land reserve of Cerrado; mine tailings; overburden; and refuse from gold smelting of a mining company in Paracatu, Minas Gerais. The total, bioaccessible and Mehlich-1-extractable As levels were determined. Based on the reproducibility and the accuracy/precision of the in vitro gastrointestinal (IVG) determination method of bioaccessible As in the reference material NIST 2710, it was concluded that this procedure is adequate to determine bioaccessible As in soil and tailing samples from gold mining areas in Brazil. All samples from the studied mining area contained low percentages of bioaccessible As.

Isotope determination of sulfur by mass spectrometry in soil samples

Sulphur plays an essential role in plants and is one of the main nutrients in several metabolic processes. It has four stable isotopes (32S, 33S, 34S, and 36S) with a natural abundance of 95.00, 0.76, 4.22, and 0.014 in atom %, respectively. A method for isotopic determination of S by isotope-ratio mass spectrometry (IRMS) in soil samples is proposed. The procedure involves the oxidation of organic S to sulphate (S-SO4(2-)), which was determined by dry combustion with alkaline oxidizing agents. The total S-SO4(2-) concentration was determined by turbidimetry and the results showed that the conversion process was adequate. To produce gaseous SO2 gas, BaSO4 was thermally decomposed in a vacuum system at 900 ºC in the presence of NaPO3. The isotope determination of S (atom % 34S atoms) was carried out by isotope ratio mass spectrometry (IRMS). In this work, the labeled material (K2(34)SO4) was used to validate the method of isotopic determination of S; the results were precise and accurate, showing the viability of the proposed method.

Potential acidity estimated by SMP pH in soils of the state of Pará

The use of SMP pH to estimate the potential acidity (H + Al) is more practical than the method of 0.5 mol L-1 calcium acetate for routine laboratory analyses. The objective was to fit an equation to estimate the H + Al from SMP pH values of soils of the State of Pará. From various regions of the state, 177 soil samples were collected, in which the SMP pH was determined in 0.01 mol L-1 CaCl2 solution and H + Al in 0.5 mol L-1 calcium acetate and the results were related by regression analysis. The equation H + Al = 77.77 + 20.61 SMP pH - 1.435 SMP pH² (R² = 0.90) expressed the H + Al values (in cmol c dm-3) best. When the SMP pH values were used in equations referring to other regions or states in Brazil, the H +Al values were over- or underestimated. The potential acidity in soils of Pará can be estimated by the method of SMP pH.

Lead and zinc in the structure of organic and mineral soil components

In addition to the more reactive forms, metals can occur in the structure of minerals, and the sum of all these forms defines their total contents in different soil fractions. The isomorphic substitution of heavy metals for example alters the dimensions of the unit cell and mineral size. This study proposed a method of chemical fractionation of heavy metals, using more powerful extraction methods, to remove the organic and different mineral phases completely. Soil samples were taken from eight soil profiles (0-10, 10-20 and 20-40 cm) in a Pb mining and metallurgy area in Adrianópolis, Paraná, Brazil. The Pb and Zn concentrations were determined in the following fractions (complete phase removal in each sequential extraction): exchangeable; carbonates; organic matter; amorphous and crystalline Fe oxides; Al oxide, amorphous aluminosilicates and kaolinite; and residual fractions. The complete removal of organic matter and mineral phases in sequential extractions resulted in low participation of residual forms of Pb and Zn in the total concentrations of these metals in the soils: there was lower association of metals with primary and 2:1 minerals and refractory oxides. The powerful methods used here allow an identification of the complete metal-mineral associations, such as the occurrence of Pb and Zn in the structure of the minerals. The higher incidence of Zn than Pb in the structure of Fe oxides, due to isomorphic substitution, was attributed to a smaller difference between the ionic radius of Zn2+ and Fe3+.

Estimation of water percolation by different methods using TDR

Detailed knowledge on water percolation into the soil in irrigated areas is fundamental for solving problems of drainage, pollution and the recharge of underground aquifers. The aim of this study was to evaluate the percolation estimated by time-domain-reflectometry (TDR) in a drainage lysimeter. We used Darcy's law with K(θ) functions determined by field and laboratory methods and by the change in water storage in the soil profile at 16 points of moisture measurement at different time intervals. A sandy clay soil was saturated and covered with plastic sheet to prevent evaporation and an internal drainage trial in a drainage lysimeter was installed. The relationship between the observed and estimated percolation values was evaluated by linear regression analysis. The results suggest that percolation in the field or laboratory can be estimated based on continuous monitoring with TDR, and at short time intervals, of the variations in soil water storage. The precision and accuracy of this approach are similar to those of the lysimeter and it has advantages over the other evaluated methods, of which the most relevant are the possibility of estimating percolation in short time intervals and exemption from the predetermination of soil hydraulic properties such as water retention and hydraulic conductivity. The estimates obtained by the Darcy-Buckingham equation for percolation levels using function K(θ) predicted by the method of Hillel et al. (1972) provided compatible water percolation estimates with those obtained in the lysimeter at time intervals greater than 1 h. The methods of Libardi et al. (1980), Sisson et al. (1980) and van Genuchten (1980) underestimated water percolation.

Estimation of field capacity from ring infiltrometer-drainage data

Field capacity (FC) is a parameter widely used in applied soil science. However, its in situ method of determination may be difficult to apply, generally because of the need of large supplies of water at the test sites. Ottoni Filho et al. (2014) proposed a standardized procedure for field determination of FC and showed that such in situ FC can be estimated by a linear pedotransfer function (PTF) based on volumetric soil water content at the matric potential of -6 kPa [θ(6)] for the same soils used in the present study. The objective of this study was to use soil moisture data below a double ring infiltrometer measured 48 h after the end of the infiltration test in order to develop PTFs for standard in situ FC. We found that such ring FC data were an average of 0.03 m³ m- 3 greater than standard FC values. The linear PTF that was developed for the ring FC data based only on θ(6) was nearly as accurate as the equivalent PTF reported by Ottoni Filho et al. (2014), which was developed for the standard FC data. The root mean squared residues of FC determined from both PTFs were about 0.02 m³ m- 3. The proposed method has the advantage of estimating the soil in situ FC using the water applied in the infiltration test.