Flow cytometry in the study of cell death

In this report we present a concise review concerning the use of flow cytometric methods to characterize and differentiate between two different mechanisms of cell death, apoptosis and necrosis. The applications of these techniques to clinical and basic research are also considered. The following cell features are useful to characterize the mode of cell death: (1) activation of an endonuclease in apoptotic cells results in extraction of the low molecular weight DNA following cell permeabilization, which, in turn, leads to their decreased stainability with DNA-specific fluorochromes. Measurements of DNA content make it possible to identify apoptotic cells and to recognize the cell cycle phase specificity of apoptotic process; (2) plasma membrane integrity, which is lost in necrotic but not in apoptotic cells; (3) the decrease in forward light scatter, paralleled either by no change or an increase in side scatter, represent early changes during apoptosis. The data presented indicate that flow cytometry can be applied to basic research of the molecular and biochemical mechanisms of apoptosis, as well as in the clinical situations, where the ability to monitor early signs of apoptosis in some systems may be predictive for the outcome of some treatment protocols.

Programmed cell death in Trypanosoma cruzi induced by Bothrops jararaca venom

Cells die through a programmed process or accidental death, know as apoptosis or necrosis, respectively. Bothrops jararaca is a snake whose venom inhibits the growth of Trypanosoma cruzi epimastigote forms causing mitochondrion swelling and cell death. The aim of the present work was to determine the type of death induced in epimastigotes of T. cruzi by this venom. Parasite growth was inhibited after venom treatment, and 50% growth inhibition was obtained with 10 µg/ml. Ultrastructural observations confirmed mitochondrion swelling and kinetoplast disorganization. Furthermore, cytoplasmic condensation, loss of mitochondrion membrane potential, time-dependent increase in phosphatidylserine exposure at the outer leaflet plasma membrane followed by permeabilization, activation of caspase like protein and DNA fragmentation were observed in epimastigotes throughout a 24 h period of venom treatment. Taken together, these results indicate that the stress induced in epimastigote by this venom, triggers a programmed cell death process, similar to metazoan apoptosis, which leads to parasite death.

Mortality due to systemic mycoses as a primary cause of death or in association with AIDS in Brazil: a review from 1996 to 2006

Deaths caused by systemic mycoses such as paracoccidioidomycosis, cryptococcosis, histoplasmosis, candidiasis, aspergillosis, coccidioidomycosis and zygomycosis amounted to 3,583 between 1996-2006 in Brazil. When analysed as the underlying cause of death, paracoccidioidomycosis represented the most important cause of deaths among systemic mycoses (~ 51.2%). When considering AIDS as the underlying cause of death and the systemic mycoses as associated conditions, cryptococcosis (50.9%) appeared at the top of the list, followed by candidiasis (30.2%), histoplasmosis (10.1%) and others. This mortality analysis is useful in understanding the real situation of systemic mycoses in Brazil, since there is no mandatory notification of patients diagnosed with systemic mycoses in the official health system.

Miltefosine induces programmed cell death in Leishmania amazonensis promastigotes

In the current study, we evaluated the mechanism of action of miltefosine, which is the first effective and safe oral treatment for visceral leishmaniasis, in Leishmania amazonensis promastigotes. Miltefosine induced a process of programmed cell death, which was determined by the externalization of phosphatidylserine, the incorporation of propidium iodide, cell-cycle arrest at the sub-G0/G1 phase and DNA fragmentation into oligonucleosome-sized fragments. Despite the intrinsic variation that is detected in Leishmania spp, our results indicate that miltefosine causes apoptosis-like death in L. amazonensis promastigote cells using a similar process that is observed in Leishmania donovani.

Computer-assisted cartography using topographic properties: precision and accuracy of local soil maps in central Mexico

Map units directly related to properties of soil-landscape are generated by local soil classes. Therefore to take into consideration the knowledge of farmers is essential to automate the procedure. The aim of this study was to map local soil classes by computer-assisted cartography (CAC), using several combinations of topographic properties produced by GIS (digital elevation model, aspect, slope, and profile curvature). A decision tree was used to find the number of topographic properties required for digital cartography of the local soil classes. The maps produced were evaluated based on the attributes of map quality defined as precision and accuracy of the CAC-based maps. The evaluation was carried out in Central Mexico using three maps of local soil classes with contrasting landscape and climatic conditions (desert, temperate, and tropical). In the three areas the precision (56 %) of the CAC maps based on elevation as topographical feature was higher than when based on slope, aspect and profile curvature. The accuracy of the maps (boundary locations) was however low (33 %), in other words, further research is required to improve this indicator.

Identification of quantitative trait loci for resistance against soybean sudden death syndrome caused by Fusarium tucumaniae

The objective of this work was to identify genomic regions that underlie resistance to Fusarium tucumaniae sp. nov., the causing agent of sudden death syndrome (SDS) in soybean in South America, using a population with a genetic background different from that previously reported for Fusarium virguliforme sp. nov. (F. solani f. sp. glycines), also responsible for SDS in soybean. Although major genes and quantitative trait loci (QTL) for SDS resistance have been identified, little is known about the same disease caused by Fusarium tucumaniae sp. nov., in South America. To identify genetic factors related to resistance to F. tucumaniae and DNA markers associated with them, a QTL analysis was performed using recombinant inbred lines. The map locations of the four loci, here identified, differed from those SDS resistance QTL previously described. It was screened a residual heterozygous line (RHL), which was heterozygous around the most effective QTL, RSDS1, and homozygous for the other genomic regions. The genetic effect of RSDS1 was confirmed using near-isogenic lines (NIL) derived from the RHL. The line which was homozygous for the Misuzudaizu genotype showed resistance levels comparable with that of the line homozygous for the Moshidou Gong 503 genotype.

Validation of microsatellite markers for assisted selection of soybean resistance to cyst nematode races 3 and 14

The objective of this work was to validate microsatellite markers associated with resistance to soybean cyst nematode (Heterodera glycines Ichinohe) races 3 and 14, in soybean (Glycine max L.) genotypes, for use in marker-assisted selection (MAS) programs. Microsatellites of soybean linkage groups A2, D2 and G were tested in two populations, and their selection efficiencies were determined. The populations were 65 F2:3 families from Msoy8001 (resistant) x Conquista (susceptible) cross, and 66 F2:3 families of S5995 (resistant) x Renascença (susceptible) cross, evaluated for resistance to races 3 and 14, respectively. Families with female index up to 30% were considered moderately resistant. Markers of A2 and G linkage groups were associated with resistance to race 3. Markers Satt309 and GMENOD2B explained the greatest proportion of phenotypic variance in the different groups. The combinations Satt309+GMENOD2B and Satt309+Satt187 presented 100% selection efficiency. Resistance to race 14 was associated with markers of G linkage group, and selection efficiency in the Satt309+Satt356 combination was 100%. The selection differential obtained by phenotypic and marker assisted selection showed that both can result in similar gains.

Inoculation methods under greenhouse conditions for evaluating soybean resistance to sudden death syndrome

The objectives of this work were to evaluate two greenhouse screening methods for sudden death syndrome (SDS) and to determine which one is best correlated with field resistance of soybean genotypes. The evaluations were done with three sets of genotypes that were classified as partially resistant, intermediate, and susceptible to SDS based on previous field evaluations. These three sets were independently evaluated for greenhouse SDS reactions using cone and tray inoculation methods. Plants were infected using grains of white sorghum [Sorghum bicolor (L.) Moench] infested with Fusarium solani f. sp. glycines. Foliar symptom severity was rated 21 days after emergence. The cone and field SDS ratings were significantly correlated and ranged from 0.69 for set 1 to 0.51 for set 3. Correlations of SDS ratings of genotypes between field and greenhouse tray ratings were significant for set 1 and not significant for set 2. The cone method showed the highest correlation with field results and is recommended to screen soybean genotypes for SDS resistance.

Molecular and morphological characterization of hydrochar produced by microwave-assisted hydrothermal carbonization of cellulose

The objective of this work was to characterize the morphology and molecular composition of the hydrochar produced by microwave-assisted hydrothermal carbonization of cellulose. The produced hydrochar consists mainly of aggregate microspheres with about 2.0 µm in diameter, with aliphatic and aromatic structures and the presence of carbonyl functional groups. The aromatic groups are formed mainly by benzofuran-like structures, being chemically different from common cellulose char. Microwave-assisted hydrothermal carbonization yields a functionalized carbon-rich material similar to that produced by the conventional hydrothermal process.

Diagnostic underestimation of atypical ductal hyperplasia and ductal carcinoma in situ at percutaneous core needle and vacuum-assisted biopsies of the breast in a Brazilian reference institution

Abstract Objective: To determine the rates of diagnostic underestimation at stereotactic percutaneous core needle biopsies (CNB) and vacuum-assisted biopsies (VABB) of nonpalpable breast lesions, with histopathological results of atypical ductal hyperplasia (ADH) or ductal carcinoma in situ (DCIS) subsequently submitted to surgical excision. As a secondary objective, the frequency of ADH and DCIS was determined for the cases submitted to biopsy. Materials and Methods: Retrospective review of 40 cases with diagnosis of ADH or DCIS on the basis of biopsies performed between February 2011 and July 2013, subsequently submitted to surgery, whose histopathological reports were available in the internal information system. Biopsy results were compared with those observed at surgery and the underestimation rate was calculated by means of specific mathematical equations. Results: The underestimation rate at CNB was 50% for ADH and 28.57% for DCIS, and at VABB it was 25% for ADH and 14.28% for DCIS. ADH represented 10.25% of all cases undergoing biopsy, whereas DCIS accounted for 23.91%. Conclusion: The diagnostic underestimation rate at CNB is two times the rate at VABB. Certainty that the target has been achieved is not the sole determining factor for a reliable diagnosis. Removal of more than 50% of the target lesion should further reduce the risk of underestimation.

Efficient synthesis of sulfonamide derivatives on solid supports catalyzed using solvent-free and microwave-assisted methods

In this work we report the synthesis of sulfonamide derivatives using a conventional procedure and with solid supports, such as silica gel, florisil, alumina, 4Å molecular sieves, montmorillonite KSF, and montmorillonite K10 using solvent-free and microwave-assisted methods. Our results show that solid supports have a catalytic activity in the formation of sulfonamide derivatives. We found that florisil, montmorillonite KSF, and K10 could be used as inexpensive alternative catalysts that are easily separated from the reaction media. Additionally, solvent-free and microwave-assisted methods were more efficient in reducing reaction time and in increasing yield.

Synthesis of quinoxaline 1,4-di-N-oxide derivatives on solid support using room temperature and microwave-assisted solvent-free procedures

We describe the synthesis of 12 new ethyl and methyl quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives on solid supports with room temperature and microwave-assisted solvent-free procedures. Results show that solid supports have good catalytic activity in the formation of quinoxaline 1,4-di-N-oxide derivatives. We found that florisil and montmorillonite KSF and K10 could be used as new, easily available, inexpensive alternatives of catalysts. Additionally, room temperature and microwave-irradiation solvent-free synthesis was more efficient than a conventional procedure (Beirut reaction), reducing reaction time and increasing yield.

Sonication-assisted preparation of CaO nanoparticles for antibacterial agents

The effect of calcination conditions on the size and killing activity of CaO nanoparticles towards L. plantarum was studied in this paper. The results showed that CaO nanoparticles with a diameter of 20 nm could be obtained under the investigated conditions. The lethal effect of CaO nanoparticles after incubation of 6 or 24 h increased with increasing calcination time. Using CaO-SA, CaO-SB, and CaO-SC after a 24-h exposure, 2.25, 3.37, and 5.97 log L. plantarum were killed, respectively, at a concentration of 100 ppm. The current results show that the use of CaO nanoparticles as antibacterial agents has significant potential in food-relevant industries.

Spectrophotometric determination of diclofenac in pharmaceutical preparations assisted by microwave oven

In this work, an effective and low-cost method for the determination of sodium or potassium diclofenac is proposed in its pure form and in their pharmaceutical preparations. The method is based on the reaction between diclofenac and tetrachloro-p-benzoquinone (p-chloranil), in methanol medium. This reaction was accelerated by irradiating of reactional mixture with microwave energy (1100 W) during 27 seconds, producing a charge transfer complex with a maximum absorption at 535 nm. The optimal reaction conditions values such as reagent concentration, heating time and stability of the reaction product were determined. Beer's law is obeyed in a concentration range from of 1.25x10-4 to 2.00x10-3 mol l-1 with a correlation coefficient of 0.9993 and molar absorptivity of 0.49 x10³ l mol-1 cm-1. The limit of detection (LOD) was 1.35x10-5 mol l-1 and the limit of quantification (LOQ) was 4.49x10-5 mol l-1. In the presence of the common excipients, such as glucose, lactose, talc, starch, magnesium stearate, sodium sulphite, titanium dioxide, polyethyleneglycol, polyvinylpirrolidone, mannitol and benzilic alcohol no interferences were observed. The analytical results obtained by applying the proposed method compare very favorably with those given by the United States Pharmacopeia standard procedure. Recoveries of diclofenac from various pharmaceutical preparations were within 95.9% to 103.3%, with standard deviations ranging from 0.2% to 1.8%.

Air flow humidification for air-assisted boom sprayer

The air-assisted ground spray is fairly widespread. However, due to the unpredictable weather conditions, the operational efficiency is impaired by stops on grounds of low humidity and high temperatures. The aim of this work was to assess an air humidification method and evaluate its impact on temperature and air humidity for the air curtain of the air-assisted sprayer. With respect to relative air humidity, it has increased in 6.59%, being the maximum change when inserting 1.92 L min-1. So, it is concluded that the pipeline humidification might significantly reduce temperature and enhance air humidity. The treatments performed in this study consisted of a varied flow of a humidity device, related to weather conditions. Temperature and relative air humidity were measured at 1.0 m height from right to left of middle point of the machine, corresponding to the end of the spray boom, in the middle and end of right spray boom. The readings were also performed at three different distances from the end of the pipeline and at 0.25 and 0.50 m from that to the soil. The results show that 0.48 L min-1 in the humidification system has promoted a better efficiency in reducing air-temperature, on average 2.52 ºC when compared to the non-humidified one.