In situ evaluation of single-cell lysis by cytosol extraction observation through fluorescence decay and dielectrophoretic trapping time

We present a new method for lysis of single cells in continuous flow, where cells are sequentially trapped, lysed and released in an automatic process. Using optimized frequencies, dielectrophoretic trapping allows exposing cells in a reproducible way to high electrical fields for long durations, thereby giving good control on the lysis parameters. In situ evaluation of cytosol extraction on single cells has been studied for Chinese hamster ovary (CHO) cells through out-diffusion of fluorescent molecules for different voltage amplitudes. A diffusion model is proposed to correlate this out-diffusion to the total area of the created pores, which is dependent on the potential drop across the cell membrane and enables evaluation of the total pore area in the membrane. The dielectrophoretic trapping is no longer effective after lysis because of the reduced conductivity inside the cells, leading to cell release. The trapping time is linked to the time required for cytosol extraction and can thus provide additional validation of the effective cytosol extraction for non-fluorescent cells. Furthermore, the application of one single voltage for both trapping and lysis provides a fully automatic process including cell trapping, lysis, and release, allowing operating the device in continuous flow without human intervention.

Isolation and partial characterization of host cell wall surface glycoproteins: their involvement in agglutination, attachment and appressorium formation by piptocephalis virginiana

Cell surface proteins obtained by alkaline extraction from isolated cell walls of Mortierella pusilla and M. candelabrum, host and nonhost, respectively, to the mycoparasite, Piptocephalis virginiana, were tested for their ability to agglutinate mycoparasite spores. The host cell wall protein extract had a high agglutinating activity (788 a.u. mg- t ) as compared with the nonhost extract (21 a.li. mg- t ). SDS-polyacrylamide gel electrophoresis of the cell wall proteins revealed four protein bands, a, b, c, and d (Mr 117, 100, 85 and 64 kd, respectively) at the host surface, but not at the nonhost surface, except for the faint band c. Deletion of proteins b or c from the host cell wall protein extract significantly reduced its agglutinating activity. Proteins band c, obtained as purified preparations by a series of procedures, were shown to be two glycoproteins. Carbohydrate analysis by gas chromatography demonstrated that glucose and Nacetylglucosamine were the major carbohydrate components of the glycoproteins. It was further shown that the agglutinating activity of the pure preparation containing both band c was 500-850 times that of the single glycoproteins, suggesting the involvement of both glycoproteins in agglutination. The results suggest that the glycoproteins band c are the two subunits of agglutinin present at the host cell surface. The two glycoproteins band c purified from the host cell wall protein extract were further examined after various treatments for their possible role in agglutination, attachment and appressorium formation by the mycoparasite. Results obtained by agglutination and attachment tests showed: (1) the two glycoprotein-s are not only an agglutinin responsible for the mycoparasite spore agglutination, but may also serve as a receptor for the specific recognition, attachment and appressorium formation by the mycoparasite; (2) treatment of the rnycoparasite spores with various sugars revealed that arabinose, glucose and N-acetylglucosamine inhibited the agglutination and attachment activity of the glycoproteins, however, the relative percentage of appressorium formation was not affected by the above sugars; (3) the two glycoproteins are relatively stable with respect to their agglutinin and receptor functions. The present results suggest that the agglutination and attachment may be mediated directly by certain sugars present at the host and mycoparasite cell surfaces while the appressorlum formation may be the response of complementary combinations of both sugar and protein, the two parts of the glycoproteins at the interacting surfaces of two fungi.

Extraction behavior of the synergistic system 2,6-bis-(benzoxazolyl)-4-dodecyloxylpyridine and 2-bromodecanoic acid using Am and Eu as radioactive tracers

In this study, the extraction properties of a synergistic system consisting of 2,6-bis-(benzoxazolyl)-4-dodecyloxylpyridine (BODO) and 2-bromodecanoic acid (HA) in tert-butyl benzene (TBB) have been investigated as a function of ionic strength by varying the nitrate ion and perchlorate ion concentrations. The influence of the hydrogen ion concentration has also been investigated. Distribution ratios between 0.03-12 and 0.003-0.8 have been found for Am(III) and Eu(HI), respectively, but there were no attempts to maximize these values. It has been shown that the distribution ratios decrease with increasing amounts of ClO4-, NO3-, and H+. The mechanisms, however, by which the decrease occurs, are different. In the case of increasing perchlorate ion concentration, the decrease in extraction is linear in a log-log plot of the distribution ratio vs. the ionic strength, while in the nitrate case the complexation between nitrate and Am or Eu increases at high nitrate ion concentrations and thereby decreases the distribution ratio in a non-linearway. The decrease in extraction could be caused by changes in activity coefficients that can be explained with specific ion interaction theory (SIT); shielding of the metal ions, and by nitrate complexation with Am and Eu as competing mechanism at high ionic strengths. The separation factor between Am and Eu reaches a maximum at similar to1 M nitrate ion concentration. Thereafter the values decrease with increasing nitrate ion concentrations.

An investigation into the extraction of americium(III), lanthanides and D-block metals by 6,6 '-bis-(5,6-dipentyl-[1,2,4]triazin-3-yl)-[2,2 ']bipyridinyl(C-5-BTBP)

The tetradentate ligand (C-5-BTBP) was able to extract americium(III) selectively from nitric acid. In octanol/kerosene the distribution ratios suggest that stripping will be possible. C-5-BTBP has unusual properties and potentially offers a means of separating metals, which otherwise are difficult to separate. For example C-5-BTBP has the potential to separate paliadium(II) from a mixture containing rhodium(III) and ruthenium(H) nitrosyl. In addition, C-5-BTBP has the potential to remove traces of cadmium from effluent or from solutions of other metals contaminated with cadmium. C-5-BTBP has potential as a reagent for the separation of americium(III) from solutions contaminated with iron(III) and nickel(II), hence offering a means of concentrating americium(III) for analytical purposes from nitric acid solutions containing high concentrations of iron(III) or nickel(II).

Chromatographic alignment of LC-MS and LC-MS/MS datasets by genetic algorithm feature extraction

Liquid chromatography-mass spectrometry (LC-MS) datasets can be compared or combined following chromatographic alignment. Here we describe a simple solution to the specific problem of aligning one LC-MS dataset and one LC-MS/MS dataset, acquired on separate instruments from an enzymatic digest of a protein mixture, using feature extraction and a genetic algorithm. First, the LC-MS dataset is searched within a few ppm of the calculated theoretical masses of peptides confidently identified by LC-MS/MS. A piecewise linear function is then fitted to these matched peptides using a genetic algorithm with a fitness function that is insensitive to incorrect matches but sufficiently flexible to adapt to the discrete shifts common when comparing LC datasets. We demonstrate the utility of this method by aligning ion trap LC-MS/MS data with accurate LC-MS data from an FTICR mass spectrometer and show how hybrid datasets can improve peptide and protein identification by combining the speed of the ion trap with the mass accuracy of the FTICR, similar to using a hybrid ion trap-FTICR instrument. We also show that the high resolving power of FTICR can improve precision and linear dynamic range in quantitative proteomics. The alignment software, msalign, is freely available as open source.

Spatial and temporal modeling of community non-timber forest extraction

This paper examines the interaction of spatial and dynamic aspects of resource extraction from forests by local people. Highly cyclical and varied across space and time, the patterns of resource extraction resulting from the spatial–temporal model bear little resemblance to the patterns drawn from focusing either on spatial or temporal aspects of extraction alone. Ignoring this variability inaccurately depicts villagers’ dependence on different parts of the forest and could result in inappropriate policies. Similarly, the spatial links in extraction decisions imply that policies imposed in one area can have unintended consequences in other areas. Combining the spatial–temporal model with a measure of success in community forest management—the ability to avoid open-access resource degradation—characterizes the impact of incomplete property rights on patterns of resource extraction and stocks.

Complexation of lanthanides, actinides and transition metal cations with a 6-(1,2,4-triazin-3-yl)-2,2′:6′,2′′-terpyridine ligand: implications for actinide(III)/lanthanide(III) partitioning

The quadridentate N-heterocyclic ligand 6-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,2,4-benzotriazin- 3-yl)-2,2′ : 6′,2′′-terpyridine (CyMe4-hemi-BTBP) has been synthesized and its interactions with Am(III),U(VI), Ln(III) and some transition metal cations have been evaluated by X-ray crystallographic analysis, Am(III)/Eu(III) solvent extraction experiments, UVabsorption spectrophotometry, NMR studies and ESI-MS. Structures of 1 : 1 complexes with Eu(III), Ce(III) and the linear uranyl (UO2 2+) ion were obtained by X-ray crystallographic analysis, and they showed similar coordination behavior to related BTBP complexes. In methanol, the stability constants of the Ln(III) complexes are slightly lower than those of the analogous quadridentate bis-triazine BTBP ligands, while the stability constant for the Yb(III)complex is higher. 1H NMR titrations and ESI-MS with lanthanide nitrates showed that the ligand forms only 1 : 1 complexes with Eu(III), Ce(III) and Yb(III), while both 1 : 1 and 1 : 2 complexes were formed with La(III) and Y(III) in acetonitrile. A mixture of isomeric chiral 2 : 2 helical complexes was formed with Cu(I), with a slight preference (1.4 : 1) for a single directional isomer. In contrast, a 1 : 1 complex was observed with the larger Ag(I) ion. The ligand was unable to extract Am(III) or Eu(III) from nitric acid solutions into 1-octanol, except in the presence of a synergist at low acidity. The results show that the presence of two outer 1,2,4-triazine rings is required for the efficient extraction and separation of An(III)from Ln(III) by quadridentate N-donor ligands.

Aqueous enzyme assisted oil extraction from oilseeds and emulsion de-emulsifying methods: a review

Regulatory, safety, and environmental issues have prompted the development of aqueousenzymatic extraction (AEE) for extracting components from oil-bearing materials. The emulsion resulting from AEE requires de-emulsification to separate the oil; when enzymes are used for this purpose, the method is known as aqueous enzymatic emulsion de-emulsification (AEED). In general, enzyme assisted oil extraction is known to yield oil having highly favourable characteristics. This review covers technological aspects of enzyme assisted oil extraction, and explores the quality characteristics of the oils obtained,focusing particularly on recent efforts undertaken to improve process economics by recovering and reusing enzymes.

Contrasting magmatic signatures in the Rairakhol and Koraput alkaline complexes, Eastern Ghats belt, India

The relation between alkaline magmatism and tectonism has been a contentious issue, particularly for the Precambrian continental regions. Alkaline complexes at the southwestern margin of Eastern Ghats belt, India, have been interpreted as rift-valley magmatism. However, those complexes occurring in granulite ensemble in the interior segments of the Eastern Ghats belt could not possibly be related to the rift-system, assumed for the western margin of the Eastern Ghats belt. Koraput complex was emplaced in a pull-apart structure, dominated by magmatic fabrics and geochemically similar to a fractionated alkaline complex, compatible with an alkalibasalt series. Rairakhol complex, on the other hand, shows dominantly solid-state deformation fabrics and geochemically similar to a fractionated calc-alkaline suite. Isotopic data for the Koraput complex indicate ca. 917 Ma alkaline magmatism from a depleted mantle source and postcrystalline thermal overprint at ca. 745 Ma, also recorded from sheared metapelitic country rocks. The calc-alkaline magmatism of the Rairakhol complex occurred around 938 Ma, from an enriched mantle source, closely following Grenvillian granulite facies imprint in the charnockitic country rocks.

In vitro evaluation of Cu and Fe bioavailability in cashew nuts by off-line coupled SEC-UV and SIMAAS

In this work Cu and Fe bioavailability in cashew nuts was evaluated using in vitro method. Extractions with simulated gastric and intestinal fluids and dialysis procedures were applied for this purpose. The proteins separation and quantification were performed by size exclusion chromatography (SEC) coupled on-line to ultra-violet (UV) and off-line to simultaneous multielement atomic absorption spectrometry (SIMAAS). The SEC-UV and SIMAAS profiles of the protein fractions obtained by alkaline extraction (NaOH) and precipitation with HCl indicated the presence of high and low molecular weight species in the range between >75 kDa and 9.3 kDa. Almost 83% of Cu and 78% of Fe were extracted during cashew nut digestion and 90% of both elements were dialyzed. With these results it is possible to assume that 75% of Cu and 70% of Fe present in cashew nut could be bioavailable. The SEC-UV and SIMAAS chromatographic profiles obtained after in vitro gastrointestinal digestion reveal that Cu and Fe not dialyzed can be associated to a compound of 9.2 kDa. (C) 2010 Elsevier B.V. All rights reserved.

Erythrocyte ghost cell-alkaline phosphatase: construction and characterization of a vesicular system for use in biomineralization studies

Alkaline phosphatase is required for the mineralization of bone and cartilage. This enzyme is localized in the matrix vesicle, which plays a role key in calcifying cartilage. In this paper we standardize a method to construction a resealed ghost cell-alkaline phosphatase system to mimic matrix vesicles and examine the kinetic behavior of the incorporated enzyme. Polidocanol-solubilized alkaline phosphatase, free of detergent, was incorporated into resealed ghost cells. This process was time-dependent and practically 50% of the enzyme was incorporated into the vesicles in 40 h of incubation, at 25 degreesC. Alkaline phosphatase-ghost cell systems were relatively homogeneous with diameters of about 300 nm and were more stable when stored at -20 degreesC.Alkaline phosphatase was completely released from the resealed ghost cell-system using only phospholipase C. These experiments confirm that the interaction between alkaline phosphatase and the lipid bilayer of resealed ghost cell is exclusively via glycosylphosphatidylinositol (GPI) anchor of the enzyme.An important point shown is that an enzyme bound to resealed ghost cell does not lose the ability to hydrolyze ATP, pyrophosphate and p-nitrophenyl phosphate (PNPP), but the presence of a ghost membrane, as a support of the enzyme, affects its kinetic properties. Moreover, calcium ions stimulate and phosphate ions inhibit the PNPPase activity of alkaline phosphatase present in resealed ghost cells. (C) 2002 Elsevier B.V. B.V. All rights reserved.

Supercritical fluid extraction from Lippia alba: global yields, kinetic data, and extract chemical composition

In this work, experimental data for the system Lippia alba + CO2 is presented. The major constituents of the L. alba volatile oil are limonene and carvone. Thus, literature data for the systems limonene + CO2 and carvone + CO2, and the Peng-Robinson equation of state (PR-EOS) were used to select the operating temperature and pressure, which maximize the global yield in L. alba extract. Global yields were determined at 80, 100, and 120 bar and 40, 45, and 50 degrees C. L. alba extracts were also obtained by conventional processes (hydrodistillation, low-pressure ethanol extraction and Soxhlet ethanol). The chemical compositions of the extracts were determined by gas and thin layer chromatography (TLC). The secretor structures of L. alba were observed by scanning electron microscopy (SEM) before and after supercritical extraction. The largest yield (similar to 7%, mass of extract/mass of dry solid) of the CO2-extract was obtained at 318 K and 100 bar. The chemical compositions of the CO2-extracts were different from those of the extracts obtained by Soxhlet and low-pressure solvent extraction (LPSE) because of the co-extraction of heavy substances by ethanol. The operating conditions that maximized the carvone and limomene yields were 80 bar and 323 K (80 mass%) and 120 bar and 323 K (17 mass%), respectively. (c) 2004 Elsevier B.V All rights reserved.

Extração do corante reactive blue 19 utilizando tensoativo não iônico

The generation of effluent from the finishing process in textile industry is a serious environmental problem and turned into an object of study in several scientific papers. Contamination with dyes and the presences of substances that are toxic to the environment characterize this difficult treatment effluent. Several processes have already been evaluated to remove and even degrade such pollutants are examples: coagulation-flocculation, biological treatment and advanced oxidative processes, but not yet sufficient to enable the recovery of dye or at least of the recovery agent. An alternative to this problem is the cloud point extraction that involves the application of nonionic surfactants at temperatures above the cloud point, making the water a weak solvent to the surfactant, providing the agglomeration of those molecules around the dyes molecules by affinity with the organic phase. After that, the formation of two phases occurred: the diluted one, poor in dye and surfactant, and the other one, coacervate, with higher concentrations of dye and surfactants than the other one. The later use of the coacervate as a dye and surfactant recycle shows the technical and economic viability of this process. In this paper, the cloud point extraction is used to remove the dye Reactive Blue from the water, using nonionic surfactant nonyl phenol with 9,5 etoxilations. The aim is to solubilize the dye molecules in surfactant, varying the concentration and temperature to study its effects. Evaluating the dye concentration in dilute phase after extraction, it is possible to analyze thermodynamic variables, build Langmuir isotherms, determine the behavior of the coacervate volume for a surfactant concentration and temperature, the distribution coefficient and the dye removal efficiency. The concentration of surfactant proved itself to be crucial to the success of the treatment. The results of removal efficiency reached values of 91,38%, 90,69%, 89,58%, 87,22% and 84,18% to temperatures of 65,0, 67,5, 70,0, 72,5 and 75,0°C, respectively, showing that the cloud point extraction is an efficient alternative for the treatment of wastewater containing Reactive Blue

Comparative study of analytical methods by direct and first-derivative UV spectrophotometry for evaluation of losartan potassium in capsules

O losartano potássico é um agente anti-hipertensivo não peptídico, que exerce sua ação por bloqueio específico dos receptores da angiotensina II. Este trabalho propôs a validação e aplicação de métodos analíticos orientados ao controle de qualidade de losartano potássico 50 mg na forma farmacêutica cápsula, utilizando a espectrofotometria direta e derivada de primeira ordem na região do UV. Baseado nas características espectrofotométricas de losartano potássico, um sinal a 205 nm do espectro de ordem zero e um sinal a 234 nm do espectro de primeira derivada foram adequados para a quantificação. Os resultados foram usados para comparar essas duas técnicas instrumentais. O coeficiente de correlação entre as respostas e as concentrações de losartano potássico na faixa de 3,0-7,0 mg L-1 e 6,0-14,0 mg L-1 para espectrofotometria direta e derivada de primeira ordem em solução aquosa, respectivamente, foi de (r) of 0,9999 para ambos os casos. Os métodos foram aplicados para quantificação de losartano potássico em cápsulas obtidas de farmácias de manipulação locais e demonstraram ser eficientes, fáceis de aplicar e de baixo custo. Além disso, não necessitam de reagentes poluentes e requerem equipamentos economicamente viáveis.

Off line extraction of phenol from human urine sample with isoamyl alcohol and determination by HPLC

This method has been developed for extraction and determination of phenol in a urine sample by high performance liquid chromatography.After acid hydrolysis, the free phenol was extracted with isoamyl alcohol solvent, followed by back extraction with 0.5 mol.L-1 sodium hydroxide solution and analyzed by an isocratic HPLC Varian System, equipped with reverse-phase column (MicroPak-C-18). The mobile phase was acetonitrile in 0.01 mol.L-1 hydrochloric acid solution, (20:80 v/v), and at a now-rate of 1.0 mL.min-1. The chromatogram was monitored at 220 nm in room temperature. The identification was based on retention time and the quantification was performed by automatic peak-area determination, corrected for the external standards method.The recovery was higher than 99.5 % for phenol and reproducibility of method was shown to be 2.3% standard deviation and 5.6% coefficient of variance (n=20). The limit detection was 0.05 mgL(-1) and a range of 0.05 to 20.0 mgL(-1) of phenol for linearity.