Prior Purification Research Articles

ASSESSMENT OF PESTICIDES IN ENVIRONMENTAL SAMPLES USING VOLTAMMETRIC MOLECULAR IMPRINTED BASED SENSORS: A REVIEW (2006-2015)

The increased concerns about toxic effects of pesticides exposure led to necessity of its monitoring using rapid, sensitive and selective analytical tools. Traditional instrumental analytical techniques are often time consuming, labor intensive and need tedious prior separation or purification steps and expensive instrumentation. Besides those electrochemical sensors can overcome disadvantages of the traditional techniques, MIP-based sensors offer high degree of selectivity in binding target analytes in the presence of their interferents making them ideal for determination of pesticides in complex environmental samples. The scope of this review is to provide a general overview on MIPs field discussing first methods of MIP preparation and then dealing with applications of MIP based sensors in assessment of pesticides in environmental samples using voltammetry as transduction mechanism.

A novel approach to the measurement of surfactant parameters in arthropod digestive juices

In arthropods, the determination of two important parameters of digestive juices, i.e. the total surfactant concentration and the critical micelle concentration (CMC), is challenging due to small sample volumes and low surfactant concentrations. In this work, we report a successful implementation of potentiometric titrations using the surfactant ion-selective electrode (SISE) and the pyrene fluorescence method (PFM) for the determination of the total surfactant concentration and CMC in the digestive juice of terrestrial isopod crustaceans Porcellio scaber. Pooled digestive juice extracts of four (SISE) or two (PFM) animals were used per measurement run. In both cases, digestive juice extracts in 100μL of deionized water were sufficient for one measurement run. The total surfactant concentration of P. scaber digestive juice was determined to be 9.2±3.5mM and the CMC was approximately 90μM. Our work presents an important improvement towards easy CMC determination in small volume samples in comparison with the commonly used stalagmometric technique, where much larger sample volumes are usually needed. To date, the total surfactant concentration was not measured in the digestive juices of arthropods other than Homarus vulgaris, Astacus leptodactylus and Cancer pagurus, for which complex separation and analytical techniques were required. Our results obtained by SISE and PFM therefore present the first successful quantification of surfactants and their CMC in small volumes of arthropod digestive juice without prior separation or purification techniques.

Co-translational formation and pharmacological characterization of beta1-adrenergic receptor/nanodisc complexes with different lipid environments

G protein-coupled receptors are of key significance for biomedical research. Streamlined approaches for their efficient recombinant production are of pivotal interest in order to explore their intrinsic conformational dynamics and complex ligand binding behavior. We have systematically optimized the co-translational association and folding of G protein-coupled receptors with defined membranes of nanodiscs by cell-free expression approaches. Each optimization step was quantified and the ligand binding active fraction of the receptor samples could drastically be improved. The strategy was exemplified with a stabilized and a non-stabilized derivative of the turkey beta1-adrenergic receptor. Systematic lipid screens with preformed nanodiscs revealed that generation of ligand binding active conformations of the analyzed beta1-adrenergic receptors strongly depends on lipid charge, flexibility and chain length. The lipid composition of the nanodisc membranes modulates the affinities to a variety of ligands of both receptor derivatives. In addition, the thermostabilization procedure had a significant impact on specific ligand affinities of the receptor and abolished or reduced the binding of certain antagonists. Both receptors were highly stable after purification with optimized nanodisc membranes. The procedure avoids any detergent contact of the receptors and sample production takes less than two days. Moreover, even non-stabilized receptors can be analyzed and their prior purification is not necessary for the formation of nanodisc complexes. The established process appears therefore to be suitable as a new platform for the functional or even structural characterization of recombinant G protein-coupled receptors associated with defined lipid environments.

Assessing the Enzyme Activity of Different Plant Extracts of Biomasses from Sub-Saharan Africa for Ethyl Biodiesel Production

This work focused on identifying plant biomasses as potential sources of lipases and their use as biocatalysts for ethyl biodiesel production. To that end, plant extracts prepared from dormant and germinated seeds of eight local (Burkina Faso) oil-bearing biomasses were tested as a biocatalyst for the hydrolysis and ethanol transesterification of sunflower oil. The optimum transesterification conditions with the most active extracts were then determined. The results of the hydrolysis and transesterification tests showed that the plant extracts from germinated Jatropha curcas (JG) and Moringa oleifera (MG) seeds contained lipases whose catalytic activity could be utilized without prior purification. The highest hydrolysis yield obtained was 88% using the JG extracts and 62% with the MG extracts after 24 h of reaction at 40 °C and at pH 7. For transesterification, the highest ethyl ester yield obtained was 94% with the JG extracts and 48% with the MG extracts after 144 h at 37 °C, following two successive transesterification reactions. Using the plant extracts in transesterification called for the stepwise addition of ethanol in a TAG:ethanol molar ratio of 1:0.3 at 6 h intervals to overcome the inhibiting effect of high ethanol concentrations on the catalytic activity of the plant extracts. In addition, a study on the influence of the reaction conditions showed that water had a negligible effect on transesterification yield when its content in the ethanol was lower than or equal to 5% (v/v) and that adding silica to the reaction medium led to an increase in transesterification yield.

Efficient conversion of crude glycerol from various industrial wastes into single cell oil by yeast Yarrowia lipolytica

In this study, crude glycerol from various industries was used to produce lipids via wild type Yarrowia lipolytica A101. We tested samples without any prior purification from five different waste products; each contained various concentrations of glycerol (42–87%) as the sole carbon source. The best results for lipid production were obtained for medium containing glycerol from fat saponification. This reached 1.69gL−1 (25% of total cell dry weight) with a biomass yield of 0.17gg−1 in the flasks experiment. The batch cultivation in a bioreactor resulted in enhanced lipid production—it achieved 4.72gL−1 with a biomass yield 0.21gg−1. Moreover, the properly selected batch of crude glycerol provides a defined fatty acid composition. In summary, this paper shows that crude glycerol from soap production could be efficiently converted to single cell oil without any prior purification.

The Impact of Leachate on the Quality of Surface and Groundwater and Proposal of Measures for Pollution Remediation

Direct discharge of municipal and industrial waste waters and leachate (originating from the illegal landfills) into recipients without prior purification is unfortunately very common practice in the region of northern Kosovo. In addition, irresponsible and incorrect selection of sites for industrial and municipal dumps, which are often located in vicinity or on actual river banks, contributes significantly to environmental pollution. Analysis of the leachate from such sites was done by direct sampling and by using TCLP (Toxicity Characteristic Leashing Procedures) method. Based on the analysis of physicochemical parameters of the filtrated water from the sites Zitkovac, Grabovac and Balaban and analysis of the samples of surface water and groundwater from the site Grabovac, possible steps for removal and reduction of the existing pollution were proposed. Potential permanent solution in form of purification of municipal and industrial waste waters as well as leachate from illegal landfills was suggested. The potential implementation of remediation with a unified system for water purification, by using Membrane Bio Reactor (MBR), which includes the process of stabilization/solidification of a residual sludge, would have as an end product a neutral powder material completely safe for the environment, suitable for a variety of applications.

Application of reporter strains for new antibiotic screening

Screening for new antibiotics remains an important area of biology and medical science. Indispensable for this type of research is early identification of antibiotic mechanism of action. Preferentially, it should be studied quickly and cost-effectively, on the stage of primary screening. In this review we describe an application of reporter strains for rapid classification of antibiotics by its target, without prior purification of an active compound and determination of chemical structure.

Effects of Minor Components of Crude Vegetable Oil on the Enzymatic Method to Analyze Positional Fatty Acid Distributions in Triacylglycerols withCandida antarctica Lipase B.

Crude soybean and rapeseed oils were subjected to the method to determine FA distributions in TAG using Candida antarctica lipase B, giving similar results to those for refined oils. Minor components in crude oils, such as percentages of FFA or phospholipids were indicated not to affect 1(3)-selective transesterification by the lipase and FA compositional analysis of the resulting 2-MAG fraction significantly. Phospholipids were confirmed not to contaminate the 2-MAG fraction. Oxidized soybean oil with a PV of 10 meq/kg also gave similar results to the ones for refined oil. The method was confirmed to be applicable for crude oils and oxidized oils with a PV smaller than 10 meq/kg without prior purification of TAG.

The use of differential scanning fluorimetry in the rational design of plastic antibodies for protein targets.

The development of molecularly imprinted polymer nanoparticles (MIP-NPs), which specifically bind biomolecules, is of great interest in the area of biosensors, sample purification, therapeutic agents and biotechnology. Polymerisation techniques such as precipitation polymerisation, solid phase synthesis and core shell surface imprinting have allowed for significant improvements to be made in developing MIP-NPs which specifically recognise proteins. However, the development of MIP-NPs for protein templates (targets) still require lengthy optimisation and characterisation using different ratios of monomers in order to control their size, binding affinity and specificity. In this work we successfully demonstrated that differential scanning fluorimetry (DSF) can be used to rapidly determine the optimum imprinting conditions and monomer composition required for MIP-NP design and polymerisation. This is based on the stability of the protein template and shift in apparent melting points (Tm) upon interaction with different functional acrylic monomers. The method allows for the characterisation of molecularly imprinted nanoparticles (MIP-NPs) due to the observed differences in melting point profiles between, protein-MIP-NPs complexes, pre-polymerisation mixtures and non-imprinted nanoparticles (NIP-NPs) without the need for prior purification. The technique is simple, rapid and can be carried out on most quantitative polymerase chain reaction (qPCR) thermal cyclers which have the required filters for SYPRO

Chemometrics-assisted determination of amiloride and triamterene in biological fluids with overlapped peaks and unknown interferences.

Amiloride (AMI) and triamterene (TRI) are both potassium-saving diuretics, which are ordinarily used as doping to enhance the performance of athletes in sports. For the similar structures and complex matrices existence, chromatography and extraction are commonly employed to realize the determination of AMI and TRI in biological fluids, which are very time-consuming and laborious. A novel method is presented to simultaneous interference-free determination of AMI and TRI in complex biological fluids samples using excitation-emission matrix fluorescence coupled with second-order calibration method based on alternating normalization-weight error algorithm. The proposed method can obtain accurate qualitative and quantitative information of the analytes, even in the presence of the interference from complex biological fluids, which requires few prior purification and separation procedures.

Expansion of NK cells from PBMCs using immobilized 4-1BBL and interleukin-21.

Adoptive transfer of NK cells has been widely applied clinically for cancer immunotherapy. However, the difficulties to obtain a large number of activated NK cells impede the successful application of such therapy. In the present study, we implemented a novel method involving the use of immobilized human 4-1BBL and interleukin-21 to amplify NK cells from the peripheral blood mononuclear cells (PBMCs) of healthy donors. Following stimulation for 21 days, we achieved considerable expansion of NK cells with high purity and strong cytotoxicity. This is the first time solid phase cytokines were used to augment NK cells, and this method has the advantage of no need to introduce feeder cells, without prior purification of NK cells and it effectively stimulated and expanded NK cells. The strategy of cell proliferation and activation could lead to a safer and more effective application of NK cells clinically.

A novel Cre recombinase reporter mouse strain facilitates selective and efficient infection of primary immune cells with adenoviral vectors.

Replication-deficient recombinant adenoviruses are potent vectors for the efficient transient expression of exogenous genes in resting immune cells. However, most leukocytes are refractory to efficient adenoviral transduction as they lack expression of the coxsackie/adenovirus receptor (CAR). To circumvent this obstacle, we generated the R26/CAG-CARΔ1(StopF) (where R26 is ROSA26 and CAG is CMV early enhancer/chicken β actin promoter) knock-in mouse line. This strain allows monitoring of in situ Cre recombinase activity through expression of CARΔ1. Simultaneously, CARΔ1 expression permits selective and highly efficient adenoviral transduction of immune cell populations, such as mast cells or T cells, directly ex vivo in bulk cultures without prior cell purification or activation. Furthermore, we show that CARΔ1 expression dramatically improves adenoviral infection of in vitro differentiated conventional and plasmacytoid dendritic cells (DCs), basophils, mast cells, as well as Hoxb8-immortalized hematopoietic progenitor cells. This novel dual function mouse strain will hence be a valuable tool to rapidly dissect the function of specific genes in leukocyte physiology.

Noninvasive genotyping of common marmoset (Callithrix jacchus) by fingernail PCR.

The common marmoset (Callithrix jacchus) is a New World primate that is a useful model for medical studies. In this study, we report a convenient, reliable, and noninvasive procedure to genotype a living common marmoset by using fingernails. This method was used to successfully genotype DNA by restriction fragment length polymorphism (RFLP) PCR without prior purification, by using the KOD FX PCR enzyme kit. Additionally, there is no sample contamination from hematopoietic chimera derived from fused placenta in utero. We compared chimeric levels between various tissues in females with male littermates using quantitative fluorescent (QF)-PCR to prepare a reliable DNA source for genetic analyses, such as genotyping, gene mapping, or genomic sequencing. The chimerism detected appeared to be restricted to lymphatic tissues, such as bone marrow, thymus, spleen, lymph nodes and blood cells. As a result, DNA from fingernails with the quick is the best DNA source for genetic research in living marmosets.

Determination of Pb and Cd in Macedonian Wines by Electrothermal Atomic Absorption Spectrometry (ETAAS)

In this study, electrothermal atomic absorption spectrometry (ETAAS) was used for determination of lead and cadmium in Macedonian white wines. Wine samples, without prior purification, were directly injected into the ETAAS system, using matrix modifiers for Pb and Cd. The standard addition method was used for quantitative analysis of Pb and Cd content. Reliability of the method was verified by determination of selected validation characteristics. The results of the measurements indicated satisfactory precision and accuracy, confirming that the method is accurate and convenient for quantitative analysis. The lead and cadmium concentrations were ranged between 2–28.5 and 0.4–5.69 μg/L, respectively. The levels were below the maximal allowed concentration in wine and compare well with those reported for similar wines from other parts of the world.

Diversity of extracellular vesicles in human ejaculates revealed by cryo-electron microscopy

Human ejaculates contain extracellular vesicles (EVs), that to a large extent are considered to originate from the prostate gland, and are often denominated “prostasomes.” These EVs are important for human fertility, for example by promoting sperm motility and by inducing immune tolerance of the female immune system to the spermatozoa. So far, the EVs present in human ejaculate have not been studied in their native state, inside the seminal fluid without prior purification and isolation procedures. Using cryo-electron microscopy and tomography, we performed a comprehensive inventory of human ejaculate EVs. The sample was neither centrifuged, fixed, filtered or sectioned, nor were heavy metals added. Approximately 1,500 extracellular structures were imaged and categorized. The extracellular environment of human ejaculate was found to be diverse, with 5 major subcategories of EVs and 6 subcategories of extracellular membrane compartments, including lamellar bodies. Furthermore, 3 morphological features, including electron density, double membrane bilayers and coated surface, are described in all subcategories. This study reveals that the extracellular environment in human ejaculate is multifaceted. Several novel morphological EV subcategories are identified and clues to their cellular origin may be found in their morphology. This inventory is therefore important for developing future experimental approaches, and to interpret previously published data to understand the role of EVs for human male fertility.

Western blot analysis of adhesive interactions under fluid shear conditions: the blot rolling assay.

Western blotting has proven to be an important technique in the analysis of receptor-ligand interactions (i.e., by ligand blotting) and for identifying molecules mediating cell attachment (i.e., by cell blotting). Conventional ligand blotting and cell blotting methods employ non-dynamic (static) incubation conditions, whereby molecules or cells of interest are placed in suspension and overlaid on membranes. However, many cell-cell and cell-matrix adhesive interactions occur under fluid shear conditions, and shear stress itself mediates and/or facilitates the engagement of these physiologically appropriate receptors and ligands. Notably, shear forces critically influence the adhesion of circulating cells and platelets to vessel walls in physiologic cell migration and hemostasis, as well as in inflammatory and thrombotic disorders, cancer metastasis, and atherosclerosis. Use of non-dynamic blotting conditions to analyze such interactions can introduce bias, overtly missing relevant effectors and/or exaggerating the relative role(s) of non-physiologic adhesion molecules. To address this shortfall, we have developed a new technique for identifying binding interactions under fluid shear conditions, the "blot rolling assay." Using this method, molecules in a complex mixture are resolved by gel electrophoresis, transferred to a membrane that is rendered semitransparent, and the membrane is then incorporated into a parallel-plate flow chamber apparatus. Under controlled flow conditions, cells or particles bearing adhesion proteins of interest are then introduced into the chamber and interactions with individual immobilized molecules (bands) can be visualized in real time. The substrate molecule(s) supporting adhesion under fluid shear can then be identified by staining with specific antibodies or by excising the relevant band(s) and performing mass spectrometry or microsequencing of the isolated material. This method thus allows for the identification, within a complex mixture and without prior isolation or purification, of both known and previously uncharacterized adhesion molecules operational under dynamic conditions.

Development of rapid direct PCR assays to identify downy and powdery mildew and grey mould in <em>Vitis vinifera</em> tissues

<p style="text-align: justify;"><strong>Aims</strong>: The development of a rapid and reliable direct PCR method to detect fungal propagules in grapevine tissues without prior DNA purification steps, and illustration of its potential use with different examples.</p><p style="text-align: justify;"><strong>Methods and results</strong>: Different grapevine samples crushed in the presence of polyvinylpolypyrrolidone (PVPP) were used as templates for direct PCR amplification with primers specifie for <em>Erysiphe necator</em>, <em>Plasmopara viticola</em>, <em>Botrytis cinerea</em> and <em>Vitis vinifera</em>. Sequencing of the PCR products confirmed the specificity of the amplifications. The sensitivity tested using conidia/sporangia dilution series was high, ranging from five sporangia for <em>P. viticola</em> to one conidium for <em>E. necator</em>. The potential of this technique is illustrated through the study of four epidemiological questions. Fungal propagules were observed in dormant buds using microscopy, but the responsible species could not be identified. Direct PCR revealed the presence of <em>E. necator</em> and<em> B. cinerea</em> in 29 % and 65 % of the buds, respectively. Downy mildew could be detected in asymptomatic leaves sampled in fields after potentially infectious events. In bunch, microscopic analysis of rachis sections showed the presence of hyphae growing in the green tissue. Direct PCR identified the presence of <em>P. viticola</em>.</p><p style="text-align: justify;"><strong>Conclusion</strong>: A direct PCR method without DNA purification was demonstrated to be a simple and reliable method for the detection and identification of fungal pathogens in grapevine tissues. This method, together with microscopy, is a very interesting tool that can be used to study various epidemiological problems in the grapevine, including important unanswered questions such as the route of infection that leads to brown rot caused by downy mildew.</p><p style="text-align: justify;"><strong>Significance and impact of the study</strong>: Direct PCR was shown to be a simple and versatile technique for the study of epidemiological questions in the grapevine. This technique could be extended to other pathosystems with minor adaptations.</p>

Characterization of coal and charcoal by alpha-particle and gamma-ray spectrometry

Abstract Although coal and charcoal have similar physical and chemical characteristics, there are several crystallographic procedures used to distinguish and characterize them. But if the matrix is crushed, there is no standard procedure to distinguish coal from charcoal. In this work, a procedure to characterize coal and charcoal samples based on the radioactive content is proposed. The first assay is by gamma-ray spectrometry, which allows a part of the radioactive content to be determined rapidly and non-destructively. Then, alpha-particle spectrometry is applied to assay the content of those radionuclides which are difficult to determine precisely by gamma-ray spectrometry. This second technique requires prior chemical purification of the carbon sample in order to separate the corresponding radionuclides of interest.

Steroid hormone measurements from different types of assays in relation to body mass index and breast cancer risk in postmenopausal women: Reanalysis of eighteen prospective studies

Epidemiological studies have examined breast cancer risk in relation to sex hormone concentrations measured by different methods: “extraction” immunoassays (with prior purification by organic solvent extraction, with or without column chromatography), “direct” immunoassays (no prior extraction or column chromatography), and more recently with mass spectrometry-based assays. We describe the associations of estradiol, estrone and testosterone with both body mass index and breast cancer risk in postmenopausal women according to assay method, using data from a collaborative pooled analysis of 18 prospective studies. In general, hormone concentrations were highest in studies that used direct assays and lowest in studies that used mass spectrometry-based assays. Estradiol and estrone were strongly positively associated with body mass index, regardless of the assay method; testosterone was positively associated with body mass index for direct assays, but less clearly for extraction assays, and there were few data for mass spectrometry assays. The correlations of estradiol with body mass index, estrone and testosterone were lower for direct assays than for extraction and mass spectrometry assays, suggesting that the estimates from the direct assays were less precise. For breast cancer risk, all three hormones were strongly positively associated with risk regardless of assay method (except for testosterone by mass spectrometry where there were few data), with no statistically significant differences in the trends, but differences may emerge as new data accumulate. Future epidemiological and clinical research studies should continue to use the most accurate assays that are feasible within the design characteristics of each study.

High-performance liquid-catalyst fuel cell for direct biomass-into-electricity conversion.

Herein, we report high-performance fuel cells that are catalyzed solely by polyoxometalate (POM) solution without any solid metal or metal oxide. The novel design of the liquid-catalyst fuel cells (LCFC) changes the traditional gas-solid-surface heterogeneous reactions to liquid-catalysis reactions. With this design, raw biomasses, such as cellulose, starch, and even grass or wood powders can be directly converted into electricity. The power densities of the fuel cell with switchgrass (dry powder) and bush allamanda (freshly collected) are 44 mW cm(-2) and 51 mW cm(-2) respectively. For the cellulose-based biomass fuel cell, the power density is almost 3000 times higher than that of cellulose-based microbial fuel cells. Unlike noble-metal catalysts, POMs are tolerant to most organic and inorganic contaminants. Therefore, almost any raw biomass can be used directly to produce electricity without prior purification.