A comparison study of cleanup techniques for oil spill treatment using magnetic nanomaterials.

Magnetic nanoparticles have been successfully used to recovery oil from oil spilled on water. Two different methods, floating and vortex, were employed to promote the interaction of four oil samples with different API (e.g., 10, 20, 28 and 45) spilled on seawater and deionized water with three magnetic materials, namely: magnetite nanoparticles (N); magnetic nanocomposites of yeast biomass provided by ethanol industry (Y); and magnetic nanocomposites of cork powder (C). The magnetic nanomaterials exposed to oil on water were taking out by a neodymium magnet, and the oil recoveries were determined by gravimetric analysis before and after lyophilization. The lyophilization was determinant to guarantee the accuracy of the experiments, and without this step, the masses of oil recovered would be overestimated due to the drag of water during the oil and magnetic material removal process. Three main factors, API, contact method and magnetic material, and two interactions (i.e., API × contact method, and contact method × magnetic material) presented a statistically significant effect on oil recovery. It was observed that oil recovery increases as API decreases, and it was possible to establish a model to predict the amount of recovered oil according to this effect. Higher oil recoveries were also obtained by magnetic nanocomposites of yeast biomass (Y), regardless of the contact method and type of water, recoveries of 23% and 100% for 45 and 10 API, respectively, employing around 20 mg of Y on 300 mg of spilled oil. These percentages correspond to 0.29 ±â€¯0.01 kg/kg and 15.98 kg/kg of recovering oil by the magnetic procedure. The increase of mass of magnetic material improved the recovery of oils with higher APIs. The reusability of the spent materials presents potential for its application in oil spill cleaning technologies.

DNA purification using dynamic solid-phase extraction on a rotationally-driven polyethylene-terephthalate microdevice.

We report the development of a disposable polyester toner centrifugal device for semi-automated, dynamic solid phase DNA extraction (dSPE) from whole blood samples. The integration of a novel adhesive and hydrophobic valving with a simple and low cost microfabrication method allowed for sequential addition of reagents without the need for external equipment for fluid flow control. The spin-dSPE method yielded an average extraction efficiency of ∼45% from 0.6 µL of whole blood. The device performed single sample extractions or accommodate up to four samples for simultaneous DNA extraction, with PCR-readiness DNA confirmed by effective amplification of a ß-globin gene. The purity of the DNA was challenged by a multiplex amplification with 16 targeted amplification sites. Successful multiplexed amplification could routinely be obtained using the purified DNA collected post an on-chip extraction, with the results comparable to those obtained with commercial DNA extraction methods. This proof-of-principle work represents a significant step towards a fully-automated low cost DNA extraction device.

Single nucleotide polymorphisms in candidate genes associated with gastrointestinal nematode infection in goats.

Cytokines are small cell-signaling proteins that play an important role in the immune system, participating in intracellular communication. Four candidate genes of the cytokine family (IL2, IL4, IL13, and IFNG) were selected to identify Single Nucleotide Polymorphisms (SNPs) that might be associated with resistance to gastrointestinal endoparasites in goats. A population of 229 goats, F2 offspring from an F1 intercross was produced by crossing pure Saanen goats, considered as susceptible to gastrointestinal endoparasites, with pure Anglo-Nubian goats, considered resistant. Blood was collected for DNA extraction and fecal samples were also collected for parasite egg count. Polymorphisms were prospected by sequencing animals with extreme phenotype for fecal egg count (FEC) distribution. The association between SNPs and phenotype was determined by using the Fisher exact test with correction for multiple tests. Three of the 10 SNPs were identified as significant (P ≤ 0.03). They were found in intron 1 of IL2 (ENSBTA00000020883), intron 3 of IL13 (ENSBTA00000015953) and exon 3 of IFNG (ENSBTA00000012529), suggesting an association between them and gastrointestinal endoparasite resistance. Further studies will help describe the effects of these markers accurately before implementing them in marker assisted selection. This study is the pioneer in describing such associations in goats.

Proteomic analysis reveals suppression of bark chitinases and proteinase inhibitors in citrus plants affected by the citrus sudden death disease.

Citrus sudden death (CSD) is a disease of unknown etiology that greatly affects sweet oranges grafted on Rangpur lime rootstock, the most important rootstock in Brazilian citriculture. We performed a proteomic analysis to generate information related to this plant pathogen interaction. Protein profiles from healthy, CSD-affected and CSD-tolerant stem barks, were generated using two-dimensional gel electrophoresis. The protein spots were well distributed over a pI range of 3.26 to 9.97 and a molecular weight (MW) range from 7.1 to 120 kDa. The patterns of expressed proteins on 2-DE gels made it possible to distinguish healthy barks from CSD-affected barks. Protein spots with MW around 30 kDa and pI values ranging from 4.5 to 5.2 were down-regulated in the CSD-affected root-stock bark. This set of protein spots was identified as chitinases. Another set of proteins, ranging in pI from 6.1 to 9.6 with an MW of about 20 kDa, were also suppressed in CSD-affected rootstock bark; these were identified as miraculin-like proteins, potential trypsin inhibitors. Down-regulation of chitinases and proteinase inhibitors in CSD-affected plants is relevant since chitinases are well-known pathogenesis-related protein, and their activity against plant pathogens is largely accepted.

An attempt to correlate fat and protein content of biological samples with residual carbon after microwave-assisted digestion.

The residual carbon content of a variety of bovine-derived samples and forage was determined by inductively coupled plasma optical emission spectrometry with radial view configuration (ICP-OES) after microwave-assisted digestion under high pressure in a closed vessel. The original carbon concentration in the samples was determined by elemental analysis. The highest amount of original carbon content (64%) was found in viscera. After digestion, up to 75% of it was destroyed. Viscera presented the highest ether extract and blood exhibited a high crude protein content of up to 99%. The efficiency in destroying the organic matter in biological materials seemed to be related to their fat content and showed no significant difficulty for protein-rich samples. The correlation coefficient between the fat content of the samples and the residual carbon after acid decomposition was 0.9173 indicating a fair fit. However, no correlation was observed between % RC and the protein content.

DNA sequencing by capillary array electrophoresis and microfabricated array systems.

To comply with the current needs for high-speed DNA sequencing analysis, several instruments and innovative technologies have been introduced by several groups in recent years. This review article discusses and compares the issues regarding high-throughput DNA sequencing by electrophoretic methods in miniaturized systems, such as capillaries, capillary arrays, and microchannels. Initially, general features of several capillary array designs (including commercial ones) will be considered, followed by similar analyses with microfabricated array electrophoretic devices and how they can contribute to the success of large sequencing projects.

Assessing metal sorption on the marine alga Pilayella littoralis.

Increasing interest in the development of biological materials for metal sorption led us to investigate the brown marine alga, Pilayella littoralis, as a biological sorbent. This work focuses on the harvest, preparation and evaluation of P. littoralis from Nahant beaches for use as a metal biosorbent. This biomass was used in batch tests with synthetic solutions. Its metal uptake properties, including metal binding capacity, the pH dependence of metal uptake and the kinetics of metal sorption, were investigated. Most metal sorption occurred within the first 5 min of exposure and the metals were optimally bound to the algae at pH 5.5. The algal binding capacities for Al(III), Cd(II), Co(II), Cr(VI), Cu(II), Fe(III), Ni(II) and Zn(II), were 2,000, 430, 560, 90, 850, 700, 390 and 450 micromol g(-1) of dried biomass, respectively. Metals were desorbed with 0.12 mol l(-1) HCl and determined by inductively coupled plasma atomic emission spectrometry (ICP-AES).

Routine DNA sequencing of 1000 bases in less than one hour by capillary electrophoresis with replaceable linear polyacrylamide solutions.

Long, accurate reads are an important factor for high-throughput de novo DNA sequencing. In previous work from this laboratory, a separation matrix of high-weight-average molecular mass (HMM) linear polyacrylamide (LPA) at a concentration of 2% (w/w) was used to separate 1000 bases of DNA sequence in 80 min with an accuracy close to 97% (Carrilho, E.; et al. Anal. Chem. 1996, 68, 3305-3313). In the present work, significantly improved speed and sequencing accuracy have been achieved by further optimization of factors affecting electrophoretic separation and data processing. A replaceable matrix containing a mixture of 2.0% (w/w) HMM (9 MDa) and 0.5% (w/w) low-weight-average molecular mass (50 kDa) LPA was employed to enhance the separation of DNA sequencing fragments in CE. Experimental conditions, such as electric field strength and column temperature, as well as internal diameter of the capillary column, have been optimized for this mixed separation matrix. Under these conditions, in combination with energy-transfer (BigDye) dye-labeled primers for high signal-to-noise ratio and a newly developed expert system for base calling, the electrophoretic separation of 1000 DNA sequencing fragments of both standard (M13mp18) and cloned single-stranded templates from human chromosome 17 could be routinely achieved in less than 55 min, with a base-calling accuracy between 98 and 99%. Identical read length, accuracy, and migration time were achieved in more than 300 consecutive runs in a single column.

A sample purification method for rugged and high-performance DNA sequencing by capillary electrophoresis using replaceable polymer solutions. A. Development of the cleanup protocol.

A method for the cleanup of Sanger DNA sequencing reaction products for capillary electrophoresis analysis with replaceable polymer solutions has been developed. A poly(ether sulfone) ultrafiltration membrane pretreated with linear polyacrylamide was first used to remove template DNA from the sequencing samples. Then, gel filtration in a spin column format (two columns per sample) was employed to decrease the concentration of salts below 10 microM in the sample solution. The method was very reproducible and increased the injected amount of the sequencing fragments 10-50-fold compared to traditional cleanup protocols. Using M13mp18 as template, the resulting cleaned-up single DNA sequencing fragments could routinely be separated to more than 1000 bases with a base-calling accuracy of at least 99% for 800 bases. The method is simple and universal and can be easily automated. In the following paper, a systematic study to determine quantitatively the effects of the sample solution components such as high-mobility ions (e.g., chloride and dideoxynucleotides) and template DNA on the injected amount and separation efficiency of the sequencing fragments is presented.

A sample purification method for rugged and high-performance DNA sequencing by capillary electrophoresis using replaceable polymer solutions. B. Quantitative determination of the role of sample matrix components on sequencing analysis.

In the previous paper, a sample cleanup procedure for DNA sequencing reaction products was developed, in which template DNA was removed by ultrafiltration and the total concentration of salts (chloride and di- and deoxynucleotides) was decreased below 10 microM using gel filtration. In this paper, a quantitative study of the effects of these sample solution components on the injected amount and separation efficiency of the sequencing fragments in capillary electrophoresis is presented. The presence of chloride and deoxynucleotides in a total concentration above 10 microM in the sample solution significantly decreased the amount of DNA sequencing fragments injected into the capillary column. However, the separation efficiency was not affected upon increasing the amount of salt. On the other hand, in the presence of only 0.1 microgram of template in the sample (one-third of the lowest quantity recommended in cycle sequencing) and at very low chloride concentration (approximately 5 microM), the separation efficiency decreased by 70%, and the injected amount of DNA sequencing fragments was 40% lower compared to the sample cleaned by the new purification method. The deleterious effect of template DNA on the separation of sequencing fragments was suppressed in the presence of salt in a concentration above 100 microM in the sample solution. Separately, it was found that both the electric field strength and duration of injection affected the resolution of DNA sequencing fragments when the cleaned up sample solution was used. Separation efficiencies of 15 x 10(6) theoretical plates/m were achieved when the sample was loaded at low electric field, e.g., 25 V/cm for 80 s or less. The results demonstrate that the sample solution components (chloride, deoxynucleotides, template DNA) and injection conditions must be controlled to achieve high performance and rugged DNA sequencing analysis.

Characterization of high molecular mass linear polyacrylamide powder prepared by emulsion polymerization as a replaceable polymer matrix for DNA sequencing by capillary electrophoresis.

In a previous paper, a 2% w/w replaceable high molecular mass linear polyacrylamide solution (high molecular mass LPA) was used to achieve long read-lengths for DNA sequencing by capillary electrophoresis (E. Carrilho et al., Anal. Chem. 1996, 68, 3305-3313). In that work, the polymer was prepared by polymerization in water at 6% w/w, followed by dilution to 2% w/w. In this study, an improved method for preparation of high molecular mass LPA was developed, based on inverse emulsion polymerization. With this polymerization procedure, the LPA results in a molecular mass of approximately 9 MDa, with characteristics of a fine powder of high purity and practically unlimited shelf life. Using size exclusion chromatography (SEC) and viscosity measurements to characterize the polymer, good batch-to-batch reproducibility was found. It was observed that the viscous polymer solutions made from these high molecular mass polymers require careful preparation and handling because the method of dissolution could affect the molecular mass distribution and the resultant separation of DNA components. Solutions containing 2% w/w of LPA made by emulsion polymerization were simple to prepare, resulting in excellent performance as a replaceable matrix for DNA sequencing by capillary electrophoresis. The viscosity of the polymer decreased exponentially when pressure was applied, allowing easy replacement from a capillary using a syringe. With a properly prepared matrix, a read-length of more than 1000 bases in 80 min with an accuracy better than 97%, and better than 99% for the first 800 bases, could be achieved.

Capillary electrophoresis for the detection of known point mutations by single-nucleotide primer extension and laser-induced fluorescence detection.

Capillary electrophoresis (CE) with laser-induced fluorescence (LIF) was used to detect known point mutations using the method of single-nucleotide primer extension (SNuPE). Three different point mutations in human mitochondrial DNA associated with Leber's hereditary optic neuropathy (LHON) were detected by annealing a primer immediately 5' to the mutation on the template and extending the primer by one fluorescently labeled dideoxy terminator complementary to the mutation. By using two or more differently labeled terminators, both the mutant and wild type could be simultaneously detected. The advantages of using CE-LIF for detecting SNuPE reactions include speed and ease of analysis, absence of radioactivity, and potential for automation.

Missense mutation R1066C in the second transmembrane domain of CFTR causes a severe cystic fibrosis phenotype: study of 19 heterozygous and 2 homozygous patients.

We report the clinical features of 21 unrelated cystic fibrosis (CF) patients from Portugal and Spain, who carry the mutation R1066C in the CFTR gene. The current age of the patients was higher in the R1066C/any mutation group (P < 0.01), as compared to the deltaF508/deltaF508 group. Poor values for lung radiological involvement (Chrispin-Norman) and general status (Shwachman-Kulcycki) were observed in the R1066C/any mutation group (P < 0.005 and P < 0.0004). A slightly, but not significantly worse lung function was found in the R1066C/any mutation group when compared with the deltaF508/deltaF508 patients. No significant differences were detected regarding the age at diagnosis, sweat Cl-values, or percentiles of height and weight between the two groups. Neither were significant differences observed regarding sex, meconium ileus (4.7% vs. 11.1%), dehydration (10.5% vs. 14.7%), or pancreatic insufficiency (PI) (100% vs. 97.8%). The proportion of patients with lung colonization by bacterial pathogens was slightly, but not significantly higher in the R1066C/any mutation group (70.0%), as compared with the deltaF508/deltaF508 group (57.5%). Other clinical complications were significantly more frequent in the R1066C/any mutation patients(P < 0.02) than in the deltaF508/deltaF508 group. The two homozygous R1066C/R1066C patients died at the ages of 3 months and 7 years. The data presented in this study clearly demonstrate that the R1066C mutation is responsible for a severe phenotype similar to that observed in homozygous deltaF508 patients. The poor clinical scores and complications of patients with the R1066C mutation are probably related to their slightly longer survival.

Rapid DNA sequencing of more than 1000 bases per run by capillary electrophoresis using replaceable linear polyacrylamide solutions.

The read length for DNA sequencing using capillary electrophoresis and replaceable linear polyacrylamide (LPA) solutions has been extended to more than 1000 bases with a run time of 80 min. This result was successfully achieved through the combined use of cycle sequencing with dye-labeled primers, improved matrix and separation conditions, and enhanced base-calling software. The influences of LPA molecular weight and concentration on separation were investigated. Additionally, the separation buffer, column temperature, and electric field were adjusted to increase the number of resolvable DNA fragments per run while maintaining an enhanced separation speed. Using low concentrations [2% (w/v)] of high molecular weight LPA polymers (> 5.5 x 10(6) Da), elevated column temperature (50 degrees C) and moderately high field (150 V/cm), rapid sequencing analysis for more than 1000 bases on a model ssM13mp18 template was obtained with 96.8% accuracy.

DNA sequencing by capillary electrophoresis using short oligonucleotide primer libraries.

Two strategies for DNA sequencing by primer walking using short oligonucleotide primer libraries have been successfully employed along with capillary electrophoresis using replaceable polymer solutions of linear polyacrylamide and fluorescence detection. A 3.5-kb stretch of the single-stranded M13mp18 template was sequenced with T7 PRISM dye-terminator/Sequenase chemistry. An in-house base-calling program offered read lengths of roughly 450 bases with an average of 97.8% accuracy.

Correction of iron interface in the spectrophotometric flow injection catalytic determination of molybdenum in plants.

Iron interference in the spectrophotometric catalytic determination of molybdenum based on the iodide-hydrogen peroxide reaction can be corrected by using sulphosalicylic acid as masking and color-forming reagent. The catalytic influence of iron ions is circumvented to the extent of about 90% and correction of any remaining iron ions is possible by monitoring the colored iron(III)-salicylate complex at 490 nm. In this way, iron is also determined. With the proposed system, molybdenum can be determined in plant and food digests within the 0-100 mug Mo 1(-1) range in the presence of up to 25 mg Fe 1(-1), at a sampling rate of about 50 determinations h(-1). The relative standard deviation of 10 consecutive measurements was estimated as < 2%. Results for samples were comparable with those obtained by graphite furnace atomic absorption spectrometry. In addition, recoveries within the range 94-100% were calculated.

Characterization and performance of a neutral hydrophilic coating for the capillary electrophoretic separation of biopolymers.

Polyvinylmethylsiloxanediol (50% vinyl) was synthesized and combined with a cross-linker for static coating onto fused-silica columns. After cross-linking and binding to the surface, linear polyacrylamide was grafted to the double bonds of the siloxanediol; subsequently, this linear polymer matrix was cross-linked with formaldehyde. The grafted neutral polymeric layer provided suppression of electroosmotic flow and minimized adsorption. This combination yielded successful open tube and polymer network separations of proteins, peptides and DNA molecules. Very high efficiencies (ca. 1 x 10(6) plates/m) were achieved for open tube protein separations, and hundreds of consecutive runs were performed with minimal change in migration times.

[Marginal leakage of amalgam restorations]. / Fenda marginal das restaurações a amálgama.

The authoress presents a revision work on a marginal microleakage around amalgam restorations. After a brief introduction in which some harmful aspects of its formation are made clear, a reference to the causes and tooth prevention against its formation follows.