Chemical composition and enzymatic digestibility of sugarcane clones selected for varied lignin content

Abstract Background The recalcitrance of lignocellulosic materials is a major limitation for their conversion into fermentable sugars. Lignin depletion in new cultivars or transgenic plants has been identified as a way to diminish this recalcitrance. In this study, we assessed the success of a sugarcane breeding program in selecting sugarcane plants with low lignin content, and report the chemical composition and agronomic characteristics of eleven experimental hybrids and two reference samples. The enzymatic digestion of untreated and chemically delignified samples was evaluated to advance the performance of the sugarcane residue (bagasse) in cellulosic-ethanol production processes. Results The ranges for the percentages of glucan, hemicellulose, lignin, and extractive (based on oven-dry biomass) of the experimental hybrids and reference samples were 38% to 43%, 25% to 32%, 17% to 24%, and 1.6% to 7.5%, respectively. The samples with the smallest amounts of lignin did not produce the largest amounts of total polysaccharides. Instead, a variable increase in the mass of a number of components, including extractives, seemed to compensate for the reduction in lignin content. Hydroxycinnamic acids accounted for a significant part of the aromatic compounds in the samples, with p-coumaric acid predominating, whereas ferulic acid was present only in low amounts. Hydroxycinnamic acids with ester linkage to the hemicelluloses varied from 2.3% to 3.6%. The percentage of total hydroxycinnamic acids (including the fraction linked to lignin through ether linkages) varied from 5.0% to 9.2%, and correlated to some extent with the lignin content. These clones released up to 31% of glucose after 72 hours of digestion with commercial cellulases, whereas chemically delignified samples led to cellulose conversion values of more than 80%. However, plants with lower lignin content required less delignification to reach higher efficiencies of cellulose conversion during the enzymatic treatment. Conclusion Some of the experimental sugarcane hybrids did have the combined characteristics of high biomass and high sucrose production with low lignin content. Conversion of glucan to glucose by commercial cellulases was increased in the samples with low lignin content. Chemical delignification further increased the cellulose conversion to values of more than 80%. Thus, plants with lower lignin content required less delignification to reach higher efficiencies of cellulose conversion during the enzymatic treatment.

CRITICAL APPLICATIONS OF SW 846 US EPA METHODS TO EVALUATION OF MARINE SAMPLES QUALITY

Technical evaluation of analytical data is of extreme relevance considering it can be used for comparisons with environmental quality standards and decision-making as related to the management of disposal of dredged sediments and the evaluation of salt and brackish water quality in accordance with CONAMA 357/05 Resolution. It is, therefore, essential that the project manager discusses the environmental agency`s technical requirements with the laboratory contracted for the follow-up of the analysis underway and even with a view to possible re-analysis when anomalous data are identified. The main technical requirements are: (1) method quantitation limits (QLs) should fall below environmental standards; (2) analyses should be carried out in laboratories whose analytical scope is accredited by the National Institute of Metrology (INMETRO) or qualified or accepted by a licensing agency; (3) chain of custody should be provided in order to ensure sample traceability; (4) control charts should be provided to prove method performance; (5) certified reference material analysis or, if that is not available, matrix spike analysis, should be undertaken and (6) chromatograms should be included in the analytical report. Within this context and with a view to helping environmental managers in analytical report evaluation, this work has as objectives the discussion of the limitations of the application of SW 846 US EPA methods to marine samples, the consequences of having data based on method detection limits (MDL) and not sample quantitation limits (SQL), and present possible modifications of the principal method applied by laboratories in order to comply with environmental quality standards.

Rapid sample preparation procedure for As speciation in food samples by LC-ICP-MS

This paper describes a rapid method for arsenic (As) speciation by LC-ICP-MS in several types of food samples. Prior to analysis, samples were milled and the As species extracted from biological tissues by sonication in only 2 min with a solution containing MeOH (10%, v/v) plus HNO3 (2%, v/v). As species were separated by LC using an anion exchange column. Method detection limits for AsB, As3+,DMA, MMA and As5+ were 1.3, 0.9, 0.6, 0.7 and 0.8 ng g(-1), respectively. Method accuracy and precision were traceable to Certified Reference Materials SRM1577 bovine liver from the National Institute of Standards and Technology, CE278 mussel tissue from the Institute of Reference Materials and Measurements and DOLT-3 dogfish liver tissue and DORM-3 fish protein from the National Research Council of Canada. Finally, the method was applied to speciate As in food samples (egg, fish muscle, beef and chicken) purchased in Brazilian markets.

Development of an enzymeless electroanalytical method for the indirect detection of creatinine in urine samples

The aim of this study is to develop a new enzymeless electroanalytical method for the indirect quantification of creatinine from urine sample. This method is based on the electrochemical monitoring of picrate anion reduction at a glassy carbon electrode in an alkaline medium before and after it has reacted with creatinine (Jaffe's reaction). By using the differential pulse voltammetry technique under the optimum experimental conditions (step potential, amplitude potential, reaction time, and temperature), a linear analytical curve was obtained for concentrations of creatinine ranging from 1 to 80 mu mol L-1, with a detection limit of 380 nmol L-1. This proposed method was used to measure creatinine in human urine without the interference of most common organic species normally present in biological fluids (e.g., uric acid, ascorbic acid, glucose, and phosphocreatinine). The results obtained using urine samples were highly similar to the results obtained using the reference spectrophotometric method (at a 95% confidence level). (C) 2012 Elsevier B.V. All rights reserved.

Evaluation of the Concentration of Nonessential and Essential Elements in Chicken, Pork, and Beef Samples Produced in Brazil

Food safety is a global concern. Meat represents the most important protein source for humans. Thus, contamination of meat products by nonessential elements is a ready source of human exposure. In addition, knowledge of the concentration of essential elements is also relevant with respect to human nutrition. The aim of the present study was to determine the concentration of 17 elements in pork, beef, and chicken produced in Brazil. Meat samples were analyzed by inductively coupled plasma mass spectrometry. The estimated daily intake for nonessential elements including arsenic (As), cadmium (Cd), lead (Pb), mercury (Hg), and antimony (Sb) through meat consumption is below the toxicological reference values. However, high levels were detected for the nonessential element cesium (Cs), mainly in beef samples, an observation that deserves future studies to identify the source of contamination and potential adverse consequences.

An environmentally friendly analytical procedure for nickel determination by atomic and molecular spectrometry after cloud point extraction in different samples

Cloud point extraction (CPE) was employed for separation and preconcentration prior to the determination of nickel by graphite furnace atomic absorption spectrometry (GFAAS), flame atomic absorption spectrometry (FAAS) or UV-Vis spectrophotometry. Di-2-pyridyl ketone salicyloylhydrazone (DPKSH) was used for the first time as a complexing agent in CPE. The nickel complex was extracted from the aqueous phase using the Triton X-114 surfactant. Under optimized conditions, limits of detection obtained with GFAAS, FAAS and UV-Vis spectrophotometry were 0.14, 0.76 and 1.5 mu g L-1, respectively. The extraction was quantitative and the enrichment factor was estimated to be 27. The method was applied to natural waters, hemodialysis concentrates, urine and honey samples. Accuracy was evaluated by analysis of the NIST 1643e Water standard reference material.

Evaluation of reference-based two-color methods for measurement of gene expression ratios using spotted cDNA microarrays

Abstract Background Spotted cDNA microarrays generally employ co-hybridization of fluorescently-labeled RNA targets to produce gene expression ratios for subsequent analysis. Direct comparison of two RNA samples in the same microarray provides the highest level of accuracy; however, due to the number of combinatorial pair-wise comparisons, the direct method is impractical for studies including large number of individual samples (e.g., tumor classification studies). For such studies, indirect comparisons using a common reference standard have been the preferred method. Here we evaluated the precision and accuracy of reconstructed ratios from three indirect methods relative to ratios obtained from direct hybridizations, herein considered as the gold-standard. Results We performed hybridizations using a fixed amount of Cy3-labeled reference oligonucleotide (RefOligo) against distinct Cy5-labeled targets from prostate, breast and kidney tumor samples. Reconstructed ratios between all tissue pairs were derived from ratios between each tissue sample and RefOligo. Reconstructed ratios were compared to (i) ratios obtained in parallel from direct pair-wise hybridizations of tissue samples, and to (ii) reconstructed ratios derived from hybridization of each tissue against a reference RNA pool (RefPool). To evaluate the effect of the external references, reconstructed ratios were also calculated directly from intensity values of single-channel (One-Color) measurements derived from tissue sample data collected in the RefOligo experiments. We show that the average coefficient of variation of ratios between intra- and inter-slide replicates derived from RefOligo, RefPool and One-Color were similar and 2 to 4-fold higher than ratios obtained in direct hybridizations. Correlation coefficients calculated for all three tissue comparisons were also similar. In addition, the performance of all indirect methods in terms of their robustness to identify genes deemed as differentially expressed based on direct hybridizations, as well as false-positive and false-negative rates, were found to be comparable. Conclusion RefOligo produces ratios as precise and accurate as ratios reconstructed from a RNA pool, thus representing a reliable alternative in reference-based hybridization experiments. In addition, One-Color measurements alone can reconstruct expression ratios without loss in precision or accuracy. We conclude that both methods are adequate options in large-scale projects where the amount of a common reference RNA pool is usually restrictive.

RNA extraction from ten year old formalin-fixed paraffin-embedded breast cancer samples: a comparison of column purification and magnetic bead-based technologies

Abstract Background The development of protocols for RNA extraction from paraffin-embedded samples facilitates gene expression studies on archival samples with known clinical outcome. Older samples are particularly valuable because they are associated with longer clinical follow up. RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue is problematic due to chemical modifications and continued degradation over time. We compared quantity and quality of RNA extracted by four different protocols from 14 ten year old and 14 recently archived (three to ten months old) FFPE breast cancer tissues. Using three spin column purification-based protocols and one magnetic bead-based protocol, total RNA was extracted in triplicate, generating 336 RNA extraction experiments. RNA fragment size was assayed by reverse transcription-polymerase chain reaction (RT-PCR) for the housekeeping gene glucose-6-phosphate dehydrogenase (G6PD), testing primer sets designed to target RNA fragment sizes of 67 bp, 151 bp, and 242 bp. Results Biologically useful RNA (minimum RNA integrity number, RIN, 1.4) was extracted in at least one of three attempts of each protocol in 86–100% of older and 100% of recently archived ("months old") samples. Short RNA fragments up to 151 bp were assayable by RT-PCR for G6PD in all ten year old and months old tissues tested, but none of the ten year old and only 43% of months old samples showed amplification if the targeted fragment was 242 bp. Conclusion All protocols extracted RNA from ten year old FFPE samples with a minimum RIN of 1.4. Gene expression of G6PD could be measured in all samples, old and recent, using RT-PCR primers designed for RNA fragments up to 151 bp. RNA quality from ten year old FFPE samples was similar to that extracted from months old samples, but quantity and success rate were generally higher for the months old group. We preferred the magnetic bead-based protocol because of its speed and higher quantity of extracted RNA, although it produced similar quality RNA to other protocols. If a chosen protocol fails to extract biologically useful RNA from a given sample in a first attempt, another attempt and then another protocol should be tried before excluding the case from molecular analysis.

APPLICABILITY OF kDNA-PCR FOR ROUTINE DIAGNOSIS OF AMERICAN TEGUMENTARY LEISHMANIASIS IN A TERTIARY REFERENCE HOSPITAL.

This study evaluated the applicability of kDNA-PCR as a prospective routine diagnosis method for American tegumentary leishmaniasis (ATL) in patients from the Instituto de Infectologia Emílio Ribas (IIER), a reference center for infectious diseases in São Paulo - SP, Brazil. The kDNA-PCR method detected Leishmania DNA in 87.5% (112/128) of the clinically suspected ATL patients, while the traditional methods demonstrated the following percentages of positivity: 62.8% (49/78) for the Montenegro skin test, 61.8% (47/76) for direct investigation, and 19.3% (22/114) for in vitro culture. The molecular method was able to confirm the disease in samples considered negative or inconclusive by traditional laboratory methods, contributing to the final clinical diagnosis and therapy of ATL in this hospital. Thus, we strongly recommend the inclusion of kDNA-PCR amplification as an alternative diagnostic method for ATL, suggesting a new algorithm routine to be followed to help the diagnosis and treatment of ATL in IIER.