Electrochemical Quantification of D-Glucose during the Production of Bioethanol from Thermo-Mechanically Pre-treated Wheat Straw

Mechanical pre-treatment (disc refining) of wheat straw, at both atmospheric and elevated pressure, is shown to be an efficient process to access fermentable monosaccharides, with the potential to integrate within the infrastructure of existing first-generation bioethanol plants. The mild, enzymatic degradation of this sustainable lignocellulosic biomass affords ca. 0.10-0.13 g/g (dry weight) of D-glucose quantifiable voltammetrically in real time, over a two hundred-fold range in experimental laboratory scales (25 mL to 5.0 L), with pressure disc refining of the wheat straw enabling almost twice the amount of D-glucose to be generated during the hydrolysis stage than experiments using atmospheric refining (0.06 – 0.09 g/g dry weight). Fermentation of the resulting hydrolysate affords 0.08 – 0.10 g/g (dry weight) of ethanol over similar scales, with ethanol productivity at ca. 37 mg/(L h). These results demonstrate that minimal cellulose decomposition occurs during pressure refining of wheat straw, in contrast to hemicellulose, and suggest that the development of green, mechanochemical processes for the scalable and cost-effective manufacture of second-generation bioethanol requires improved cellulose decomposition

Transcriptome analysis of grain development in hexaploid wheat

Background: Hexaploid wheat is one of the most important cereal crops for human nutrition. Molecular understanding of the biology of the developing grain will assist the improvement of yield and quality traits for different environments. High quality transcriptomics is a powerful method to increase this understanding. Results: The transcriptome of developing caryopses from hexaploid wheat ( Triticum aestivum, cv. Hereward) was determined using Affymetrix wheat GeneChip (R) oligonucleotide arrays which have probes for 55,052 transcripts. Of these, 14,550 showed significant differential regulation in the period between 6 and 42 days after anthesis ( daa). Large changes in transcript abundance were observed which were categorised into distinct phases of differentiation ( 6 - 10 daa), grain fill ( 12 - 21 daa) and desiccation/maturation ( 28 - 42 daa) and were associated with specific tissues and processes. A similar experiment on developing caryopses grown with dry and/or hot environmental treatments was also analysed, using the profiles established in the first experiment to show that most environmental treatment effects on transcription were due to acceleration of development, but that a few transcripts were specifically affected. Transcript abundance profiles in both experiments for nine selected known and putative wheat transcription factors were independently confirmed by real time RT-PCR. These expression profiles confirm or extend our knowledge of the roles of the known transcription factors and suggest roles for the unknown ones. Conclusion: This transcriptome data will provide a valuable resource for molecular studies on wheat grain. It has been demonstrated how it can be used to distinguish general developmental shifts from specific effects of treatments on gene expression and to diagnose the probable tissue specificity and role of transcription factors

Reply to “Analysis of NO and its metabolites by mass spectrometry. Comment on ‘Detection of nitric oxide in tissue samples by ESI-MS’”

Comment by Tsikas et al. (Analyst, 2011, DOI: 10.1039/c0an00411a) on the preliminary work by Shen and colleagues (Analyst, 2010, 135, 302) describing the use of ESI-MS/MS for the detection of methylpiperazinobenzenediamine, as a probe for NO in tissue samples, have been addressed in this communication. The article concludes that the points raised by Tsikas represent a logical progression to the research reported and indicate the way forward in realising appropriate analytical methodology

Toward a microfluidic system for proteomics

There has been much interest in the application of microfluidic systems to proteomics in recent years. This is because of the many unique advantages afforded by the reduced dimensions of microfluidic systems compared to classical methods. The reduction in the reaction vessel dimensions leads to a high degree of control, higher sensitivity, better selectivity and reduced analysis time. Initial experimental results have shown the possibility for developing a novel approach for proteomic analysis. A highly efficient protein digestion microdevice was fabricated using commercially available immobilized trypsin on agarose beads, packed into a silica capillary and connected directly to an electrospray mass spectrometer via a ‘microtight T’ connector, from which aqueous acetic acid (0.2 %) was pumped to the mass spectrometer ion source. Six proteins with molecular masses ranging from 2846 to 77703 Da were digested separately, each within eight minutes using this system. In a second set of experiments a short monolithic separation column was fabricated and placed after the immobilized trypsin capillary. This system demonstrated partial separation of the tryptic peptides generated, and detection limits in the pmol range were obtained by utilization of this separation column. The methodology was then transferred to a glass microchip and a novel system was fabricated. The system consists of an on-chip protein digestion channel and on-chip monolithic ion exchange separation column. The digestion system performance was evaluated using single proteins and mixtures of protein. Significant reduction in the digestion time was observed in comparison to the traditional digestion method and a complete digestion was obtained for cytochrome C in less than a minute while a protein mixture was digested within five minutes. The on-chip separation column was initially evaluated using the tryptic digest of cytochrome C. High column efficiency was obtained as indicated from the peak width at half height measurement data (varying between 19.0 to 24.9 s). A detection limit of 3.7 pmol was obtained; lower detection limits may be obtained if a nano-electrospray source were to be used. The separation column was also evaluated by separating a tryptic digest of a mixture of four proteins. Results showed at least 40 % improvement in the sequence coverage due to the separation achieved. In another experiment, the system was successfully used for protein digestion and a first dimension separation followed by an off-line second dimension separation. This was demonstrated using a tryptic digest of BSA and a number of additional peptides were identified as a result of the first dimension separation carried out using the microfluidic system. The system was also tested with a biological sample and initial results gave positive indications; however, this has to be confirmed after optimization of the protein extraction method. The developed system can be used along with the commercially available on-chip reverse phase columns to perform on-chip protein digestion and two dimensional separation of the generated peptides in a high throughput system for the analysis of biological samples. Another novel microfluidic system was also fabricated. The system consists of an on-chip protein digestion channel and on-chip monolithic reverse phase separation column. The microfluidic system was evaluated using cytochrome C, BSA and a mixture of four proteins. The separation and digestion were completed within one hour. However, the column performance was lower than that of the on-chip monolithic ion exchange column previously fabricated as indicated from the peak width at half height measurement data (varying between 30.0 to 115.0 s).EThOS - Electronic Theses Online ServiceJami'at al-Sultan Qabus (Sponsor)GBUnited Kingdo

Proteomic analysis of B-cell receptor signaling in chronic lymphocytic leukaemia reveals a possible role for kininogen

CLL is an incurable disease with variable prognosis. The hyper reactivity of the B-cell receptor (BCR) to unknown antigen ligation plays a pivotal role in CLL-cell survival. We aimed to investigate the BCR signalling pathway using proteomics to identify novel proteins which may have clinical relevance in this disease.Three CLL samples were selected based upon BCR responsiveness, demonstrated by upregulation of phospho-ERK following in vitro stimulation. The differential expression of proteins, upon artificial stimulation of the BCR, was examined in these samples using two-dimensional gel electrophoresis in combination with mass spectrometry. Proteins of interest were subsequently examined using immunoblotting. Proteomic analysis revealed that kininogen, a critical protein of kinin-kallikrein system, was upregulated in all 3 clinical samples upon BCR stimulation. There are 2 forms of kininogen: HMWK and LMWK. The upregulation of LMWK upon BCR stimulation was confirmed by immunoblotting in all 3 of these samples. In a pilot series of 52 unselected CLL samples, 71% demonstrated basal LMWK expression. There was a trend towards shorter median survival in LMWK positive cases (147. months versus 253. months for LMWK negative cases; p=. 0.125). Kininogen may be a novel therapeutic target in CLL and the possible association with prognosis warrants further investigation. Biological significance: We have identified the upregulation of LMWK upon BCR stimulation of CLL samples. There is no previous published research to suggest a link between kininogen and normal B-cells or CLL cells. In 52 unselected CLL samples, 71% demonstrated basal LMWK expression. There was a trend towards shorter median survival in LMWK positive cases. The absence of LMWK protein expression on normal B-cells suggests that this could be a biomarker for CLL and further research should be undertaken. © 2013 Elsevier B.V

Detection of nitric oxide in tissue samples by ESI-MS

A novel method to determine nitric oxide (NO) in biological tissue samples with minimal interference from the cellular detritus is described. Methylpiperazinobenzenediamine, consisting of an o-phenylenediamine and a methyl piperazine group, was chosen as a probe for the detection of NO by mass spectrometry (MS) in biological tissue samples. The o-phenylenediamine group reacts with NO to form a characteristic benzotriazole. The product was identified using electrospray ionization mass spectrometry (ESI-MS) and the method validated within the range of 95-1900 nM. NO levels associated with tissue biopsies (similar to 10 mg) from rat vasculature and intestine tissue biopsies have been successfully determined. The different rates of NO generated from tissue samples under hypoxic and normoxic conditions have been studied by this simple and sensitive method

Proteomic Identification of Putative Biomarkers of Radiotherapy Resistance: A Possible Role for the 26S Proteasome?1

PURPOSE: We aimed to identify putative predictive protein biomarkers of radioresistance. EXPERIMENTAL DESIGN: Three breast cancer cell lines (MCF7, MDA-MB-231, and T47D) were used as in vitro models to study radioresistance. Inherent radiosensitivities were examined using a clonogenic survival assay. It was revealed that each cell line differed in their response to radiotherapy. These parental breast cancer cell lines were used to establish novel derivatives (MCF7RR, MDA-MB-231RR, and T47DRR) displaying significant resistance to ionizing radiation. Derivative cells were compared with parental cells to identify putative biomarkers associated with the radioresistant phenotype. To identify these biomarkers, complementary proteomic screening approaches were exploited encompassing two-dimensional gel electrophoresis in combination with mass spectrometry, liquid chromatography coupled with tandem mass spectrometry and quantitative proteomics using iTRAQ technology. RESULTS: A large number of potential biomarkers were identified, and several of these were confirmed using Western blot analysis. In particular, a decrease in the expression of the 26S proteasome was found in all radioresistant derivatives when compared with the respective parent cells. Decreased expression of this target was also found to be associated with radioresistant laryngeal tumors (P = .05) in a small pilot immunohistochemical study. CONCLUSIONS: These findings suggest that the 26S proteasome may provide a general predictive biomarker for radiotherapy outcome