Ravi C. Dwivedi

Proteomic analysis of Clostridium thermocellum core metabolism: relative protein expression profiles and growth phase-dependent changes in protein expression.

BackgroundClostridium thermocellum produces H2 and ethanol, as well as CO2, acetate, formate, and lactate, directly from cellulosic biomass. It is therefore an attractive model for biofuel production via consolidated bioprocessing. Optimization of end-product yields and titres is crucial for making biofuel production economically feasible. Relative protein expression profiles may provide targets for metabolic engineering, while understanding changes in protein expression and metabolism in response to carbon limitation, pH, and growth phase may aid in reactor optimization. We performed shotgun 2D-HPLC-MS/MS on closed-batch cellobiose-grown exponential phase C. thermocellum cell-free extracts to determine relative protein expression profiles of core metabolic proteins involved carbohydrate utilization, energy conservation, and end-product synthesis. iTRAQ (isobaric tag for relative and absolute quantitation) based protein quantitation was used to determine changes in core metabolic proteins in response to growth phase.ResultsRelative abundance profiles revealed differential levels of putative enzymes capable of catalyzing parallel pathways. The majority of proteins involved in pyruvate catabolism and end-product synthesis were detected with high abundance, with the exception of aldehyde dehydrogenase, ferredoxin-dependent Ech-type [NiFe]-hydrogenase, and RNF-type NADH:ferredoxin oxidoreductase. Using 4-plex 2D-HPLC-MS/MS, 24% of the 144 core metabolism proteins detected demonstrated moderate changes in expression during transition from exponential to stationary phase. Notably, proteins involved in pyruvate synthesis decreased in stationary phase, whereas proteins involved in glycogen metabolism, pyruvate catabolism, and end-product synthesis increased in stationary phase. Several proteins that may directly dictate end-product synthesis patterns, including pyruvate:ferredoxin oxidoreductases, alcohol dehydrogenases, and a putative bifurcating hydrogenase, demonstrated differential expression during transition from exponential to stationary phase.ConclusionsRelative expression profiles demonstrate which proteins are likely utilized in carbohydrate utilization and end-product synthesis and suggest that H2 synthesis occurs via bifurcating hydrogenases while ethanol synthesis is predominantly catalyzed by a bifunctional aldehyde/alcohol dehydrogenase. Differences in expression profiles of core metabolic proteins in response to growth phase may dictate carbon and electron flux towards energy storage compounds and end-products. Combined knowledge of relative protein expression levels and their changes in response to physiological conditions may aid in targeted metabolic engineering strategies and optimization of fermentation conditions for improvement of biofuels production.

Assessment of the reproducibility of random hexapeptide peptide library-based protein normalization.

The wide dynamic range of proteins in biological samples poses a challenge for the detection of low-abundance proteins. Recently, combinatorial hexapeptide peptide libraries have been suggested as an approach to normalization of proteins in such mixtures. We examined the reproducibility of a commercial hexapeptide ligand library for quantitative and comparative serum proteomic analysis. We also compared this technology with IgY-based affinity depletion.

The effects of infliximab therapy on the serum proteome of rheumatoid arthritis patients

IntroductionAlthough the clinical effects of infliximab therapy in rheumatoid arthritis have been documented extensively, the biological effects of this intervention continue to be defined. We sought to examine the impact of infliximab therapy on the serum proteome of rheumatoid arthritis patients by means of a mass spectrometry-based approach.MethodsSera from 10 patients with rheumatoid arthritis were obtained prior to and following 12 weeks of infliximab therapy using a standard clinical protocol. The sera were immunodepleted of the 12 highest abundance proteins, labeled by the iTRAQ (isobaric tagging for relative and absolute protein quantification) technique, and analyzed by mass spectrometry to identify proteomic changes associated with treatment.ResultsAn average of 373 distinct proteins were identified per patient with greater than 95% confidence. In the 3 patients demonstrating the most robust clinical responses, changes of greater than 20% in the serum levels were observed in 39 proteins following treatment. The majority of these proteins were regulated directly or indirectly by tumour necrosis factor-alpha (TNF-α) and nuclear factor-kappa-B, with acute-phase proteins being uniformly down-regulated. A number of proteins, including members of the SERPIN family and S100A8, were down-regulated irrespective of clinical response.ConclusionsThe present study demonstrates that a robust clinical response to infliximab is associated with the down-regulation of a spectrum of serum proteins regulated by TNF-α, and provides a possible basis for defining the broader biological effects of the treatment in vivo.

Mitochondrial dysfunction resulting from the absence of mitochondrial porin in Neurospora crassa

Porin, the voltage-dependent anion-selective channel (VDAC) in the mitochondrial outer membrane, contributes to metabolism and apoptosis. VDAC function was investigated in Neurospora, an obligate aerobe with a single porin. Porinless strains are viable, with cold-sensitive growth, cytochrome deficiencies and overexpression of alternative oxidase. iTRAQ labeling of mitochondria from a porinless strain and its progenitor revealed a small group of proteins with altered expression levels in the mutant organelles. Porinless Neurospora appears to compensate not by inducing alternative pores, but by altering electron flow and nucleotide metabolism. Transcriptional and post-transcriptional mechanisms contribute to the response, reflecting the extent of porin influence.

Systematic review of carcinoma arising in pharyngeal diverticula: A 112-year analysis

Carcinoma is a rare complication of pharyngeal diverticula. There is a paucity of information about its incidence, presentation, management, and treatment outcomes. A systematic review and analysis of all reported cases has been carried out.

Should the treatment paradigms for oral and oropharyngeal cancers be changed now: the role of human papilloma virus?

all situations, but they are recommended for trainees in the listed specialties. The purchase of this piece of equipment needs to follow careful consideration and discussion with senior surgeons. Price is an issue, but it is tax deductible in Australia and in the UK. The starting point is 2.5¥ to 3.5¥ magnification, on compound or prismatic lenses of appropriate working distance, using headlamps where necessary (i.e. compound lenses, magnification above 4.0¥). Benefits will show immediately in trauma management, but comfort in the operating room will only be achieved after a relatively short learning curve. Once the surgeon is comfortable, careful maintenance will lead to a long working life of this piece of equipment.

Low-cost dual-action aural foreign-body extractor

Adequate visualization, appropriate equipment, a cooperative patient, and a skilled physician are keys to successful foreign body removal. The first attempt at removal is critical because success rates markedly decrease after the first failed attempt. Laryngoscope, 119:351–354, 2009

Reversed-Phase HPLC of Peptides – A Valuable Sample Preparation Tool in Bottom-Up Proteomics: Separation Selectivity in Single and Multi-dimensional Separation Modes

Reversed-phase HPLC is a leading tool for peptide fractionation in proteomics. Various combinations of stationary and mobile phases are employed depending on the purpose of the separation and the detection procedure. This diversity results in significant changes in separation selectivity, which are currently not well understood. Our ongoing work, focused on collection of peptide retention data and the development of sequence specific algorithms for peptide retention prediction, reveals these differences and helps define sets of conditions where predictive models can be considered independent from chromatographic platform used. We describe our observations on peptide separation selectivity variations for a wide range of RP-HPLC conditions: C18 sorbents of different pore sizes, using trifuoroacetic/formic/heptafluorobutyric acids as ion-pairing modifiers, RP separation at pH 10, and RP separation with an alternative perfluorinated chemistry of the bonded phase. Both the charge distribution within the peptide chain and the ion-pair formation mechanisms were found to be major factors in determining the peptide separation selectivity in RP-HPLC.

Activity-Based Protein Profiling of Intraoperative Serine Hydrolase Activities during Cardiac Surgery

The processes involved in the initiation of acute kidney injury (AKI) following cardiopulmonary bypass (CPB) are thought to occur during the intraoperative period. Such a rapid development might indicate that some of the inductive events are not dependent on de novo protein synthesis, raising the possibility that changes in activities of pre-existing enzymes could contribute to the development of AKI. Activity-based protein profiling (ABPP) was used to compare the serine hydrolase enzyme activities present in the urines of CPB patients who subsequently developed AKI versus those who did not (non-AKI) during the intra- and immediate postoperative periods. Sequential urines collected from a nested case-control cohort of AKI and non-AKI patients were reacted with a serine hydrolase activity probe, fluorophosphonate-TAMRA, and separated by SDS-PAGE. The patterns and levels of probe-labeled proteins in the two groups were initially comparable. However, within 1 h of CPB there were significant pattern changes in the AKI group. Affinity purification and mass spectrometry-based analysis of probe-labeled enzymes in AKI urines at 1 h CPB and arrival to the intensive care unit (ICU) identified 28 enzymes. Quantitative analysis of the activity of one of the identified enzymes, kallikrein-1, revealed some trends suggesting differences in the levels and temporal patterns of enzyme activity between a subset of patients who developed AKI and those who did not. A comparative analysis of affinity-purified probe reacted urinary proteins from these patient groups during the intraoperative period suggested the presence of both shared and unique enzyme patterns. These results indicate that there are intraoperative changes in the levels and types of serine hydrolase activities in patients who subsequently develop AKI. However, the role of these activity differences in the development of AKI remains to be determined.

Development of an SRM method for absolute quantitation of MYDGF/C19orf10 protein.

To develop a MS‐based selected reaction monitoring (SRM) assay for quantitation of myeloid‐derived growth factor (MYDGF) formerly chromosome 19 open reading frame (C19orf10).