Reference gene and tropomyosin expression in mud crab Scylla olivacea,Scylla paramamosain and Scylla tranquebarica

Molecular Biology Reports - Tropomyosin, a muscle tissue protein is a major allergen in most of shellfish including mud crab. Quantitative real time-PCR (qRT-PCR) using a stable reference gene is...     

u-Genome: a database on genome design in unicellular genomes

Unicellular eukaryotes were among the first ones to be selected for complete genome sequencing because of the small size of their genomes and their interactions with humans and a broad range of animals and plants. Currently, ten completely sequenced unicellular genome sequences have been publicly released and as the number of available unicellular genomes increases, comparative genomics analysis within this group of organisms becomes more and more instructive. However, such an analysis is difficult to carry out without a suitable platform gathering not only the original annotations but also relevant information available in public databases or obtained by applying common bioinformatics methods. With the aim of solving these difficulties, we have developed a web-accessible database named u-Genome, the unicellular genome design database. The database is unique in featuring three datasets namely (1) orthologous proteins (2) paralogous proteins and (3) statistical distributions on exons, introns, intergenic DNA and correlations between them. A tool, Uniview, designed to visualize the gene structures for individual genes in the genome is also integrated. This database is of importance in understanding unicellular genome design and architecture and evolution related studies. The database is available through a web interface at http://sege.ntu.edu.sg/wester/ugenome.     

Characterization of the human gastric fluid proteome reveals distinct pH-dependent protein profiles: implications for biomarker studies

Gastric fluid is a source of gastric cancer biomarkers. However, very little is known about the normal gastric fluid proteome and its biological variations. In this study, we performed a comprehensive analysis of the human gastric fluid proteome using samples obtained from individuals with benign gastric conditions. Gastric fluid proteins were prefractionated using ultracentrifuge filters (3 kDa cutoff) and analyzed by two-dimensional gel electrophoresis (2-DE) and multidimensional LC-MS/MS. Our 2-DE analysis of 170 gastric fluid samples revealed distinct protein profiles for acidic and neutral samples, highlighting pH effects on protein composition. By 2D LC-MS/MS analysis of pooled samples, we identified 284 and 347 proteins in acidic and neutral samples respectively (FDR ≤1%), of which 265 proteins (72.4%) overlapped. However, unlike neutral samples, most proteins in acidic samples were identified from peptides in the filtrate (i.e., <3 kDa). Consistent with this finding, immunoblot analysis of six potential gastric cancer biomarkers rarely detected full-length proteins in acidic samples. These findings have important implications for biomarker studies because a majority of gastric cancer patients have neutral gastric fluid compared to noncancer controls. Consequently, sample stratification, choice of proteomic approaches, and validation strategy can profoundly affect the interpretation of biomarker findings. These observations should help to refine gastric fluid biomarker studies.     

Metabolic adaptation to a disruption in oxygen supply during myocardial ischemia and reperfusion is underpinned by temporal and quantitative changes in the cardiac proteome

Despite decades of intensive research, there is still no effective treatment for ischemia/reperfusion (I/R) injury, an important corollary in the treatment of ischemic disease. I/R injury is initiated when the altered biochemistry of cells after ischemia is no longer compatible with oxygenated microenvironment (or reperfusion). To better understand the molecular basis of this alteration and subsequent incompatibility, we assessed the temporal and quantitative alterations in the cardiac proteome of a mouse cardiac I/R model by an iTRAQ approach at 30 min of ischemia, and at 60 or 120 min reperfusion after the ischemia using sham-operated mouse heart as the baseline control. Of the 509 quantified proteins identified, 121 proteins exhibited significant changes (p-value<0.05) over time and were mostly clustered in eight functional groups: Fatty acid oxidation, Glycolysis, TCA cycle, ETC (electron transport chain), Redox Homeostasis, Glutathione S-transferase, Apoptosis related, and Heat Shock proteins. The first four groups are intimately involved in ATP production and the last four groups are known to be important in cellular antioxidant activity. During ischemia and reperfusion, the short supply of oxygen precipitates a pivotal metabolic switch from aerobic metabolism involving fatty acid oxidation, TCA, and phosphorylation to anaerobic metabolism for ATP production and this, in turn, increases reactive oxygen species (ROS) formation. Therefore the implication of these 8 functional groups suggested that ischemia-reperfusion injury is underpinned in part by proteomic alterations. Reversion of these alterations to preischemia levels took at least 60 min, suggesting a refractory period in which the ischemic cells cannot adjust to the presence of oxygen. Therefore, therapeutics that could compensate for these proteomic alterations during this interim refractory period could alleviate ischemia-reperfusion injury to enhance cellular recovery from an ischemic to a normoxic microenvironment. Among the perturbed proteins, Park7 and Ppia were selected for further investigation of their functions under hypoxia. The results show that Park7 plays a key role in regulating antioxidative stress and cell survival, and Ppia may function in coping with the unfolded protein stress in the I/R condition.     

Longitudinal Liver Stiffness Assessment in Patients with Chronic Hepatitis C Undergoing Antiviral Therapy

Background/Aims

Liver stiffness (LS) measurement by means of transient elastography (TE) is accurate to predict fibrosis stage. The effect of antiviral treatment and virologic response on LS was assessed and compared with untreated patients with chronic hepatitis C (CHC).

Methods

TE was performed at baseline, and at weeks 24, 48, and 72 in 515 patients with CHC.

Results

323 treated (62.7%) and 192 untreated patients (37.3%) were assessed. LS experienced a significant decline in treated patients and remained stable in untreated patients at the end of study (P<0.0001). The decline was significant for patients with baseline LS ≥ 7.1 kPa (P<0.0001 and P 0.03, for LS ≥9.5 and ≥7.1 kPa vs lower values, respectively). Sustained virological responders and relapsers had a significant LS improvement whereas a trend was observed in nonresponders (mean percent change −16%, −10% and −2%, for SVR, RR and NR, respectively, P 0.03 for SVR vs NR). In multivariate analysis, high baseline LS (P<0.0001) and ALT levels, antiviral therapy and non-1 genotype were independent predictors of LS improvement.

Conclusions

LS decreases during and after antiviral treatment in patients with CHC. The decrease is significant in sustained responders and relapsers (particularly in those with high baseline LS) and suggests an improvement in liver damage.     

Effects of Hydrogen Peroxide on Wound Healing in Mice in Relation to Oxidative Damage

It has been established that low concentrations of hydrogen peroxide (H₂O₂) are produced in wounds and is required for optimal healing. Yet at the same time, there is evidence that excessive oxidative damage is correlated with poor-healing wounds. In this paper, we seek to determine whether topical application of H₂O₂ can modulate wound healing and if its effects are related to oxidative damage. Using a C57BL/6 mice excision wound model, H₂O₂ was found to enhance angiogenesis and wound closure at 10 mM but retarded wound closure at 166 mM. The delay in closure was also associated with decreased connective tissue formation, increased MMP-8 and persistent neutrophil infiltration. Wounding was found to increase oxidative lipid damage, as measured by F₂-isoprostanes, and nitrative protein damage, as measured by 3-nitrotyrosine. However H₂O₂ treatment did not significantly increase oxidative and nitrative damage even at concentrations that delay wound healing. Hence the detrimental effects of H₂O₂ may not involve oxidative damage to the target molecules studied.     

C radiolabeling of proteins to monitor biodistribution of ingested proteins

The economical preparation of microgram quantities of ¹⁴C-labeled proteins by in vacuo methylation with methyl iodide is described. The ¹⁴C radiolabeling was achieved by the covalent attachment of [¹⁴C]methyl groups onto amino and imidazole groups by reaction in vacuo with [¹⁴C]methyl iodide. The method was tested by investigating the biodistribution of ¹⁴C in rats that were fed ¹⁴C-labeled human soluble cluster of differentiation 14 (CD14) protein, a receptor for bacterial lipopolysaccharide. Two other control proteins, bovine serum albumin (BSA) and casein, were also labeled with ¹⁴C and used for comparative analysis to determine the following: (i) the efficacy and cost efficiency of the in vacuo radiolabeling procedure and (ii) the extent of incorporation of the ¹⁴C label into the organs of orogastrically fed 10-day-old Sprague–Dawley rats. [¹⁴C]BSA, [¹⁴C]casein, and [¹⁴C]CD14 were individually prepared with specific radioactivities of 34,400, 18,800, and 163,000 disintegrations per minute (dpm)/μg, respectively. It was found that the accumulation of ¹⁴C label in the organs of [¹⁴C]CD14-fed rats, most notably the persistence of ¹⁴C in the stomach 480 min postgavage, was temporally and spatially distinct from [¹⁴C]BSA and [¹⁴C]casein-fed rats.     

Identifying protein complexes from heterogeneous biological data

With the increasing availability of diverse biological information for proteins, integration of heterogeneous data becomes more useful for many problems in proteomics, such as annotating protein functions, predicting novel protein–protein interactions and so on. In this paper, we present an integrative approach called InteHC (Inte grative H ierarchical C lustering) to identify protein complexes from multiple data sources. Although integrating multiple sources could effectively improve the coverage of current insufficient protein interactome (the false negative issue), it could also introduce potential false‐positive interactions that could hurt the performance of protein complex prediction. Our proposed InteHC method can effectively address these issues to facilitate accurate protein complex prediction and it is summarized into the following three steps. First, for each individual source/feature, InteHC computes the matrices to store the affinity scores between a protein pair that indicate their propensity to interact or co‐complex relationship. Second, InteHC computes a final score matrix, which is the weighted sum of affinity scores from individual sources. In particular, the weights indicating the reliability of individual sources are learned from a supervised model (i.e., a linear ranking SVM). Finally, a hierarchical clustering algorithm is performed on the final score matrix to generate clusters as predicted protein complexes. In our experiments, we compared the results collected by our hierarchical clustering on each individual feature with those predicted by InteHC on the combined matrix. We observed that integration of heterogeneous data significantly benefits the identification of protein complexes. Moreover, a comprehensive comparison demonstrates that InteHC performs much better than 14 state‐of‐the‐art approaches. All the experimental data and results can be downloaded from http://www.ntu.edu.sg/home/zhengjie/data/InteHC . Proteins 2013; 81:2023–2033. © 2013 Wiley Periodicals, Inc.     

A Quantitative,Comparative Study of Element Variations Found within the Range of Blood Cells from the Tropical Ascidian Phallusia philippinensis,using the Nuclear Microscope

The present study examines the concentrations of vanadium, bromine and sulphur contained within cryofixed/freeze-dried blood cells of the ascidian Phallusia philippinensis. Elemental profiles of seven cell types were obtained using the National University of Singapore nuclear microscope, in order to ascertain the cell types predominantly involved in accumulation. Morula cells were found to contain the following mean values (in ppm dry weight); 7878 vanadium, 34484 bromine and 61078 sulphur. Signet ring cells contained 5191 vanadium, 23945 bromine and 15281 sulphur. Compartment cells had 606 vanadium, 20700 bromine and 24309 sulphur. Other less abundant cell types such as lymphocytes, macrogranular amoebocytes, carotenoid pigment cells and granular amoebocytes were also analysed and found to contain (in ppm) 4384, 6652, 2366 and 10246 vanadium, 19652, 15630, 5964 and 11735 bromine and 13289, 15309, 3106 and 42968 sulphur, respectively. Sulphur occurred in high levels in all cell types, which could indicate its involvement in the vanadium concentration process, while bromine, incorporated into complexes, may be utilised for anti-fouling rather than as a deterrent to predators.