The cytochrome P450s of Leptographium qinlingensis: Gene characteristics, phylogeny, and expression in response to terpenoids.

Leptographium qinlingensis is one of the major fungal associates of the Chinese white pine beetle (Dendroctonus armandi) and is an active participant in the large-scale death of Pinus armandi. Beetles and associated fungi have evolved efficient systems for overcoming the toxicity of host defense chemicals, which consist of a multitude of monoterpenes and diterpenes. As fungal cytochromes (CYPs) can detoxify and degrade various xenobiotic compounds, we identified 11 CYPs from L. qinlingensis to analyze their potential function in detoxifying or degrading host chemical defense. These 11 CYPs of L. qinlingensis belong to 6 clans and cluster into 3 clades, clade 2, clade 8 and clade 10, based on their phylogenetic relationships. Moreover, we also analyzed the transcript levels of CYPs following treatment with terpenes or expression in mycelia grown on terpenoids as a carbon source. The results in this paper showed that several CYPs were upregulated after treatment with terpenes or growth on terpenoids as the only carbon source. Our research provides some insight into the function of CYPs from bark beetle symbiotic fungi in the detoxification of pine defense compounds or their relationships with the utilization of terpenoids.

Receiver-Side TCP Countermeasure in Cellular Networks.

Cellular-based networks keep large buffers at base stations to smooth out the bursty data traffic, which has a negative impact on the user's Quality of Experience (QoE). With the boom of smart vehicles and phones, this has drawn growing attention. For this paper, we first conducted experiments to reveal the large delays, thus long flow completion time (FCT), caused by the large buffer in the cellular networks. Then, a receiver-side transmission control protocol (TCP) countermeasure named Delay-based Flow Control algorithm with Service Differentiation (DFCSD) was proposed to target interactive applications requiring high throughput and low delay in cellular networks by limiting the standing queue size and decreasing the amount of packets that are dropped in the eNodeB in Long Term Evolution (LTE). DFCSD stems from delay-based congestion control algorithms but works at the receiver side to avoid the performance degradation of the delay-based algorithms when competing with loss-based mechanisms. In addition, it is derived based on the TCP fluid model to maximize the network utility. Furthermore, DFCSD also takes service differentiation into consideration based on the size of competing flows to shorten their completion time, thus improving user QoE. Simulation results confirmed that DFCSD is compatible with existing TCP algorithms, significantly reduces the latency of TCP flows, and increases network throughput.

Proteomic analysis of circulating monocytes in Chinese premenopausal females with extremely discordant bone mineral density.

Osteoporosis (OP) is a major public health problem, mainly characterized by low bone mineral density (BMD). Circulating monocytes (CMCs) may serve as progenitors of osteoclasts and produce a wide variety of factors important to bone metabolism. However, the specific action mechanism of CMCs in the pathogenesis of OP is far from clear. We performed a comparative protein expression profiling study of CMCs in Chinese premenopausal females with extremely discordant BMD, identified a total of 38 differentially expressed proteins, and confirmed with Western blotting five proteins: ras suppressor protein1 (RSU1), gelsolin (GSN), manganese-containing superoxide dismutase (SOD2), glutathione peroxidase 1(GPX1), and prolyl 4-hydroxylase beta subunit (P4HB). These proteins might affect CMCs' trans-endothelium, differentiation, and/or downstream osteoclast functions, thus contribute to differential osteoclastogenesis and finally lead to BMD variation. The findings promote our understanding of the role of CMCs in BMD determination, and provide an insight into the pathogenesis of human OP.

How is mRNA expression predictive for protein expression? A correlation study on human circulating monocytes.

A key assumption in studying mRNA expression is that it is informative in the prediction of protein expression. However, only limited studies have explored the mRNA-protein expression correlation in yeast or human tissues and the results have been relatively inconsistent. We carried out correlation analyses on mRNA-protein expressions in freshly isolated human circulating monocytes from 30 unrelated women. The expressed proteins for 71 genes were quantified and identified by 2-D electrophoresis coupled with mass spectrometry. The corresponding mRNA expressions were quantified by Affymetrix gene chips. Significant correlation (r=0.235, P<0.0001) was observed for the whole dataset including all studied genes and all samples. The correlations varied in different biological categories of gene ontology. For example, the highest correlation was achieved for genes of the extracellular region in terms of cellular component (r=0.643, P<0.0001) and the lowest correlation was obtained for genes of regulation (r=0.099, P=0.213) in terms of biological process. In the genome, half of the samples showed significant positive correlation for the 71 genes and significant correlation was found between the average mRNA and the average protein expression levels in all samples (r=0.296, P<0.01). However, at the study group level, only five studied genes had significant positive correlation across all the samples. Our results showed an overall positive correlation between mRNA and protein expression levels. However, the moderate and varied correlations suggest that mRNA expression might be sometimes useful, but certainly far from perfect, in predicting protein expression levels.

High-throughput analysis of rat liver plasma membrane proteome by a nonelectrophoretic in-gel tryptic digestion coupled with mass spectrometry identification.

In-gel digestion is commonly used after proteins are resolved by polyacrylamide gel electrophoresis (SDS-PAGE, 2-DE). It can also be used on its own in conjunction with tandem mass spectrometry (MS/MS) for the direct analysis of complex proteins. Here, we describe a strategy combining isolation of purified plasma membrane, efficient digestion of plasma membrane proteins in polyacrylamide gel, and high-sensitivity analysis by advanced mass spectrometry to create a new rapid and high-throughput method. The plasma membrane protein mixture is directly incorporated into a polyacrylamide gel matrix, After formation of the gel, proteins in the gel section are digested with trypsin, and the resulting peptides are subjected to reversed-phase, high-performance liquid chromatography followed by electrospray ion-trap tandem mass analysis. Using this optimized strategy, we have identified 883 rat liver membrane proteins, of which 490 had a gene ontology (GO) annotation indicating a cellular component, and 294 (60%) of the latter were known integral membrane proteins or membrane proteins. In total, 333 proteins are predicted by the TMHMM 2.0 algorithm to have transmembrane domains (TMDs) and 52% (175 of 333) proteins to contain 2-16 TMDs. The identified membrane proteins provide a broad representation of the rat plasma membrane proteome with little bias evident due to protein p I and molecular weight (MW). Also, membrane proteins with a high GRAVY score (grand average hydrophobicity score) were identified, and basic and acidic membrane proteins were evenly represented. This study not only offered an efficient and powerful method in shotgun proteomics for the identification of proteins of complex plasma membrane samples but also allowed in-depth study of liver membrane proteomes, such as of rat models of liver-related disease. This work represents one of the most comprehensive proteomic analyses of the membrane subproteome of rat liver plasma membrane in general.

Integration of a two-phase partition method into proteomics research on rat liver plasma membrane proteins.

To comprehensively identify proteins of the rat liver plasma membrane (PM), we have adopted a proteomics strategy that utilizes sucrose density centrifugation in conjunction with aqueous two-phase partition for plasma membrane isolation, followed by SDS-PAGE, mass spectrometry and bioinformatics. Western blot analysis showed that this method results in highly purified plasma membrane fractions, which is a key to successful plasma membrane proteomics. The PM proteins were separated by SDS-PAGE and digested with trypsin. Through nano-ESI-LC MS/MS analysis we identified 428 rat liver membrane proteins, of which 304 had a gene ontology (GO) annotation indicating a cellular component, and 204 (67%) of the latter were known integral membrane proteins or membrane-associated proteins. In addition to proteins known to be associated with the plasma membrane, several hypothetical proteins have also been identified. This study not only provides a tool to study plasma membrane proteins with low levels of contamination, but also provides a data set for proteins of high to moderate abundance in rat liver plasma membranes, thus allowing for more comprehensive characterization of membrane proteins and a better understanding of membrane dynamics.

Comparative proteomics analysis of human lung squamous carcinoma.

Two-dimensional polyacrylamide gel electrophoresis (2-DE) profiles of human lung squamous carcinoma tissue and paired surrounding normal bronchial epithelial tissue were compared. Selected differential protein-spots were identified with peptide mass fingerprinting based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database searching. Well-resolved and reproducible 2-DE patterns of both the tumor and the normal tissues were acquired. The average deviations of spot position were 0.873+/-0.125mm in IEF direction and 1.025+/-0.213mm in SDS-PAGE direction, respectively. For the tumor tissues, a total of 1349+/-67 spots were detected and 1235+/-48 spots were matched with an average matching rate of 91.5%. For the corresponding normal tissues, a total of 1297+/-73 spots were detected and 1183+/-56 spots were matched with an average matching rate of 91.2%. A total of 1069+/-45 spots were matched between the tumor and the normal tissues. Forty differential proteins between tumor and normal tissues were characterized. Some proteins were the products of oncogenes and others were involved in the regulation of cell cycle and signal transduction. These data are valuable for mass identification of differentially expressed proteins involved in lung carcinogenesis, establishing human lung cancer proteome database and screening molecular marker to further study human lung squamous carcinoma.

Proteomic comparison of two-dimensional gel electrophoresis profiles from human lung squamous carcinoma and normal bronchial epithelial tissues.

Differential proteome profiles of human lung squamous carcinoma tissue compared to paired tumor-adjacent normal bronchial epithelial tissue were established and analyzed by means of immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). The results showed that well-resolved, reproducible 2-DE patterns of human lung squamous carcinoma and adjacent normal bronchial epithelial tissues were obtained under the condition of 0.75-mg protein-load. The average deviation of spot position was 0.733+/-0.101 mm in IEF direction, and 0.925+/-0.207 mm in SDS-PAGE direction. For tumor tissue, a total of 1241+/-88 spots were detected, 987+/-65 spots were matched with an average matching rate of 79.5%. For control, a total of 1190+/-72 spots were detected, and 875+/-48 spots were matched with an average matching rate of 73.5%. A total of 864+/-34 spots were matched between tumors and controls. Forty-three differential proteins were characterized: some proteins were related to oncogenes, and others involved in the regulation of cell cycle and signal transduction. It is suggested that the differential proteomic approach is valuable for mass identification of differentially expressed proteins involved in lung carcinogenesis. These data will be used to establish human lung cancer proteome database to further study human lung squamous carcinoma.

[Differential proteomic analysis of human lung adenocarcinoma cell line A-549 and of normal cell line HBE].

To explore the differential proteomic expressions between human lung adenocarcinoma cell line A-549 and normal cell line HBE, a series of methods, including immobilized pH gradient-two dimensional polyacrylamide gel electrophoresis, silver staining, PDQuest 2-DE software analysis, peptide mass fingerprinting based on matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and SWISS-PROT database searching, were used to separate and identify the differential proteomic expressions between A-549 and HBE. The results showed that the good 2-DE pattern including high resolution and reproducibility was obtained. After silver staining, the 2-DE image analysis by PDQuest 2-DE software detected average (890 +/- 38) spots in A-549, and (757 +/- 27) spots in HBE. The average positional deviation of the matched spots between A-549 and HBE 2-DE maps was (2.85 +/- 0.48) mm in IEF direction, and (2.69 +/- 0.37) mm in SDS-PAGE direction. The differential proteomic expression analysis found that there were 535 matched spots between A-549 and HBE 2-DE maps, 355 spots that were not matched in A-549, 222 spots that were not matched in HBE. 18 differential spots (8 spots in A-549 and 10 spots in HBE) were cut off from silver staining gel at random, digested in gel with TPCK-trypsin, measured with MALDI-TOF-MS and searched in the SWISS-PROT database with PeptIdent software. 18 protein were preliminarily identified. These proteins were related to cell signal transduction, cell metabolism, proliferation and differentiation etc. There was a significant difference at protein level between human lung adenocarcinoma cell line A-549 and normal cell line HBE. It suggests that the differential expression analysis of proteomes may be useful to further study of the related proteins and the molecular markers of lung adenocarcinoma.

Identification of Protein Spots in Silver-stained Two-dimensional Gels by MALDI-TOF Mass Peptide Map Analysis.

Protein spots in silver-stained two dimensional gels were analyzed and identified by employing an improved procedure of mass spectrometric peptide mapping, including i) In-gel reduction, alkylation and enzymatic digestion ii) Extraction and desalting by using a pipette tip containing a small C18 micro-column (ZipTip(TM)) iii) Direct MALDI-TOF mass analysis and protein database searching. The results demonstrate that single silver-stained protein spots in a 2-DE-gel could be identified rapidly by this procedure and the use of the ZipTip(TM) pipette tip could increase evidently the sensitivity of the MALDI-TOF analysis. By using this the procedure, 10 protein spots in a silver-stained gel of 2-DE of crude venom of the spider S.Huwena were analyzed and identified.