AYUMS: an algorithm for completely automatic quantitation based on LC-MS/MS proteome data and its application to the analysis of signal transduction

BACKGROUND: Comprehensive description of the behavior of cellular components in a quantitative manner is essential for systematic understanding of biological events. Recent LC-MS/MS (tandem mass spectrometry coupled with liquid chromatography) technology, in combination with the SILAC (Stable Isotope Labeling by Amino acids in Cell culture) method, has enabled us to make relative quantitation at the proteome level. The recent report by Blagoev et al. (Nat. Biotechnol., 22, 1139–1145, 2004) indicated that this method was also applicable for the time-course analysis of cellular signaling events. Relative quatitation can easily be performed by calculating the ratio of peak intensities corresponding to differentially labeled peptides in the MS spectrum. As currently available software requires some GUI applications and is time-consuming, it is not suitable for processing large-scale proteome data. RESULTS: To resolve this difficulty, we developed an algorithm that automatically detects the peaks in each spectrum. Using this algorithm, we developed a software tool named AYUMS that automatically identifies the peaks corresponding to differentially labeled peptides, compares these peaks, calculates each of the peak ratios in mixed samples, and integrates them into one data sheet. This software has enabled us to dramatically save time for generation of the final report. CONCLUSION: AYUMS is a useful software tool for comprehensive quantitation of the proteome data generated by LC-MS/MS analysis. This software was developed using Java and runs on Linux, Windows, and Mac OS X. Please contact [email protected] if you are interested in the application. The project web page is

Fluid shear stress modulation of hepatocyte like cell function

Freshly isolated human adult hepatocytes are considered to be the gold standard tool for in vitro studies. However, primary hepatocyte scarcity, cell cycle arrest and the rapid loss of cell phenotype limit their widespread deployment. Human embryonic stem cells and induced pluripotent stem cells provide renewable sources of hepatocyte-like cells (HLCs). Despite the use of various differentiation methodologies, HLCs like primary human hepatocytes exhibit unstable phenotype in culture. It has been shown that the functional capacity can be improved by adding back elements of human physiology, such as cell co-culture or through the use of natural and/or synthetic surfaces. In this study, the effect of fluid shear stress on HLC performance was investigated. We studied two important liver functions, cytochrome P450 drug metabolism and serum protein secretion, in static cultures and those exposed to fluid shear stress. Our study demonstrates that fluid shear stress improved Cyp1A2 activity by approximately fivefold. This was paralleled by an approximate ninefold increase in sensitivity to a drug, primarily metabolised by Cyp2D6. In addition to metabolic capacity, fluid shear stress also improved hepatocyte phenotype with an approximate fourfold reduction in the secretion of a foetal marker, alpha-fetoprotein. We believe these studies highlight the importance of introducing physiologic cues in cell-based models to improve somatic cell phenotype

The Drosophila melanogaster PeptideAtlas facilitates the use of peptide data for improved fly proteomics and genome annotation

<p>Abstract</p> <p>Background</p> <p>Crucial foundations of any quantitative systems biology experiment are correct genome and proteome annotations. Protein databases compiled from high quality empirical protein identifications that are in turn based on correct gene models increase the correctness, sensitivity, and quantitative accuracy of systems biology genome-scale experiments.</p> <p>Results</p> <p>In this manuscript, we present the <it>Drosophila melanogaster </it>PeptideAtlas, a fly proteomics and genomics resource of unsurpassed depth. Based on peptide mass spectrometry data collected in our laboratory the portal <url>http://www.drosophila-peptideatlas.org</url> allows querying fly protein data observed with respect to gene model confirmation and splice site verification as well as for the identification of proteotypic peptides suited for targeted proteomics studies. Additionally, the database provides consensus mass spectra for observed peptides along with qualitative and quantitative information about the number of observations of a particular peptide and the sample(s) in which it was observed.</p> <p>Conclusion</p> <p>PeptideAtlas is an open access database for the <it>Drosophila </it>community that has several features and applications that support (1) reduction of the complexity inherently associated with performing targeted proteomic studies, (2) designing and accelerating shotgun proteomics experiments, (3) confirming or questioning gene models, and (4) adjusting gene models such that they are in line with observed <it>Drosophila </it>peptides. While the database consists of proteomic data it is not required that the user is a proteomics expert.</p

Self-assembled monolayers of alendronate on Ti6Al4V alloy surfaces enhance osteogenesis in mesenchymal stem cells

Phosphonates have emerged as an alternative for functionalization of titanium surfaces by the formation of homogeneous self-assembled monolayers (SAMs) via Ti-O-P linkages. This study presents results from an investigation of the modification of Ti6Al4V alloy by chemisorption of osseoinductive alendronate using a simple, effective and clean methodology. The modified surfaces showed a tailored topography and surface chemistry as determined by SEM microscopy and RAMAN spectroscopy. X-ray photoelectron spectroscopy revealed that an effective mode of bonding is created between the metal oxide surface and the phosphate residue of alendronate, leading to formation of homogenous drug distribution along the surface. In-vitro studies showed that alendronate SAMs induce differentiation of hMSC to a bone cell phenotype and promote bone formation on modified surfaces. Here we show that this novel method for the preparation of functional coatings on titanium-based medical devices provides osseoinductive bioactive molecules to promote enhanced integration at the site of implantation

Growth inhibition of oral mutans streptococci and candida by commercial probiotic lactobacilli - an in vitro study

<p>Abstract</p> <p>Background</p> <p>Probiotic bacteria are suggested to play a role in the maintenance of oral health. Such health promoting bacteria are added to different commercial probiotic products. The aim of the study was to investigate the ability of a selection of lactobacilli strains, used in commercially available probiotic products, to inhibit growth of oral mutans streptococci and <it>C. albicans in vitro</it>.</p> <p>Methods</p> <p>Eight probiotic lactobacilli strains were tested for growth inhibition on three reference strains and two clinical isolates of mutans streptococci as well as two reference strains and three clinical isolates of <it>Candida albicans </it>with an agar overlay method.</p> <p>Results</p> <p>At concentrations ranging from 10⁹to 10⁵CFU/ml, all lactobacilli strains inhibited the growth of the mutans streptococci completely with the exception of <it>L. acidophilus </it>La5 that executed only a slight inhibition of some strains at concentrations corresponding to 10⁷and 10⁵CFU/ml. At the lowest cell concentration (10³CFU/ml), only <it>L. plantarum </it>299v and <it>L. plantarum </it>931 displayed a total growth inhibition while a slight inhibition was seen for all five mutans streptococci strains by <it>L. rhamnosus </it>LB21, <it>L. paracasei </it>F19, <it>L. reuteri </it>PTA 5289 and <it>L. reuteri </it>ATCC 55730. All the tested lactobacilli strains reduced candida growth but the effect was generally weaker than for mutans streptococci. The two <it>L. plantarum </it>strains and <it>L. reuteri </it>ATCC 55730 displayed the strongest inhibition on <it>Candida albicans</it>. No significant differences were observed between the reference strains and the clinical isolates.</p> <p>Conclusion</p> <p>The selected probiotic strains showed a significant but somewhat varying ability to inhibit growth of oral mutans streptococci and <it>Candida albicans in vitro</it>.</p

An Integrative -omics Approach to Identify Functional Sub-Networks in Human Colorectal Cancer

Emerging evidence indicates that gene products implicated in human cancers often cluster together in “hot spots” in protein-protein interaction (PPI) networks. Additionally, small sub-networks within PPI networks that demonstrate synergistic differential expression with respect to tumorigenic phenotypes were recently shown to be more accurate classifiers of disease progression when compared to single targets identified by traditional approaches. However, many of these studies rely exclusively on mRNA expression data, a useful but limited measure of cellular activity. Proteomic profiling experiments provide information at the post-translational level, yet they generally screen only a limited fraction of the proteome. Here, we demonstrate that integration of these complementary data sources with a “proteomics-first” approach can enhance the discovery of candidate sub-networks in cancer that are well-suited for mechanistic validation in disease. We propose that small changes in the mRNA expression of multiple genes in the neighborhood of a protein-hub can be synergistically associated with significant changes in the activity of that protein and its network neighbors. Further, we hypothesize that proteomic targets with significant fold change between phenotype and control may be used to “seed” a search for small PPI sub-networks that are functionally associated with these targets. To test this hypothesis, we select proteomic targets having significant expression changes in human colorectal cancer (CRC) from two independent 2-D gel-based screens. Then, we use random walk based models of network crosstalk and develop novel reference models to identify sub-networks that are statistically significant in terms of their functional association with these proteomic targets. Subsequently, using an information-theoretic measure, we evaluate synergistic changes in the activity of identified sub-networks based on genome-wide screens of mRNA expression in CRC. Cross-classification experiments to predict disease class show excellent performance using only a few sub-networks, underwriting the strength of the proposed approach in discovering relevant and reproducible sub-networks

PAH mineralization and bacterial organotolerance in surface sediments of the Charleston Harbor estuary

Semi-volatile organic compounds (SVOCs) in estuarine waters can adversely affect biota but watershed sources can be difficult to identify because these compounds are transient. Natural bacterial assemblages may respond to chronic, episodic exposure to SVOCs through selection of more organotolerant bacterial communities. We measured bacterial production, organotolerance and polycyclic aromatic hydrocarbon (PAH) mineralization in Charleston Harbor and compared surface sediment from stations near a known, permitted SVOC outfall (pulp mill effluent) to that from more pristine stations. Naphthalene additions inhibited an average of 77% of bacterial metabolism in sediments from the more pristine site (Wando River). Production in sediments nearest the outfall was only inhibited an average of 9% and in some cases, was actually stimulated. In general, the stations with the highest rates of bacterial production also were among those with the highest rates of PAH mineralization. This suggests that the capacity to mineralize PAH carbon is a common feature amongst the bacterial assemblage in these estuarine sediments and could account for an average of 5.6% of bacterial carbon demand (in terms of production) in the summer, 3.3% in the spring (April) and only 1.2% in winter (December)