Peptide Database Research Articles

Post-Translational Modifications of Natural Antimicrobial Peptides and Strategies for Peptide Engineering

Natural antimicrobial peptides (AMPs) are gene-coded defense molecules discovered in all the three life domains: Eubacteria, Archaea, and Eukarya. The latter covers protists, fungi, plants, and animals. It is now recognized that amino acid composition, peptide sequence, and post-translational modifications determine to a large extent the structure and function of AMPs. This article systematically describes post-translational modifications of natural AMPs annotated in the antimicrobial peptide database (http://aps.unmc.edu/AP). Currently, 1147 out of 1755 AMPs in the database are modified and classified into more than 17 types. Through chemical modifications, the peptides fold into a variety of structural scaffolds that target bacterial surfaces or molecules within cells. Chemical modifications also confer desired functions to a particular peptide. Meanwhile, these modifications modulate other peptide properties such as stability. Elucidation of the relationship between AMP property and chemical modification inspires peptide engineering. Depending on the objective of our design, peptides may be modified in various ways so that the desired features can be enhanced whereas unwanted properties can be minimized. Therefore, peptide design plays an essential role in developing natural AMPs into a new generation of therapeutic molecules.

Proteomic Profiles of White Sucker (Catostomus commersonii) Sampled from within the Thunder Bay Area of Concern Reveal Up-Regulation of Proteins Associated with Tumor Formation and Exposure to Environmental Estrogens

White sucker (Catostomus commersonii) sampled from the Thunder Bay Area of Concern were assessed for health using a shotgun approach to compile proteomic profiles. Plasma proteins were sampled from male and female fish from a reference location, an area in recovery within Thunder Bay Harbour, and a site at the mouth of the Kaministiquia River where water and sediment quality has been degraded by industrial activities. The proteins were characterized using reverse-phase liquid chromatography tandem to a quadrupole-time-of-flight (LC-Q-TOF) mass spectrometer and were identified by searching in peptide databases. In total, 1086 unique proteins were identified. The identified proteins were then examined by means of a bioinformatics pathway analysis to gain insight into the biological functions and disease pathways that were represented and to assess whether there were any significant changes in protein expression due to sampling location. Female white sucker exhibited significant (p = 0.00183) site-specific changes in the number of plasma proteins that were related to tumor formation, reproductive system disease, and neurological disease. Male fish plasma had a significantly different (p < 0.0001) number of proteins related to neurological disease and tumor formation. Plasma concentrations of vitellogenin were significantly elevated in females from the Kaministiquia River compared to the Thunder Bay Harbour and reference sites. The protein expression profiles indicate that white sucker health has benefited from the remediation of the Thunder Bay Harbour site, whereas white sucker from the Kaministiquia River site are impacted by ongoing contaminant discharges.

Urinary Proteome Analysis at 5-Year Followup of Patients With Nonoperated Ureteropelvic Junction Obstruction Suggests Ongoing Kidney Remodeling

Severe ureteropelvic junction obstruction is treated surgically. However, for milder cases most clinical teams adopt a watchful waiting approach and only operate in the presence of significant decline of renal function combined with severe hydronephrosis. Little is known about the long-term consequences of ureteropelvic junction obstruction. Using capillary electrophoresis coupled with mass spectrometry, we analyzed the urinary proteome of 42 patients with ureteropelvic junction obstruction 5 years postoperatively or 5 years following spontaneous resolution. At 5-year followup urinary proteomes were similar between patients with early surgical correction of ureteropelvic junction obstruction and age matched controls. In contrast, urinary proteomes differed significantly between conservatively followed patients and controls. Analyses of the proteomic differences suggested ongoing renal or ureteral remodeling in the conservatively followed patients that was not visible clinically. Long-term followup studies are warranted in patients with ureteropelvic junction obstruction, especially those followed conservatively, to determine whether the observed changes in the urinary proteomes become clinically relevant at a later stage.

De Novo Design of Antiviral and Antibacterial Peptides with Varying Loop Structures

Although the rate of new HIV infections has been declining, AIDS continues to be one of the leading causes of death worldwide. The lack of an effective HIV vaccine makes it necessary to develop alternative strategies, such as the development of topical microbicides, to prevent transmission. Antimicrobial peptides represent promising microbicide candidates. Previously, we succeeded in enhancing the anti-HIV activities of several peptides that form helical structures based on the bioinformatic results learned from the antimicrobial peptide database. This study showed that Lys-to-Arg alterations also improved the HIV inhibitory activity of thanatin which is known to form a β-hairpin structure. Using a previously reported de novo designed HIV inhibitory peptide GLR-19 as the starting template, loop structures of varying sizes were generated by restraining a disulfide bond at different positions. The thanatin-mimicking constructs are referred to as GLRC peptides since they are composed of only four amino acid residues G, L, R, and C. While GLRC-2, the peptide with a medium-sized loop structure, was most potent against HIV-1 and HSV-2, GLRC-3, with the small loop structure, was most potent against Escherichia coli K12. Thus, the efficacy of the GLRC peptides is microbe dependent. Further terminal sequence truncation of GLRC-2 reduced antimicrobial activity against both viruses and bacteria. It appears that the high antiviral potency of GLRC-2 is related to high hydrophobicity, although a wide-range correlation is lacking. In addition, GLRC-2, which is more active against viruses, is also more resistant to the action of chymotrypsin. Therefore, GLRC-2, a novel peptide that acquired not only higher stability but also higher anti-HIV activity than the GLR-19 template, serves as the starting point for additional rounds of peptide engineering.

The Importance of Amino Acid Composition in Natural AMPs: An Evolutional, Structural, and Functional Perspective

Antimicrobial peptides (AMPs) are critical components of natural host defense systems against infectious pathogens (Zasloff, 2002; Boman, 2003; Hancock and Sahl, 2006). They are ubiquitous in nature and have been found in nearly all forms of life, ranging from single-celled bacteria to multicellular organisms such as plants and animals. AMPs are short peptides (5–100 amino acids) with an average net charge of +3 (Wang, 2010). They can display broad or narrow-spectrum antimicrobial activities. The fact that AMPs are effective against multidrug resistance pathogens, including suppression of biofilm formation, deserves our attention (Menousek et al., 2012). In addition to direct bacterial elimination, these peptides have regulatory effects on immune systems. Consequently, AMPs are also referred to as host defense peptides (Hancock and Sahl, 2006). To decode the key elements behind the functional diversity of AMPs, we have been taking time and efforts in constructing a comprehensive database that annotates such information. The first version of the Antimicrobial Peptide Database (APD; http://aps.unmc.edu/AP/main.html) was established in 2003 (Wang and Wang, 2004) and the database has since been further developed (Wang et al., 2009). The APD contained 1973 entries as of May 2012. To facilitate our bioinformatic analysis, we will register a peptide into the APD if it is (1) from natural sources; (2) with minimal inhibitory concentration (MIC) of less than 100 μM or 100 μg/mL; (3) less than 100 amino acid residues; and (4) with a characterized amino acid sequence (Wang, 2010). The APD allows users to extract important parameters (e.g., charge, hydrophobicity, motif, and structure) that determine peptide function. In particular, our database enables the generation of the amino acid composition for a select peptide or a family of AMPs with a common feature. This bioinformatic tool thus uncovers the amino acid use in natural AMPs from different sources, with different functions, or three-dimensional structures. This opinion article highlights the critical roles of the amino acid composition in naturally occurring AMPs in terms of evolutional, structural, and functional significance. Moreover, its application in designing and predicting new AMPs will also be discussed.

Brainpeps: the blood–brain barrier peptide database

Peptides are able to cross the blood-brain barrier (BBB) through various mechanisms, opening new diagnostic and therapeutic avenues. However, their BBB transport data are scattered in the literature over different disciplines, using different methodologies reporting different influx or efflux aspects. Therefore, a comprehensive BBB peptide database (Brainpeps) was constructed to collect the BBB data available in the literature. Brainpeps currently contains BBB transport information with positive as well as negative results. The database is a useful tool to prioritize peptide choices for evaluating different BBB responses or studying quantitative structure-property (BBB behaviour) relationships of peptides. Because a multitude of methods have been used to assess the BBB behaviour of compounds, we classified these methods and their responses. Moreover, the relationships between the different BBB transport methods have been clarified and visualized.

DAMPD: a manually curated antimicrobial peptide database.

The demand for antimicrobial peptides (AMPs) is rising because of the increased occurrence of pathogens that are tolerant or resistant to conventional antibiotics. Since naturally occurring AMPs could serve as templates for the development of new anti-infectious agents to which pathogens are not resistant, a resource that contains relevant information on AMP is of great interest. To that extent, we developed the Dragon Antimicrobial Peptide Database (DAMPD, http://apps.sanbi.ac.za/dampd) that contains 1232 manually curated AMPs. DAMPD is an update and a replacement of the ANTIMIC database. In DAMPD an integrated interface allows in a simple fashion querying based on taxonomy, species, AMP family, citation, keywords and a combination of search terms and fields (Advanced Search). A number of tools such as Blast, ClustalW, HMMER, Hydrocalculator, SignalP, AMP predictor, as well as a number of other resources that provide additional information about the results are also provided and integrated into DAMPD to augment biological analysis of AMPs.

Peptide structure prediction using distributed volunteer computing networks

Recent investigations to develop novel antimicrobial, antibiotical drugs have focused on the development of artificial protein peptides. As short peptides are naturally involved in many important biological processes in the cell and therefore target many kinds of cells. To functionalize peptides it is vital to design peptides, which can differentially target bacterial and eucariotic cells. Although the length of the peptides investigated in this study was limited to 16 amino acids, the number of possible peptide sequences is still too large to synthesize them in a trial- and error manner, therefore requiring a method for directed, but also high-througput peptide design. By predicting the structure of peptide proteins, this design process can be supported through structure-function analysis and peptide-membrane interaction simulation. In this investigation we could predict peptide structures de-novo, i.e. with the sequence information alone, using a massively parallel simulation scheme. We sample a sizable fraction of the peptide’s conformational space using Monte-Carlo simulations in the free-energy forcefield PFF02 on the volunteer computing network POEM@HOME. This forcefield models the protein’s native conformation as the global minimum of the free-energy. We could identify peptides of different topologies in a completely automated manner, which allows for the high-throughput screening of large peptide databases for their structural features, which would allow the rapid protopying of peptides needed for novel peptide design.

Identification of a Protein with Antioxidant Activity that is Important for the Protection against Beer Ageing

This study was carried out with fresh Australian lager beer which was sampled directly off the production line, the same samples aged for 12 weeks at 30 °C, and the vintage beer which was kept at 20 °C for 5 years. Characteristic Australian lager flavour was maintained in the fresh and vintage beers but was lost in the aged beer. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and free thiol group labelling analyses of beer proteins found that this flavour stability correlated with the presence of an unknown 10 kilodaltons (kDa) protein with a higher level of free thiols. The protein was purified by size-exclusion chromatography, then peptide sequencing and database matching identified it as the barley lipid transfer protein (LTP1). Further characterisation using diphenylpicrylhydrazyl (DPPH) free radical scavenging and a Saccharomyces cerevisiae-based antioxidant screening assay demonstrated that the LTP1 protein was active in DPPH reduction and antioxidant activity. The absence of free thiol in the aged beer indicates that the thiol functional groups within the LTP1 protein were saturated and suggests that it is important in the flavour stability of beer by maintaining reduction capacity during the ageing process.

WebWatch

BioTechniquesVol. 51, No. 2 WebWatchOpen AccessWebWatchKevin AhernKevin Ahern*E-mail Address: ahernk@orst.eduSearch for more papers by this authorPublished Online:3 Apr 2018https://doi.org/10.2144/000113713AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail The GMO ShowTo go GMO or not to go GMO? That is the question asked increasingly around the world. Probably nowhere is it asked more seriously than in Europe, home of this informative GMO safety page. The Federal Ministry of Education and Research in Germany hosts this large, news-based GMO Safety site that takes no obvious position on the controversy. At press time, containment of genetically engineered plants and the effects of transgenic technology on water quality, nematodes, and earthworms dominated the news. Informative videos abound and debates on both sides of the topic are easy to find. An important aspect of the site, dubbed ‘coexistence,’ is aimed at encouraging parallel, compatible systems of GMOs and organic agriculture in food processing.[www.gmo-safety.eu]Poison PeptidesHome to information on over 1750 peptides, the Antimicrobial Peptide Database is a popular resource for anyone interested in novel agents for resistant microbes. These intriguing biomolecules, each with fewer than 100 amino acid residues, are produced by bacteria, fungi, plants, and animals, and are inhibitory at concentrations of less than 100 µM. Not surprisingly, many of them are antibacterial and/or antifungal, but a small percentage actually work against viruses while others have anti-tumor properties. The site contains structural and sequence information on various peptides. There is also a peptide designer tool to improve the antimicrobial activity of peptides and a predictor that will take an input sequence, characterize it, and match it against peptides in the database.[http://aps.unmc.edu/AP/main.php]Bite-sizedOur pearly whites may sometimes cause us pain, but as a potential source of stem cells, they may ultimately save us pain if those cells can be harnessed to regenerate new teeth, bone, and cartilage. A more complete understanding of teeth at the molecular level is an important goal and the University of Helsinki's Gene Expression in Tooth Web site is definitely a step in the right direction. The site is a compilation of efforts from contributing researchers, with data originating mostly from published reports. Over 40 different research sections subdivide the information, including growth factors, signaling molecules, methods, extracellular and intracellular molecules, and genes expressed in specific tissue domains, such as the enamel epithelia, dental papilla, and the basement membrane. A vast collection of references and genetic diseases/traits round out the offerings, giving any visitor a lot to chew on.[http://bite-it.helsinki.fi]Garden Gone WildA botanical effort by one of the most enthused, nonprofessional botanists you will find anywhere, US Wildflowers is a garden of many delights. Featuring information and images on almost 300 different species, the site is a labor of love for Gerry Williamson, a self-described hobbyist whose fascination with wildflowers drove him to put together this impressive collection. Here you will find information about wildflowers in all 50 states, easily accessed through a pop-up menu of state names. The site contains many links to other useful pages and most of these are also state-specific. The photos are beautiful and were also a strong motivation for the author to design the site in the first place.[http://uswildflowers.com]Spotted Jewelweed's bright color makes it a favorite of hummingbirds.© 2009 Gerry Williamson.On FirstIt is said that science is both the easiest and the most difficult subject to write about. It is easy because there is no shortage of fascinating stories to tell, but difficult because of the need to carefully choose language to explain complicated concepts in ways that everyone can understand. Finding that sweet spot of effective scientific communication is FirstScience.com, an up-to-the-minute news site whose focus on fun takes it in many directions. In the news section, articles are intriguing (“Solving the Dilemma of Gender Imbalance,” for example), while the blogs are aimed at grabbing readers (“The 2000 Year-old Computer”). There are poems and quotes, games and quizzes, sights and sounds, and a free newsletter and video news to boot. Information is sorted into nine categories and a search engine helps retrieve information from a huge archive.[www.firstscience.com/site/home.asp]World Wide WebdirectoryTo thoroughly cover the academic world of biology by spanning both the virtual world of the Web and the physical world is no small task. Up for the challenge is Biology WebDirectory, a tidily organized collection of links to thousands of interesting, relevant pages. From the U.S. alone, there are over 25,000 selections, for example. With representation of 16 countries in the Americas, Europe, and Asia, Biology WebDirectory is truly a global resource. Information is bundled into 11 broad categories in lists hundreds of pages long, and can be easily retrieved by the excellent keyword-based search engine.[www.biologydir.com]FiguresReferencesRelatedDetails Vol. 51, No. 2 Follow us on social media for the latest updates Metrics History Published online 3 April 2018 Published in print August 2011 Information© 2011 Author(s)PDF download

To tag or not to tag: A comparative evaluation of immunoaffinity-labeling and tandem mass spectrometry for the identification and localization of posttranslational protein carbonylation by 4-hydroxy-2-nonenal, an end-product of lipid peroxidation

Posttranslational carbonylation of proteins by the covalent attachment of the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) is a biomarker of oxidative stress. Tandem mass spectrometry (MS/MS) has become an essential tool for characterization of this modification. Chemical tagging methods have been used to facilitate the immunoaffinity-based enrichment or even quantification of HNE-modified peptides and proteins. With MS/MS spectra of the untagged modified peptides considered as references, a comparative evaluation is presented focusing on the impact of affinity-tagging with four carbonyl-specific reagents (2,4-dinitrophenyl hydrazine, biotin hydrazide, biotinamidohexanoic acid hydrazide and N'-aminooxymethylcarbonyl-hydrazino D-biotin) on collision-induced dissociation of the tagged HNE-carbonylated peptides. Our study has shown that chemical labeling may not be carried out successfully for all the peptides and with all the reagents. The attachment of a tag usually cannot circumvent the occurrence of strong neutral losses observed with untagged species and, in addition, fragmentation of the introduced tag may also happen. Chemical tagging of certain peptides may, nevertheless, afford more sequence ions upon MS/MS than the untagged carbonylated peptide, especially when Michael addition of the lipid peroxidation product occurs on cysteine residues. Therefore, tagging may increase the confidence of identifications of HNE-modified peptides by database searches.

Differential Proteins of the Optic Ganglion in Octopus vulgaris Under Methanol Stress Revealed Using Proteomics

An analytical approach using the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique separated the proteome from the optic ganglia of Octopus vulgaris (OVOG). Approximately 600 protein spots were detected from the extraction when applying 150 μg protein to a 2D-PAGE gel in the pH range 5.0-8.0. Compared to the control, significant changes of 18 protein spots were observed in OVOG under the stress of native seawater containing 2% methanol for 72 h. Among these spots, we found that eight were down-regulated and ten were up-regulated in the gels, which were further identified using both peptide mass fingerprinting and database searches. Significant proteins such as glyceraldehyde-3-phosphate dehydrogenase, alpha subunit of succinyl-CoA synthetase, alcohol dehydrogenase, and long-chain specific acyl-CoA dehydrogenase were up-regulated proteins, whereas putative ABC transporter was a down -regulated protein. These differential proteins at the level of subcellular localization were further classified using LOCtree software with a hierarchical system of support vector machines. We found that most of the differential proteins in the gel could be identified as mitochondrial proteins, suggesting that these protective or marker proteins might help to prevent methanol poisoning via the mitochondria in the optical ganglia. The results indicated that both beta-tubulin and beta-actin were potential biomarkers as up-regulated proteins for monitoring methanol toxicosis associated with fish foods such as octopus and shark.

Human urinary peptide database for multiple disease biomarker discovery

Human urine is an ideal candidate for use in clinical diagnostics. It is easily available, as untrained personnel can collect it. It correlates well with the pathophysiology of a number of diseases, making it a useful source for clinical proteomics. In this article, we give an update of the human urinary peptide database derived from over 13,000 data sets of CE-MS by now. Urine samples from both patients and healthy subjects were analyzed by CE-MS; these included 47 different pathophysiological conditions. Besides defining biomarkers by their experimental parameters, information on their sequences provides fundamental data into the pathological pathways of diseases. Therefore, we have sequenced 953 urinary peptides by using state-of-the-art top-down MS/MS. Identified biomarkers of all clinical proteomic CE-MS studies including their regulation are also listed in this work. Biomarker discovery can be used in the management of a wide range of diseases, by combining these data sets of the database. Taking this approach, we can reveal details, at a molecular level, on the pathogenesis of a number of diseases, in particular those associated with urine production and excretion.

Angiotensin-I-converting enzyme inhibitory peptides: Chemical feature based pharmacophore generation

A validated 3D pharmacophore model was generated for a series of ACE inhibitory peptides, which consisted of five features (two hydrophobic functions, two hydrogen bond acceptors, and a negative ionizable function). The built model was able to correctly predict the activity of known ACE inhibitors. The model was then used as query to search 3D databases of peptides. Three novel peptides (I, II and III) were synthesized and biologically evaluated in vitro. It appears that the in vitro activity of peptides I, II and III was consistent with their molecular modeling results. Our results provided confidence for the utility of the pharmacophore model to retrieve novel ACE inhibitory peptides with desired biological activity by virtual screening.

Machine learning study for the prediction of transdermal peptide

In order to develop a computational method to rapidly evaluate transdermal peptides, we report approaches for predicting the transdermal activity of peptides on the basis of peptide sequence information using Artificial Neural Network (ANN), Partial Least Squares (PLS) and Support Vector Machine (SVM). We identified 269 transdermal peptides by the phage display technique and use them as the positive controls to develop and test machine learning models. Combinations of three descriptors with neural network architectures, the number of latent variables and the kernel functions are tried in training to make appropriate predictions. The capacity of models is evaluated by means of statistical indicators including sensitivity, specificity, and the area under the receiver operating characteristic curve (ROC score). In the ROC score-based comparison, three methods proved capable of providing a reasonable prediction of transdermal peptide. The best result is obtained by SVM model with a radial basis function and VHSE descriptors. The results indicate that it is possible to discriminate between transdermal peptides and random sequences using our models. We anticipate that our models will be applicable to prediction of transdermal peptide for large peptide database for facilitating efficient transdermal drug delivery through intact skin.

Identification of Thrombospondin-1 and L-Selectin as Potential Plasma Biomarkers of Silent Cerebral Infarct In Children with Sickle Cell Disease Using a Proteomic-Based Approach

AbstractAbstract 259 Objectives:Silent cerebral infarction (SCI) occurs in approximately 27% of children with sickle cell disease (SCD) by age 6 years, and is associated with decreased neurocognitive function and a 14-fold increased risk of progression to overt stroke. While several clinical parameters, such as increased white blood cell (WBC) and platelet counts and decreased hemoglobin (Hb) or hematocrit, have been reported in the literature to be associated with SCI, to date no validated biomarkers exist to predict SCI in patients with SCD. Furthermore, recent unpublished data from the Silent Infarct Transfusion (SIT) Trial has identified systolic blood pressure and total hemoglobin as risk factors. The aim of this study was to identify candidate biomarker plasma proteins that correlate with SCI in patients with SCD. Methods:We used a proteomic discovery approach involving three sequential separation steps to compare the plasma proteomes of 15 children with SCD (7 with SCI and 8 without SCI), aged 5–15 years. Baseline steady-state plasma samples were obtained from the SIT Trial Biologic Repository and matched for age, Hb and WBC. Plasma samples were Hb depleted in the first dimension, separated using immunoaffinity depletion and reverse phase liquid chromatography fractionation, and then trypsin-digested for characterization using label-free quantification on a LTQ-Orbitrap (Thermo) mass spectrometer. The resulting MS/MS data were analyzed using PASS (Integrated Analysis, Bethesda, MD) with X! Tandem searches (www.thegpm.org; version 2008.12.01) of the International Protein Index peptide database (human, 3.19). We measured candidate proteins in a validation cohort of 116 children with SCD (n=65 SCI, 51 non-SCI) and 24 age-matched, healthy African American control subjects using enzyme-linked immunosorbent assays (thrombospondin 1 [TSP1], L-selectin, RandD Systems, Minneapolis, MN) and immunoassays (E- and P-selectin, Mesoscale Discovery, Maryland). All samples were run in duplicate according to the manufacturers' protocols. Statistical differences in biomarker plasma concentrations between groups were compared by the Mann-Whitney U test. Results:TSP1 (5 peptides) and L-selectin (3 peptides) were among 335 proteins that showed differential detection between the SCI and non-SCI groups based on the spectral counts. TSP1 is an extracellular matrix glycoprotein that is involved with platelet aggregation, inhibition of neovascularization and tumorigenesis and has been shown to promote the adherence of sickle erythrocytes to the vascular endothelium. L-selectin is an adhesion molecule that mediates leukocyte interaction with the vascular endothelium. In a validation cohort of 116 children with SCD (n=65 SCI, 51 non-SCI) and 24 age matched, healthy African American control subjects, TSP1 and L-selectin were both significantly increased in SCI vs. non-SCI groups (median 8.5 vs. 6.2 μ g/ml for TSP1, P =0.03; 1.5 vs. 1.4 μ g/ml for L-selectin, P =0.03). As expected, neither TSP nor L-selectin were elevated in the age-matched normal controls (median=4.6 μ g/ml for TSP1, P =0.10, 1.2 μ g/ml for L-selectin, P =0.10). The specificity of the L-selectin results was verified by demonstrating that E-selectin and P-selectin were not increased in the SCI group. TSP1 was correlated with baseline oxygen saturation in both the SCI and non-SCI groups (r=-0.51, and r=-0.35, P<0.001). L-selectin correlated with systolic blood pressure in the SCI group only (r=0.3, P<0.02). Conclusions:TSP1 and L-selectin may represent the first two plasma biomarkers of SCI in children with SCD. Although further studies are needed, these and other potential biomarkers may provide insight into the pathophysiology of SCI, and may fill an important clinical need in identifying children with SCD who are at risk for SCI. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Correction: Frequent MAGE Mutations in Human Melanoma

Reference 26 is missing from the final version of the paper. It should be: The Peptide Database (accessed 2010) http://www.cancerimmunity.org/peptidedatabase/Tcellepitopes.htm

Integration of deep transcriptome and proteome analyses reveals the components of alkaloid metabolism in opium poppy cell cultures

BackgroundPapaver somniferum (opium poppy) is the source for several pharmaceutical benzylisoquinoline alkaloids including morphine, the codeine and sanguinarine. In response to treatment with a fungal elicitor, the biosynthesis and accumulation of sanguinarine is induced along with other plant defense responses in opium poppy cell cultures. The transcriptional induction of alkaloid metabolism in cultured cells provides an opportunity to identify components of this process via the integration of deep transcriptome and proteome databases generated using next-generation technologies.ResultsA cDNA library was prepared for opium poppy cell cultures treated with a fungal elicitor for 10 h. Using 454 GS-FLX Titanium pyrosequencing, 427,369 expressed sequence tags (ESTs) with an average length of 462 bp were generated. Assembly of these sequences yielded 93,723 unigenes, of which 23,753 were assigned Gene Ontology annotations. Transcripts encoding all known sanguinarine biosynthetic enzymes were identified in the EST database, 5 of which were represented among the 50 most abundant transcripts. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of total protein extracts from cell cultures treated with a fungal elicitor for 50 h facilitated the identification of 1,004 proteins. Proteins were fractionated by one-dimensional SDS-PAGE and digested with trypsin prior to LC-MS/MS analysis. Query of an opium poppy-specific EST database substantially enhanced peptide identification. Eight out of 10 known sanguinarine biosynthetic enzymes and many relevant primary metabolic enzymes were represented in the peptide database.ConclusionsThe integration of deep transcriptome and proteome analyses provides an effective platform to catalogue the components of secondary metabolism, and to identify genes encoding uncharacterized enzymes. The establishment of corresponding transcript and protein databases generated by next-generation technologies in a system with a well-defined metabolite profile facilitates an improved linkage between genes, enzymes, and pathway components. The proteome database represents the most relevant alkaloid-producing enzymes, compared with the much deeper and more complete transcriptome library. The transcript database contained full-length mRNAs encoding most alkaloid biosynthetic enzymes, which is a key requirement for the functional characterization of novel gene candidates.

Analysis of expressed sequence tags in developing secondary xylem and shoot of Acacia mangium

Acacia mangium is a fast-growing tree widely planted in tropical countries because of its rapid growth, high wood density, high fiber quality, and good adaptability. Despite its importance as a fiber source in the pulp and paper industry, a large-scale analysis of expressed sequence tags (ESTs) has not been performed in A. mangium. In this study, we sequenced 10 752 clones of a normalized complementary DNA (cDNA) library prepared from A. mangium developing secondary xylem and shoot, and obtained a total of 8963 ESTs. The ESTs were assembled into 6220 unigenes comprising 1614 contigs and 4606 singletons. The unigene set was then subjected to various bioinformatic analyses. BlastN searches of the unigene set against the Gene Index Databases of soybean, Medicago truncatula, Lotus japonicus, grape, poplar, spruce, and pine demonstrated that the largest number of unigenes shared homologies with the soybean Gene Indices. BlastX searches against the TAIR9 peptide database enabled us to annotate the unigenes. Based on the annotation, we discussed whether the unigenes involved in the cell cycle, cell growth, shoot apical meristem development, and cell wall biosynthesis were present. This new genomic resource will accelerate the functional genomics of wood formation and molecular breeding to improve the wood properties of A. mangium.

Proteomics of toxic oil syndrome in humans: Phenotype distribution in a population of patients

Objectives Toxic oil syndrome (TOS) is a disease that appeared in Spain in 1981. Epidemiological work traced the origin to the ingestion of aniline-adulterated rapeseed oil, fraudulently marketed and sold as edible oil. It affected more than 20,000 people with over 400 deaths in the first 2 years. In 2001 evidence was presented that genetic factors could play a role in the susceptibility of individuals to the disease. Thus, a prospective study on the differences in gene expression in sera between control versus TOS-affected populations, both originally exposed to the toxic oil, was undertaken in our laboratory. Methods Differential protein expression was analyzed by two-dimensional electrophoresis (2-DE). Problems related with serum analysis by 2-DE were addressed to improve protein detection in the gel images. Three new commercial systems for albumin depletion were tested to optimize the detection of minor proteins. The use of nonionic reductants or the presence of thiourea in the gels, were also tested. Results From the resulting optimized images, a group of 329 major gel spots was located, matched and compared with serum samples. Thirty-five of these protein spots were found to be under- or over-expressed in TOS patients (threefold increase or decrease). Proteins in these spots were identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) peptide map fingerprinting and database search. Several haptoglobin (Hp) isoforms were found to be differentially expressed, showing expression phenotypes that could be related with TOS. Resolution of the homologous α-1s and α-1f chains, with a mass difference of only 0.043 Da, was obtained after guanidation of the protein with O-methylisourea. We applied this procedure to the study of the distribution of the Hp alleles HP 2, HP 1s and HP 1f in control versus TOS-affected populations. The MALDI-TOF proteotyping method was validated by a parallel analysis of the serum samples by 2-DE. Conclusions Data obtained from 54 TOS cases and 48 controls indicate significant differences in the distribution of Hp phenotypes in the two populations. Haptoglobin phenotypes have been reported to have biological and clinical consequences and have been described as risk factors for several diseases. Consequently, it was concluded that haptoglobin polymorphism could play a role in TOS.