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Myasthenia gravis (MG) is an autoimmune disease in which autoantibodies, most commonly directed against the acetylcholine receptor (AChR), impair neuromuscular transmission and cause muscle weakness. In this study, we utilized two-dimensional difference in-gel electrophoresis (2D-DIGE) to analyze the muscle's proteomic profile at different stages of experimental autoimmune myasthenia gravis (EAMG). We identified twenty-two differentially expressed proteins, mainly related to metabolic and stress-response pathways. Interestingly, these identified proteins have also been associated with other contraction-impairing muscle pathologies (e.g. inclusion body myositis), suggesting a similar response of the muscle to such conditions.

The renewed interest in controlling Staphylococcus aureus infections using their natural enemies, bacteriophages, has led to the isolation of a limited number of virulent phages so far. These phages are all members of the Twortlikevirus , displaying little variance. We present two novel closely related (95.9% DNA homology) lytic myoviruses, Romulus and Remus, with double-stranded DNA (dsDNA) genomes of 131,333 bp and 134,643 bp, respectively. Despite their relatedness to Staphylococcus phages K, G1, ISP, and Twort and Listeria phages A511 and P100, Romulus and Remus can be proposed as isolates of a new species within the Twortlikevirus genus. A distinguishing feature for these phage genomes is the unique distribution of group I introns compared to that in other staphylococcal myoviruses. In addition, a hedgehog/intein domain was found within their DNA polymerase genes, and an insertion sequence-encoded transposase exhibits splicing behavior and produces a functional portal protein. From a phage therapy application perspective, Romulus and Remus infected approximately 70% of the tested S. aureus isolates and displayed promising lytic activity against these isolates. Furthermore, both phages showed a rapid initial adsorption and demonstrated biofilm-degrading capacity in a proof-of-concept experiment.

Bacteriophage ZF40 is the only currently available, temperate Myoviridae phage infecting the potato pathogen Pectobacterium carotovorum subsp . carotovorum . Despite its unusual tail morphology, its major tail sheath and tube proteins remained uncharacterized after the initial genome annotation. Using ESI tandem mass-spectrometry, 24 structural proteins of the ZF40 virion were identified, with a sequence coverage ranging between 15,8 and 87,8%. The putative function of sixteen proteins could be elucidated based on secondary structure analysis and conservative domain searches. The experimental annotation of 35% of the encoded gene products within the structural region of the genome represents a complete view of the virion structure, which can serve as the basis for future structural analysis as a model phage.

Enlarged white adipose tissue (WAT) is a feature of obesity and leads to changes in its paracrine and endocrine function. Dysfunction of WAT cells is associated with obesity-associated disorders like type 2 diabetes and cardiovascular diseases. Resveratrol (RSV), a natural polyphenolic compound, mimics beneficial effects of calorie restriction. As such, RSV seems a promising therapeutic target for obesity-associated disorders. The effect of RSV on the human adipokine profile is still elusive. Therefore, a proteomic study together with bioinformatical analysis was performed to investigate the effect of RSV on the secretion profile of mature human SGBS adipocytes. RSV incubation resulted in elevated basal glycerol release and reduced intracellular TG content. This increased intracellular lipolysis was accompanied by profound changes in the adipocyte secretion profile. Extracellular matrix proteins were down-regulated while processing proteins were mostly up-regulated after RSV treatment. Interestingly, RSV induced secretion of proteins protective against cellular stress and proteins involved in the regulation of apoptosis. Furthermore, we found a RSV-induced up-regulation of adiponectin and ApoE accompanied by a down-regulation of PAI-1 and PEDF secretion which may improve anti-inflammatory processes and increased insulin sensitivity. These effects may contribute to alleviate obesity-induced metabolic complications. In addition, two novel RSV-regulated adipocyte-secreted proteins were identified.

We report the study of phage AF, the first member of the canonical lambdoid phage group infecting Pseudomonas putida . Its 42.6 kb genome is related to the “epsilon15-like viruses” and the “BPP-1-like viruses”, a clade of bacteriophages shaped by extensive horizontal gene transfer. The AF virions display exopolysaccharide (EPS)-degrading activity, which originates from the action of the C-terminal domain of the tail spike (Gp19). This protein shows high similarity to the tail spike of the T7-like P. putida -infecting phage φ15. These unrelated phages have an identical host spectrum and EPS degradation characteristics, designating the C-terminal part of Gp19 as sole determinant for these functions. While intact AF particles have biofilm-degrading properties, Gp19 and non-infectious AF particles do not, emphasizing the role of phage amplification in biofilm degradation.

The bacterium 'Dickeya solani', an aggressive biovar 3 variant of Dickeya dianthicola, causes rotting and blackleg in potato. To control this pathogen using bacteriophage therapy, we isolated and characterized two closely related and specific bacteriophages, vB_DsoM_LIMEstone1 and vB_DsoM_LIMEstone2. The LIMEstone phages have a T4-related genome organization and share DNA similarity with Salmonella phage Vil. Microbiological and molecular characterization of the phages deemed them suitable and promising for use in phage therapy. The phages reduced disease incidence and severity on potato tubers in laboratory assays. In addition, in a field trial of potato tubers, when infected with 'Dickeya solani', the experimental phage treatment resulted in a higher yield. These results form the basis for the development of a bacteriophage-based biocontrol of potato plants and tubers as an alternative for the use of antibiotics. EMA and the experimental work were funded through a PhD scholarship by the Katholieke Universiteit Leuven and the Institute for Agricultural and Fisheries Research Flanders (ILVO). PJC hold a post-doctoral fellowship of the Fonds voor Wetenschappelijk Onderzoek (FWO Flanders). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

A more detailed insight into disease mechanisms of multiple sclerosis (MS) is crucial for the development of new and more effective therapies. MS is a chronic inflammatory autoimmune disease of the central nervous system. The aim of this study is to identify novel disease associated proteins involved in the development of inflammatory brain lesions, to help unravel underlying disease processes. Brainstem proteins were obtained from rats with MBP induced acute experimental autoimmune encephalomyelitis (EAE), a well characterized disease model of MS. Samples were collected at different time points: just before onset of symptoms, at the top of the disease and following recovery. To analyze changes in the brainstem proteome during the disease course, a quantitative proteomics study was performed using two-dimensional difference in-gel electrophoresis (2D-DIGE) followed by mass spectrometry. We identified 75 unique proteins in 92 spots with a significant abundance difference between the experimental groups. To find disease-related networks, these regulated proteins were mapped to existing biological networks by Ingenuity Pathway Analysis (IPA). The analysis revealed that 70% of these proteins have been described to take part in neurological disease. Furthermore, some focus networks were created by IPA. These networks suggest an integrated regulation of the identified proteins with the addition of some putative regulators. Post-synaptic density protein 95 (DLG4), a key player in neuronal signalling and calcium-activated potassium channel alpha 1 (KCNMA1), involved in neurotransmitter release, are 2 putative regulators connecting 64% of the identified proteins. Functional blocking of the KCNMA1 in macrophages was able to alter myelin phagocytosis, a disease mechanism highly involved in EAE and MS pathology. Quantitative analysis of differentially expressed brainstem proteins in an animal model of MS is a first step to identify disease-associated proteins and networks that warrant further research to study their actual contribution to disease pathology. This work was supported by Alma-In-Silico NR. EMR INT4.-1.3.-2008-03/003, LSM tUL impuls phase II and Hasselt University. JJAH was supported by The Research Foundation – Flanders (FWO). PL is a FRS-FNRS Research Associate. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

BACKGROUND: The flagellotropic phage 7-7-1 infects motile cells of Agrobacterium sp H13-3 by attaching to and traveling along the rotating flagellar filament to the secondary receptor at the base, where it injects its DNA into the host cell. Here we describe the complete genomic sequence of 69,391 base pairs of this unusual bacteriophage. METHODS: The sequence of the 7-7-1 genome was determined by pyro(454)sequencing to a coverage of 378-fold. It was annotated using MyRAST and a variety of internet resources. The structural proteome was analyzed by SDS-PAGE coupled electrospray ionization-tandem mass spectrometry (MS/MS). RESULTS: Sequence annotation and a structural proteome analysis revealed 127 open reading frames, 84 of which are unique. In six cases 7-7-1 proteins showed sequence similarity to proteins from the virulent Burkholderia myovirus BcepB1A. Unique features of the 7-7-1 genome are the physical separation of the genes encoding the small (orf100) and large (orf112) subunits of the DNA packaging complex and the apparent lack of a holin-lysin cassette. Proteomic analysis revealed the presence of 24 structural proteins, five of which were identified as baseplate (orf7), putative tail fibre (orf102), portal (orf113), major capsid (orf115) and tail sheath (orf126) proteins. In the latter case, the N-terminus was removed during capsid maturation, probably by a putative prohead protease (orf114). ispartof: Virology Journal vol:9 issue:1 pages:102-1 ispartof: location:England status: published

Purpose/Objective: A more detailed insight into disease mechanisms of multiple sclerosis (MS) is crucial for the development of new and more effective theories. MS is a chronic inflammatory autoimmune disease of the central nervous system. The aim of this study is to identify novel disease associated proteins that are functionally involved in the MS brain pathology. Materials and methods: In a previous proteomics study, brainstem proteins were obtained from Lewis rats with MBP induced acute experimental autoimmune encephalomyelitis (EAE), a well characterized disease model of MS. Samples were collected at different time points: just before onset of symptoms, at the top of the disease and following recovery. To analyze changes in the brainstem proteome during the disease course, a quantitative proteomics study was performed using two-dimensional difference in-gel electrophoresis (2D-DIGE) followed by mass spectrometry. Results: We identified 75 proteins with a significant abundance difference between the different disease stages. Regulated proteins were mapped to existing biological networks by Ingenuity Pathway Analysis (IPA). Post-synaptic density protein 95 (DLG4), a key player in neuronal signalling and calcium-activated potassium channel alpha 1(KCNMA1), involved in neurotransmitter release, are 2 putative regulators connecting 64% of the proteins identified. The involvement of KCNMA1 in macrophage functionality was studied in vitro by using a specific functional blocker for KCNMA1, paxillin. We show that blocking of KCNMA1 altered myelin phagocytosis and proinflammatory cytokine release, disease mechanisms which are highly involved in EAE and MS pathology. We are currently investigating possible influences of this blocker on functionality of other disease relevant cells and processes using in vitro and in vivo models. Conclusions: This study will elucidate to what extent modulation via this ion channel affects disease processes in the context of EAE/MS. Publication appears at doi: 10.1111/imm.12002

Seven metabolites of 4-chlorotestosterone acetate were identified in urine of cattle that received a single injection of the drug. The steroids were isolated by means of a series of clean-up steps carried out before and after enzymatic hydrolysis. The obtained extract was fractionated by high-performance liquid chromatography and each fraction was examined both by high-performance thin-layer chromatography and by capillary gas chromatography—mass spectrometry of the m -ethoxime—trimethylsilyl derivatives. The metabolites were tentatively identified by studying the mass spectra of selected peaks not found in blank samples. The structures of two metabolites, viz. 4-chloroandrost-4-ene-3,17-dione and 4-chloroandrost-4-ene-3α,17β-diol were confirmed by chemical synthesis. The synthesized metabolites and 4-chloro-17α-testosterone, a third metabolite which was identified tentatively, were located on the thin-layer chromatograms obtained. This study led to the conclusion that the illegal use of 4-chlorotestosterone acetate can be detected by identifying one or more of its metabolites in urine.

The increasing antibiotic resistance in bacterial populations requires alternatives for classical treatment of infectious diseases and therefore drives the renewed interest in phage therapy. Methicillin resistant Staphylococcus aureus (MRSA) is a major problem in health care settings and live-stock breeding across the world. This research aims at a thorough microbiological, genomic, and proteomic characterization of S. aureus phage ISP, required for therapeutic applications. Host range screening of a large batch of S. aureus isolates and subsequent fingerprint and DNA microarray analysis of the isolates revealed a substantial activity of ISP against 86% of the isolates, including relevant MRSA strains. From a phage therapy perspective, the infection parameters and the frequency of bacterial mutations conferring ISP resistance were determined. Further, ISP was proven to be stable in relevant in vivo conditions and subcutaneous as well as nasal and oral ISP administration to rabbits appeared to cause no adverse effects. ISP encodes 215 gene products on its 138,339 bp genome, 22 of which were confirmed as structural proteins using tandem electrospray ionization-mass spectrometry (ESI-MS/MS), and shares strong sequence homology with the 'Twort-like viruses'. No toxic or virulence-associated proteins were observed. The microbiological and molecular characterization of ISP supports its application in a phage cocktail for therapeutic purposes. PC holds a postdoctoral fellowship of the 'Fonds voor Wetenschappelijk Onderzoek-Vlaanderen' (FWO-Vlaanderen, Belgium). This study was performed by members of the research community 'PhageBiotics' and was funded by the Federal Public Service of Health, Food Chain Safety and Environment, Belgium (contract RT 08/6 PHAGE). DNA fingerprinting and hybridization experiments were supported by grant MED 19 of the Royal High Institute for Defence, Belgium. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Formation of a protected biofilm environment is recognized as one of the major causes of the increasing antibiotic resistance development and emphasizes the need to develop alternative antibacterial strategies, like phage therapy. This study investigates the in vitro degradation of single-species Pseudomonas putida biofilms, PpG1 and RD5PR2, by the novel phage phi 15, a 'T7-like virus' with a virion-associated exopolysaccharide (EPS) depolymerase. Phage phi 15 forms plaques surrounded by growing opaque halo zones, indicative for EPS degradation, on seven out of 53 P. putida strains. The absence of haloes on infection resistant strains suggests that the EPS probably act as a primary bacterial receptor for phage infection. Independent of bacterial strain or biofilm age, a time and dose dependent response of phi 15-mediated biofilm degradation was observed with generally a maximum biofilm degradation 8 h after addition of the higher phage doses (10(4) and 10(6) pfu) and resistance development after 24 h. Biofilm age, an in vivo very variable parameter, reduced markedly phage-mediated degradation of PpG1 biofilms, while degradation of RD5PR2 biofilms and phi 15 amplification were unaffected. Killing of the planktonic culture occurred in parallel with but was always more pronounced than biofilm degradation, accentuating the need for evaluating phages for therapeutic purposes in biofilm conditions. EPS degrading activity of recombinantly expressed viral tail spike was confirmed by capsule staining. These data suggests that the addition of high initial titers of specifically selected phages with a proper EPS depolymerase are crucial criteria in the development of phage therapy. This research was partially supported by the K.U. Leuven (STRT1/10/021TBA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study. The authors thank Prof. M. Vaneechoutte (Laboratory Bacteriology, Ghent University Hospital, Belgium), Prof. R. De Mot (Centre of Microbial and Plant Genetics, K.U.Leuven, Belgium) and Prof. D. Springael (Division Soil and Water Management, K.U.Leuven, Belgium) for providing bacteria for this study. The authors are also grateful to Dr. Hans-W. Ackermann (Laval University, Quebec, Canada) for expert electron microscopy and to Dr. Y. Briers and Y. Born for providing information on the capsule staining assay (Institute of Food Science and Nutrition, ETH Zurich, Switzerland). Anneleen Cornelissen holds a predoctoral fellowship of the 'Instituut voor de aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen' (I.W.T., Belgium). Pieter-Jan Ceyssens is a postdoctoral fellow of the 'Fonds voor Wetenschappelijk Onderzoek-Vlaanderen' (FWO-Vlaanderen, Belgium).

The leaf proteome of 3-week-old Arabidopsis thaliana seedlings exposed for 1 week to low, environmentally realistic Cd concentrations was investigated. The data indicated that at 1 μM Cd, A. thaliana plants adapted their metabolism to cope with the Cd exposure. As a result, only moderate protein changes were observed. However, at 10 μM Cd, severe stress was indicated by growth reduction and chlorosis of rosette leaves at the macroscopic level and by lipid peroxidation and enhanced peroxidase activity at the cellular level. Of the 730 reproducible proteins among all gels, 21 were statistically upregulated in response to Cd. These proteins can be functionally grouped into 5 classes: proteins involved in (1) oxidative stress response, (2) photosynthesis and energy production, (3) protein metabolism, (4) gene expression and finally, (5) proteins with various or unknown function. In order to provide greater insight into the mechanisms induced on Cd exposure, a working model is proposed.

Giant bacteriophages phi KZ and EL of Pseudomonas aeruginosa contain 62 and 64 structural proteins, respectively, identified by ESI-MS/MS on total virion particle proteins. These identifications verify gene predictions and delineate the genomic regions dedicated to phage assembly and capsid formation (30 proteins were identified from a tailless phi KZ mutant). These data form the basis for future structural studies and provide insights into the relatedness of these large phages. The 4 KZ structural proteome strongly correlates to that of Pseudomonas chlororaphis bacteriophage 201 phi 2-1. Phage EL is more distantly related, shown by its 26 non-conserved structural proteins and the presence of genomic inversions.

The presence of missing values in gel-based proteomics data represents a real challenge if an objective statistical analysis is pursued. Different methods to handle missing values were evaluated and their influence is discussed on the selection of important proteins through multivariate techniques. The evaluated methods consisted of directly dealing with them during the multivariate analysis with the nonlinear estimation by iterative partial least squares (NIPALS) algorithm or imputing them by using either k-nearest neighbor or Bayesian principal component analysis (BPCA) before carrying out the multivariate analysis. These techniques were applied to data obtained from gels stained with classical postrunning dyes and from DIGE gels. Before applying the multivariate techniques, the normality and homoscedasticity assumptions on which parametric tests are based on were tested in order to perform a sound statistical analysis. From the three tested methods to handle missing values in our datasets, BPCA imputation of missing values showed to be the most consistent method. This research has been carried out in the framework of EU COST action 924. R. P. extends the acknowledgement to the International Relations Office of the K.U. Leuven (IRO Scholarship). Dr. S. C. C. is supported by a postdoctoral fellowship of the K. U. Leuven.

Cereals contain proteinaceous inhibitors of endo-beta-1,4-xylanases (E.C.3.2.1.8, xylanases). Since these xylanase inhibitors (XIs) are only active against xylanases of microbial origin and do not interact with plant endogenous xylanases, they are believed to act as a defensive barrier against phytopathogenic attack. So far, three types of XIs have been identified, i.e. Triticum aestivum XI (TAXI), xylanase inhibiting protein (XIP), and thaumatin-like XI (TLXI) proteins. In this study the variation in XI forms present in wheat grain was elucidated using high-resolution 2-DE in combination with LC-ESI-MS/MS and biochemical techniques. Reproducible 2-DE fingerprints of TAXI-, XIP-, and TLXI-type XIs, selectively purified from whole meal of three European wheat cultivars using cation exchange chromatography followed by affinity chromatography, were obtained using a pH-gradient of 6 to 11 and a molecular mass range of 10 to 60 kDa. Large polymorphic XI families, not known to date, which exhibit different pI- and/or molecular mass values, were visualised by colloidal CBB staining. Identification of distinct genetic variants by MS/MS-analysis provides a partial explanation for the observed XI heterogeneity. Besides genetic diversity, PTMs, such as glycosylation, account for the additional complexity of the 2-DE patterns.

The virulent Pseudomonas aeruginosa bacteriophage LUZ24 (45,625 bp) was isolated from hospital sewage. It belongs to the family of the Podoviridae , and carries a bidirectionally transcribed dsDNA genome delineated by two direct terminal repeats of 184 bp. In vitro transcriptional analysis identified seven σ 70 promoters, revealing a bias towards stronger promoter strength in the late genomic region. Reverse transcription demonstrated in vivo splicing of a 668 bp Group I intron embedded inside the DNA polymerase gene. Using mass spectrometry, nine structural proteins were identified as part of the phage particle. The lytic characteristics of LUZ24 are evaluated against its genomic content, which displays an overall 71% sequence similarity to the temperate phage PaP3.

Oligodendrocytes are glial cells responsible for the synthesis and maintenance of myelin in the central nervous system (CNS). Oligodendrocytes are vulnerable to damage occurring in a variety of neurological diseases. Understanding oligodendrocyte biology is crucial for the dissemination of de- and remyelination mechanisms. The goal of the present study is the construction of a protein database of mature rat oligodendrocytes. Post-mitotic oligodendrocytes were isolated from mature Wistar rats and subjected to immunocytochemistry. Proteins were extracted and analyzed by means of two-dimensional gel electrophoresis and two-dimensional liquid chromatography, both coupled to mass spectrometry. The combination of the gel-based and gel-free approach resulted in confident identification of a total of 200 proteins. A minority of proteins were identified in both proteomic strategies. The identified proteins represent a variety of functional groups, including novel oligodendrocyte proteins. The results of this study emphasize the power of the applied proteomic strategy to study known or to reveal new proteins and to investigate their regulation in oligodendrocytes in different disease models.

Phylogenetic analysis of the superfamily of D-2-hydroxyacid dehydrogenases identified the previously unrecognized cluster of glyoxylate/hydroxypyruvate reductases (GHPR). Based on the genome sequence of Rhizobium etli, the nodulating endosymbiont of the common bean plant, we predicted a putative 3-phosphoglycerate dehydrogenase to exhibit GHPR activity instead. The protein was overexpressed and purified. The enzyme is homodimeric under native conditions and is indeed capable of reducing both glyoxylate and hydroxypyruvate. Other substrates are phenylpyruvate and ketobutyrate. The highest activity was observed with glyoxylate and phenylpyruvate, both having approximately the same kcat/Km ratio. This kind of substrate specificity has not been reported previously for a GHPR. The optimal pH for the reduction of phenylpyruvate to phenyllactate is pH 7. These data lend support to the idea of predicting enzymatic substrate specificity based on phylogenetic clustering.

Neurological diseases, including multiple sclerosis (M.S.), often provoke changes in the functioning of the endothelial and epithelial brain barriers and give rise to disease-associated alterations of the cerebrospinal fluid (CSF) proteome. In the present study, pooled and ultrafiltered CSF of M. S. and non-M.S. patients was digested with trypsin and analyzed by off-line strong cation-exchange chromatography (SCX) coupled to on-line reversed-phase LC-ESI-MS/MS. In an alternative approach, the trypsin-treated subproteomes were analyzed directly by LC-ESI-MS/MS and gas-phase fractionation in the mass spectrometer. Taken together, both proteomic approaches in combination with a three-step evaluation process including the search engines Sequest and Mascot, and the validation software Scaffold, resulted in the identification of 148 proteins. Sixty proteins were identified in CSF for the first time by mass spectrometry. For validation purposes, the concentration of cystatin A was determined in individual CSF and serum samples of M. S. and non-M. S. patients using ELISA. ispartof: Journal of proteome research vol:5 issue:7 pages:1647-1657 ispartof: location:United States status: published

Natural populations thriving in heavy-metal-contaminated ecosystems are often subjected to selective pressures for increased resistance to toxic metals. In the present study we describe a population of the ectomycorrhizal fungus Suillus luteus that colonized a toxic Cu mine spoil in Norway. We hypothesized that this population had developed adaptive Cu tolerance and was able to protect pine trees against Cu toxicity. We also tested for the existence of cotolerance to Cu and Zn in S. luteus . Isolates from Cu-polluted, Zn-polluted, and nonpolluted sites were grown in vitro on Cu- or Zn-supplemented medium. The Cu mine isolates exhibited high Cu tolerance, whereas the Zn-tolerant isolates were shown to be Cu sensitive, and vice versa. This indicates the evolution of metal-specific tolerance mechanisms is strongly triggered by the pollution in the local environment. Cotolerance does not occur in the S. luteus isolates studied. In a dose-response experiment, the Cu sensitivity of nonmycorrhizal Pinus sylvestris seedlings was compared to the sensitivity of mycorrhizal seedlings colonized either by a Cu-sensitive or Cu-tolerant S. luteus isolate. In nonmycorrhizal plants and plants colonized by the Cu-sensitive isolate, root growth and nutrient uptake were strongly inhibited under Cu stress conditions. In contrast, plants colonized by the Cu-tolerant isolate were hardly affected. The Cu-adapted S. luteus isolate provided excellent insurance against Cu toxicity in pine seedlings exposed to elevated Cu levels. Such a metal-adapted Suillus - Pinus combination might be suitable for large-scale land reclamation at phytotoxic metalliferous and industrial sites.

Multiple sclerosis is an autoimmune inflammatory demyelinating disease of the central nervous system. Disease mechanisms in multiple sclerosis at the molecular level remain poorly understood and no reliable proteinaceous disease markers are available yet. The goal of the present study is the construction of a protein database of two-dimensional gel electrophoresis (2-DE) separated cerebrospinal fluid (CSF) proteins from multiple sclerosis patients. By means of liquid chromatography tandem mass spectrometry 65 different proteins were identified from 300 spots. Eighteen of these proteins have not been reported previously on 2-DE gels of CSF. Here we report on the identification of these proteins and discuss their potential relation to multiple sclerosis.

The objective of this intercomparison study was to evaluate the qualitative aspects and the interlaboratory performance of the method selected to be recommended as the official Community reference confirmatory method for the analysis of beta-agonists in animal feed. This method contains three possible options, i.e. a narrow range method for clenbuterol-type compounds based either on HPLC or on GCMS as the end-determination step and a broad range GCMS method for clenbuterol-type and salbutamol-type-beta-agonists. Three types of animal feed materials were provided: a series of blank materials and two series of materials contaminated with clenbuterol and salbutamol at a low and a high level, respectively. The results showed that the majority of the laboratories were able to identify blank, low and high level materials both for clenbuterol and salbutamol. For clenbuterol the narrow range GCMS method has been shown to be the most satisfactory. Although the participants had comments on the purity of the extracts obtained by means of the broad range method it was found appropriate as a multi-residue method which is able to measure simultaneously clenbuterol-type and salbutamol-type beta-agonists. A statistical evaluation of the quantitative measurement was also performed.

Solubilization of barley (Hordeum vulgare L.) thylakoid membranes with sodium dodecylsulphate plus sodium deoxycholate with or without Triton X-100 and subsequent fractionation in the polyacrylamide gel electrophoresis system described in this paper resulted: (1) in the resolution of the chlorophyll-proteins and chlorophyll-protein complexes commonly known as CP1a, CP1, LHCP(1), LHCP(2), CPa and LHCP(3); (2) in the highly increased stability of CP1 and CP1a, as judged by their chlorophyll content, (3) at the expense of the free pigment concentration (4) which could be reduced to a negligible amount. Some 40% of the total chlorophyll contained in the mature higher plant thylakoid membrane is associated with CP1 and CP1 a and as already suggested before [19] no significant amount of free chlorophyll occurs in vivo.

We present a detailed analysis of the genome architecture, structural proteome and infection-related properties of three Pseudomonas phages, designated LUZ7, LIT1 and PEV2 These podoviruses encapsulate 72 5 to 749 kb genomes and lyse their host after 25 min aerobic infection PEV2 can successfully infect under anaerobic conditions, but its latent period is tripled, the lysis proceeds far slower and the burst size decreases significantly While the overall genome structure of these phages resembles the well-studied coliphage N4, these Pseudomonas phages encode a cluster of tail genes which displays significant similarity to a Pseudomonas aeruginosa (cryptic) prophage region Using ESI-MS/MS, these tail proteins were shown to be part of the phage particle, as well as ten other proteins including a giant 370 kDa virion RNA polymerase These phages are the first described representatives of a novel kind of obligatory lytic P aeruginosa-infecting phages, belonging to the widespread "N4-like viruses" genus (C) 2010 Elsevier Inc All rights reserved We would also thank Dr Jane Burns at Children's Hospital, Seattle, for the gift of over 200 clinical pseudomonads. Pieter-Jan Ceyssens holds a postdoctoral scholarship (PDM) of the 'Bijzonder Onderzoeksfonds' at the K.U. Leuven

Cronobacter sakazakii is a Gram-negative pathogen found in milk-based formulae that causes infant meningitis. Bacteriophages have been proposed to control bacterial pathogens; however, comprehensive knowledge about a phage is required to ensure its safety before clinical application. We have characterized C. sakazakii phage vB_CsaM_GAP32 (GAP32), which possesses the second largest sequenced phage genome (358,663 bp). A total of 571 genes including 545 protein coding sequences and 26 tRNAs were identified, thus more genes than in the smallest bacterium, Mycoplasma genitalium G37. BLASTP and HHpred searches, together with proteomic analyses reveal that only 23.9% of the putative proteins have defined functions. Some of the unique features of this phage include: a chromosome condensation protein, two copies of the large subunit terminase, a predicted signal-arrest-release lysin; and an RpoD-like protein, which is possibly involved in the switch from immediate early to delayed early transcription. Its closest relatives are all extremely large myoviruses, namely coliphage PBECO4 and Klebsiella phage vB_KleM-RaK2, with whom it shares approximately 44% homologous proteins. Since the homologs are not evenly distributed, we propose that these three phages belong to a new subfamily. The authors thank Franco Pagotto, and Roger Stephan for providing bacterial strains: This work was financially supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) (NSERC IRCRJ 127376-07).