The discriminatory power of MALDI-TOF mass spectrometry to differentiate between isogenic teicoplanin-susceptible and teicoplanin-resistant strains of methicillin-resistant Staphylococcus aureus.

To explore the discriminatory power of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for detecting subtle differences in isogenic isolates, we tested isogenic strains of Staphylococcus aureus differing in their expression of resistance to methicillin or teicoplanin. More important changes in MALDI-TOF MS spectra were found with strains differing in methicillin than in teicoplanin resistance. In comparison, very minor or no changes were recorded in pulsed-field gel electrophoresis profiles or peptidoglycan muropeptide digest patterns of these strains, respectively. MALDI-TOF MS might be useful to detect subtle strain-specific differences in ionizable components released from bacterial surfaces and not from their peptidoglycan network.

Sternal wound infection after heart operations in pediatric patients associated with nasal carriage of Staphylococcus aureus.

A cluster of six pediatric cases of deep-seated Staphylococcus aureus infection after heart operations prompted us to perform molecular typing of the S. aureus isolates by pulsed-field gel electrophoresis. This revealed the presence of genotypically distinct isolates in four of the six patients. Isolates of two patients were genotypically identical. All patients carried S. aureus in the anterior nares. In each patient, the banding pattern of deoxyribonucleic acid in these isolates was indistinguishable from that in strains isolated from blood or wound cultures. Molecular typing with pulsed-field gel electrophoresis ruled out nosocomial transmission of S. aureus between four patients; at the same time, it provided evidence for an association between nasal colonization and postoperative wound infection. Epidemiologic investigation of potential links between two patients with identical isolates did not provide any evidence for nosocomial transmission of S. aureus between these patients. Because nasal colonization with S. aureus may be a risk factor for surgical wound infection in pediatric patients undergoing heart operations, preoperative decolonization appears to be warranted.

Comparison of double-locus sequence typing (DLST) and multilocus sequence typing (MLST) for the investigation of Pseudomonas aeruginosa populations.

Reliable molecular typing methods are necessary to investigate the epidemiology of bacterial pathogens. Reference methods such as multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) are costly and time consuming. Here, we compared our newly developed double-locus sequence typing (DLST) method for Pseudomonas aeruginosa to MLST and PFGE on a collection of 281 isolates. DLST was as discriminatory as MLST and was able to recognize "high-risk" epidemic clones. Both methods were highly congruent. Not surprisingly, a higher discriminatory power was observed with PFGE. In conclusion, being a simple method (single-strand sequencing of only 2 loci), DLST is valuable as a first-line typing tool for epidemiological investigations of P. aeruginosa. Coupled to a more discriminant method like PFGE or whole genome sequencing, it might represent an efficient typing strategy to investigate or prevent outbreaks.

Development and evaluation of double locus sequence typing for molecular epidemiological investigations of Clostridium difficile.

Despite the development of novel typing methods based on whole genome sequencing, most laboratories still rely on classical molecular methods for outbreak investigation or surveillance. Reference methods for Clostridium difficile include ribotyping and pulsed-field gel electrophoresis, which are band-comparing methods often difficult to establish and which require reference strain collections. Here, we present the double locus sequence typing (DLST) scheme as a tool to analyse C. difficile isolates. Using a collection of clinical C. difficile isolates recovered during a 1-year period, we evaluated the performance of DLST and compared the results to multilocus sequence typing (MLST), a sequence-based method that has been used to study the structure of bacterial populations and highlight major clones. DLST had a higher discriminatory power compared to MLST (Simpson's index of diversity of 0.979 versus 0.965) and successfully identified all isolates of the study (100 % typeability). Previous studies showed that the discriminatory power of ribotyping was comparable to that of MLST; thus, DLST might be more discriminatory than ribotyping. DLST is easy to establish and provides several advantages, including absence of DNA extraction [polymerase chain reaction (PCR) is performed on colonies], no specific instrumentation, low cost and unambiguous definition of types. Moreover, the implementation of a DLST typing scheme on an Internet database, such as that previously done for Staphylococcus aureus and Pseudomonas aeruginosa ( http://www.dlst.org ), will allow users to easily obtain the DLST type by submitting directly sequencing files and will avoid problems associated with multiple databases.

Simulations of electrophoretic collisions of DNA knots with gel obstacles

Gel electrophoresis can be used to separate nicked circular DNA molecules of equal length but forming different knot types. At low electric fields, complex knots drift faster than simpler knots. However, at high electric field the opposite is the case and simpler knots migrate faster than more complex knots. Using Monte Carlo simulations we investigate the reasons of this reversal of relative order of electrophoretic mobility of DNA molecules forming different knot types. We observe that at high electric fields the simulated knotted molecules tend to hang over the gel fibres and require passing over a substantial energy barrier to slip over the impeding gel fibre. At low electric field the interactions of drifting molecules with the gel fibres are weak and there are no significant energy barriers that oppose the detachment of knotted molecules from transverse gel fibres.

Unique genome replication mechanism of the archaeal virus AFV1.

The exceptional genomic content and genome organization of the Acidianus filamentous virus 1 (AFV1) that infects the hyperthermophilic archaeon Acidianus hospitalis suggest that this virus might exploit an unusual mechanism of genome replication. An analysis of replicative intermediates of the viral genome by two-dimensional (2D) agarose gel electrophoresis revealed that viral genome replication starts by the formation of a D-loop and proceeds via strand displacement replication. Characterization of replicative intermediates using dark-field electron microscopy, in combination with the 2D agarose gel electrophoresis data, suggests that recombination plays a key role in the termination of AFV1 genome replication through the formation of terminal loops. A terminal protein was found to be attached to the ends of the viral genome. The results allow us to postulate a model of genome replication that relies on recombination events for initiation and termination.

Does wheat genetically modified for disease resistance affect root-colonizing pseudomonads and arbuscular mycorrhizal fungi?

This study aimed to evaluate the impact of genetically modified (GM) wheat with introduced pm3b mildew resistance transgene, on two types of root-colonizing microorganisms, namely pseudomonads and arbuscular mycorrhizal fungi (AMF). Our investigations were carried out in field trials over three field seasons and at two locations. Serial dilution in selective King's B medium and microscopy were used to assess the abundance of cultivable pseudomonads and AMF, respectively. We developed a denaturing gradient gel electrophoresis (DGGE) method to characterize the diversity of the pqqC gene, which is involved in Pseudomonas phosphate solubilization. A major result was that in the first field season Pseudomonas abundances and diversity on roots of GM pm3b lines, but also on non-GM sister lines were different from those of the parental lines and conventional wheat cultivars. This indicates a strong effect of the procedures by which these plants were created, as GM and sister lines were generated via tissue cultures and propagated in the greenhouse. Moreover, Pseudomonas population sizes and DGGE profiles varied considerably between individual GM lines with different genomic locations of the pm3b transgene. At individual time points, differences in Pseudomonas and AMF accumulation between GM and control lines were detected, but they were not consistent and much less pronounced than differences detected between young and old plants, different conventional wheat cultivars or at different locations and field seasons. Thus, we conclude that impacts of GM wheat on plant-beneficial root-colonizing microorganisms are minor and not of ecological importance. The cultivation-independent pqqC-DGGE approach proved to be a useful tool for monitoring the dynamics of Pseudomonas populations in a wheat field and even sensitive enough for detecting population responses to altered plant physiology.

Clinical proteomics : a focus on transformed human T and B lymphocytes

Résumé large public La protéomique clinique est une discipline qui vise l'étude des protéines dans un but diagnostique ou thérapeutique. Nous avons utilisé cette approche pour étudier les lymphocytes T «tueurs » ou cytotoxiques qui font partie des globules blancs du système sanguin et agissent dans la lutte contre les infections et les tumeurs. Ces cellules sont impliquées dans l'immunothérapie cellulaire qui se fonde sur la capacité naturelle des ces lymphocytes à repérer les cellules tumorales et à les détruire. L'introduction du gène de la télomérase dans les lymphocytes T résulte en une prolongation de leur longévité, ce qui en ferait des candidats intéressants pour l'immunothérapie cellulaire. Il subsiste cependant des doutes quant aux conséquences de l'utilisation de ces lymphocytes «immortalisés ». Pour répondre à cette question, nous avons comparé le profile protéique de lymphocytes T cytotoxiques «jeunes » et vieux » avec celui des lymphocytes «immortalisés ». Nous avons trouvé que ces derniers présentent une double face et partagent à la fois les caractéristiques de la jeunesse et de la vieillesse. Dans une seconde étude de protéomique clinique, nous nous sommes penchés sur les lymphocytes B «immortalisés » cette fois-ci non pas avec la télomérase, mais avec le virus d'Epstein-Barr. Ces derniers sont utilisés comme modèle dans l'étude de la leucodystrophie, une maladie génétique rare qui affecte le cerveau. Notre but est d'identifier des marqueurs biologiques potentiels qui pourraient aider le diagnostic et le traitement de cette maladie neurodégénérative. Nous avons pour ce faire comparé les profiles protéiques des lymphocytes B «immortalisés » provenant d'individus sains et malades. Malheureusement, notre analyse n'a pas révélé de différences notoires entre ces deux classes de lymphocytes. Ceci nous permet toutefois de conclure que la maladie n'affecte pas la synthèse des protéines de manière prépondérante dans ces cellules sanguines. En résumé, le travail présenté dans cette thèse montre à la fois le potentiel et les limites de l'analyse des protéines lymphocytaires, dans différentes situations biologiques. Résumé La protéomique clinique ouvre la porte vers de multiples horizons relatifs au traitement de diverses maladies. Ce domaine particulier alliant la protéomique à la médecine, implique l'intervention de la biologie moléculaire et cellulaire. Dans notre étude, nous nous sommes d'abord intéressés aux lymphocytes T CD8+ cytotoxiques dans le contexte de l'immunothérapie adoptive. Le fondement de cette thérapie repose sur la capacité naturelle de ces lymphocytes à reconnaître les cellules tumorales et à les détruire chez les patients atteints de cancer. L'introduction du gène de la transcriptase réverse de la télomérase (hTERT) dans les lymphocytes T humains permet de rallonger leur durée de vie, sans toutefois induire d'altérations liées à la transformation. Cependant, des incertitudes subsistent quant à la ressemblance physiologique et biochimique entre ces cellules surexprirnant la télomérase et les cellules normales. Afin de répondre à cette question, nous avons comparé l'expression des protéines de lymphocytes humains T CD8+ «jeunes » et «vieux »avec celle de lymphocytes transduits avec hTERT. Nous avons trouvé que les lymphocytes T surexprimant la télomérase ont un profile protéique intermédiaire, avec certaines expressions protéiques similaires aux jeunes cellules T et d'autres se rapprochant des cellules vieilles. Dans la seconde partie de notre étude, nous nous sommes intéressés aux lymphocytes B transformés avec le virus d'Epstein-Barr provenant de patients atteints d'une maladie génétique rare du cerveau, la leucodystrophie. Dans cette maladie, des mutations dans le facteur de transcription eIF2B, impliqué dans la synthèse protéique, ont été trouvées. Afin d'analyser les conséquences de ces mutations et de trouver des biomarqueurs spécifiques à cette maladie, nous avons effectué une analyse protéomique des lymphoblastes provenant de malades et d'individus sains. Nous avons trouvé que les mutations dans le complexe ubiquitaire eIF2B n'affectent pas de manière significative l'expression des protéines des lymphoblastes mutés. En conclusion, notre travail illustre le potentiel et les limitations des technologies protéomiques utilisées pour disséquer l'implication des protéines dans différentes situations biologiques. Summary Clinical proteomics opens the door to multiple applications related to the treatment of diseases. This particular field is at the crossroad of proteomics and medicine and involves tools from cellular and molecular biology. We focused first our investigations on cytotoxic T cells in the context of adoptive immunotherapy, which is an interesting and evolving field. The basis of this therapy relies on the natural capacity of cytotoxic CD8+ T lymphocytes in recognizing tumor cells and destroying them in cancer patients. As their number is reduced, the idea would be to use transformed T lymphocytes with extended life span. Overexpression of telomerase into human T lymphocytes results in the extension of their replicative life span, but it still remains unclear whether these cells are physiologically indistinguishable from normal ones. To address this question, we compared the proteome of young and aged CD8+ T lymphocytes with that of T cells transduced with hTERT and found that the latter cells displayed an intermediate protein pattern, sharing similar protein expression with young, but also with aged T cells. We were then interested in studying Epstein-Barr virus transformed B lymphocytes in the context of a rare human brain genetic disorder called leukodystrophy. In this disease, mutations in the ubiquitous factor eIF2B involved in protein synthesis and its regulation have been reported. In order to analyze the functional consequences of the mutations and to find out specific biomarkers of eIF2B-related disorders, proteomic and peptidomic studies were carried out on lymphoblasts from eIF2Bmutated patients versus healthy patients. Following two-dimensional gel electrophoresis and mass fingerprints, mutations in the eIF2B complex did not appear to significantly affect the proteome of the mutated lymphoblasts extracts. To conclude, our work emphasizes the potentials and the limitations of the proteomic technologies used to analyze the role of lymphocyte proteins in different biological situations.

Sedimentation of macroscopic rigid knots and its relation to gel electrophoretic mobility of DNA knots.

We address the general question of the extent to which the hydrodynamic behaviour of microscopic freely fluctuating objects can be reproduced by macrosopic rigid objects. In particular, we compare the sedimentation speeds of knotted DNA molecules undergoing gel electrophoresis to the sedimentation speeds of rigid stereolithographic models of ideal knots in both water and silicon oil. We find that the sedimentation speeds grow roughly linearly with the average crossing number of the ideal knot configurations, and that the correlation is stronger within classes of knots. This is consistent with previous observations with DNA knots in gel electrophoresis.

Immuno-reactive proteins from Mycobacterium immunogenum useful for serodiagnosis of metalworking fluid hypersensitivity pneumonitis.

Metalworking fluid-associated hypersensitivity pneumonitis (MWF-HP) is a pulmonary disease caused by inhaling microorganisms present in the metalworking fluids used in the industrial sector. Mycobacterium immunogenum is the main etiological agent. Among the clinical, radiological and biological tools used for diagnosis, serological tests are important. The aim of this study was to identify immunogenic proteins in M. immunogenum and to use recombinant antigens for serological diagnosis of MWF-HP. Immunogenic proteins were detected by two-dimensional Western blot and candidate proteins were identified by mass spectrometry. Recombinant antigens were expressed in Escherichia coli and tested by enzyme-linked immunosorbent assay (ELISA) with the sera of 14 subjects with MWF-HP and 12 asymptomatic controls exposed to M. immunogenum. From the 350 spots visualized by two-dimensional gel electrophoresis with M. immunogenum extract, 6 immunogenic proteins were selected to be expressed as recombinant antigens. Acyl-CoA dehydrogenase antigen allowed for the best discrimination of MWF-HP cases against controls with an area under the receiver operating characteristics (ROC) curve of 0.930 (95% CI=0.820-1), a sensitivity of 100% and a specificity of 83% for the optimum threshold. Other recombinant antigens correspond to acyl-CoA dehydrogenase FadE, cytosol aminopeptidase, dihydrolipoyl dehydrogenase, serine hydroxymethyltransferase and superoxide dismutase. This is the first time that recombinant antigens have been used for the serodiagnosis of hypersensitivity pneumonitis. The availability of recombinant antigens makes it possible to develop standardized serological tests which in turn could simplify diagnosis, thus making it less invasive.

Proteomic analysis of cytokine induced proteins in human intestinal epithelial cells: implications for inflammatory bowel diseases.

A role for cytokine regulated proteins in epithelial cells has been suggested in the pathogenesis of inflammatory bowel diseases (IBD). The aim of this study was to identify such cytokine regulated targets using a proteomic functional approach. Protein patterns from (35)S-radiolabeled homogenates of cultured colon epithelial cells were compared before and after exposure to interferon-gamma, interleukin-1beta and interleukin-6. Proteins were separated by two-dimensional polyacrylamide gel electrophoresis. Both autoradiographies and silver stained gels were analyzed. Proteins showing differential expression were identified by tryptic in-gel digestion and mass spectrometry. Metabolism related proteins were also investigated by Western blot analysis. Tryptophanyl-tRNA synthetase, indoleamine-2,3-dioxygenase, heterogeneous nuclear ribonucleoprotein JKTBP, interferon-induced 35kDa protein, proteasome subunit LMP2 and arginosuccinate synthetase were identified as cytokine modulated proteins in vitro. Using purified epithelial cells from patients, overexpression of indoleamine-2,3-dioxygenase, an enzyme involved in tryptophan metabolism, was confirmed in Crohn's disease as well as in ulcerative colitis, as compared to normal mucosa. No such difference was found in diverticulitis. Potentially, this observation opens new avenues in the treatment of IBD.

Capsule permeability via polymer and protein ingress/egress.

Static incubation tests, where microcapsules and beads are contacted with polymer and protein solutions, have been developed for the characterization of permselective materials applied for bioartificial organs and drug delivery. A combination of polymer ingress, detected by size-exclusion chromatography, and protein ingress/ egress, assessed by gel electrophoresis, provides information regarding the diffusion kinetics, molar mass cutoff(MMCO) and permeability. This represents an improvement over existing permeability measurements that are based on the diffusion of a single type of solute. Specifically, the permeability of capsules based on alginate, cellulose sulfate, polymethylene-co-guanidine were characterized as a function of membrane thickness. Solid alginate beads were also evaluated. The MMCO of these capsules was estimated to be between 80 and 90 kDa using polymers, and between 116-150 kDa with proteins. Apparently, the globular shape of the proteins (radius of gyration (Rg) of 4.2-4.6 nm) facilitates their passage through the membrane, comparatively to the polysaccharide coil conformation (Rg of 6.5-8.3 nm). An increase of the capsule membrane thickness reduced these values. The MMCO of the beads, which do not have a membrane limiting their permselective properties, was higher, between 110 and 200 kDa with dextrans, and between 150 and 220 kDa with proteins. Therefore, although the permeability estimated with biologically relevant molecules is generally higher due to their lower radius of gyration, both the MMCO of synthetic and natural watersoluble polymers correlate well, and can be used as in vitro metrics for the immune protection ability of microcapsules and microbeads. This article shows, to the authors' knowledge, the first reported concordance between permeability measures based on model natural and biological macromolecules.

Proteomics fingerprinting of phagosome maturation and evidence for the role of a Galpha during uptake.

Phagocytosis, whether of food particles in protozoa or bacteria and cell remnants in the metazoan immune system, is a conserved process. The particles are taken up into phagosomes, which then undergo complex remodeling of their components, called maturation. By using two-dimensional gel electrophoresis and mass spectrometry combined with genomic data, we identified 179 phagosomal proteins in the amoeba Dictyostelium, including components of signal transduction, membrane traffic, and the cytoskeleton. By carrying out this proteomics analysis over the course of maturation, we obtained time profiles for 1,388 spots and thus generated a dynamic record of phagosomal protein composition. Clustering of the time profiles revealed five clusters and 24 functional groups that were mapped onto a flow chart of maturation. Two heterotrimeric G protein subunits, Galpha4 and Gbeta, appeared at the earliest times. We showed that mutations in the genes encoding these two proteins produce a phagocytic uptake defect in Dictyostelium. This analysis of phagosome protein dynamics provides a reference point for future genetic and functional investigations.

Production of an affinity-purified antibody against an aldehyde-treated neurofilament protein for use in immunocytochemistry.

Fixation enhances cellular morphology and reduces loss of molecules during tissue processing. Antibodies against fixation-resistant epitopes are very useful, because they allow an immunocytochemical detection in tissue of better preserved morphology. However, fixatives can alter antigenicity and adversely affect the result of immunohistochemical procedures. To address this problem, this study examined the feasibility of generating antibodies to a paraformaldehyde-fixed antigen for use in immunohistochemical procedures. The large subunit of neurofilament proteins was selected for this study. Crude neurofilament proteins were isolated and separated by SDS-polyacrylamide gel electrophoresis. The large subunit of neurofilaments (NF-H) was electroeluted from the electrophoresis gel and exposed to paraformaldehyde, and used for immunization of a rabbit. The rabbit antiserum was affinity purified on CNBr-sepharose immobilized neurofilament proteins. On Western blots, the antibody reacted with the NF-H protein in a phosphorylation-dependent manner. In aldehyde-fixed cerebellum, the antibody strongly stained axons. In contrast, in alcohol-fixed cryostat sections the immunocytochemical detection was substantially reduced. The procedure presented in this study, involving a simple pretreatment of the immunogen, allows for the generation of an antibody that may be used in immunohistochemical studies where localization of the immunogen may be reduced or even lost by aldehyde fixation.

Purification and characterization of (Na+ + K+)-ATPase from toad kidney

This report describes the partial purification and the characteristics of (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) from an amphibian source. Toad kidney microsomes were solubilized with sodium deoxycholate and further purified by sodium dodecyl sulphate treatment and sucrose gradient centrifugation, according to the methods described by Lane et al. [(1973) J. Biol. Chem. 248, 7197--7200], Jørgensen [(1974) Biochim. Biophys. Acta 356, 36--52] and Hayashi et al. [(1977) Biochim. Biophys. Acta 482, 185--196]. (Na+ + K+)-ATPase preparations with specific activities up to 1000 mumol Pi/mg protein per h were obtained. Mg2+-ATPase only accounted for about 2% of the total ATPase activity. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed three major protein bands with molecular weights of 116 000, 62 000 and 26 000. The 116 000 dalton protein was phosphorylated by [gamma-32P]ATP in the presence of sodium but not in the presence of potassium. The 62 000 dalton component stained for glycoproteins. The Km for ATP was 0.40 mM, for Na+ 12.29 mM and for K+ 1.14 mM. The Ki for ouabain was 35 micron. Temperature activation curves showed two activity peaks at 37 degrees C and at 50 degrees C. The break in the Arrhenius plot of activity versus temperature appeared at 15 degrees C.