Evaluation of the MRSA/SA ELITe MGB Assay for the Detection of Staphylococcus aureus in Bone and Joint Infections.

Bone and joint infections represent a potentially devastating complication of prosthetic orthopedic joint replacement, thus requiring both rapid and appropriate antibiotic treatment. Staphylococcus aureus is one of the most common pathogens involved in this pathology. Being able to assert its presence is the first step of efficient patient management. This monocenter study evaluated the MRSA/SA ELITe MGB assay for the molecular detection of S. aureus and methicillin-resistant S. aureus (MRSA) in bone and joint biopsy specimens and synovial fluids. This test, together with conventional techniques, including standard cultures and the 16S rRNA amplification assay, was performed on 208 successive perioperative samples collected prospectively for 1 year obtained from 129 patients. Using conventional techniques, we detected a microbial pathogen in 76 samples from 58 patients, 40 of which were identified as S. aureus. The limit of detection (LOD) of the MRSA/SA ELITe MGB assay was experimentally determined for bone and joint biopsy specimens and synovial fluids using negative samples spiked with S. aureus ATCC 43300. The sensitivities of S. aureus detection with the MRSA/SA ELITe MGB assay were 82.5% (33/40 samples) and 97.5% (39/40 samples) using the manufacturer's LOD and an experimentally determined LOD, respectively. Interestingly, using the osteoarticular specific LOD, 15 additional samples were determined to be positive for S. aureus DNA with the MRSA/SA ELITe MGB assay; in all cases, these samples were obtained from patients considered to be infected with S. aureus according to their clinical and microbiological records. The results were available within 24 h, which could help to expedite therapeutic decisions.

The complex relationship between effort and heart rate: a hint from dynamic analysis.

OBJECTIVE: Dynamic analysis can be used to study the changes of self-regulated biological processes driven by external stimuli. Recently, the changes of heart rate during effort tests has successfully been adjusted using a simple first-order differential equation model driven by body power expenditure. Although this approach produces valid estimates and yields pertinent indices for the analysis of such measurements, it suffers from an inability to model the saturation of the heart-rate increase at high power expenditures and the change of heart-rate equilibrium following effort. APPROACH: We propose a new analysis allowing the estimation of changes of the heart rate in response to effort (gain) as a function of the power expenditure value. MAIN RESULTS: When applied to the measured heart rates of 30 amateur athletes performing a maximum graded-effort treadmill test, the proposed model was able to predict 99% of the heart rate change measured during exercise. The estimated gains decreased with a power increase above the first ventilatory threshold. This trend was stronger above the second ventilatory threshold and was strongly correlated with the maximum oxygen consumption. SIGNIFICANCE: The proposed approach yields a highly precise model of heart rate dynamics during variable effort that reflects the changes of metabolic energy systems at play during exercise.

Validity of dynamical analysis to characterize heart rate and oxygen consumption during effort tests.

Performance is usually assessed by simple indices stemming from cardiac and respiratory data measured during graded exercise test. The goal of this study is to characterize the indices produced by a dynamical analysis of HR and VO2 for different effort test protocols, and to estimate the construct validity of these new dynamical indices by testing their links with their standard counterparts. Therefore, two groups of 32 and 14 athletes from two different cohorts performed two different graded exercise testing before and after a period of training or deconditioning. Heart rate (HR) and oxygen consumption (VO2) were measured. The new dynamical indices were the value without effort, the characteristic time and the amplitude (gain) of the HR and VO2 response to the effort. The gain of HR was moderately to strongly associated with other performance indices, while the gain for VO2 increased with training and decreased with deconditioning with an effect size slightly higher than VO2 max. Dynamical analysis performed on the first 2/3 of the effort tests showed similar patterns than the analysis of the entire effort tests, which could be useful to assess individuals who cannot perform full effort tests. In conclusion, the dynamical analysis of HR and VO2 obtained during effort test, especially through the estimation of the gain, provides a good characterization of physical performance, robust to less stringent effort test conditions.

Ultra-endurance events in tropical environments and countermeasures to optimize performances and health.

Physical performance in a tropical environment, combining high heat and humidity, is a difficult physiological challenge that requires specific preparation. The elevated humidity of a tropical climate impairs the thermoregulatory mechanisms by limiting the rate of sweat evaporation. Hence, a proper management of whole-body temperature is required to complete an ultra-endurance event in such an environment. In these long-duration events, which can last from 8 to 20 h, held in hot and humid settings, performance is tightly linked to the ability in maintaining an optimal hydration status. Indeed, the rate of withdrawal in these longer races was associated with lower water intake, and the majority of finishers exhibited alterations in electrolyte balance (e.g., sodium). Hence, this work reviews the effects on performance of high heat and humidity in two representative ultra-endurance sports, ultramarathons and long-distance triathlons, and several countermeasures to counteract the impact of these harsh environmental stresses and maintain a high level of performance, such as hydration, cooling strategies and heat acclimation.

Pre-radiation chemotherapy improves survival in pediatric diffuse intrinsic pontine gliomas.

BACKGROUND: The median survival of patients with diffuse intrinsic pontine glioma (DIPG) remains less than 1 year. The BSG 98 pre-irradiation chemotherapy protocol showed a significant increase in overall survival. In contrast to current treatment strategies, patients did not have to undergo surgical stereotactic biopsy, which can sometimes lead to complications, to be included in this protocol. MATERIALS AND METHODS: We retrospectively reviewed all the cases of DIPG that were treated in our department from September 15, 2004 to September 15, 2014. We compared the group of patients who followed our BSG 98 protocol to those who were treated with new targeted therapy protocols where systematic biopsy was required. RESULTS: Patients in the BSG 98 protocol were treated with BCNU, cisplatin, and methotrexate, followed by radiation at disease progression. Targeted therapy protocols included radiation therapy along with treatment by erlotinib, cilengitide, or an association of nimotuzumab and vinblastine. Sixteen patients were treated with the BSG 98 protocol, and 9 patients were treated with new targeted therapy protocols. Median overall survival was significantly higher in the BSG 98 group compared to the targeted therapy group (16.1 months (95 % CI, 10.4-19.0) vs 8.8 months (95 % CI 1.4-12.3); p = 0.0003). An increase in the median progression-free survival was observed (respectively, 8.6 vs 3.0 months; p = 0.113). CONCLUSION: The present study confirms that the BSG 98 protocol is one of the most effective current treatment strategies for DIPG. It may be used as the control arm in randomized trials investigating the use of innovative treatments and may be proposed to families who are averse to biopsy.

Evaluation of the Microsemi CRP, an automated hematology analyzer for rapid 3-part WBC differential and CRP using whole blood.

INTRODUCTION: We evaluated the basic performance of Microsemi CRP, an unique automated hematology analyzer which can simultaneously measure CBC including 3-part WBC differential (3-Diff) and CRP using whole blood treated with EDTA-2K anticoagulant. METHOD: We found that it produced generally the acceptable results for all parameters performed (repeatability, reproducibility, linearity, interference effect, carry over, and correlation) using control materials, fresh human whole bloods, and serum samples. RESULTS: CBC data examined using Microsemi CRP showed the good correlation with the previous model, Micros CRP200 (r ≧ 0.9), and also those obtained using the routine analyzer, ADVIA 2120i (r ≧ 0.989). Concerning the 3-Diff, both GRA (%) and LYM (%) showed the excellent correlation coefficient between Microsemi CRP and Micros CRP200 (r ≧ 0.992) as well as ADVIA 2120i (r ≧ 0.957). MON (%) showed good correlation between Microsemi CRP and Micros CRP200 (r = 0.959), but lower correlation between Microsemi CRP and ADVIA 2120 i (r = 0.471). CRP data showed the good correlation with HITACHI7600 (r ≧ 0.997) and Micros CRP200 (r ≧ 0.997). CONCLUSION: From these findings, we concluded that Microsemi CRP seemed the convenient laboratory analyzer in the setting of point of care testing (POCT) especially at NICU or primary care unit.

Assessing implications between genotypic and phenotypic variables through lattice analysis.

A previous paper assessed a "Molecular Mapping of Twenty-Four Features of Down Syndrome on Chromosome 21" (Delabar et al., 1993), by analyzing the genotypes/phenotypes of patients suffering from partial trisomy. The mapping was defined through implications--each feature was mapped to the conjunction of cytogenetic bands that were shared by all patients having that feature. In the present paper, we extend that approach to determine how far those implications depart from defining equivalences. Finding equivalences is important. Local equivalences permit a genetic characterization of a feature. And if global equivalences held for all features, that set of bands would be sufficient to characterize the various phenotypes observed in individuals with partial trisomy 21. To extend the earlier approach, we examine the structure of equivalences as well as the structure of implications. We examine both conjunctions of bands and conjunctions of features. The use of Galois lattices permits simultaneous evaluation of both kinds of structures. Each Galois lattice is labeled with a basis (minimal generating set) of implications going from conjunctions of features into bands and those going from conjunctions of bands into features. Analysis reveals that about half of the conjunctions of bands that characterize the genetic structure embody equivalences. This allows us to improve the genetic description of features and to specify minimal sets of questions that need to be investigated to make the global genetic description more precise.

Substrate recognition domains within extracellular signal-regulated kinase mediate binding and catalytic activation of mitogen-activated protein kinase phosphatase-3.

Mitogen-activated protein (MAP) kinase phosphatase-3 (MKP-3) is a dual specificity phosphatase that inactivates extracellular signal-regulated kinase (ERK) MAP kinases. This reflects tight and specific binding between ERK and the MKP-3 amino terminus with consequent phosphatase activation and dephosphorylation of the bound MAP kinase. We have used a series of p38/ERK chimeric molecules to identify domains within ERK necessary for binding and catalytic activation of MKP-3. These studies demonstrate that ERK kinase subdomains V-XI are necessary and sufficient for binding and catalytic activation of MKP-3. These domains constitute the major COOH-terminal structural lobe of ERK. p38/ERK chimeras possessing these regions display increased sensitivity to inactivation by MKP-3. These data also reveal an overlap between ERK domains interacting with MKP-3 and those known to confer substrate specificity on the ERK MAP kinase. Consistent with this, we show that peptides representing docking sites within the target substrates Elk-1 and p90(rsk) inhibit ERK-dependent activation of MKP-3. In addition, abolition of ERK-dependent phosphatase activation following mutation of a putative kinase interaction motif (KIM) within the MKP-3 NH(2) terminus suggests that key sites of contact for the ERK COOH-terminal structural lobe include residues localized between the Cdc25 homology domains (CH2) found conserved between members of the DSP gene family.

Loss of aggression, after transfer onto a C57BL/6J background, in mice carrying a targeted disruption of the neuronal nitric oxide synthase gene.

Phenotypic differences among mice with disrupted genes and those with wild-type alleles have not provided the necessary evidence for desired gene/phenotype correlations. These differences could be due to "passenger genes" from the donor 129 strains that are used to produce stem cells. Three variations of attack behavior were measured, using mice carrying a disruption of the neural nitric oxide synthase gene. In the first population, the disrupted gene had been maintained on a mixed background including C57BL/6J and 129 alleles. We have developed a second population in which the disrupted gene was transferred onto a C57BL/6J background during five backcross generations. On the mixed C57BL/6J-129 background, mice homozygous for disrupted Nos1 alleles attacked more frequently, had shorter attack latencies, and presented a greater number of attacks than mice carrying nondisrupted alleles. On the C57BL/6J background, no significant difference persisted between the carriers of the disrupted gene and their noncarrier siblings. The noncarriers on the mixed C57BL/6J-129 background, and the carriers or noncarriers on the C57BL/6J background, did not differ from C57BL/6J. The frequency of attacking males was identical in the homozygous carriers of the disrupted gene, in the mixed C57BL/6J-129 background, and in the 129/SvPas, which approximates the 129/SvJae strain from which the stem cells were derived to produce the disrupted Nos1 gene. These results suggest that Nos1 disruption was not implicated in attack behavior. A possible passenger-gene effect from the 129 donor strain is discussed.

Catalytic activation of the phosphatase MKP-3 by ERK2 mitogen-activated protein kinase.

MAP kinase phosphatase-3 (MKP-3) dephosphorylates phosphotyrosine and phosphothreonine and inactivates selectively ERK family mitogen-activated protein (MAP) kinases. MKP-3 was activated by direct binding to purified ERK2. Activation was independent of protein kinase activity and required binding of ERK2 to the noncatalytic amino-terminus of MKP-3. Neither the gain-of-function Sevenmaker ERK2 mutant D319N nor c-Jun amino-terminal kinase-stress-activated protein kinase (JNK/SAPK) or p38 MAP kinases bound MKP-3 or caused its catalytic activation. These kinases were also resistant to enzymatic inactivation by MKP-3. Another homologous but nonselective phosphatase, MKP-4, bound and was activated by ERK2, JNK/SAPK, and p38 MAP kinases. Catalytic activation of MAP kinase phosphatases through substrate binding may regulate MAP kinase activation by a large number of receptor systems.

The mitogen-activated protein kinase phosphatase-3 N-terminal noncatalytic region is responsible for tight substrate binding and enzymatic specificity.

We have reported recently that the dual specificity mitogen-activated protein kinase phosphatase-3 (MKP-3) elicits highly selective inactivation of the extracellular signal-regulated kinase (ERK) class of mitogen-activated protein (MAP) kinases (Muda, M., Theodosiou, A., Rodrigues, N., Boschert, U., Camps, M., Gillieron, C., Davies, K., Ashworth, A., and Arkinstall, S. (1996) J. Biol. Chem. 271, 27205-27208). We now show that MKP-3 enzymatic specificity is paralleled by tight binding to both ERK1 and ERK2 while, in contrast, little or no interaction with either c-Jun N-terminal kinase/stress activated protein kinase (JNK/SAPK) or p38 MAP kinases was detected. Further study revealed that the N-terminal noncatalytic domain of MKP-3 (MKP-3DeltaC) binds both ERK1 and ERK2, while the C-terminal MKP-3 catalytic core (MKP-3DeltaN) fails to precipitate either of these MAP kinases. A chimera consisting of the N-terminal half of MKP-3 with the C-terminal catalytic core of M3-6 also bound tightly to ERK1 but not to JNK3/SAPKbeta. Consistent with a role for N-terminal binding in determining MKP-3 specificity, at least 10-fold higher concentrations of purified MKP-3DeltaN than full-length MKP-3 is required to inhibit ERK2 activity. In contrast, both MKP-3DeltaN and full-length MKP-3 inactivate JNK/SAPK and p38 MAP kinases at similarly high concentrations. Also, a chimera of the M3-6 N terminus with the MKP-3 catalytic core which fails to bind ERK elicits non selective inactivation of ERK1 and JNK3/SAPKbeta. Together, these observations suggest that the physiological specificity of MKP-3 for inactivation of ERK family MAP kinases reflects tight substrate binding by its N-terminal domain.

Induction of the mitogen-activated protein kinase phosphatase MKP3 by nerve growth factor in differentiating PC12.

In PC12 sympathetic neurons activation and nuclear translocation of ERK family MAP kinases plays an essential role in processes underlying nerve growth factor (NGF)-dependent differentiation. We have recently cloned MKP-3 as a novel dual specificity phosphatase displaying selectivity towards inactivation of the ERK1 and ERK2 MAP kinases. Here we report that in PC12 cells, MKP-3 undergoes powerful and specific up-regulation by NGF while a number of mitogens and cellular stresses are ineffective. NGF-stimulated MKP-3 expression appears after 1 h, is maximal at 3 h, and is sustained for 5 days. This coincides with a critical period of neurite outgrowth and terminal differentiation. Consistent with a role mediating inhibition of PC12 cell MAP kinases, NGF-stimulated ERK2 activation was suppressed considerably following pretreatment with fibroblast growth factor and 9-cis-retinal, two additional differentiation factors found to induce powerfully MKP-3 expression. Given the clear cytosolic localization of MKP3 in PC12 cells and sympathetic neurons, these results suggest a critical role for inactivating ERK MAP kinases in non-nuclear compartments during essential stages of NGF-mediated PC12 differentiation.

Bcl-2 undergoes phosphorylation by c-Jun N-terminal kinase/stress-activated protein kinases in the presence of the constitutively active GTP-binding protein Rac1.

We have studied the phosphorylation of the Bcl-2 family of proteins by different mitogen-activated protein (MAP) kinases. Purified Bcl-2 was found to be phosphorylated by the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) p54-SAPKbeta, and this is specific insofar as the extracellular signal-regulated kinase 1 (ERK1) and p38/RK/CSBP (p38) catalyzed only weak modification. Bcl-2 undergoes similar phosphorylation in COS-7 when coexpressed together with p54-SAPKbeta and the constitutive Rac1 mutant G12V. This is seen by both 32PO4 labeling and the appearance of five discrete Bcl-2 bands with reduced gel mobility. As anticipated, both intracellular p54-SAPKbeta activation and Bcl-2 phosphorylation are blocked by co-transfection with the MAP kinase specific phosphatase MKP3/PYST1. MAP kinase specificity is also seen in COS-7 cells as Bcl-2 undergoes only weak phosphorylation when co-expressed with enzymatically activated ERK1 or p38. Four critical residues undergoing phosphorylation in COS-7 cells were identified by expression of the quadruple Bcl-2 point mutant T56A,S70A,T74A, S87A. Sequencing phosphopeptides derived from tryptic digests of Bcl-2 indicates that purified GST-p54-SAPKbeta phosphorylates identical sites in vitro. This is the first report of Bcl-2 phosphorylation by the JNK/SAPK class of MAP kinases and could indicate a key modification allowing control of Bcl-2 function by cell surface receptors, Rho family GTPases, and/or cellular stresses.

[Evaluation of reticulocyte count on Cell-Dyn 3500 automate: comparison with a flow cytometry technique]. / Evaluation de la numération des réticulocytes sur automate Cell-Dyn 3500: comparaison avec une technique par cytométrie en flux.

Cell-Dyn 3500 automate (Abbott) provides reticulocyte count using a recent software, after ribosomal RNA colouring with methylene blue reagent. The aim of this study was to evaluate results from Cell-Dyn 3500, and to compare them with the routine laboratory technique by flow cytometry (FacScan, Becton-Dickinson). Cell-Dyn 3500 analytical parameters (inter- and intra-assay precision, sample-to-sample contamination) were good and in accord to manufacturer's specifications. Reticulocyte values from blood samples kept at 4 degrees C during 3 days were unchanged. Usual values in 145 normal patients were found between 71.3 and 78.3 G/l with Cell-Dyn 3500, between 119.3 and 130.2 G/l with FacScan. Both techniques were compared in 87 patients with various anemias: Cell-Dyn 3500 showed more frequently high reticulocyte values in regenerative anemias (hemorrhagic), but was less performant for classification of aregenerative anemias. Reticulocyte count with Cell-Dyn 3500 is a semi automatic, easy to perform and swift technique, using no toxic reagent but unitary test tubes kept at room temperature. The test cost is however high, and Cell-Dyn 3500 cannot be used during the analysis.

Molecular cloning and functional characterization of a novel mitogen-activated protein kinase phosphatase, MKP-4.

Extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38/RK/CSBP (p38) mitogen-activated protein (MAP) kinases are target enzymes activated by a wide range of cell-surface stimuli. Recently, a distinct class of dual specificity phosphatase has been shown to reverse activation of MAP kinases by dephosphorylating critical tyrosine and threonine residues. By searching the expressed sequence tag data base (dbEST) for homologues of known dual specificity phosphatases, we identified a novel partial human sequence for which we isolated a full-length cDNA (termed MKP-4). The deduced amino acid sequence of MKP-4 is most similar to MKP-X/PYST2 (61% identity) and MKP-3/PYST1 (57% identity), includes two N-terminal CH2 domains homologous to the cell cycle regulator Cdc25 phosphatase, and contains the extended active site sequence motif VXVHCXAGXSRSXTX3AYLM (where X is any amino acid) conserved in dual specificity phosphatases. MKP-4 produced in Escherichia coli catalyzes vanadate-sensitive breakdown of p-nitrophenyl phosphate as well as in vitro inactivation of purified ERK2. When expressed in COS-7 cells, MKP-4 blocks activation of MAP kinases with the selectivity ERK > p38 = JNK/SAPK. This cellular specificity is similar to MKP-3/PYST1, although distinct from hVH-5/M3-6 (JNK/SAPK = p38 >>> ERK). Northern analysis reveals a highly restricted tissue distribution with a single MKP-4 mRNA species of approximately 2.5 kilobases detected only in placenta, kidney, and embryonic liver. Immunocytochemical analysis showed MKP-4 to be present within cytosol although punctate nuclear staining co-localizing with promyelocytic protein was also observed in a subpopulation (10-20%) of cells. Chromosomal localization by analysis of DNAs from human/rodent somatic cell hybrids and a panel of radiation hybrids assign the human gene for MKP-4 to Xq28. The identification and characterization of MKP-4 highlights the emergence of an expanding family of structurally homologous dual specificity phosphatases possessing distinct MAP kinase specificity and subcellular localization as well as diverse patterns of tissue expression.

Mapping regions of G alpha q interacting with PLC beta 1 using multiple overlapping synthetic peptides.

The heterotrimeric G-protein alpha-chain G alpha q plays a critical role mediating receptor-linked activation of the beta isoforms of PLC which hydrolyse membrane inositol-containing phospholipids to generate the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. Despite knowledge of the three-dimensional structure of two G-protein alpha-chains (G alpha t and G alpha i1) as well as high regional amino acid conservation between members of the G-protein alpha-chain family, the precise molecular domains of G alpha q mediating activation of PLC beta 1 are unknown. To map sites responsible for effector interaction we employed 188 peptides each of 15 residues and corresponding to overlapping regions of the complete G alpha q sequence. These were tested for their ability to inhibit G alpha q-dependent activation of recombinant PLC beta 1 using an in vitro reconstitution assay. Peptides from two regions of G alpha q mediated up to 100% inhibition of GTP gamma S-stimulated PLC beta 1 activity, and representative peptides from each of these regions were half-maximally effective at 69.3 +/- 27.4 microM (n = 4) (G alpha q: 251-265) and 110.0 +/- 41.9 microM (n = 4) (G alpha q: 306-319). G alpha q regions described by inhibitory peptides are conserved selectively in other G-protein alpha-chains linked to PLC beta 1 activation (G alpha 11, G alpha 14) and correspond spatially to sites of effector interaction identified in G alpha s by scanning mutagenesis and in transducin using site-specific antibodies and peptides. Computer transducin using site-specific antibodies and peptides. Computer homology modelling of G alpha q based on the crystal structure of transducin indicates that regions interacting with PLC beta 1 form two parallel alpha-helices lying at the surface of the G alpha q structure. These observations provide the first description of two regions within G alpha q critically important for activating PLC beta 1, and moreover, indicate that effector binding domains identified in transducin and G alpha s are also conserved spatially in G alpha q.

["I non-exist". Paradoxes of death in adolescence]. / "J'inexiste". Les paradoxes de la mort á l'adolescence.

This text presents, from the case study of the analytical treatment of a young girl after a very serious suicide attempt, a theoretical reflection on the question of death during adolescence and its place and function in the psyche. The hypothesis suggested is that resorting to the "idea of death" constitutes an attempt to neutralise the excitement created by the excess of representational activities. A parallel is suggested between the way death and pain are used as a protective shield.