Do patients need advice and information to prevent infections - results of a single centre structured survey.

Background: Healthcare-associated infections are a major burden for hospitals, leading to morbidity and mortality and unnecessary medical costs. They can probably be reduced through what is known as patient empowerment. This study aims to address the question of whether patients are interested in receiving infection prevention and control information. Methods: Patients were asked in structured interviews whether they would like more information on infection prevention and control. Inclusion criteria comprised 2 groups of patients. Group 1 were patients undergoing elective total endoprosthesis (TEP) and Group 2 were patients tested positive for meticillin-resistant Staphylococcus aureus (MRSA). Results: The response rate was 38.4 % (163/425 patients). Approximately 75 % of the patients were interested in information on infection prevention and control. The topics of interest differed between the two patient groups: MRSA patients had a higher need for infection prevention and control information. TEP patients showed a high acceptance of antiseptic body wash and a willingness to pay for it themselves. Information given to patients should be group-specific and timely. Conclusion: Our data suggest a lack of information on infection prevention and control among patients and underline the importance of patient empowerment. The willingness of patients to pay personally for antiseptic wash should be assessed further.

Spatially resolved proteomics in osteoarthritis: State of the art and new perspectives.

Osteoarthritis (OA) is one of the most common diseases worldwide caused by chronic degeneration of the joints. Its high prevalence and the involvement of several tissues define OA as a highly heterogeneous disease. New biological markers to evaluate the progression of the pathology and improve its prognosis are needed. Among all the different -omic strategies applied to OA, solution phase bottom-up proteomics has made an extensive contribution to the field of biomarker research. However, new technologies for protein analysis should be considered for a better understanding of the disease. This review focuses on complementary proteomic methodologies and new technologies for translational research of OA and other rheumatic pathologies, especially mass spectrometry imaging and protein imaging methods not applied by the OA community yet.

Polydnavirus replication: the EP1 segment of the parasitoid wasp Cotesia congregata is amplified within a larger precursor molecule.

Polydnaviruses are unique viruses: they are essential for successful parasitism by tens of thousands of species of parasitoid wasps. These viruses are obligatorily associated with the wasps and are injected into the host during oviposition. Molecular analyses have shown that each virus sequence in the segmented polydnavirus genome is present in the wasp DNA in two forms: a circular form found in the virus particles and an integrated form found in the wasp chromosomes. Recent studies performed on polydnaviruses from braconid wasps suggested that the circular forms were excised from the chromosome. The different forms of the EP1 circle of Cotesia congregata polydnavirus during the pupal-adult development of the parasitoid wasp were analysed. Unexpectedly, an off-size fragment formerly used to diagnose the integration of the EP1 sequence into wasp genomic DNA was found to be amplified in female wasps undergoing virus replication. The EP1 sequence is amplified within a larger molecule comprising at least two virus segments. The amplified molecule is different from the EP1 chromosomally integrated form and is not encapsidated into virus particles. These findings shed light on a new step towards EP1 circle production: the amplification of virus sequences preceding individual circle excision.

Chromosome segregation.

The ability to visualise specific genes and proteins within bacterial cells is revolutionising knowledge of chromosome segregation. The essential elements appear to be the driving force behind DNA replication, which occurs at fixed cellular positions, the condensation of newly replicated DNA by a chromosome condensation machine located at the cell 1/4 and 3/4 positions, and molecular machines that act at midcell to allow chromosome separation after replication and movement of the sister chromosomes away from the division septum prior to cell division. This review attempts to provide a perspective on current views of the bacterial chromosome segregation mechanism and how it relates to other cellular processes.

Circles: the replication-recombination-chromosome segregation connection.

Crossing over by homologous recombination between monomeric circular chromosomes generates dimeric circular chromosomes that cannot be segregated to daughter cells during cell division. In Escherichia coli, homologous recombination is biased so that most homologous recombination events generate noncrossover monomeric circular chromosomes. This bias is lost in ruv mutants. A novel protein, RarA, which is highly conserved in eubacteria and eukaryotes and is related to the RuvB and the DnaX proteins, gamma and tau, may influence the formation of crossover recombinants. Those dimeric chromosomes that do form are converted to monomers by Xer site-specific recombination at the recombination site dif, located in the replication terminus region of the E. coli chromosome. The septum-located FtsK protein, which coordinates cell division with chromosome segregation, is required for a complete Xer recombination reaction at dif. Only correctly positioned dif sites present in a chromosomal dimer are able to access septum-located FtsK. FtsK acts by facilitating a conformational change in the Xer recombination Holliday junction intermediate formed by XerC recombinase. This change provides a substrate for XerD, which then completes the recombination reaction.

FtsK functions in the processing of a Holliday junction intermediate during bacterial chromosome segregation.

In bacteria with circular chromosomes, homologous recombination can generate chromosome dimers that cannot be segregated to daughter cells at cell division. Xer site-specific recombination at dif, a 28-bp site located in the replication terminus region of the chromosome, converts dimers to monomers through the sequential action of the XerC and XerD recombinases. Chromosome dimer resolution requires that dif is positioned correctly in the chromosome, and the activity of FtsK, a septum-located protein that coordinates cell division with chromosome segregation. Here, we show that cycles of XerC-mediated strand exchanges form and resolve Holliday junction intermediates back to substrate irrespective of whether conditions support a complete recombination reaction. The C-terminal domain of FtsK is sufficient to activate the exchange of the second pair of strands by XerD, allowing both intra- and intermolecular recombination reactions to go to completion. Proper positioning of dif in the chromosome and of FtsK at the septum is required to sense the multimeric state of newly replicated chromosomes and restrict complete Xer reactions to dimeric chromosomes.

Unambiguous demonstration of triple-helix-directed gene modification.

Triple-helix-forming oligonucleotides (TFOs), which can potentially modify target genes irreversibly, represent promising tools for antiviral therapies. However, their effectiveness on endogenous genes has yet to be unambiguously demonstrated. To monitor endogenous gene modification by TFOs in a yeast model, we inactivated an auxotrophic marker gene by inserting target sequences of interest into its coding region. The genetically engineered yeast cells then were treated with psoralen-linked TFOs followed by UV irradiation, thus generating highly mutagenic covalent crosslinks at the target site whose repair could restore gene function; the number of revertants and spectrum of mutations generated were quantified. Results showed that a phosphoramidate TFO indeed reaches its target sequence, forms crosslinks, and generates mutations at the expected site via a triplex-mediated mechanism: (i) under identical conditions, no mutations were generated by the same TFO at two other loci in the target strain, nor in an isogenic control strain carrying a modified target sequence incapable of supporting triple-helix formation; (ii) for a given target sequence, whether the triplex was formed in vivo on an endogenous gene or in vitro on an exogenous plasmid, the nature of the mutations generated was identical, and consistent with the repair of a psoralen crosslink at the target site. Although the mutation efficiency was probably too low for therapeutic applications, our results confirm the validity of the triple-helix approach and provide a means of evaluating the effectiveness of new chemically modified TFOs and analogs.

Preparation for TEM of layered samples with fragile microstructure and weak layer interface.

The objective of this work was to prepare for transmission electron microscopy (TEM) a layered structure of materials with fragile microstructure. The samples consisted of two layers of different materials, silicon nitride and borosilicate glass, loosely bonded together. The low strength of the sample resulted in fragmentation during more conventional preparation. However, it was possible to prepare the fragments by mounting them in a titanium specimen carrier with aluminium strips as support. After grinding and polishing, a technique of low-angle ion milling was used to obtain electron beam transparent areas at the nitride/glass interface.

Asymmetric recognition of psoralen interstrand crosslinks by the nucleotide excision repair and the error-prone repair pathways.

Psoralen is an asymmetric photoreactive intercalator with a furane and a pyrone side. When intercalated at 5'-TpA-3' sites and upon UVA irradiation, the psoralen can react with the thymine residues on both strands, introducing an interstrand crosslink. Using psoralen-coupled triple-helix-forming oligonucleotides, psoralen interstrand crosslinks can be site-specifically introduced in the coding sequence of URA3, a yeast auxotrophic marker carried on plasmid vectors. In addition, crosslinks introduced via a triple-helix-forming oligonuleotide are oriented with the furane side of the psoralen associated with a specific strand of the target sequence. Here, the transformation efficiency, the mutation frequency and the mutational spectra of site-specifically placed and oriented crosslinks were examined in yeast cells. We found that the nature of the targeted mutations depended on the crosslink orientation: bypass of the pyrone-adducted thymine yielded T-->A or T-->C substitutions and A insertions, while bypass of the furane-adducted thymine yielded T-->G substitutions and G insertions. Thus, the structure of the damage strongly influences the choice of the nucleotide incorporated during translesion synthesis. In addition, the observed pattern of mutagenesis suggests a coupling to transcription, similar to the one observed in mammalian cells. Finally, the substitutions affected only the coding strand when the pyrone link of the psoralen crosslink was on this strand, whereas they affected both strands when the pyrone link was on the transcribed strand, suggesting that the incision preference of psoralen crosslinks, which has been observed with purified uvrABC proteins in bacteria, is conserved in live eucaryotic cells.

Covalent crosslinks introduced via a triple helix-forming oligonucleotide coupled to psoralen are inefficiently repaired.

Triple helix-forming oligonucleotides (TFOs) represent potentially powerful tools to artificially modulate gene activity. In particular, they can be used to specifically introduce a lesion into a selected target sequence: interstrand crosslinks and monoadducts can be introduced via TFOs coupled to psoralen. The efficiency of these strategies depends on the cell ability to repair these lesions, an issue which is still controversial. Here we show, using psoralen-coupled TFOs and the yeast as a convenient cellular test system, that interstrand crosslinks are quantitatively poorly repaired, resulting in an efficient modification of target gene activity. In addition, these lesions result in the introduction of mutations in a high proportion of cells. We show that these mutations are generated by the Error-Prone Repair pathway, alone or in combination with Nucleotide Excision Repair. Taken together, these results suggest that TFOs coupled to psoralen could be used to inactivate a gene with significant efficiency.

Histone acetyltransferase activity of CBP is controlled by cycle-dependent kinases and oncoprotein E1A.

Transforming viral proteins such as E1A force cells through the restriction point of the cell cycle into S phase by forming complexes with two cellular proteins: the retinoblastoma protein (Rb), a transcriptional co-repressor, and CBP/p300, a transcriptional co-activator. These two proteins locally influence chromatin structure: Rb recruits a histone deacetylase, whereas CBP is a histone acetyltransferase. Progression through the restriction point is triggered by phosphorylation of Rb, leading to disruption of Rb-associated repressive complexes and allowing the activation of S-phase genes. Here we show that CBP, like Rb, is controlled by phosphorylation at the G1/S boundary, increasing its histone acetyltransferase activity. This enzymatic activation is mimicked by E1A.

Highly efficient oligonucleotide transfer into intact yeast cells using square-wave pulse electroporation.

Here, we present a rapid and reproducible procedure based on square-wave pulse electroporation that allows efficient penetration of synthetic oligonucleotides into intact yeast cells. This procedure was successfully used to modify the yeast genome with small amounts of oligonucleotide.

[Simulation study of the influence of collimator defects on the uniformity of scintigraphic images]. / Etude en simulation de l'influence des défauts des collimateurs sur l'uniformité des images scintigraphiques.

The mechanical tolerances in building collimators for scintillation cameras are studied. A simulation method has been used to quantify the effects of defects in hole inclination and hole diameter on the uniformity of planar and tomographic images. The calculation takes into account the geometry of the hexagonal hole collimator, the camera intrinsic resolution, the object size, the pixel size, the effect of low-pass filtering, as well as the type, size and position of the defect. For instance, a 0.03 mm diameter defect on several holes located in the central region of a very high resolution collimator can result in a 12% uniformity artefact in tomographic imaging of an 18 cm diameter cylinder, using a 3.45 mm resolution camera, 4.5 mm pixel size, and Hamming filtering with a Nyquist frequency cut-off. A 0.17 degree inclination defect of a few holes can result in the same uniformity artefact. These results show that the building of a collimator has to be very precise.

Flooding-dose of various amino acids for measurement of whole-body protein synthesis in the rat.

These studies were undertaken to investigate the influence of the precursor amino acid on the measurement of whole-body protein turnover by the flooding-dose method. Whole-body protein synthesis rates were estimated in 70 g rats using an intravenous injection of L-(U(14)C) Threonine, L-(U(14)C) Lysine (200µmoles/100g; 0.15µCi/µmol or 1000µmoles/100 g; 0.15µCi/µmol), L-(U(14)C) Phenylalanine, L-(1(14)C) Leucine or L-(U(14)C) Histidine (200µmoles/100 g; 0.15µCi/µmol). Forty two rats were divided into seven groups. Each group received a large dose of one of the labelled amino acids. In each group, one animal was killed every 2 min between 5 and 15 min after the injection. Whole-body protein fractional rate was determined from the slope of the linear regression of Sb(t) (protein specific radioactivity) against Si'(t) ×t, where Si'(t) is the average specific radioactivity of free tissue amino acids between 0 andt. Whole-body protein fractional synthesis rates were 41.4, 25.6, 31.1, 31.4 and 22.8%/day with threonine, lysine, phenylalanine, leucine and histidine respectively. These data suggest that the estimation of whole-body protein synthesis rate varies according to the amino acid used because of the heterogeneity of the protein pool.

Whole body protein synthesis: studies with different amino acids in the rat.

These studies were undertaken to determine to what extent constant infusion measurements and plasma sampling could provide sensible answers for rates of whole body protein turnover and also which amino acid would be the most representative probe of whole body protein turnover. Whole body protein synthesis rates were estimated in 70-g rats with L-[U-14C]threonine, L-[U-14C]lysine, L-[U-14C]tyrosine, L-[U-14C]phenylalanine, and L-[1-14C]leucine by either simultaneous tracer infusion of four amino acids or by injections of large quantities of 14C-labeled amino acids. In the infusion experiment, indirect estimates of whole body protein turnover based on free amino acid specific radioactivity and stochastic modeling were compared with direct measurement of the incorporation of the tracer into proteins. These two methods of analysis provided similar results for each amino acid, although in each case fractional synthesis rates were lower (by between 26 and 63%) when calculations were based on plasma rather than tissue specific radioactivity. With the flooding-dose method, whole body fractional protein synthesis rates were 41.4, 25.6, 31.1, and 31.4% with threonine, lysine, phenylalanine, and leucine, respectively. These values were similar to those obtained by the continuous infusion method using tissue specific radioactivity for threonine and lysine. For leucine, however, the flooding-dose method provided an intermediate value between the two estimates derived either from the plasma or the tissue specific radioactivity in the infusion method.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of dietary leucine content on the activities of branched-chain amino acid aminotransferase (EC 2.6.1.42) and branched-chain alpha-keto acid dehydrogenase (EC 1.2.4.4) complex in tissues of preruminant lambs.

1. Branched-chain amino acid aminotransferase (EC 2.6.1.42; BCAAT) and branched-chain alpha-keto acid dehydrogenase (EC 1.2.4.4; BCKDH) activities were measured preruminant lamb liver, longissimus dorsi muscle, kidney, jejunum and adipose tissue, 2 h after a meal with or without an excess of leucine. 2. Skeletal muscle contained about 70% of the total basal BCAAT activities of the tissues studied whereas liver contained about 60% of the total BCKDH activities of these tissues. 3. BCAAT activities were very low in preruminant lamb tissues. BCKDH was more phosphorylated in tissues of preruminant lambs than in rats, especially in liver. These low catalytic potentialities might contribute to a low rate of branched-chain amino acid catabolism in sheep. 4. Ingestion of an excess of leucine led to an increase in liver and jejunum BCAAT activities and activation of BCKDH in jejunum.

Correlation between structure of polyoxotungstates and their inhibitory activity on polymerases.

The 21-tungsto-9-antimonate (TA, HPA 23), a polyoxotungstate, has shown a significant antiviral activity in vivo and in vitro. It inhibits viral and bacterial DNA polymerases. In this paper, several compounds of two polyoxotungstic families, tungstoantimonates and tungstoarsenates, have been used to specify the mechanism of polymerase inhibition. It has been demonstrated that the inhibitory activity of polyoxotungstates is not related to the occupation of their coordinates sites by cations, nor to the nature of these bound cations. Kinetic studies and binding assays have shown that polyoxotungstates bind to the polymerases in competition with the nucleic acid template. This result seems to be related to their polyanionic nature. Furthermore, the size and charge of these compounds may play a prominent part in their affinity for the polymerases.