USA300, A strain of community-associated methicillin-resistant Staphylococcus aureus, crossing Belgium's borders: outbreak of skin and soft tissue infections in a hospital in Belgium.

OBJECTIVE: to investigate an outbreak of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 in a hospital setting, and the effect of infection control measures. DESIGN: outbreak investigation and retrospective chart review. SETTING: local inpatient and outpatient clinic. PATIENTS: all patients with a history of skin and soft tissue infections with culture-confirmed methicillin-resistant Staphylococcus aureus USA 300 infection from September, 2014, through June, 2015. INTERVENTIONS: an outbreak investigation with a "search and destroy" policy was carried out. A review of infection control practices was conducted. Chart reviews were conducted to study the management and outcomes of the patients. Infection control measures included education and cultures of skin colonization sites (anterior nares, pharynx, perineum). Specific decontamination schemes for uncomplicated and complicated carriers were enforced. Separate decontamination schemes for neonates and children under 5 years of age were implemented. RESULTS: between September 2014 and June 2015, 12 clinical cases and six carriers were identified. Of the twelve clinical presentations with positive cultures, eight were children. Of the four patients who had a relapse, three were children (75%). After outbreak investigation and infection control measures have been implemented, three persistent carriers remained. A policy of periodic screening, consultation, and watchful waiting for skin infections was instituted for these patients. No new cases linked to the CA-MRSA outbreak have since been reported. CONCLUSION: we report the first Belgian outbreak of CA-MRSA USA300 in this article. A strict search and destroy strategy and continued surveillance are required in the management of CA-MRSA USA300.

Dynamic analyses of mother-child interactions in functional and dysfunctional dyads: a synergetic approach.

Describes the application of a new analytical approach (derived from synergetics, a complex dynamic systems theory) to home observational data of mother-child interactions in average dyads and dyads with children referred for disruptive behavior problems at home and school (n = 11 in each group). Results show that (1) the two groups differed in their daily interactions in predictable ways, and (2) the most frequent patterns of interactions observed in the two groups brought them back repeatedly to behave in similar ways toward each other. The findings are in keeping with a body of literature on mother-child interactions. However, they add to it by providing multivariate. graphical representations of these interactions and by offering a conceptual framework within which to move from an observational to an inferential level of analysis. At that level, the transactional processes that are characteristic of functional and dysfunctional relationships may become apparent.

Pressure-induced inactivation of E. coli beta-galactosidase: influence of pH and temperature.

In order to assess the feasibility of a high-pressure immunodesorption process using a beta-galactosidase-anti-beta-galactosidase complex as a model, the influence of high hydrostatic pressure on the activation of E. coli beta-galactosidase has been investigated. The irreversible activity loss of beta-galactosidase was studied as a function of pH and temperature for pressures comprised between atmospheric pressure and 500 megapascal (MPa; 1 MPa = 10 bar). This enabled us to establish a practical pressure-temperature diagram of stability for this enzyme. The stability domains determined thus appeared to be strongly dependent on the pH under atmospheric pressure of phosphate buffer employed for pressurisation. Therefore, to interpret meaningfully this result, the influence of pressure on the pH-activity curve of beta-galactosidase was investigated by using a high-pressure stopped-flow device. It appeared that the pH-activity curve of this enzyme was also reversibly affected by pressures lower than 150 MPa. An interpretation of these results in relation to the high-pressure induced changes of ionisation constants is proposed. For our practical purpose, the implications for the elaboration of a high-pressure immunodesorption process using beta-galactosidase as a tag, are discussed.

The characteristic phenotype of distal 9q3 trisomy is due to duplication of band 9q32.

In this report we describe a 6-month-old female child with inverted duplication of bands 9q32-9q33. The phenotypic findings are identical to the clinical syndrome previously reported to be associated with 9q31-9q32 duplication. The findings in the present child indicate that the minimal segment of overlap in this partial trisomy syndrome is 9q32.

Autoprocessing of the human immunodeficiency virus type 1 protease precursor expressed in Escherichia coli from a synthetic gene.

A gene encoding an N-terminally extended precursor of 107 residues of the human immunodeficiency virus type 1 protease (PR107) was chemically synthesized and cloned into a bacterial expression vector, under the control of the araB promoter. PR107 was expressed alone or fused in phase to the amino or carboxy terminus of the bacterial beta-galactosidase (beta-gal). The yield of protease and beta-gal was found to be significantly higher when the gene for PR107 was cloned upstream of the Escherichia coli lacZ gene (PR107-beta-gal). Comparisons of the level of cloned protein expression between protease precursor and mature form suggested that this enhanced expression was due to the additional 5' sequence of the PR107 gene, and occurred at the post-transcriptional level. Autoprocessing of protease precursor and its release from the beta-gal fusion protein were analysed using wild-type and mutated cleavage sites. Mutations were introduced at amino acids downstream of the F-P scissile bond, at positions P4' and P5' in the C-terminal site (TLNF*PISP), and at position P3' in a consensus N-terminal site (TLNF*PQITL) placed at the protease-beta-gal junction. The data obtained suggested that (i) autoprocessing at the carboxy-terminal F-P bond was not significantly influenced by the presence of the N-terminal precursor sequence, (ii) P4' and P5' substitutions in the C-terminal site had no effect on cleavage, and (iii) P3' in the N-terminal site tolerated a wide variety of substitutions.

Production of a full length Tat protein in E. coli and its purification.

A full length tat gene was constructed by a combination of polymerase chain reaction (PCR) for the first exon and chemical synthesis for the second exon. This gene was expressed in E. coli under the control of the strongly regulated araB promoter, either directly or fused to a secretion signal encoding sequence. We then defined a rapid, three-step procedure for the purification of the Tat protein.

Functional and structural effects of an Ala to Val mutation in the adenovirus serotype 2 fibre.

H2ts125 is a fibre-defective, temperature-sensitive mutant of adenovirus serotype 2. H2ts125 fibre is unstable at the non-permissive temperature (ts phenotype), and does not migrate in the same way as the wild-type fibre in an SDS/polyacrylamide gel (elm phenotype). Sequence analysis has shown that H2ts125 carries two mutations on the fibre gene: Leu105 to Phe, and Ala434 to Val. Analysis of the structural modifications occurring in H2ts125 fibre was performed using peptide finger-printing and antipeptide sera as immunological probes. We found that all the detectable structural alterations in the mutant fibre were due to the substitution on codon 434. In addition, the ts phenotype was rescued by a wild-type DNA fragment containing the 3' moiety of the fibre gene and overlapping the 434th codon. Morphological analysis of fibre molecules observed under the electron microscope showed minor but statistically significant differences in the fibre length between mutant and wild-type. The mutant fibre was found to be slightly longer (308.8 +/- 1.9 A) than the wild-type fibre (300.1 +/- 2.1 A). Thus both ts and elm phenotypes were carried by the same Ala434 to Val mutation which probably resulted from a change in the three-dimensional structure of the fibre protein, and not from some proteolytic cleavage.

Structural study of adenovirus type 2 fibre using anti-fibre and anti-peptide sera.

Peptides corresponding to the N- and C-extremities of the adenovirus 2 fibre polypeptide were synthesized, coupled to tetanus toxoid and injected into rabbits. Two sera were obtained: the anti-NTT serum and the anti-CTT serum. These sera and an anti-native-fibre serum were used to study fragments generated by hydrochloric acid cleavage of the fibre. The 44-Kd fragment corresponding to the 2/3 N-terminal part of the molecule retained its antigenic reactivity. This is consistent with a shaft structure for this part of the fibre. The anti-peptide sera were used to orientate the fibre, i.e., to determine the site of anchorage of this protein in the penton base. First, immunorevelation of blots of enzymatic digests of native or dissociated penton suggested that the N-extremity of the fibre was involved in the assembly of this protein in the penton base. Second, attempts were made to determine the accessibility of the fibre ends in the penton structure by ELISA assays and by immunorevelation of penton in Western blots. The results agreed with the proposed orientation derived from study of the enzymatic digests. Since the 2 anti-peptide sera and the peptides were unable to affect viral adsorption, it was not possible to determine how the fibre is orientated with respect to the cell receptor. However, the anti-peptide sera were found to inhibit viral production slightly.

Inhibition of antigen-specific activation of an L3T4+ T cell line by cyclosporine with maintenance of macrophage-mediated antigen presentation.

Cyclosporine has clearly been shown to directly inhibit T lymphocyte activation by monoclonal antibodies or mitogens where nominal antigen and accessory cells are not present. However, when T lymphocytes are stimulated by antigen, as occurs in allograft rejection, T lymphocytes and accessory cells must interact with one another. Under the latter circumstances, the issue of whether cyclosporine acts on T lymphocyte, accessory cell, or both is not resolved. This issue is addressed in this study. To assess the effect of cyclosporine on T cell activation, macrophages were incubated with heat-killed Listeria and then fixed in paraformaldehyde. These fixed macrophages retained their ability to present antigen to T cells but were not affected by subsequent treatment with cyclosporine. When cyclosporine and a L3T4+ T lymphocyte line were added simultaneously to fixed, antigen-pulsed macrophages, the drug inhibited antigen-specific T cell activation with a half maximal inhibitory concentration of 10 ng/ml. To our knowledge, this is the first evidence that low doses of cyclosporine inhibit antigen-specific T cell activation where the drug's effects on antigen-presenting cells have been excluded. To assess the effects of cyclosporine on macrophage-mediated antigen-presentation, macrophages were exposed simultaneously to cyclosporine and antigen, and then fixed. Antigen-presentation was not inhibited unless extremely large doses (9000 ng/ml) of cyclosporine were used. In our experimental system, any new inhibitory activity acquired by live cyclosporine-treated macrophages could be explained by residual drug. Finally, cyclosporine did not inhibit the induction of macrophage Ia expression nor antigen-presenting function after stimulation in vitro with lymphokine.

Chelating, DNA-binding and DNA-cleaving properties of a synthetic model for bleomycin.

We have previously described a simplified model of the complexing part of bleomycin, namely methyl 2-(2-aminoethyl)-aminomethyl-pyridine-6-carboxyl-histidinate (AMPHIS), and disubstituted bithiazoles structurally related to the 'tripeptide S' moiety of bleomycin. The present work is devoted to the study of a new derivative, [3-[2-[2-(2-aminoethyl)-aminomethyl-pyridine-6-carboxyl-histidyl-3 -aminobutyryl-glycyl]-2',4-bithiazole-4-carboxamido]-propyl]- dimethylsulphonium iodide (AMBI-A2), which includes both AMPHIS and a judiciously chosen synthetic bithiazole. This compound, the synthesis of which is described here, has been shown to mimic the chelating and binding properties of the parent drug bleomycin A2, but to cleave DNA at higher concentration.

Subcellular distribution of spin-labeled bithiazoles and bleomycin in living KB cells: an ESR study.

Spin-labeled derivatives of bithiazole and bleomycin were studied with respect to their uptake and localization in KB cells in vivo. It was found that the amidification of the C-terminal carboxylic group of bleomycinic acid was essential for the penetration of the probes in the cells and that the subcellular localization depended on the number and spacing of positively charged groups.

Flow cytophotometric and time-lapse cinematographic study of human cells infected by adenovirus type 2 wild-type and two DNA-negative temperature-sensitive mutants.

Flow cytophotometry and time-lapse cinematography were used to study viral DNA synthesis and alteration of the cellular DNA content of human cells infected by adenovirus type 2 wild-type and two DNA-negative temperature-sensitive mutants. Cell populations with DNA contents greater than 4n (where n represents the normal haploid DNA content of cells) were found after infection and their origin is discussed with regard to cell cycle changes, viral DNA replication and cell fusion or endomitosis.

Subcellular distribution of nitroxide spin-labelled 9-aminoacridine in living KB cells.

A spin-labelled derivative of 9-aminoacridine, the AATEMPO, was studied with respect to its localization in KB cells in vivo. It was found that both nuclear and mitochondrial DNAs were targets for this intercalating dye. The observed speed of intercalation and the absence of a blocked signal in cellular membranes suggested that no receptor -or carrier- proteins were implied in the penetration process. No changes in cell membrane fluidity were observed following the administration of m-AMSA, the unlabelled structural analog, to the living cells, which seemed to exclude the plasma membrane as a possible site of action of 9-aminoacridines. Side effects of phenol/chloroform mixtures and high-salt concentrations on the intercalation phenomenon were also described.

Biochemical and genetical characterization of a fiber-defective temperature-sensitive mutant of type 2 adenovirus.

The adenovirus type 2 fiber mutant H2 ts 125 synthesized an unstable, temperature-sensitive fiber polypeptide with an apparent mol. wt. smaller by 2500 than the wild-type (62 K). The polypeptide of 59.5 K was found to be stable at the permissive temperature (33 degrees C). H2 ts 125 fiber synthesized in reticulocyte lysates had the same apparent mol. wt. of 59.5 K as the mutant fiber produced in vivo. Neither structural nor functional differences between wild-type and mutant fibers were detected in the N-terminal and C-terminal sequences, excluding the occurrence of a new initiation or termination codon. Restriction analysis of H2 ts 125 DNA also ruled out the hypothesis of a deletion mutant. The 59.5 K mutant fiber unit was normally glycosyated, N-acetylated, assembled into 6S oligomeric fiber and incorporated into virions. DNA sequencing of the H2 ts 125 fiber gene revealed two point mutations at nucleotides 3970 (C*TT leads to T*TT) and 4958 (GC*T leads to GT*T), corresponding to two amino acid changes at positions 105 and 434, respectively. The 105 mutation consisted of a conservative change Leu leads to Phe; the 434 interchange was Ala leads to Val, usually considered as nonconservative. The possibility of a donor site for splicing created by the mutation at codon GTT was eliminated on the basis of S1 nuclease analysis data. All these results suggested that either one or both mutations concerned highly organized domain(s) of the fiber polypeptide chain, resulting in aberrant mobility in SDS-polyacrylamide gels and temperature-sensitivity.

Characterization of adenovirus protein IX.

Protein IX from adenovirus type 2 was purified by two methods, one from groups of nine hexons obtained by disrupting purified virus by heating in the presence of deoxycholate, and the other by a previously published method. The purified protein was used to obtain a monospecific antiserum. Protein IX was found to possess both sub-group- and type-specific antigenic determinants which were apparently accessible within the groups of nine hexons. Approximately 15 molecules of IX were found per group of nine hexons and from considerations of symmetry it seemed possible that IX was located at the 'corner to edge' contacts between hexons in the icosahedron. The protein in infected cells was found to possess approximately neutral charge as determined by immunoelectrophoresis. This was consistent with the amino acid composition, which showed it to be rich in serine, alanine and leucine with approximately half of its glutamic and aspartic acid residues amidified, and the isoelectric point of 6.0, as determined by two dimensional gel analysis. No free N-terminal amino acid was detectable. It is suggested that a unique tryptophan residue is located at around position 70 from the blocked N-terminus, on the basis of chemical cleavage by BNPS-skatole. Based on one tryptophan residue a total of 107 amino acids and a mol. wt. of 11200 was deduced. Analysis of 35S-methionine-labelled infected cell extracts in a two-dimensional gel system showed that the synthesis of polypeptide IX could be detected early in infection, i.e. in the presence of an inhibitor of DNA synthesis.