General practitioners' stay-at-work practices in patients with musculoskeletal disorders: using Intervention Mapping to develop a training program.

OBJECTIVES: To describe current stay-at-work practices among Danish general practitioners (GPs) in relation to patients with musculoskeletal disorders, to identify potential avenues for improvement, and to suggest a training program for the GPs. DESIGN AND SETTING: We followed the principles of Intervention Mapping. Data were collected by means of literature searches, focus group interviews with GPs, and interaction with stakeholder representatives from the Danish labour market. RESULTS: GPs' current stay-at-work practices were influenced by systemic, organisational, and legislative factors, and by personal determinants, including knowledge and skills relating to stay-at-work principles and musculoskeletal disorders, recognition of the patient's risk of long-term work disability, their role as a GP, and expectations of interactions with other stay-at-work stakeholders. GPs described themselves as important partners and responsible for the diagnostic and holistic assessments of the patient but placed themselves on the side line relying on the patient or workplace stakeholders to act. Their practices are influenced both by patients, employers, and by other stakeholders. We propose a training course for GPs that incorporate both concrete tools and behaviour change techniques. CONCLUSIONS: We have identified varied perspectives on the roles and responsibilities of GPs, as well as legislative and organisational barriers, and proposed a training program. Not all barriers identified can be addressed by a training course, and some questions are left unanswered, among others - who are best suited to help patients staying at work?

Musculoskeletal disorders are highly prevalent and one of the most common causes for visiting a GP.In many countries, GPs are important in facilitating that patients stay at work, when they are experiencing musculoskeletal pain and disability.In our research, GPs place themselves on the side line as coaches relying on the patient or workplace to act.Barriers such as role identity, systemic and organisational issues prevent GPs from being more involved in stay-at-work practices.GPs' with knowledge about stay-at-work practices may empower patients to better self-management.

Deletion and duplication of specific sequences in the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coli.

Small, defined in-frame deletions and in-frame duplications of specific sequences were made within the faeG gene encoding the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coli. The cellular localization and proteolytic stability of the different mutated fimbrial subunit proteins were determined, and compared with those of the wild-type protein. Based upon these results, we predict a functional role of specific structures in the K88ab fimbrial subunit protein in subunit-subunit interactions as well as in interactions between FaeG and the other proteins encoded by the K88ab operon. The results obtained were further compared with results obtained from operon deletions, linker insertion mutagenesis and the current model for biogenesis of K88 fimbriae. One of the mutated fimbrial subunit genes was used to construct a secreted in-frame fusion between FaeG and a characterized epitope (lacking cysteine) from the Hepatitis B pre-S2 protein. Such fusion proteins might be useful in the design of recombinant vaccines.

Dra-nupC-pdp operon of Bacillus subtilis: nucleotide sequence, induction by deoxyribonucleosides, and transcriptional regulation by the deoR-encoded DeoR repressor protein.

The genes encoding deoxyriboaldolase (dra), nucleoside uptake protein (nupC), and pyrimidine nucleoside sequences were determined. Sequence analysis showed that the genes were localized immediately downstream of the hut operon. Insertional gene disruption studies indicated that the three genes constitute an operon with the gene order dra-nupC-pdp. A promoter mapping immediately upstream of the dra gene was identified, and downstream of the pdp gene the nucleotide sequence indicated the existence of a factor-independent transcription terminator structure. In wild-type cells growing in succinate minimal medium, the pyrimidine nucleoside phosphorylase and deoxyriboaldolase levels were five- to eightfold higher in the presence of thymidine and fourfold higher in the presence of deoxyadenosine. By the use of lacZ fusions, the regulation was found to be at the level of transcription. The operon expression was subject to glucose repression. Upstream of the dra gene an open reading frame of 313 amino acids was identified. Inactivation of this gene led to an approximately 10-fold increase in the levels of deoxyriboaldolase and pyrimidine nucleoside phosphorylase, and no further induction was seen upon the addition of deoxyribonucleosides. The upstream gene most likely encodes the regulator for the dra-nupC-pdp operon and was designated deoR (stands for deoxyribonucleoside regulator).

A xyloglucan-specific endo-beta-1,4-glucanase from Aspergillus aculeatus: expression cloning in yeast, purification and characterization of the recombinant enzyme.

A full-length c-DNA encoding a xyloglucan-specific endo -beta-1, 4-glucanase (XEG) has been isolated from the filamentous fungus Aspergillus aculeatus by expression cloning in yeast. The colonies expressing functional XEG were identified on agar plates containing azurine-dyed cross-linked xyloglucan. The cDNA encoding XEG was isolated, sequenced, cloned into an Aspergillus expression vector, and transformed into Aspergillus oryzae for heterologous expression. The recombinant enzyme was purified to apparent homogeneity by anion-exchange and gel permeation chromatography. The recombinant XEG has a molecular mass of 23,600, an isoelectric point of 3.4, and is optimally stable at a pH of 3.4 and temperature below 30 degreesC. The enzyme hydrolyzes structurally diverse xyloglucans from various sources, but hydrolyzes no other cell wall component and can therefore be considered a xyloglucan-specific endo -beta-1, 4-glucanohydrolase. XEG hydrolyzes its substrates with retention of the anomeric configuration. The Kmof the recombinant enzyme is 3.6 mg/ml, and its specific activity is 260 micromol/min per mg protein. The enzyme was tested for its ability to solubilize xyloglucan oligosaccharides from plant cell walls. It was shown that treatment of plant cell walls with XEG yields only xyloglucan oligosaccharides, indicating that this enzyme can be a powerful tool in the structural elucidation of xyloglucans.

Cloning and characterization of two structurally and functionally divergent rhamnogalacturonases from Aspergillus aculeatus.

Two rhamnogalacturonases from the filamentous fungus Aspergillus aculeatus have been cloned and characterized. A cDNA library from A. aculeatus was constructed, and a novel rhamnogalacturonase B was isolated by expression cloning in yeast. For this purpose a new plate screening assay was developed, specific for the detection of rhamnogalacturonase activity. The rhamnogalacturonase A, known from previous reports, was shown not to be expressed in yeast in an active form. Therefore, rhamnogalacturonase A was purified, peptide sequences were obtained, and full-length cDNAs encoding the enzyme were isolated using a polymerase chain reaction-generated product as a probe. Comparison of the deduced primary structures indicates that the two rhamnogalacturonases are structurally different. This is further supported by the finding that polyclonal antibodies raised against native rhamnogalacturonase A do not cross-react with rhamnogalacturonase B. The cloned genes were transformed into Aspergillus oryzae for high level expression. The recombinant enzymes were purified and characterized, revealing significant differences in glycosylation pattern and substrate specificity as well as in pH and temperature optima and stability. Data from the hydrolysis of apple rhamnogalacturonan with the recombinant rhamnogalacturonases suggest that the two enzymes exert their action at different sites in the backbone.

Functional cloning of an endo-arabinanase from Aspergillus aculeatus and its heterologous expression in A. or oryzae and tobacco.

Functional cloning in yeast has been used to isolate full-length cDNAs encoding an endo-alpha-1,5-L-arabinanase from the filamentous fungus Aspergillus aculeatus. Screening of a cDNA library constructed in a yeast expression vector for transformants that hydrolysed AZCL-arabinan identified 44 Saccharomyces cerevisiae clones all harbouring the same arabinanase-encoding cDNA. The cloned cDNA was expressed in A. oryzae and the recombinant enzyme was purified and characterized. The mode of action of the enzyme was studied by analysis of the digestion pattern towards debranched arabinan. The digestion profile obtained strongly suggests that the enzyme is an endo-arabinanase. In addition, the feasibility using Nicotiana tabacum as an alternative host for arabinanase expression was investigated.

Neural network modeling of a dolphin's sonar discrimination capabilities.

The capability of an echolocating dolphin to discriminate differences in the wall thickness of cylinders was previously modeled by a counterpropagation neural network using only spectral information from the echoes. In this study, both time and frequency information were used to model the dolphin discrimination capabilities. Echoes from the same cylinders were digitized using a broadband simulated dolphin sonar signal with the transducer mounted on the dolphin's pen. The echoes were filtered by a bank of continuous constant-Q digital filters and the energy from each filter was computed in time increments of 1/bandwidth. Echo features of the standard and each comparison target were analyzed in pairs by a counterpropagation neural network, a backpropagation neural network, and a model using Euclidean distance measures. The backpropagation network performed better than both the counterpropagation network, and the Euclidean model, using either spectral-only features or combined temporal and spectral features. All models performed better using features containing both temporal and spectral information. The backpropagation network was able to perform better than the dolphins for noise-free echoes with Q values as low as 2 and 3. For a Q of 2, only temporal information was available. However, with noisy data, the network required a Q of 8 in order to perform as well as the dolphin.

Pectin methyl esterase from Aspergillus aculeatus: expression cloning in yeast and characterization of the recombinant enzyme.

Seventeen full-length cDNAs encoding pectin methyl esterase I (PME I) have been isolated from the filamentous fungus Aspergillus aculeatus by expression cloning in yeast. Yeast colonies expressing functional PME I were identified on agar plates containing highly esterified pectin, and a cDNA encoding PME I was isolated. The deduced amino acid sequence of PME I is highly similar (74% identity) to the PME from Aspergillus niger. A full-length cDNA encoding PME I was cloned into an Aspergillus expression vector and transformed into Aspergillus oryzae for heterologous expression, purification and characterization of the recombinant enzyme. The recombinant PME I had a molecular mass of 36.2 kDa, an isoelectric point of pH 3.8, a pH optimum of 4.6 and a temperature optimum of 45 degrees C. The authentic PME I was purified from A. aculeatus culture supernatant and subjected to amino acid sequencing. The peptide sequences covered 138 amino acid residues and were in complete agreement with the deduced PME I sequence. Both recombinant and authentic PME I were glycosylated, but the composition of the glycan moieties was different. PME I was able to remove 75-85% of the methyl groups in highly methylated pectin, and it did not remove acetyl groups from acetylated polysaccharides. When the enzyme was added together with polygalacturonases to pectin, a rapid depolymerization was observed. By comparison, polygalacturonases alone showed a very limited degradation of the methylated substrate. This demonstrates that PME I acts in synergy with polygalacturonases in the degradation of plant cell wall pectin.