Early experience of a local pathway on the waiting time for MRI in patients presenting to a UK district general hospital with suspected cauda equina syndrome.

AIM: This study evaluated the impact of the Salisbury Protocol for Assessment of Cauda Equina Syndrome (SPACES) on the waiting time for MRI in patients presenting with suspected Cauda Equina Syndrome (sCES) within a UK district general hospital. PATIENTS AND METHODS: All consecutive patients undergoing an MRI scan in our hospital, for sCES, over a 12 month period, prior to and following the introduction of SPACES, were identified. Patient's gender, age, MRI diagnosis, time from MRI request to imaging and outcome were recorded. RESULTS: In the year prior to the introduction of SPACES, 66 patients underwent MRI for sCES, out of which 10.6% had cauda equina compression (CEC), 63.5% had other spinal pathology and 25% had a normal scan. In the year after introduction of SPACES, 160 patients underwent MRI for sCES out of which 6.2% had CEC, 70.7% had other spinal pathology and 23% had a normal scan. Despite the referrals for sCES increasing by more than 2-fold following the introduction of SPACES, the median time from MRI request to scan decreased from 9.1 to 4.2 hours (p = 0.106, Mann-Whitney-U) and the number of patients transferred to the regional hub hospital decreased from 7 to 3. CONCLUSION: Implementation of SPACES for patients with sCES resulted in a substantial reduction in waiting time for MRI and decreased the number of transfers to the regional hub hospital. Based on our early experience, we encourage other centres within the UK to introduce such a pathway locally, to improve the management of patients with sCES.

Characterization of fluoranthene- and pyrene-degrading Mycobacterium-like strains by RAPD and SSU sequencing.

Bacterial isolates were obtained from two sites in New Zealand contaminated with polycyclic aromatic hydrocarbons. Isolates capable of degrading polycyclic aromatic hydrocarbons were characterized in two mycobacterial groups according to phenotypic properties. These groups were supported by random amplified polymorphic DNA analysis. Nucleotide sequences of 16S ribosomal RNA genes from isolates representing each group were determined and compared with other mycobacterial 16S ribosomal RNA sequences. The taxonomic relationships of these isolates are considered.

A molecular view of microbial diversity in a dynamic landfill in Québec.

An Aroclor 1260 (polychlorinated biphenyl, PCB)-laden soil and one heavily contaminated with polycyclic aromatic hydrocarbons (PAHs) from a secure, engineered landfill site in Québec were analyzed for microbial diversity using a clone library of the 16S rDNA sequences. Phylogenetic analysis revealed that three phyla and their major subdivisions of the domain Bacteria were highly represented in these samples despite the high pollution, particularly by PAHs. None of the 16S rDNA sequences obtained matched known sequences from cultivated bacterial species or from 16S rDNA sequences amplified directly from other environmental samples.

Biochemical and biophysical analysis of plasmid pMJ600-encoded tellurite [TeO2(3-)] resistance.

Escherichia coli AB1157, and the transconjugant AB1157 (pMJ600), were used to study the mechanism of tellurite resistance conferred by pMJ600, which contains the tellurite resistance determinant cloned from the IncHI-2 conjugative plasmid pMER610. The transconjugant can tolerate a 100-fold higher concentration of potassium tellurite [K2TeO3] than the plasmid-free strain. Both strains were found to accumulate tellurite irreversibly at equivalent rates, with elemental tellurium being deposited intracellularly, direct efflux of tellurite was not found to contribute to the resistance mechanism. However, under these conditions, growth, protein synthesis and oxygen uptake ceased in AB1157, but-were unaffected in the transconjugant. No NADH- or NADPH-linked reduction of tellurite was detected in crude cell extracts of either strain; however, cell extracts of both reduced tellurite at alkaline pH in the absence of any co-factors.

Accumulation and intracellular fate of tellurite in tellurite-resistant Escherichia coli: a model for the mechanism of resistance.

The tellurite accumulation properties of three Escherichia coli strains containing different tellurium-resistance determinants of Gram-negative origin, from plasmids pMER610, pHH1508a and RK2, were compared. In all three cases membrane-associated tellurium crystallization was observed, and neither reduced uptake nor increased export contributed to the resistance. Specific membrane-proximal reduction is proposed as the mechanism of resistance to tellurite coded by all three determinants, despite their lack of sequence homology.

In vivo and in vitro cloning and phenotype characterization of tellurite resistance determinant conferred by plasmid pTE53 of a clinical isolate of Escherichia coli.

A determinant encoding resistance against potassium tellurite (Te(r)) was discovered in a clinical isolate of Escherichia coli strain KL53. The strain formed typical black colonies on solid LB medium with tellurite. The determinant was located on a large conjugative plasmid designated pTE53. Electron-dense particles were observed in cells harboring pTE53 by electron microscopy. X-Ray identification analysis identified these deposits as elemental tellurium and X-ray diffraction analysis showed patterns typical of crystalline structures. Comparison with JCPDS 4-0554 (Joint Committee on Powder Diffraction Standards) reference data confirmed that these crystals were pure tellurium crystals. In common with other characterized Te(r) determinants, accumulation studies with radioactively labeled tellurite showed that reduced uptake of tellurite did not contribute to the resistance mechanism. Tellurite accumulation rates for E. coli strain AB1157 harboring pTE53 were twice higher than for the plasmid-free host strain. In addition, no efflux mechanism was detected. The potassium tellurite resistance determinant of plasmid pTE53 was cloned using both in vitro and in vivo techniques in low-copy-number vectors pACYC184 and mini-Mu derivative pPR46. Cloning of the functional Te(r) determinant into high-copy cloning vectors pTZ19R and mini-Mu derivatives pBEf and pJT2 was not successful. During in vivo cloning experiments, clones with unusual "white colony" phenotypes were found on solid LB with tellurite. All these clones were Mucts62 lysogens. Their tellurite resistance levels were in the same order as the wild type strains. Clones with the "white" phenotype had a 3.6 times lower content of tellurium than the tellurite-reducing strain. Transformation of a "white" mutant with a recombinant pACYC184 based Te(r) plasmid did not change the phenotype. However, when one clone was cured from Mucts62 the "white" phenotype reverted to the wild-type "black" phenotype. It was suggested that the "white" phenotype was the result of an insertional inactivation of an unknown chromosomal gene by Mucts62, which reduced the tellurite uptake.

Stable isotope probing: technical considerations when resolving (15)N-labeled RNA in gradients.

RNA based stable isotope probing (SIP) facilitates the detection and identification of active members of microbial populations that are involved in the assimilation of an isotopically labeled compound. (15)N-RNA-SIP is a new method that has been discussed in recent literature but has not yet been tested. Herein, we define the limitations to using (15)N-labeled substrates for SIP and propose modifications to compensate for some of these shortcomings. We have used (15)N-RNA-SIP as a tool for analysing mixed bacterial populations that use nitrogen substrates. After incubating mixed microbial communities with (15)N-ammonium chloride or (15)N(2) we assessed the fractionation resolution of (15)N-RNA by isopycnic centrifugation in caesium trifluoroacetate (CsTFA) gradients. We found that the more isotopic label incorporated, the further the buoyant density (BD) separation between (15)N- and (14)N-RNA, however it was not possible to resolve the labeled from unlabeled RNA definitively through gradient fractionation. Terminal-restriction fragment length polymorphism (T-RFLP) analysis of the extracted RNA and fluorescent in situ hybridisation (FISH) analysis of the enrichment cultures provided some insight into the organisms involved in nitrogen fixation. This approach is not without its limitations and will require further developments to assess its applicability to other nitrogen-fixing environments.

Composition of nifH in a wastewater treatment system reliant on N(2) fixation.

High levels of nitrogen fixation have been observed in the wastewaters of pulp and paper mills. In this study, we show that nitrogen fixation in a model pulp and paper wastewater treatment system is supported by a high density of nifH sequences that are of low diversity. Quantitative PCR revealed a ratio of nifH to 16S rDNA of 1.14 +/- 0.76 which shows that very high levels of the nifH gene were enriched to support the high rates of nitrogen fixation that occur in this wastewater. Changes in wastewater composition and dissolved oxygen levels did not affect the nifH levels and allowed stable wastewater treatment. The nifH sequences identified display a similar profile to those seen in forest soil environments where nifH sequences derived from alpha-proteobacteria and beta-proteobacteria are also prevalent.

The Objective Structured Clinical Examination and student collusion: marks do not tell the whole truth.

OBJECTIVE: To determine whether the marks in the third year Objective Structured Clinical Examination (OSCE) were affected by the collusion reported by the students themselves on an electronic discussion board. DESIGN: A review of the student discussion, examiners' feedback and a comparison of the marks obtained on the 2 days of the OSCE. PARTICIPANTS: 255 third year medical students. SETTING: An OSCE consisting of 15 stations, administered on three sites over 2 days at a UK medical school. RESULTS: 40 students contributed to the discussion on the electronic discussion board. The main points raised were perceived inequity between students who did, or did not, have prior knowledge of the station content, and the lack of honesty and professionalism of their peers. Most contributors claimed to have received, or knew of others receiving, prior knowledge, but none confessed to passing on information. No significant difference (p = 0.16) was observed in the overall mark for the OSCE on day 1 (mean 390 (SD 37)) and day 2 (mean 397 (38)). On day 2, marks were considerably greater for four stations and markedly lower for three stations. It was not obvious why collusion should affect these station marks. A clear indication of the effects of collusion could only be obtained from a single subsection of an individual station (pathology) where 82 students on day 2 incorrectly gave the diagnosis from day 1. CONCLUSION: Marks do not provide a sound inference of student collusion in an OSCE and may mask the aspects of professional development of students.

Quantification of phnAc and nahAc in contaminated new zealand soils by competitive PCR.

Unculturable polycyclic aromatic hydrocarbon (PAH)-degrading bacteria are a significant reservoir of the microbial potential to catabolize low-molecular-weight PAHs. The population of these bacteria is larger than the population of nah-like bacteria that are the dominant organisms in culture-based studies. We used the recently described phn genes of Burkholderia sp. strain RP007, which feature only rarely in culture-based studies, as an alternative genotype for naphthalene and phenanthrene degradation and compared this genotype with the genotypically distinct but ubiquitous nah-like class in different soils. Competitive PCR quantification of phnAc and nahAc, which encode the iron sulfur protein large (alpha) subunits of PAH dioxygenases in nah-like and phn catabolic operons, revealed that the phn genotype can have a greater ecological significance than the nah-like genotype.

Glutathione S-transferase-encoding gene as a potential probe for environmental bacterial isolates capable of degrading polycyclic aromatic hydrocarbons.

Homologs of the glutathione S-transferase (GST)-encoding gene were identified in a collection of aromatic hydrocarbon-degrading Sphingomonas spp. isolated from New Zealand, Antarctica, and the United States by using PCR primers designed from the GST-encoding gene of Sphingomonas paucimobilis EPA505. Sequence analysis of PCR fragments generated from these isolates and of the GST gene amplified from DNA extracted from polycyclic aromatic hydrocarbon (PAH)-contaminated soil revealed a high degree of conservation, which may make the GST-encoding gene a potentially useful marker for PAH-degrading bacteria.

Conserved and hybrid meta-cleavage operons from PAH-degrading Burkholderia RP007.

We have compared the sequence and gene order of meta-cleavage pathway operons from alpha- and gamma-subgroups of the Proteobacteria with operons from Burkholderia sp. strain RP007 which belongs to the beta-subgroup of the Proteobacteria. Burkholderia RP007 was isolated for its ability to degrade phenanthrene and contains two meta-cleavage operons. One exhibits a comparable gene order to previously characterised gamma-subgroup Proteobacterial (Pseudomonas) meta operons, whilst the other has distinctive features present in both alpha- and gamma-subgroup Proteobacterial (Sphingomonas and Pseudomonas) meta operons. Gene sequence conservation, highlighted by examining the phylogeny of Proteobacterial catechol 2,3-dioxygenase sequences, reveals that sequences generally cluster in a manner which correlates with the taxonomic grouping of the Proteobacterial subgroup from which they originated.

Novel carbazole degradation genes of Sphingomonas CB3: sequence analysis, transcription, and molecular ecology.

The degradation of aromatic compounds by bacteria is dependent upon specific catabolic operons. The unique car locus isolated from Sphingomonas CB3 encodes the first four enzymes involved in the catabolism of the azaarene carbazole. These include a class II three-component dioxygenase enzyme system, a dihydrodiol dehydrogenase, an extradiol (meta-cleavage) dioxygenase, and a hydrolase. Homology of deduced amino acid sequences is closer to corresponding biphenyl catabolic genes than to previously characterised carbazole degradation genes. Gene arrangement is also identical to that found in some bph loci. The car genes are transcribed when carbazole is utilised as a sole carbon source, and although biphenyl does not serve as a growth substrate for Sphingomonas CB3 it is able to act as a non-metabolisable inducer of the car locus. Ecologically the car genes were detected in polycyclic aromatic hydrocarbon (PAH) contaminated soil associated with a former town gas site.

The phn genes of Burkholderia sp. strain RP007 constitute a divergent gene cluster for polycyclic aromatic hydrocarbon catabolism.

Cloning and molecular ecological studies have underestimated the diversity of polycyclic aromatic hydrocarbon (PAH) catabolic genes by emphasizing classical nah-like (nah, ndo, pah, and dox) sequences. Here we report the description of a divergent set of PAH catabolic genes, the phn genes, which although isofunctional to the classical nah-like genes, show very low homology. This phn locus, which contains nine open reading frames (ORFs), was isolated on an 11.5-kb HindIII fragment from phenanthrene-degrading Burkholderia sp. strain RP007. The phn genes are significantly different in sequence and gene order from previously characterized genes for PAH degradation. They are transcribed by RP007 when grown at the expense of either naphthalene or phenanthrene, while in Escherichia coli the recombinant phn enzymes have been shown to be capable of oxidizing both naphthalene and phenanthrene to predicted metabolites. The locus encodes iron sulfur protein alpha and beta subunits of a PAH initial dioxygenase but lacks the ferredoxin and reductase components. The dihydrodiol dehydrogenase of the RP007 pathway, PhnB, shows greater similarity to analogous dehydrogenases from described biphenyl pathways than to those characterized from naphthalene/phenanthrene pathways. An unusual extradiol dioxygenase, PhnC, shows no similarity to other extradiol dioxygenases for naphthalene or biphenyl oxidation but is the first member of the recently proposed class III extradiol dioxygenases that is specific for polycyclic arene diols. Upstream of the phn catabolic genes are two putative regulatory genes, phnR and phnS. Sequence homology suggests that phnS is a LysR-type transcriptional activator and that phnR, which is divergently transcribed with respect to phnSFECDAcAdB, is a member of the sigma54-dependent family of positive transcriptional regulators. Reverse transcriptase PCR experiments suggest that this gene cluster is coordinately expressed and is under regulatory control which may involve PhnR and PhnS.

Early effects of a new problem-based clinically oriented curriculum on students' perceptions of teaching.

OBJECTIVES: To compare the course experiences of medical students in a new problem-based (PBL) undergraduate medical course with those of their peers in a conventional curriculum. DESIGN: Whole class questionnaire survey using a pre-validated research instrument. SETTING: University of Liverpool, UK. SUBJECTS: First and second year medical students RESULTS: New curriculum students were more satisfied with their course when compared to their conventional course peers. Problem solving, team working and motivation scores were significantly higher amongst new course (PBL) students. New course students were more anxious about clarity of objectives and standard of work required. CONCLUSIONS: Early evidence suggests that curriculum reform from conventional teaching to a small group problem analysis programme results in improvement in student satisfaction with teaching and the development of appropriate learning skills.

The use of the nominal group technique as an evaluative tool in medical undergraduate education.

OBJECTIVES: In the present state of flux affecting UK medical undergraduate education, there is a pressing need for evaluative methods which will identify relevant outcomes both expected and unanticipated. The student perspective is now legitimately accepted to form part of any evaluative exercise but qualitative methods commonly used for this purpose are expensive in time and analytical skills. The nominal group technique (NGT) has been used for various purposes, including course evaluation, and appears well suited to this application. It combines qualitative and quantitative components in a structured interaction, which minimizes the influences of the researcher, and of group dynamics. The sequence and mechanics of the NGT process are described as applied to an end of first year evaluation in a novel undergraduate course. Doubts have been raised as to whether the results of NGT can be generalized to the larger group. In this paper, this problem is overcome by compiling a questionnaire based on the NGT items which was distributed throughout the class. DESIGN: Nominal group technique with questionnaire development. SETTING: The medical school at The University of Liverpool. SUBJECTS: Medical students. RESULTS: Previous claims made on behalf of the NGT, such as the focus on the student voice, the minimizing of leadership influence and the richness of the data, are upheld in this report. Broad agreement was found with the NGT items but two items (10%) did not display any consensus. CONCLUSIONS: The questionnaire extension of the NGT provides back-up evidence of the reliability of the data derived from the technique and enables it to be applied to the larger groups typical of undergraduate medicine.

Entropy-driven hydrogen bonding: stereodynamics of a protonated, N,N-chiral "proton sponge".

The C2-symmetric ("[DL]") and achiral ("[meso]") diastereoisomers of the hydrogen iodide salt of 1,8-bis-(N-benzyl-N-methylamino)naphthalene ([2H]-[I] ) interconvert in solution. Direct interconversion of the diastereoisomers of [2H]+ must involve hydrogen bond fission (to give "[nonHB-2H+]") and rotation-inversion of the non-protonated nitrogen centre. The global activation parameters (deltaH++ and deltaS++) for diastereoisomer interconversion in [D7]DMF have been determined from rate data obtained by temperature-drop and magnetisation-transfer 13C NMR spectroscopy over a temperature range of 170 degrees C. The process is found to have a high entropy of activation in both directions (deltaS++=163(+/-4) and 169(+/-4) JK(-1)mol(-1)) and this is suggested to arise through hydrogen bonding of the ammonium centre in [nonHB-2H+] with the solvent ([D7]DMF). Comparison of the enthalpy of activation (deltaH++) with that earlier found for diastereoisomer interconversion of the free-base form 2 suggests that the intramolecular hydrogen bond in [2H]+ is roughly equal in enthalpic strength (deltaH) with that made with the solvent ([D7]DMF) in the non-hydrogen-bonded intermediate [nonHB-2H+]. As such, the hydrogen bonding in [2H]+ may be considered as predominantly an entropically driven process, without any unusual enthalpic strength.

Structural and mechanistic studies on the activation and propagation of a cationic allylpalladium procatalyst in 1,6-diene cycloisomerization.

[Pd(eta3-C3H5)(MeCN)2]OTf acts as an efficient procatalyst for the cycloisomerisation of dimethyl hept-1,6-dienyl-4,4-dicarboxylate (1a) in CHCl3. The reaction displays a pronounced and variable induction period and gives dimethyl 3-methylene-4-methylcyclopentane-1,1-dicarboxylate (2a) as the kinetic product. The thermodynamically more favourable tri- and tetra-substituted alkenes dimethyl 3,4-dimethylcylopent-2-ene-1,1-dicarboxylate (3a) and dimethyl 3,4-dimethylcylopent-3-ene-1,1-dicarboxylate (4a) are also generated directly (3a) or by isomerisation (3a and 4a) of 2a. The mechanism of procatalyst activation and the ensuing cycloisomerisation reaction was investigated by NMR spectroscopy (1H, 2H, 13C) and GC analysis of the products arising from isotopically labelled substrates (13C, 2H). Three general mechanisms were considered: hydrometallation, cyclometallation and C-H insertion. These last two were shown to be incompatible with the results. The first, which involves generation and propagation of a palladium hydride species ("Pd-H"), was found to be consistent with both the isotopic distribution and stereochemistry of the reaction product and is supported by the observation of intermolecular transfer of a single 2H label. Due to the high catalytic activity of the palladium hydride and its slow generation, the cycloisomerisation process ultimately yields a mixture of alkene products (2a, 3a and 4a) with incomplete consumption of the procatalyst [Pd(eta3-C3H5)(MeCN)2]OTf. The mechanism by which the catalytically active palladium hydride is generated from the procatalyst was studied in detail by NMR spectroscopic analysis of stoichiometric reactions between diene 1a and [Pd(eta3-C3H5)(MeCN)2]OTf. This demonstrated that a carbopalladated complex, namely, [Pd[7,7-(CO2Me)2-(1,2,5,9,10-eta5)-dec-1,9-diene)](OTf)] (15a), is formed in small quantities by unfavourable displacement of acetonitrile by the diene, followed by a rapid and irreversible beta-migratory insertion reaction. Although attempts to isolate 15a from the reaction mixture were not successful (due to its slow decomposition, low concentration and competing cycloisomerisation), an alternative synthesis in the absence of acetonitrile allowed its isolation and characterisation. However, pure samples of 15a are completely ineffective as a procatalyst system for cycloisomerisation of 1a. Further investigation revealed that treatment of 15a with one equivalent of water results in quantitative beta-H elimination to generate triene 16a (C(1)-allylated 1a). Thus, addition of catalytic quantities of water to a solution of 1a in CHCl3 containing 5 mol% 15a and 10 mol% MeCN results in generation of an active "Pd-H" catalyst for cycloisomerisation. Although procatalyst activation is facilitated by traces of water, no exchange of protons is observed between "Pd-H" and H2O under catalytic turnover. The slow generation of 15a and the requirement for traces of water for beta-H elimination accounts for variability in the induction period when [Pd(eta3-C3H5)(MeCN)2]OTf is employed as procatalyst.