Otitis externa: what is the problem with getting it right? A mixed-methods study in primary and secondary care.

OBJECTIVE: Otitis externa accounts for 1.1-1.3 per cent of patient presentations in primary care and 25 per cent of urgent referrals to ENT. This study aimed to explore otitis externa clinical decision-making at the primary-secondary care interface, otitis externa prevalence and recent trends in antimicrobial resistance in otitis externa related bacterial isolates and ototopical prescribing. METHOD: This is a mixed-methods study drawing on data from primary and secondary care and open National Health Service sources. RESULTS: A total of 101 general practitioner survey respondents reported frequently prescribing oral antibiotics for otitis externa. General practitioner consultations for otitis externa increased 25 per cent over 15 years. General practitioner ototopical preparations cost the National Health Service £7 410 440 in 2006 and £11 325 241 in 2016. A total of 162 consecutive hospital otitis externa-related bacterial isolates yielded 128 pseudomonas species, with 18 that were resistant to gentamicin and 7 that were resistant to ciprofloxacin. Ten guidelines reviewed showed systematic inconsistencies. CONCLUSION: General practitioners reported regularly prescribing oral antibiotics for otitis externa. Antimicrobial drug resistance is common in otitis externa. The available guidance is suboptimal.

Radiology of acute mastoiditis and its complications: a pictorial review and interpretation checklist.

BACKGROUND: Mastoiditis is an otological emergency, and cross-sectional imaging has a role in the diagnosis of complications and surgical planning. Advances in imaging technology are becoming increasingly sophisticated and, by the same token, the ability to accurately interpret findings is essential. METHODS: This paper reviews common and rare complications of mastoiditis using case-led examples. A radiologist-derived systematic checklist is proposed, to assist the ENT surgeon with interpreting cross-sectional imaging in emergency mastoiditis cases when the opinion of a head and neck radiologist may be difficult to obtain. RESULTS: A 16-point checklist (the 'mastoid 16') was used on a case-led basis to review the radiological features of both common and rare complications of mastoiditis; this is complemented with imaging examples. CONCLUSION: Acute mastoiditis has a range of serious complications that may be amenable to treatment, once diagnosed using appropriate imaging. The proposed checklist provides a systematic approach to identifying complications of mastoiditis.

Going where other methods cannot: A systematic mapping review of 25 years of qualitative research in Otolaryngology.

OBJECTIVE: To map the use of qualitative methods within otolaryngology, providing examples and identifying gaps in the literature. DESIGN: Systematic mapping review of journal-based literature from 1990 to 2015 using Medline, Embase, PsycINFO and CENTRAL. Included studies were categorised according to clinical subspecialty, research aims and qualitative approach. RESULTS: Of 4,061 identified articles, 388 were deemed relevant to qualitative research in ENT. The number of qualitative publications has risen markedly over the last 25 years (r = 0.802), particularly since 2010. The most commonly used method was semi-structured interviews 62.1% (241/388). Head and neck cancer (41.8% (162/388)) and otology (40.2% (156/388)) publish more qualitative research than rhinology (7.0% (27/388)) and laryngology (6.7% (26/388)). CONCLUSIONS: Qualitative research in otolaryngology has increased over time, but laryngology and rhinology remain under-represented. Most studies use interviews, underutilising the strengths of other qualitative methods. There is considerable scope for further application of qualitative methods in otolaryngology.

A post-genomic view of the mitochondrion in malaria parasites.

Mitochondria in Plasmodium parasites have many characteristics that distinguish them from mammalian mitochondria. Selective targeting of malaria parasite mitochondrial physiology has been exploited in successful antimalarial chemotherapy. At present, our understanding of the functions served by the parasite mitochondrion is somewhat limited, but the availability of the genomic sequences makes it possible to develop a framework of possible mitochondrial functions by providing information on genes encoding mitochondrially targeted proteins. This review aims to provide an overview of mitochondrial physiology in this post-genomic era. Although in many cases direct experimental proof for their mitochondrial functions may not be available at present, descriptions of these potential mitochondrial proteins can provide a basis for experimental approaches.

Flexibility of the neck region of the rieske iron-sulfur protein is functionally important in the cytochrome bc1 complex.

The crystal structure of the mitochondrial cytochrome bc1 complex suggests that movement of the extramembrane (head) domain of the Rieske iron-sulfur protein (ISP) is involved in electron transfer. Such movement requires flexibility in the neck region of ISP. To test this hypothesis, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc1 complexes with altered ISP necks (residues 39-48) were generated and characterized. Mutants with increased rigidity of the neck, generated by a double-proline substitution at Ala-46 and Ala-48 (ALA-PLP) or by a triple-proline substitution of ADV at residues 42-44 (ADV-PPP), have retarded (50%) or no photosynthetic growth, respectively. However, the mutant with a shortened neck, generated by deleting ADV (DeltaADV), has a photosynthetic growth rate comparable to that of complement cells, indicating that the length of the ISP neck is less critical than its flexibility in support of photosynthetic growth. The DeltaADV and ALA-PLP mutant membranes have 10 and 30% of the cytochrome bc1 complex activity found in the complement membrane, respectively, whereas the ADV-PPP mutant membrane contains no cytochrome bc1 complex activity. The loss of cytochrome bc1 complex activity in the DeltaADV membrane is attributed to improper docking of the head domain of ISP on cytochrome b, as indicated by a drastic change in the EPR characteristics of the Rieske iron-sulfur cluster. The loss of cytochrome bc1 complex activity in the ALA-PLP and ADV-PPP mutant membranes results from the decreased mobility of the ISP head domain due to the increased rigidity of the ISP neck. The ALA-PLP mutant complex has a larger activation energy than the wild-type complex, suggesting that movement of the head domain decreases the activation energy barrier of the cytochrome bc1 complex. Using the conditions developed for the isolation of the His-tagged complement cytochrome bc1 complex, a two-subunit complex (cytochromes b and c1) was obtained from the DeltaADV and ADV-PPP mutants, indicating that mutations at the neck region of ISP weaken the interactions among cytochrome b, ISP, and subunit IV.

Intrinsic uncoupling of cytochrome c oxidase may cause the maternally inherited mitochondrial diseases MELAS and LHON.

Mutations in the human mtDNA gene encoding subunit III of cytochrome c oxidase (CO) have been reported to cause MELAS and LHON. Poracoccus denitrificans cells expressing substitutions homologous to these MELAS- and LHON-causing mutations had lower growth yield than wild type cells and lower efficiency of proton pumping by CO (e.g. lower H+/e ratio and lower deltapsi), but had similar CO activity. These results indicate that both substitutions (F263L > A212T) cause intrinsic uncoupling, which may be the direct cause of the diseases. These results also suggest that subunit III is involved in proton pumping.

The involvement of serine 175 and alanine 185 of cytochrome b of Rhodobacter sphaeroides cytochrome bc1 complex in interaction with iron-sulfur protein.

An approach involving cysteine replacement of potentially noncritical amino acid residues, followed by chemical modification studies, was used to investigate structure-function of the "cd helix" of cytochrome b from Rhodobacter sphaeroides. Three amino acid residues, Ser-155, Ser-175, and Ala-185, which span this region of cytochrome b, were selected for this study. The S155C substitution yields cells unable to support photosynthetic growth, indicating that Ser-155 is a critical amino acid residue. Further mutational studies of Ser-155 indicate that the size of the amino acid side chain at this position is critical for photosynthetic growth of R. sphaeroides. On the other hand, the S175C and A185C substitutions yield cells with photosynthetic growth rates and enzyme kinetics of the bc1 complexes very similar to those of the unmutated complex, indicating that Ser-175 and Ala-185 are noncritical residues. Thus, engineered cysteines at these two positions of cytochrome b are suitable for membrane topology and domain/subunit interaction studies. Cys-175 does not react with a sulfhydryl-modifying reagent, N-ethylmaleimide (NEM), either in sealed, inside-out chromatophores or in detergent-disrupted chromatophores, indicating that position 175 of cytochrome b is inaccessible from both sides of the membrane and is probably buried within the protein complex. Cys-185 reacts with NEM only after detergent disruption of the sealed, inside-out chromatophores, indicating that this position of cytochrome b is accessible on the outer (periplasmic) surface of the membrane. These results place the cd helix of cytochrome b on the periplasmic side of the chromatophore membrane. When purified A185C-substituted bc1 complex was treated with NEM, about 87% of the activity was abolished due to NEM modification of Cys-185. The signature of the Rieske iron-sulfur center is broadened upon NEM modification of A185C, with the gx signal shifting from g = 1.80 to g = 1.75, suggesting that Ala-185 of cytochrome b interacts with the iron-sulfur protein. When purified S175C-substituted bc1 complex is treated with NEM, no change in the activity is observed, since Cys-175 is inaccessible to NEM. However, when the iron-sulfur protein is removed from the S175C-substituted bc1 complex, Cys-175 becomes accessible to NEM, indicating that Ser-175 of cytochrome b is shielded by the iron-sulfur protein in the bc1 complex.

The involvement of threonine 160 of cytochrome b of Rhodobacter sphaeroides cytochrome bc1 complex in quinone binding and interaction with subunit IV.

The cytochrome b subunit (subunit I) of the ubiquinolcytochrome c reductase (bc1 complex) is thought to participate in the formation of two quinone/quinol reaction centers, an oxidizing center (Qo) and a reducing center, in accordance with the quinone cycle mechanism. Threonine 160 is a highly conserved residue in a segment of subunit I that was shown to bind quinone and is placed near the putative Qo site in current models of the bc1 complex. Rhodobacter sphaeroides cells expressing bc1 complexes with Ser or Tyr substituted for Thr160 grow photosynthetically at a reduced rate, and cells expressing the mutated complexes produce an "elevated" level of the bc1 complex. The Ser substitution also affects the interaction of subunit IV with subunit I. Replacement of Thr160 by Ser results in about a 70% loss of the activity in the purified complex, whereas substitution by Tyr lowers the activity by more than 80%. Both replacements lower the apparent Km for ubiquinol. Electron paramagnetic resonance (EPR) spectroscopy shows that in the Ser substituted complex, the environments of the Rieske iron-sulfur cluster in subunit III and the high potential cytochrome b (b562) in subunit I have been modified. The spectra of the Ser160 and Tyr160 iron-sulfur clusters have become redox-insensitive, with a line shape resembling that of the native complex in the fully reduced state. The EPR signal of b562 in the Ser160 complex is shifted from g = 3.50 to g = 3.52, but otherwise the line shape is very similar to the spectrum of the native complex. Most of these results are consistent with current ideas regarding the structure and function of Qo in the bc1 complex, except for the alteration of the b562 EPR feature, because this heme is not thought to be located in proximity to Qo. Immunoblotting analysis showed that the Ser or Tyr substituted complex contained significantly less than a stoichiometric amount of subunit IV. The enzymatic activity of mutated bc1 complex was found to be activable by the addition of purified subunit IV. These results indicate that Thr160 plays an important role in the structure and/or function of the bc1 complex.

Molecular genetic and protein chemical characterization of the cytochrome ba3 from Thermus thermophilus HB8.

Thermus thermophilus HB8 cells grown under reduced dioxygen tensions contain a substantially increased amount of heme A, much of which appears to be due to the presence of the terminal oxidase, cytochrome ba3. We describe a purification procedure for this enzyme that yields approximately 100 mg of pure protein from 2 kg of wet mass of cells grown in < or = 50 microM O2. Examination of the protein by SDS-polyacrylamide gel electrophoresis followed by staining with Coomassie Blue reveals one strongly staining band at approximately 35 kDa and one very weakly staining band at approximately 18 kDa as reported earlier (Zimmermann, B.H., Nitsche, C.I., Fee, J. A., Rusnak, F., and Münck, E. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5779-5783). By contrast, treatment of the gels with AgNO3 reveals that the larger polypeptide stains quite weakly while the smaller polypeptide stains very strongly. These results suggested the presence of two polypeptides in this protein. Using partial amino acid sequences from both proteins to obtain DNA sequence information, we isolated and sequenced a portion of the Thermus chromosome containing the genes encoding the larger protein, subunit I (cbaA), and the smaller protein, subunit II (cbaB). The two polypeptides were isolated using reversed phase liquid chromatography, and their mole percent amino acid compositions are consistent with the proposed translation of their respective genes. The two genes appear to be part of a larger operon, but we have not extended the sequencing to identify initiation and termination sequences. The deduced amino acid sequence of subunit I includes the six canonical histidine residues involved in binding the low spin heme B and the binuclear center Cu(B)/heme A. These and other conserved amino acids are placed along the polypeptide among alternating hydrophobic and hydrophilic segments in a pattern that shows clear homology to other members of the heme- and copper-requiring terminal oxidases. The deduced amino acid sequence of the subunit II contains the CuA binding motif, including two cysteines, two histidines, and a methionine, but, in contrast to most other subunits II, it has only one region of hydrophobic sequence near its N terminus. Alignment of these two polypeptides with other cytochrome c and quinol oxidases, combined with secondary structure analysis and previous spectral studies, clearly establish cytochrome ba3 as a bona fide member of the superfamily of heme- and copper-requiring oxidases. The alignments further indicate that cytochrome ba3 is phylogenetically distant from other cytochrome c and quinol oxidases, and they substantially decrease the number of conserved amino acid residues.

An enhanced broad-host-range vector for gram-negative bacteria: avoiding tetracycline phototoxicity during the growth of photosynthetic bacteria.

A mobilizable, broad-host-range (bhr) plasmid was derived from the widely used IncP1 vector pRK415. The new vector, pRKD418, contains an additional resistance gene and an enlarged multiple cloning site (MCS) region. The optimal growth of pRK415-containing bacteria under photosynthetic conditions generally requires the use of optical filters to protect the selective antibiotic tetracycline (Tc) from photooxidation with the resulting production of toxic photoproducts; pRK415 is not stably maintained in the absence of selective pressure. The addition of a trimethoprim-resistant dihydrofolate reductase-encoding gene provided for optimal photosynthetic growth in the presence of a selective antibiotic without any special apparatus. The presence of an antibiotic marker not found in commonly used cloning vectors in many cases facilitates the subcloning of inserts into the bhr plasmid. The new MCS region provides further cloning flexibility with at least sixteen available restriction sites. Easily constructed derivative plasmids, exemplified by pRKD418KmE, provide a convenient screening procedure for the detection of recombinants during subcloning.

Molecular modeling studies on the proposed NaCl-induced dimerization of Chromatium vinosum high-potential iron protein.

Previous work (Dunham, W.R., Hagen, W.R., Fee, J.A., Sands, R.H., Dunbar, J.B., Humblet, C. (1991) An investigation of Chromatium vinosum high-potential iron-sulfur protein by EPR and Mössbauer spectroscopy; evidence for a freezing-induced dimerization in NaCl solutions, Biochimica Biophysica Acta 1079, 253-262) suggested that under specific solution conditions and slow freezing times, samples of oxidized Chromatium vinosum (Cv) high-potential, iron-sulfur protein (HiPIP) form dimeric structures that exhibit characteristic spin-spin interaction in the EPR spectrum. In that study, it was also shown that two HiPIP molecules could approach each other along their Fe1-S4 axes to a distance of approximately 13-14 A, as required by an analysis of the spin-spin physics. This is made possible because of a flattened surface on one side of the molecule within which S4 may, depending on side-chain motions, interact with solvent (Carter, C.W., Jr., Kraut, J., Freer, S.T., Alden, R.A., Sieker, L.C., Adman, E.T., Jensen, L.H. (1972) A comparison of Fe4S4 clusters in high potential iron protein and in ferredoxin, Proc. Natl. Acad. Sci. USA 69, 3527-3529). Here we describe a computer generated, hypothetical model of this proposed dimeric structure which suggests an energetically favorable interaction between two Cv HiPIP molecules and could account for the experimental observations. Two Cv HiPIP molecules brought together along their Fe1-S4 axes and maintained at a center-to-center distance of 14 A can be rotated with respect to each other so as to create complementary interactions between two glutamine residues, two phenylalanine residues, and two leucine residues, and an energetically unfavorable interaction between two arginine residues. Energy minimization calculations using the program XPLOR indicate that this arrangement may provide an overall energetically favorable interaction between the two HiPIP molecules that is strengthened by site-specific binding of Na and Cl ions.

Cytochrome caa3 from the thermophilic bacterium Thermus thermophilus: a member of the heme-copper oxidase superfamily.

The subject of this short review is the cytochrome c oxidase (caa3) from the thermophilic bacterium Thermus thermophilus. First, some of the extensive physical and enzymological results obtained with this enzyme are reviewed, and two experiments are described, involving isotope substitutions in combination with Mössbauer and ENDOR spectroscopies, which have provided novel insight into the active sites of the enzyme. Second, we summarize recent molecular genetic work showing that Thermus cytochrome caa3 is a bona fide member of the superfamily of heme-copper oxidases. Finally, we present a rough three-dimensional model and speculate about certain features of the metal-binding sites.

Cytochrome oxidase genes from Thermus thermophilus. Nucleotide sequence of the fused gene and analysis of the deduced primary structures for subunits I and III of cytochrome caa3.

Cytochrome caa3, a cytochrome c oxidase from Thermus thermophilus, has been purified and extensively characterized as a two-subunit enzyme containing the metal centers characteristic of cytochrome c oxidases (cytochromes a and a3; copper centers CuA and CuB) and an additional cytochrome c (Fee, J. A., Kuila, D., Mather, M. W., and Yoshida, T. (1986) Biochim. Biophys. Acta 853, 153-185). We have now cloned and sequenced the genes encoding the subunits of this enzyme. The smaller subunit consists of a typical oxidase subunit II sequence fused to a cytochrome c domain (Mather, M. W., Springer, P., and Fee, J. A. (1991) J. Biol. Chem. 266, 5025-5035). The larger subunit, the A-protein, is encoded by a fusion gene lying immediately downstream of the subunit IIc gene. The 5' portion of this gene encodes an oxidase subunit I homolog, whereas the 3' portion is homologous to oxidase subunits III. The A-protein from the purified enzyme appears too small from SDS-polyacrylamide gel electrophoresis and quantitative amino acid analyses to be a complete subunit I/III fusion, but it is currently not known if proteolytic processing occurs. Analyses of the sequences of oxidase subunits are presented which clearly identify T. thermophilus cytochrome caa3 as a bona fide member of the greater family of heme- and copper-requiring oxidases. As one consequence, it is confirmed that the set of invariant histidine residues (potential ligands of the metal centers) in cytochrome c oxidase subunits I and II is reduced to 8. Possible topological and helix packing models are developed based on considerations of homology, hydropathy, and variability.

Development of plasmid cloning vectors for Thermus thermophilus HB8: expression of a heterologous, plasmid-borne kanamycin nucleotidyltransferase gene.

While several Thermus genes have been cloned and T. thermophilus has been shown to be transformable, molecular genetic studies of these thermophiles have been hampered by the absence of selectable cloning vectors. We have constructed a selectable plasmid by random insertion of a heterologous gene encoding a thermostable kanamycin nucleotidyltransferase activity into a cryptic, multicopy plasmid from T. thermophilus HB8. This plasmid should serve as a suitable starting point for the development of a gene expression system for T. thermophilus.

Cytochrome oxidase genes from Thermus thermophilus. Nucleotide sequence and analysis of the deduced primary structure of subunit IIc of cytochrome caa3.

Cytochrome caa3, a cytochrome c oxidase from Thermus thermophilus, is a two-subunit enzyme containing the four canonical metal centers of cytochrome c oxidases (cytochromes a and a3; copper centers CuA and CuB) and an additional cytochrome c. The smaller subunit contains heme C and was termed the C-protein. We have cloned the genes encoding the subunits of the oxidase and determined the nucleotide sequence of the C-protein gene. The gene and deduced primary amino acid sequences establish that both the gene and the protein are fusions with a typical subunit II sequence and a characteristic cytochrome c sequence; we now call this subunit IIc. The protein thus appears to represent a covalent joining of substrate (cytochrome c) to its enzyme (cytochrome c oxidase). In common with other subunits II, subunit IIc contains two hydrophobic segments of amino acids near the amino terminus that probably form transmembrane helices. Variability analysis of the Thermus and other subunit II sequences suggests that the two putative transmembrane helices in subunit II may be located on the surface of the hydrophobic portion of the intact cytochrome oxidase protein complex. Also in common with other subunits II is a relatively hydrophilic intermembrane domain containing a set of conserved amino acids (2 cysteines and 2 histidines) which have previously been proposed by others to serve as ligands to the CuA center. We compared the subunit IIc sequence with that of related proteins. N2O reductase of Pseudomonas stutzeri, a multi-copper protein that appears to contain a CuA site (Scott, R.A., Zumft, W.G., Coyle, C.L., and Dooley, D.M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4082-4086), contains a 59-residue sequence element that is homologous to the "CuA sequence motif" found in cytochrome oxidase subunits II, including all four putative copper ligands. By contrast, subunit II of the Escherichia coli quinol oxidase, cytochrome bo, also contains a region homologous to the CuA motif, but it lacks the proposed metal binding histidine and cysteine residues; this is consistent with the apparent absence of CuA from cytochrome bo.

Plasmid-associated aggregation in Thermus thermophilus HB8.

Thermus thermophilus HB8, a moderate thermophile, exhibits visible aggregation when growing on a rich broth. Strain HB8 also contains two cryptic plasmids. We isolated cured strains from HB8 and observed that loss of the 47-MDa plasmid was correlated with loss of aggregation. An enrichment procedure was developed for aggregating cells and used to demonstrate that aggregation was restored upon transformation of a cured strain with plasmid DNA. The aggregation phenotype of transformed cells was variably stable; most did not retain either the plasmid or the phenotype for prolonged periods of growth. Hybridization experiments using a partial sequence from the 47-MDa plasmid suggested the presence of a repeated DNA sequence on this plasmid and on the chromosome. This is the first report of a phenotype associated with a plasmid from a Thermus strain.