The Equivalence of Remote Electronic and Paper Patient Reported Outcome (PRO) Collection.

Individual patient level Patient Reported Outcomes (PROs) are increasingly important in clinical practice. Web-based collection enables clinicians to remotely collect scores at regular intervals, away from the clinic setting. In this randomized crossover study, 47 patients, having undergone hip surgery, were allocated to two groups. Group 1 completed the web-based scores followed by the paper equivalents one week later; Group 2 completed the scores the other way around. The Intraclass Correlation Coefficient (ICC) for the Oxford Hip Score was 0.99, 0.98 to 0.99 (ICC, 95% CI) and the ICCs for the other scores were between 0.95 and 0.97. We conclude that remote ePRO collection using this web-based system reveals excellent equivalence to paper PRO collection of the Oxford Hip, McCarthy, UCLA and howRu scores.

An analysis of the origins of a cooperative binding energy of dimerization.

The cooperativity between binding of cell wall precursor analogs (ligands) to and antibiotic dimerization of the clinically important vancomycin group antibiotics was investigated by nuclear magnetic resonance. When dimerization was weak in the absence of a ligand, the increase in the dimerization constant in the presence of a ligand derived largely from changes associated with tightening of the dimer interface. When dimerization was strong in the absence of a ligand, the increase in the dimerization constant in the presence of a ligand derived largely from changes associated with tightening of the ligand-antibiotic interface. These results illustrate how, when a protein has a loose structure, the binding energy of another molecule to the protein can derive in part from changes occurring within the protein.

Potential infection-control benefit for Ireland from pre-movement testing of cattle for tuberculosis.

Pre-movement testing for bovine tuberculosis (BTB) was compulsory in Ireland until 1996. We determined the proportion of herd restrictions (losing BTB-free status) attributable to the recent introduction of an infected bovid; described events between restoration of BTB-free status (de-restriction) and the next herd-level test for BTB; estimated the proportion of undetected infected cattle present at de-restriction; identified high-risk movements between herds (movements most likely to involve infected cattle); and determined the potential yield of infected cattle discovered (or herds that would not lose their BTB-free status) by pre-movement testing, relative to the numbers of cattle and herds tested. We used national data for all 6252 herds with a new BTB restriction in the 12 months from 1 April 2003 and 3947 herds declared BTB-free in the 12 months from 1 October 2001. We identified higher-risk animals from our logistic generalized estimating-equation models. We attributed 6-7% of current herd restrictions to the recent introduction of an infected animal. There were considerable changes to herd structure between de-restriction and the next full-herd test, and infection was detected in 10% of herds at the first assessment (full-herd test or abattoir surveillance) following de-restriction. Following movement from a de-restricted herd, the odds of an animal being positive at the next test increased with increasing time in the source herd prior to movement, increasing time between de-restriction and the next full-herd test and increasing severity of the source herd restriction. The odds decreased with increasing size of the source herd. We estimated that 15.9 destination-herd restrictions per year could be prevented for every 10,000 cattle tested pre-movement and that 3.3 destination-herd restrictions per year could be prevented for every 100 source herds tested pre-movement. The yield per pre-movement test can be increased by focusing on high-risk movements; however, this would result in a substantial decrease in the total number of potential restrictions identified.

The effect of varying levels of population control on the prevalence of tuberculosis in badgers in Ireland.

We examined the effect of varying levels of badger population control on the prevalence of Mycobacterium bovis infection in badgers in four counties of Ireland. In the 'Removal' and 'Buffer' areas, proactive culling was conducted to substantially reduce and subsequently maintain badger populations at a low level for five years. In the 'Reference' areas, localised reactive culling was conducted in association with herd breakdowns. The infection status of badgers was determined using bacteriology. A total of 2696 badgers were recruited into the study, and 19.0% were found to be infected with M. bovis. The two population control strategies had differing effects on the subsequent prevalence of tuberculosis in badger populations. Proactive culling led to a long term decrease in the prevalence of tuberculosis in the re-emergent populations. Although there was an overall decline in the disease prevalence, no consistent trend in disease prevalence as a result of reactive culling was observed.

A Bayesian population PK-PD model of ispinesib-induced myelosuppression.

The goal of the present analysis is to fit a Bayesian population pharmacokinetic pharmacodynomic (PK-PD) model to characterize the relationship between the concentration of ispinesib and changes in absolute neutrophil counts (ANC). Ispinesib, a kinesin spindle protein (KSP) inhibitor, blocks assembly of a functional mitotic spindle, leading to G2/M arrest. A first time in human, phase I open-label, non-randomized, dose-escalating study evaluated ispinesib at doses ranging from 1 to 21 mg/m(2). PK-PD data were collected from 45 patients with solid tumors. The pharmacokinetics of ispinesib were well characterized by a two-compartment model. A semimechanistic model was fit to the ANC. The PK and PD data were successfully modelled simultaneously. This is the first presentation of simultaneously fitting a PK-PD model to ANC using Bayesian methods. Bayesian methods allow for the use of prior information for some system-related parameters. The model may be used to examine different schedules, doses, and infusion times.

Post-operative blood salvage with autologous retransfusion in primary total hip replacement.

Clinical, haematological or economic benefits of post-operative blood salvage with autologous blood re-transfusion have yet to be clearly demonstrated for primary total hip replacement. We performed a prospective randomised study to analyse differences in postoperative haemoglobin levels and homologous blood requirements in two groups of patients undergoing primary total hip replacement. A series of 158 patients was studied. In one group two vacuum drains were used and in the other the ABTrans autologous retransfusion system. A total of 58 patients (76%) in the re-transfusion group received autologous blood. There was no significant difference in the mean post-operative haemoglobin levels in the two groups. There were, however, significantly fewer patients with post-operative haemoglobin values less than 9.0 g/dl and significantly fewer patients who required transfusion of homologous blood in the re-transfusion group. There was also a small overall cost saving in this group.

The learning curve with a new cephalomedullary femoral nail.

INTRODUCTION: The Cephalomedullary Nail (CMN) (Zimmer, Warsaw) was introduced in 2010 as part of a multicenter trial to evaluate its performance. At one year the CMN had results in keeping with other intramedullary devices with good union rates and low complication rates. In the second and third years of use an increased rate of implant failure was observed, towards the higher end of the 1-5% nail breakage rate seen in other studies. This study aims to evaluate if there any common features in this cohort of patients. MATERIALS AND METHODS: This is a retrospective cohort study looking at patients who underwent femoral fracture fixation using the cephalomedullary nail between January 2011 and June 2014. The primary outcome measure was implant failure; secondary outcomes were; fracture reduction and bisphosphonate use. RESULTS: 201 patients were included (135 female, 66 male) with an average age of 81 years. Ten (5%) nail breakages occurred in the study period at an average of 39 weeks (24-92); 9 were 125° nails 1 was a 130° nail and all fractured at the lag screw junction. CONCLUSIONS: Implant failure is a recognised complication of intramedullary nailing in cases of non-union. The increased rate of implant failure in our department required a change to a 130° CMN implant and a 3.2mm diameter guide wire for placement of the lag screw. We continue to monitor this difficult group of patients very closely.

Kinetic analysis of high-mobility-group proteins HMG-1 and HMG-I/Y binding to cholesterol-tagged DNA on a supported lipid monolayer.

High-mobility-group proteins HMG-1 and HMG-I/Y bind to multiple sites within a 268 bp A/T-rich enhancer element of the pea plastocyanin gene ( PetE ). Within a 31 bp region of the enhancer, the binding site for HMG-1 overlaps with the binding site for HMG-I/Y. The kinetics of binding and the affinities of HMG-1 and HMG-I/Y for the 31 bp DNA were determined using surface plasmon resonance. Due to very high non-specific interactions of the HMG proteins with a carboxymethyl-dextran matrix, a novel method using a cholesterol tag to anchor the DNA in a supported lipid monolayer on a thin gold film was devised. The phosphatidylcholine monolayer produced a surface that reduced background interactions to a minimum and permitted the measurement of highly reproducible protein-DNA interactions. The association rate constant ( k (a)) of HMG-I/Y with the 31 bp DNA was approximately 5-fold higher than the rate constant for HMG-1, whereas the dissociation constant ( K (D)) for HMG-I/Y (3.1 nM) was approximately 7-fold lower than that for HMG-1 (20.1 nM). This suggests that HMG-I/Y should bind preferentially at the overlapping binding site within this region of the PetE enhancer.

The structure of an asymmetric dimer relevant to the mode of action of the glycopeptide antibiotics.

BACKGROUND: Glycopeptide antibiotics of the vancomycin group are of crucial clinical importance in the treatment of methicillin resistant Staphylococcus aureus (MRSA)--the often lethal 'super-bug'--characterized by its resistance to a wide range of antibiotics in common use. The antibiotics exert their physiological action by blocking cell wall synthesis through recognition of nascent cell wall mucopeptides terminating in the sequence -D-Ala-D-Ala. Evidence suggests that the antibiotics are able to enhance their biological activity by the formation of homodimers, and this is supported by the observation that dimerization and peptide binding in vitro are cooperative phenomena. The basis of this enhancement is not understood at the molecular level. RESULTS: The first detailed structure of a dimeric glycopeptide antibiotic, that of eremomycin, is presented based upon solution NMR data. The overall structure of the dimer complex is asymmetric. The source of this asymmetry--a parallel alignment and mutual interaction of the disaccharides--appears to promote dimerization through specific sugar-sugar recognition. CONCLUSIONS: A molecular basis for the observed cooperativity of cell wall peptide binding by eremomycin is evident from these studies of the dimer. The carboxylate anion of the cell wall component, which is crucial to binding, forms an amide-mediated ion-pair interaction to the alkylammonium ion of the ring 6 sugar in the other half of the dimer making the structure and positioning of this sugar important in mediating cooperativity.

Observations on the quantitation of the phosphate content of peptides by fast-atom bombardment mass spectrometry.

Equimolar mixtures of the phosphorylated and dephosphorylated forms of several peptides have been subjected to fast-atom bombardment mass spectrometry (FABMS), to investigate whether the stoichiometry of phosphorylation can be determined from the relative molecular-ion abundances of the phospho and dephospho derivatives. It is concluded that quantitation can be achieved for peptides with large positive or negative hydrophobicity/hydrophilicity indices (delta F values) where addition of a phosphate group does not alter the distribution of the peptide within the matrix significantly. For peptides with small positive or negative delta F values, phosphopeptides tend to be partially suppressed by their dephosphorylated counterparts. Suppression can be partially or totally overcome by conversion of the peptide to a hydrophobic derivative, and by the selection of an appropriate matrix. Alternatively, addition of a very strong acid, perchloric acid, can even reverse the original suppression effect. This last effect is believed to be due to the increased ionic strength in the matrix, which forces a relatively hydrophilic analyte to the matrix surface; and the ability of such a phosphorylated analyte to form a more stable gas-phase cation.

Aurothioglucose inhibits induced NF-kB and AP-1 activity by acting as an IL-1 functional antagonist.

We have designed a series of recombinant CAT genes to study IL-1 signal transduction in murine fibroblast NIH 3T3 cells. We demonstrate that the HSV thymidine kinase (tk) promoter does not respond to IL-1, but that IL-1 induction of this promoter is observed after insertion of either NF-kB or AP-1 binding sites upstream of the HSV tk cap-site. We have studied the effects of indomethacin, dexamethasone and aurothioglucose (which have been used in the treatment of patients affected by rheumatoid arthritis) in the IL-1 inducible CAT assay. We show that aurothioglucose or dexamethasone is able to inhibit IL-1 induced CAT activity whereas a non-steroidal anti-inflammatory drug (indomethacin) is inactive. Order of addition experiments indicate that aurothioglucose, which has disease-modifying activity in treated patients, acts as an IL-1 functional antagonist in this system.

Human MAFA has alternatively spliced variants.

Human mast cell function-associated antigen (MAFA) cDNA has been cloned. This molecule is similar to the rat form having an intracellular domain containing a putative immunoreceptor tyrosine inhibition motif and an extracellular C type lectin-like domain. However, in contrast to rat MAFA, the amino acid sequence suggests the presence of two additional extracellular N-linked glycosylation sites. In addition, alternative mRNA transcripts are observed that differ substantially from those found in the rat.

A 15N nuclear magnetic resonance study of the biosynthesis of quinoxaline antibiotics.

Uniformly 15N-labelled triostin A and echinomycin have been prepared by growing the producing organisms on enriched media and their 15N nuclear magnetic resonance spectra partially assigned by a combination of nuclear Overhauser effect and scalar coupling constant measurements. Selective feeding experiments using unlabelled L-tryptophan-supplemented media have shown that N-1 and N-4 of the quinoxaline rings have their origins in the indole and amino groups of tryptophan, respectively.

Surface plasmon resonance analysis at a supported lipid monolayer.

Methods for the formation of supported lipid monolayers on top of a hydrophobic self assembled monolayer in a surface plasmon resonance instrument are described. Small unilamellar vesicles absorb spontaneously to the surface of the hydrophobic self-assembled monolayer to form a surface which resembles the surface of a cellular membrane. Lipophilic ligands, such as small acylated peptides or glycosylphosphatidylinositol-anchored proteins, were inserted into the absorbed lipid and binding of analytes to these ligands was analysed by surface plasmon resonance. Conditions for the formation of lipid monolayers have been optimised with respect to lipid type, chemical and buffer compatibility, ligand stability and reproducibility.

Amino acid sequence at the site on protein phosphatase inhibitor-2, phosphorylated by glycogen synthase kinase-3.

The primary structure surrounding the residue on Inhibitor-2 phosphorylated by glycogen synthase kinase-3 has been determined. The sequence is: Lys-Ile-Asp-Glu-Pro-Ser-Thr(P)-Pro-Tyr-His-Ser. This finding will facilitate studies of the effects of hormones on the phosphorylation state of Inhibitor-2 in vivo.

Is the hydrophobic effect stabilizing or destabilizing in proteins? The contribution of disulphide bonds to protein stability.

It has been recently concluded that the hydrophobic effect, hitherto regarded as a major driving force in the folding of proteins, destabilizes the folded state relative to the unfolded state. We summarize the properties of the hydrophobic effect obtained from solvent transfer experiments and show that the recent conclusion is an artifact of crosslinking in the unfolded state, caused by disulphide bonds, metals or cofactors. We show that, for the proteins in the data set, crosslinks surprisingly destabilize folded structures entropically, but stabilize them enthalpically to a greater extent. We also calculate non-polar surface areas of these unfolded proteins. These surface areas are decreased by crosslinks. The unfolded state of proteins lacking constraints, such as myoglobin, is well approximated by a mixture of residues containing alpha-helical and beta-sheet dihedral angles. Surface areas of unfolded proteins cannot be obtained by summing the surface areas of individual residues, since this ignores any unavoidable side-chain-side-chain interactions.

Molecular basis for methoxyamine initiated mutagenesis. 1H nuclear magnetic resonance studies of base-modified oligodeoxynucleotides.

In order to reach a more detailed understanding of the mechanism of the mutagenic action of methoxyamine and of N4-methoxycytidine and its 2'-deoxyribo-analogue, the solution structures of the self-complementary octanucleotide, d(CGAATTCG) and its analogues, d(CGAATCCG), d(CGAATMCG) and d(CGAATPCG) (designated 8mer-AT, 8mer-AC, 8mer-AM, and 8mer-AP, respectively), were investigated by 1H nuclear magnetic resonance spectroscopy; M is N4-methoxycytosine (mo4C) and P is an analogue, the bicyclic dihydropyrimido[4,5-c][1,2]oxazin-7-one, in which the N-O bond is held in the anti configuration with respect to N3 of the cytosine ring. Correlated spectroscopy and nuclear Overhauser spectroscopy allowed assignment of the base, anomeric and H2'/H2" protons in 8mers-AT, -AM and -AP, and showed that all three had features consistent with a regular B-DNA duplex structure. Duplex-to-coil transition temperatures were determined to be 52(+/- 2) degrees C (8mer-AT), 51(+/- 2) degrees C (8mer-AP), 32(+/- 2) degrees C (8mer-AM); on the chemical shift timescale, the melting transition was fast for 8mer-AT and 8mer-AP, but slow for 8mer-AM. Imino proton spectra were indicative of Watson-Crick base-pairing in 8mers-AT, -AP and -AM. The 8mer-AP duplex had a structure and melting characteristics virtually identical with those of the 8mer-AT duplex. The preferred syn configuration of the methoxyl group in M had a destabilising effect on the 8mer-AM duplex. At low temperatures, the A.M base-pair was in fast equilibrium between Watson-Crick and wobble configurations, with the methoxyl function anti-oriented, but the melting transition was accompanied by isomerization of the methoxyl group to the syn conformation. This syn-anti isomerization was the rate-determining step in the duplex-to-coil transition. The 8mer-AC oligomer did not form a stable duplex.

Protein folding in the absence of the solvent ordering contribution to the hydrophobic interaction.

Despite considerable effort there is no consensus as to what interaction, or set of interactions, provides the dominant force that drives protein folding and specifies folded protein structures. A key thermodynamic observation is that a large drop in heat capacity (delta Cp) usually accompanies folding in water. Various factors may contribute to this effect, especially changes in the structure of the solvent upon exposure of both non-polar and polar groups in the unfolded state. The unfavourable Gibbs free energy of solvating non-polar groups, in particular, is thought to provide a central driving force for folding (the hydrophobic effect) but the role of solvent ordering in this remains a matter of controversy. We report here a series of experiments that show that a protein can fold into its native conformation under conditions where solvent ordering effects are demonstrably negligible. In methanol/water mixtures ubiquitin unfolds reversibly with a delta Cp value that falls close to zero above about 30% (v/v) methanol. We are able to reason, on the basis of these data, that the net contribution to the heat capacity change arising primarily from the protein structure itself is not significant and that contributions from changes in solvent ordering are rendered negligible by the change in composition. Nuclear magnetic resonance measurements, however, indicate that non-polar side-chains do still become exposed to solvent in the denatured state under these conditions. The combination of these results and model compound studies suggests that the elimination of ordering effects is an intrinsic property of the mixed solvent. We can, therefore, conclude that the solvent ordering component of the hydrophobic effect is not an obligatory factor in determining the three-dimensional structure into which the protein will fold.

Dissecting the structure of a partially folded protein. Circular dichroism and nuclear magnetic resonance studies of peptides from ubiquitin.

The nature and interaction of structural elements in a partially ordered form of ubiquitin, the A-state, which is populated at low pH in 40 to 60% aqueous methanol, have been investigated. Two synthetic peptides have been studied under the same conditions: U(1-21), corresponding to the N-terminal beta-hairpin in the native (N) and A-states of ubiquitin and U(1-35), which includes this hairpin plus an alpha-helix. Circular dichroism studies indicate that, although these peptides are largely unfolded in water, their structural content in 30 and 60% methanol is comparable with the corresponding native secondary structure. Sequence-specific assignments of the 1H n.m.r. spectra of U(1-35) in aqueous methanol and subsequent secondary structure determination confirm the conservation in detail of native-like secondary structure. Corresponding resonances in spectra of U(1-35), U(1-21) and the A-state itself were found to have closely similar chemical shifts, suggesting that the beta-hairpin exists independently in the partially folded protein, with little or no influence from the rest of the molecule. This is confirmed by the virtual absence in nuclear Overhauser enhancement spectroscopy and rotating frame nuclear Overhauser enhancement spectroscopy spectra of nuclear Overhauser enhancement effects between structural elements. c.d. and n.m.r. evidence suggests that structure in the C-terminal half of the molecule in the A-state is largely non-native. Thus, although methanol is necessary to assure its stability in the absence of wider native interactions, the structure of the beta-hairpin, including the register of its hydrogen bonding, appears to be determined entirely by its own sequence. This intrinsic structural preference in the first part of the ubiquitin sequence is much stronger than in the C-terminal half, a conclusion reflected in the results from a variety of secondary structure prediction algorithms.

Molecular basis for methoxyamine-initiated mutagenesis: 1H nuclear magnetic resonance studies of oligonucleotide duplexes containing base-modified cytosine residues.

Methoxyamine, N4-methoxycytidine and its 2'-deoxyribo analogue are transition mutagens. The mechanism by which the latter acts after incorporation into or generation within DNA has been ascribed to the ability of the base analogue to pair effectively with both adenine and guanine. To obtain a detailed understanding of these interactions, the solution structures of the self-complementary octanucleotide d(CGGATCCG) and its analogues d(CGGATTCG), d(CGGATMCG) and d(CGGATPCG) (designated 8mer-GC, -GT, -GM and -GP, respectively) were investigated by 1H nuclear magnetic resonance spectroscopy; M is N4-methoxycytosine (mo4C) and P is an analogue, the bicyclic dihydropyrimido[4,5-c][1,2] oxazin-7-one. A variable temperature study showed the order of stability as 8mer GC > GP > GT > GM. Nuclear Overhauser spectroscopy permitted the assignment of the base, anomeric and H2'/H2" protons in these 8mers. All had spectra consistent with regular B-DNA duplex structures. Imino proton spectra showed that the 8mers GC, GP and GM involved Watson-Crick base-pairing but that the G.P and to a greater extent G.M base-pairs were in slow exchange on the nuclear magnetic resonance time-scale with the wobble configuration. Indeed, the G.M pair showed an additional exchange process interpreted in terms of the presence of syn and anti conformers of the methoxy group in the wobble pair. This accounts for the destabilization of M compared with the P-containing duplex. The observations are compared with those made earlier on the corresponding AT, AP and AM octamers. It is evident that M and P can form stable base-pairs with both A and G with essentially Watson-Crick geometry. This confirms the earlier, although unsubstantiated explanation for the transition mutational propenstty of methoxyamine which, in turn, was based on the fact that methoxycytosine bases have tautomeric constants (KT) much nearer to unity than the normal bases. The same general explanation for hydroxylamine and hydrazine-induced mutations is correspondingly rendered more certain.