Biomedical subjects

Paul Clarke

Publications and source records attributed to Paul Clarke.

At least 19 recordsLinked to original sources

PubMed2006

Factors determining the potential for onward transmission of variant Creutzfeldt-Jakob disease via surgical instruments.

While the number of variant Creutzfeldt-Jakob disease (vCJD) cases continues to decline, concern has been raised that transmission could occur directly from one person to another through routes including the transfer of blood and shared use of surgical instruments. Here we firstly present data on the surgical procedures undertaken on vCJD patients prior to onset of clinical symptoms, which supports the hypothesis that cases via this route are possible. We then apply a mathematical framework to assess the potential for self-sustaining epidemics via surgical procedures. Data from hospital episode statistics on the rates of high- and medium-risk procedures in the UK were used to estimate model parameters, and sensitivity to other unknown parameters about surgically transmitted vCJD was assessed. Our results demonstrate that a key uncertainty determining the scale of an epidemic and whether it is self-sustaining is the number of times a single instrument is re-used, alongside the infectivity of contaminated instruments and the effectiveness of cleaning. A survey into the frequency of re-use of surgical instruments would help reduce these uncertainties.

Creutzfeldt-Jakob Syndrome↗

PubMed2006

Urinary biomarker profiling in transitional cell carcinoma.

Urinary biomarkers or profiles that allow noninvasive detection of recurrent transitional cell carcinoma (TCC) of the bladder are urgently needed. We obtained duplicate proteomic (SELDI) profiles from 227 subjects (118 TCC, 77 healthy controls and 32 controls with benign urological conditions) and used linear mixed effects models to identify peaks that are differentially expressed between TCC and controls and within TCC subgroups. A Random Forest classifier was trained on 130 profiles to develop an algorithm to predict the presence of TCC in a randomly selected initial test set (n = 54) and an independent validation set (n = 43) several months later. Twenty two peaks were differentially expressed between all TCC and controls (p < 10(-7)). However potential confounding effects of age, sex and analytical run were identified. In an age-matched sub-set, 23 peaks were differentially expressed between TCC and combined benign and healthy controls at the 0.005 significance level. Using the Random Forest classifier, TCC was predicted with 71.7% sensitivity and 62.5% specificity in the initial set and with 78.3% sensitivity and 65.0% specificity in the validation set after 6 months, compared with controls. Several peaks of importance were also identified in the linear mixed effects model. We conclude that SELDI profiling of urine samples can identify patients with TCC with comparable sensitivities and specificities to current tumor marker tests. This is the first time that reproducibility has been demonstrated on an independent test set analyzed several months later. Identification of the relevant peaks may facilitate multiplex marker assay development for detection of recurrent disease.

Adult↗

PubMed2006

Vitamin K prophylaxis for preterm infants: a randomized, controlled trial of 3 regimens.

OBJECTIVE: Preterm infants may be at particular risk from either inadequate or excessive vitamin K prophylaxis. Our goal was to assess vitamin K status and metabolism in preterm infants after 3 regimens of prophylaxis. METHODS: Infants <32 weeks' gestation were randomized to receive 0.5 mg (control) or 0.2 mg of vitamin K1 intramuscularly or 0.2 mg intravenously after delivery. Primary outcome measures were serum vitamin K1, its epoxide metabolite (vitamin K1 2,3-epoxide), and undercarboxylated prothrombin assessed at birth, 5 days, and after 2 weeks of full enteral feeds. Secondary outcome measures included prothrombin time and factor II concentrations. RESULTS: On day 5, serum vitamin K1 concentrations in the 3 groups ranged widely (2.9-388.0 ng/mL) but were consistently higher than the adult range (0.15-1.55 ng/mL). Presence of vitamin K1 2,3-epoxide on day 5 was strongly associated with higher vitamin K1 bolus doses. Vitamin K1 2,3-epoxide was detected in 7 of 29 and 4 of 29 infants from the groups that received 0.5 mg intramuscularly and 0.2 mg intravenously, respectively, but in none of 32 infants from group that received 0.2 mg intramuscularly. After 2 weeks of full enteral feeding, serum vitamin K1 was lower in the infants who received 0.2 mg intravenously compared with the infants in the control group. Three infants from the 0.2-mg groups had undetectable serum vitamin K1 as early as the third postnatal week but without any evidence of even mild functional deficiency, as shown by their normal undercarboxylated prothrombin concentrations. CONCLUSIONS: Vitamin K1 prophylaxis with 0.2 mg administered intramuscularly maintained adequate vitamin K status of preterm infants until a median age of 25 postnatal days and did not cause early vitamin K1 2,3-epoxide accumulation. In contrast, 0.2 mg administered intravenously and 0.5 mg administered intramuscularly led to vitamin K1 2,3-epoxide accumulation, possibly indicating overload of the immature liver. To protect against late vitamin K1 deficiency bleeding, breastfed preterm infants given a 0.2-mg dose of prophylaxis should receive additional supplementation when feeding has been established.

Female↗

PubMed2006

FoxB of Pseudomonas aeruginosa functions in the utilization of the xenosiderophores ferrichrome, ferrioxamine B, and schizokinen: evidence for transport redundancy at the inner membrane.

Expression of the inner membrane protein FoxB (PA2465) of Pseudomonas aeruginosa in mutants of Sinorhizobium meliloti that are defective in the utilization of ferrichrome, ferrioxamine B, and schizokinen resulted in the restoration of siderophore utilization. Mutagenesis of foxB in P. aeruginosa did not abolish siderophore utilization, suggesting that the function is redundant.

Bacterial Proteins↗

PubMed2006

Immunogenicity and induction of immunological memory of the heptavalent pneumococcal conjugate vaccine in preterm UK infants.

Data on the immunogenicity and memory induction of pneumococcal conjugate vaccines in very preterm infants is limited. We vaccinated 69 full term and 68 preterm infants (median gestational age (GA) 30 weeks) with a 7-valent pneumococcal conjugate vaccine (PCV7) at 2/3/4 months of age, followed by a plain polysaccharide booster at 12 months of age. IgG-GMC (ELISA) was significantly lower in preterm infants to six vaccine serotypes (ST) at 2 months and 5 months of age, to five ST at 12 months of age and to three ST at 13 months of age. A significantly lower proportion of preterm infants achieved IgG levels>or=0.35 microg/ml to ST 4, 6B and 9V at 5 months and to ST 4, 6B, 18C, 19F and 23F at 12 months of age. Fold rises following the polysaccharide booster were comparable to those of term infants. At least 93% of both cohorts achieved IgG>or=0.35 microg/ml to all STs following booster vaccination. Pneumococcal conjugate vaccine at an accelerated schedule of 2/3/4 months of age is likely to provide protection against pneumococcal disease for preterm infants. Antibody concentrations wane over the first year of life in both preterm and term infants and booster vaccination is therefore likely to be important.

Antibodies, Bacterial↗

PubMed2006

Response of steroid-treated former preterm infants to a single dose of meningococcal C conjugate vaccine.

Attenuated antibody responses have been reported in preterm infants who received neonatal dexamethasone treatment. The duration of immunosuppression may extend into later infancy. This study assessed the immune response of former preterm infants to a single meningococcal serogroup C conjugate (MCC) immunisation given after infancy. A cohort of 49 toddlers born at less than 33 weeks' gestation were given an initial dose of MCC vaccine at a median age of 13 months; 11 had received dexamethasone in the neonatal period. Sera obtained 4 weeks post immunisation were analysed for serum bactericidal antibody (SBA) and serogroup C-specific IgG antibody concentrations. Immune responses were compared with those of an historical cohort of 70 term toddlers given a single dose of the same vaccine at age 13 months. An SBA titre of > or =8 was taken to indicate a protective response. Following a single MCC dose, the SBA geometric mean titre (GMT) for former preterm infants was 249 (95% C.I. 111, 558), not significantly different from that of the historical term cohort whose SBA GMT was 141 (95% C.I. 89, 224) (p=0.06). A significantly lower proportion of former preterm infants achieved a protective SBA titre of > or =8 compared with term infants, 37/48 (77%) versus 64/70 (91%), (p=0.03). For steroid-treated and non steroid-treated subgroups, SBA GMTs were 1237 (95% C.I. 250, 6132) and 154 (62, 385), respectively, and numbers achieving an SBA titre of > or =8 were 10/11 (91%) and 27/37 (73%), (p=0.42). Most children born at <33 weeks' gestation mount a protective immune response to a single MCC vaccine dose given at age 13 months, but fewer former preterm infants attain a protective SBA titre of 8 compared with term infants. Previous neonatal dexamethasone treatment does not appear to attenuate immune response after infancy.

Antibodies, Bacterial↗

PubMed2006

Mechanical ventilation of very low birth weight infants: is volume or pressure a better target variable?

OBJECTIVE: To compare the efficacy and safety of volume-controlled (VC) ventilation to time-cycled pressure-limited (TCPL) ventilation in very low birth weight infants with respiratory distress syndrome (RDS). STUDY DESIGN: Newborns weighing between 600 and 1500 g and with a gestational age of 24 to 31 weeks who had RDS were randomized to receive either VC or TCPL ventilation and treated with a standardized protocol. The 2 modalities were compared by determining the time required to achieve a predetermined success criterion, on the basis of either the alveolar-arterial oxygen gradient <100 mm Hg or the mean airway pressure <8 cm H(2)O. Secondary outcomes included mortality, duration of mechanical ventilation, and complications commonly associated with ventilation. RESULTS: The mean time to reach the success criterion was 23 hours in the VC group versus 33 hours in the TCPL group (P = .15). This difference was more striking in babies weighing <1000g (21 versus 58 hours; P = .03). Mean duration of ventilation with VC was 255 hours versus 327 hours with TCPL (P = .60). There were 5 deaths in the VC group and 10 deaths in the TCPL group (P = .10). The incidence of other complications was similar. CONCLUSION: VC ventilation is safe and efficacious in very low birth weight infants and may have advantages when compared with TCPL, especially in smaller infants.

Female↗

PubMed2006

Nasal continuous positive airway pressure for neonatal back transfer.

We report the use of nasal continuous positive airway pressure (nCPAP) for neonatal back transfer of 51 babies. nCPAP may be used to provide safe respiratory support during back transportation of infants. Babies transferred on nCPAP were significantly smaller and of younger postmenstrual age than historical comparators who had to wait for transfer until independent of nCPAP. nCPAP transferred babies spent significantly less time on the intensive care unit.

Continuous Positive Airway Pressure↗

PubMed2006

Identification and characterization of an iron-regulated gene, chtA, required for the utilization of the xenosiderophores aerobactin, rhizobactin 1021 and schizokinen by Pseudomonas aeruginosa.

Pseudomonas aeruginosa utilizes several xenosiderophores under conditions of iron limitation, including the citrate hydroxamate siderophore aerobactin. Analysis of the P. aeruginosa genome sequence revealed the presence of two genes, chtA (PA4675) and PA1365, encoding proteins displaying significant similarity to the aerobactin outer-membrane receptor, IutA, of Escherichia coli. The chtA and PA1365 genes were mutated by insertional inactivation and it was demonstrated that ChtA is the outer-membrane receptor for aerobactin. ChtA also mediated the utilization of rhizobactin 1021 and schizokinen, which are structurally similar to aerobactin. In contrast to the utilization of other xenosiderophores by P. aeruginosa, there was no apparent redundancy in the utilization of aerobactin, rhizobactin 1021 and schizokinen. The utilization of citrate hydroxamate siderophores by P. aeruginosa was demonstrated to be TonB1 dependent. A Fur box was identified in the region directly upstream of chtA and it was demonstrated by the in vivo Fur titration assay that this region is capable of binding Fur and accordingly that expression of chtA is iron regulated. The PA1365 mutant was unaffected in the utilization of citrate hydroxamate siderophores.

Bacterial Outer Membrane Proteins↗

PubMed2005

Influences of blood sample processing on low-molecular-weight proteome identified by surface-enhanced laser desorption/ionization mass spectrometry.

BACKGROUND: Profiling approaches in proteomics, such as surface-enhanced laser desorption/ionization (SELDI) mass spectrometry, are used in disease marker discovery. The aim of this study was to investigate the potential influence of selected preanalytical factors on the results obtained. METHODS: Plasma samples anticoagulated with EDTA, citrate, or heparin, and serum samples from healthy volunteers were profiled by SELDI on CM10, immobilized metal affinity capture (IMAC) array with copper, and H50 chip surfaces. Using linear mixed-effects models, we examined the influence of elapsed time between venipuncture and sample separation (immediate to 24 h) and the type of serum tube used (Greiner Vacuette activator, gel serum separator, or plain tubes). We analyzed purified platelets, as well as platelet-poor and platelet-rich plasma samples treated with calcium and/or thrombin to determine the platelet contribution, directly or via the clotting process, to the profiles generated. We then used cluster analysis to identify samples with similar peak profiles. RESULTS: Different plasma types and sera could be distinguished on the basis of cluster analyses of their spectral profiles. Elapsed time between venipuncture and separation of plasma and serum from blood samples altered the profiles obtained, particularly for serum samples and particularly on IMAC chips. The type of serum collection tube also affected the profiles because of differences in clotting time. In vitro manipulation of platelets revealed that specific peaks in IMAC profiles of serum appeared to be derived directly from platelets. Several other peaks, including some of those exhibiting time-dependent changes, arose during the clotting process. CONCLUSION: Preanalytical variables, such as sample handling, can markedly influence results.

Adult↗

PubMed2005

RirA is the iron response regulator of the rhizobactin 1021 biosynthesis and transport genes in Sinorhizobium meliloti 2011.

The genes encoding the biosynthesis and transport of rhizobactin 1021, a siderophore produced by Sinorhizobium meliloti, are negatively regulated by iron. Mutagenesis of rirA, the rhizobial iron regulator, resulted in abolition of the iron responsive regulation of the biosynthesis and transport genes. Bioassay analysis revealed that the siderophore is produced in the presence of iron in a rirA mutant. RNA analysis and GFP fusions supported the conclusion that RirA is the mediator of iron-responsive transcriptional repression of the two transcripts encoding the biosynthesis and transport genes. RirA in S. meliloti appears to fulfil the role often observed for Fur in other bacterial species. The regulator was found to mediate the iron-responsive expression of two additional genes, smc02726 and dppA1, repressing the former while activating the latter. The rirA mutant nodulated the host plant Medicago sativa (alfalfa) and fixed nitrogen as effectively as the wild type.

Bacterial Proteins↗

PubMed2005

Projections of the future course of the primary vCJD epidemic in the UK: inclusion of subclinical infection and the possibility of wider genetic susceptibility.

The incidence of variant Creutzfeldt-Jakob disease (vCJD) in the United Kingdom appears to be in decline, with only four deaths reported this year (to 6 September 2004). However, results of a survey of lymphoreticular tissues have suggested a substantially higher prevalence of vCJD than expected from the clinical data alone. There are two plausible explanations for this discrepancy: first, a proportion of those infected will not develop clinical disease (subclinical infection); and second, the genetic group in which no clinical cases of vCJD have yet occurred is susceptible. Using mathematical models for the primary transmission of bovine spongiform encephalopathy to humans, we explore the impact of these hypotheses on case predictions. Under the first hypothesis, the results suggest relatively few future cases will arise via primary transmission, but that these cases are a small proportion of those infected, with most having subclinical infection. Under the second hypothesis, results suggest a maximum fivefold increase in cases, but this hypothesis is unable to account for the discrepancy between clinical cases and the estimated prevalence. Predictions of future cases of vCJD therefore remain uncertain, particularly given the recent identification of additional cases infected via blood transfusion.

Carrier State↗

PubMed2005

Novel mobilizable prokaryotic two-hybrid system vectors for high-throughput protein interaction mapping in Escherichia coli by bacterial conjugation.

Since its initial description, the yeast two-hybrid (Y2H) system has been widely used for the detection and analysis of protein-protein interactions. Mating-based strategies have been developed permitting its application for automated proteomic interaction mapping projects using both exhaustive and high-throughput strategies. More recently, a number of prokaryotic two-hybrid (P2H) systems have been developed but, despite the many advantages such Escherichia coli-based systems have over the Y2H system, they have not yet been widely implemented for proteomic interaction mapping. This may be largely due to the fact that high-throughput strategies employing bacterial transformation are not as amenable to automation as Y2H mating-based strategies. Here, we describe the construction of novel conjugative P2H system vectors. These vectors carry a mobilization element of the IncPalpha group plasmid RP4 and can therefore be mobilized with high efficiency from an E.coli donor strain encoding all of the required transport functions in trans. We demonstrate how these vectors permit the exploitation of bacterial conjugation for technically simplified and automated proteomic interaction mapping strategies in E.coli, analogous to the mating-based strategies developed for the Y2H system.

Bacterial Proteins↗

PubMed2005

Solid-phase immunoassays in mechanism-based drug discovery: their application in the development of inhibitors of the molecular chaperone heat-shock protein 90.

High-throughput screening of chemical libraries and the subsequent rapid progress of hit compounds through an iterative developmental test cascade are essential parts of modern molecular mechanism-based drug discovery. These processes depend on the use of efficient assay technologies and equipment. Enzyme-linked immunosorbent assays have historically been carried out in 96-well microtitre plates. Improvements in reagents and assay technologies mean that solid-phase immunoassays can be adapted for higher throughput to play an important role in modern drug discovery. The molecular chaperone heat-shock protein (Hsp) 90 is an important anticancer drug target because it maintains the conformation, stability, and function of many important oncogenic client proteins, including those involved with signal transduction, cell proliferation, survival, differentiation, motility angiogenesis, and metastasis. Using the standard inhibitors of the adenosine triphosphatase (ATPase) activity of Hsp90, geldanamycin (GA) and 17-allylamino-17- demethoxygeldanamycin (17AAG), novel solid-phase immunoassays have been validated using a time-resolved fluorescence (TRF) end point. Their utility for confirming the mechanism of action of Hsp90 inhibition in secondary cell-based assays has been shown and applied to the novel Hsp90 inhibitor CCT018159. Adaptation of these assays for later studies using human tumour xenografts and samples obtained from a Phase 1 trial of 17AAG is also described. Finally, comparison is made between the use and applicability of this type of immunoassay and other techniques such as western blotting, immunohistochemistry, and flow cytometry analysis.

Animals↗

PubMed2005

Vitamin K status of preterm infants with a prolonged prothrombin time.

AIM: To investigate the vitamin K status of preterm infants who have a prolonged prothrombin time (PT) in the first month of life. METHODS: Measures of vitamin K status were assessed in 21 preterm infants who were found to have an abnormal PT, despite 0.2-0.5 mg vitamin K(1) prophylaxis at birth. RESULTS: All infants had normal or supraphysiological vitamin K(1) concentrations and undetectable or, in one infant, insignificant PIVKA-II, indicating adequate vitamin K status. CONCLUSION: In preterm infants born at <32 wk gestation who received > or = 0.2 mg vitamin K(1) after delivery, a prolonged PT in the first month of life is unlikely to be due to vitamin K deficiency.

Biomarkers↗

PubMed2005

Who you live with and where you live: setting the context for health using multiple membership multilevel models.

STUDY OBJECTIVE: Previous studies into the effect of area of residence on individuals' health have not accounted for changing residency over time, although few people remain resident in the same area throughout their life. Furthermore, few studies of area effects on health have accounted for the clustering of health at the household level. These methodological problems may have led previous studies to under estimate or over estimate the size of area level effects. This study uses multiple membership multilevel models to investigate whether longitudinal analyses of area effects on health need to take account of clustering at the household level. SETTING AND PARTICIPANTS: A longitudinal survey (1991-1999) of a nationally representative sample of British households (5511 households with 10 264 adult members). DESIGN: Two level (individuals within households or areas) and three level (individuals within households within areas) multiple membership models of SF-36 physical and mental health functioning scores at wave nine were analysed adjusting for age, gender, education, marital, employment, and smoking status from previous waves. RESULTS: Physical and mental health functioning seem to cluster within households. Accounting for changes in household membership over time increases estimates of the clustering in functioning at the household level. The clustering of functioning within area wards is reduced when the clustering within households and risk factors for functioning are taken into account. CONCLUSIONS: Clustered sampling units within study designs should be taken account of in individual level analyses. Changes in these units over time should be accounted for in longitudinal analysis.

Adolescent↗

PubMed2005

Social dynamics of health inequalities: a growth curve analysis of aging and self assessed health in the British household panel survey 1991-2001.

OBJECTIVES: To study how social inequalities change as people age, this paper presents a growth curve model of self assessed health, which accommodates changes in occupational class and individual health with age. DESIGN: Nationally representative interview based longitudinal survey of adults in Great Britain. SETTING: Representative members of private households of Great Britain in 1991. PARTICIPANTS: Survey respondents (n = 6705), aged 21-59 years in 1991 and followed up annually until 2001. MAIN OUTCOME MEASURE: Self assessed health. RESULTS: On average, self assessed health declines slowly from early adulthood to retirement age. No significant class differences in health were observed at age 21. Health inequalities emerged later in life with the gap between mean levels of self assessed health of those in managerial and professional occupations and routine occupations widening approaching retirement. Individual variability in health trajectories increased between ages 40 and 59 years so that this widening of mean differences between occupational classes was not significant. When the analysis is confined to people whose occupational class remained constant over time, a far greater difference in health trajectories between occupational classes was seen. CONCLUSIONS: The understanding of social inequalities in health at the population level is enriched by an analysis of individual variation in age related declines by social position.

Adult↗

PubMed2004

Identification of rhtX and fptX, novel genes encoding proteins that show homology and function in the utilization of the siderophores rhizobactin 1021 by Sinorhizobium meliloti and pyochelin by Pseudomonas aeruginosa, respectively.

Rhizobactin 1021 is a hydroxymate siderophore produced by the soil bacterium Sinorhizobium meliloti 2011. A regulon comprising rhtA, encoding the outer membrane receptor protein for the ferrisiderophore; the biosynthesis operon rhbABCDEF; and rhrA, the Ara-C-like regulator of the receptor and biosynthesis genes has been previously described. We report the discovery of a gene, located upstream of rhbA and named rhtX (for "rhizobactin transport"), which is required, in addition to rhtA, to confer the ability to utilize rhizobactin 1021 on a strain of S. meliloti that does not naturally utilize the siderophore. Rhizobactin 1021 is structurally similar to aerobactin, which is transported in Escherichia coli via the IutA outer membrane receptor and the FhuCDB inner membrane transport system. E. coli expressing iutA and fhuCDB was found to also transport rhizobactin 1021. We demonstrated that RhtX alone could substitute for FhuCDB to transport rhizobactin 1021 in E. coli. RhtX shows similarity to a number of uncharacterized proteins which are encoded proximal to genes that are either known to be or predicted to be involved in iron acquisition. Among these is PA4218 of Pseudomonas aeruginosa, which is located close to the gene cluster that functions in pyochelin biosynthesis and outer membrane transport. PA4218 was mutated by allelic replacement, and the mutant was found to have a pyochelin utilization-defective phenotype. It is proposed that PA4218 be named fptX (for "ferripyochelin transport"). RhtX and FptX appear to be members of a novel family of permeases that function as single-subunit transporters of siderophores.

Amino Acid Sequence↗