Uracil-DNA glycosylase (UNG) is the key enzyme responsible for initiation of base excision repair. We have used both kinetic and binding assays for comparative analysis of UNG enzymes from humans and ...herpes simplex virus type 1 (HSV-1). Steady-state fluorescence assays showed that hUNG has a much higher specificity constant (kcat/Km) compared with the viral enzyme due to a lower Km. The binding of UNG to DNA was also studied using a catalytically inactive mutant of UNG and non-cleavable substrate analogs (2′-deoxypseudouridine and 2′-α-fluoro-2′-deoxyuridine). Equilibrium DNA binding revealed that both human and HSV-1 UNG enzymes bind to abasic DNA and both substrate analogs more weakly than to uracil-containing DNA. Structure determination of HSV-1 D88N/H210N UNG in complex with uracil revealed detailed information on substrate binding. Together, these results suggest that a significant proportion of the binding energy is provided by specific interactions with the target uracil. The kinetic parameters for human UNG indicate that it is likely to have activity against both U·A and U·G mismatches in vivo. Weak binding to abasic DNA also suggests that UNG activity is unlikely to be coupled to the subsequent common steps of base excision repair.
Abstract
Background
Hospital antimicrobial stewardship strategies, such as ‘Start Smart, Then Focus’ in the UK, balance the need for prompt, effective antibiotic treatment with the need to limit ...antibiotic overuse using ‘review and revise’. However, only a minority of review decisions are to stop antibiotics. Research suggests that this is due to both behavioural and organizational factors.
Objectives
To develop and optimize the Antibiotic Review Kit (ARK) intervention. ARK is a complex digital, organizational and behavioural intervention that supports implementation of ‘review and revise’ to help healthcare professionals safely stop unnecessary antibiotics.
Methods
A theory-, evidence- and person-based approach was used to develop and optimize ARK and its implementation. This was done through iterative stakeholder consultation and in-depth qualitative research with doctors, nurses and pharmacists in UK hospitals. Barriers to and facilitators of the intervention and its implementation, and ways to address them, were identified and then used to inform the intervention’s development.
Results
A key barrier to stopping antibiotics was reportedly a lack of information about the original prescriber’s rationale for and their degree of certainty about the need for antibiotics. An integral component of ARK was the development and optimization of a Decision Aid and its implementation to increase transparency around initial prescribing decisions.
Conclusions
The key output of this research is a digital and behavioural intervention targeting important barriers to stopping antibiotics at review (see http://bsac-vle.com/ark-the-antibiotic-review-kit/ and http://antibioticreviewkit.org.uk/). ARK will be evaluated in a feasibility study and, if successful, a stepped-wedge cluster-randomized controlled trial at acute hospitals across the NHS.
To use a large current prospective cohort of infants <29 weeks to compare bronchopulmonary dysplasia (BPD) rates in black and white infants.
The Prematurity and Respiratory Outcome Program (PROP) ...enrolled 835 infants born in 2011-2013 at <29 weeks of gestation; 728 black or white infants survived to 36 weeks postmenstrual age (PMA). Logistic regression was used to compare BPD outcomes (defined as supplemental oxygen requirement at 36 weeks PMA) between the races, adjusted for gestational age (GA), antenatal steroid use, intubation at birth, and surfactant use at birth.
Of 707 black or white infants with available BPD outcomes, BPD was lower in black infants (38% vs 45%), even though they were of significantly lower GA. At every GA, BPD was more common in white infants. The aOR for BPD was 0.60 (95% CI, 0.42-0.85; P = .004) for black infants compared with white infants after adjusting for GA. Despite the lower rate of BPD, black infants had a higher rate of first-year post-prematurity respiratory disease (black, 79%; white, 63%).
In this large cohort of recently born preterm infants at <29 weeks GA, compared with white infants, black infants had a lower risk of BPD but an increased risk of persistent respiratory morbidity.
Objective To assess the reliability, validity and responsiveness of the Australian/Canadian (AUSCAN) Osteoarthritis Hand Index in both Likert (LK) and Visual Analogue (VA)-scaled formats.
Methods Two ...separate studies were conducted; the first addressing reliability and validity issues and the second addressing index responsiveness. In a group of 50 patients with osteoarthritis (OA) of the hand, test–retest reliability was assessed at a 1-week interval and internal consistency from single administrations of the Index. Construct validity was evaluated against several other outcome measures including the Functional Index for Hand Osteoarthritis (FIHOA), separate patient and physician global assessments, Doyle Index, grip strength, pinch grip, and Health Assessment Questionnaire. A 6-week washout retreatment design was used in a group of 44 OA hand patients to assess index responsiveness and comparative responsiveness against the FIHOA.
Results Reliability and construct validity coefficients confirm the reliability and construct validity of both the AUSCAN LK3.0 and AUSCAN VA3.0 Indices. The washout retreatment study establishes index responsiveness and suggests that the AUSCAN LK3.0 and AUSCAN VA3.0 Indices may be more responsive than the FIHOA.
Conclusions The patient self-completed AUSCAN LK3.0 and AUSCAN VA3.0 Indices are reliable, valid and responsive and can be recommended as primary outcome measures for future hand OA clinical trials.Copyright 2002 OsteoArthritis Research Society International. Published by Elsevier Science Ltd. All rights reserved.
Objective: To evaluate the effectiveness of viscosupplementation with single-injection hylan G-F20 (Synvisc-One) in knee osteoarthritis (OA), during routine clinical care, in a 52 week observational ...study.
Research design and methods: The LOBRAS study involved a 1 year long, multi-center, quasi-experimental, repeated measures, observational study. Consenting patients in Australia fulfilling inclusion/exclusion criteria under the care of a medical specialist in routine clinical practice were enrolled. Prior to, and for 52 weeks following, intra-articular single-injection hylan G-F20, patients were repeatedly evaluated using the WOMAC NRS4.1 Index and the SF-36 questionnaire. The WOMAC NRS4.1 was administered by mobile phone (with paper back-up), and the SF-36 was administered on paper. Patients were monitored for adverse events.
Main outcome measures: Western Ontario and McMaster (WOMAC) OA Index, and the Short Form 36 questionnaire (SF-36 v2).
Results: A total of 131 patients with knee OA were enrolled, of whom 119 provided both pre- and post-intervention WOMAC data. Statistically significant improvements (with a maximum of p ≤ .025) from baseline to Week 12, Month 6 and Week 52 were detected, by intention-to-treat (ITT) and per-protocol (PP) analyses, in WOMAC Pain, Stiffness, Function, PGA, and Total Score, SF-36 PCS, and WOMAC-derived HUI3. Adverse event (AE) monitoring detected treatment-related AEs in 5.3% of patients.
Conclusions: The effectiveness of single-injection hylan G-F20 in routine clinical care is supported by the detection of statistically significant, clinically important improvements in WOMAC Pain, Stiffness, Function, Total, and PGA outcomes, and statistically significant improvements in SF-36 PCS and WOMAC-derived HUI3 outcomes at multiple time points. Limitations of this study include lack of a control group or blinding. No predictive indicators of the response to treatment were identified. In general single-injection hylan G-F20 was well tolerated with very few patients experiencing any treatment-related adverse events. Collectively, these observations attest to the effectiveness of single-injection hylan G-F20 and complement previous observations in routine clinical care.
Background A DNA-prime/human adenovirus serotype 5 (HuAd5) boost vaccine encoding Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP) and Pf apical membrane antigen-1 (PfAMA1), elicited ...protection in 4/15 (27%) of subjects against controlled human malaria infection (CHMI) that was statistically associated with CD8+ T cell responses. Subjects with high level pre-existing immunity to HuAd5 were not protected, suggesting an adverse effect on vaccine efficacy (VE). We replaced HuAd5 with chimpanzee adenovirus 63 (ChAd63), and repeated the study, assessing both the two-antigen (CSP, AMA1 = CA) vaccine, and a novel three-antigen (CSP, AMA1, ME-TRAP = CAT) vaccine that included a third pre-erythrocytic stage antigen malaria multiple epitopes (ME) fused to the Pf thrombospondin-related adhesive protein (TRAP) to potentially enhance protection. Methodology This was an open label, randomized Phase 1 trial, assessing safety, tolerability, and VE against CHMI in healthy, malaria naïve adults. Forty subjects (20 each group) were to receive three monthly CA or CAT DNA priming immunizations, followed by corresponding ChAd63 boost four months later. Four weeks after the boost, immunized subjects and 12 infectivity controls underwent CHMI by mosquito bite using the Pf3D7 strain. VE was assessed by determining the differences in time to parasitemia as detected by thick blood smears up to 28-days post CHMI and utilizing the log rank test, and by calculating the risk ratio of each treatment group and subtracting from 1, with significance calculated by the Cochran-Mantel-Haenszel method. Results In both groups, systemic adverse events (AEs) were significantly higher after the ChAd63 boost than DNA immunizations. Eleven of 12 infectivity controls developed parasitemia (mean 11.7 days). In the CA group, 15 of 16 (93.8%) immunized subjects developed parasitemia (mean 12.0 days). In the CAT group, 11 of 16 (63.8%) immunized subjects developed parasitemia (mean 13.0 days), indicating significant protection by log rank test compared to infectivity controls (p = 0.0406) and the CA group (p = 0.0229). VE (1 minus the risk ratio) in the CAT group was 25% compared to -2% in the CA group. The CA and CAT vaccines induced robust humoral (ELISA antibodies against CSP, AMA1 and TRAP, and IFA responses against sporozoites and Pf3D7 blood stages), and cellular responses (IFN-gamma FluoroSpot responses to CSP, AMA1 and TRAP) that were not associated with protection. Conclusions This study demonstrated that the ChAd63 CAT vaccine exhibited significant protective efficacy, and confirmed protection was afforded by adding a third antigen (T) to a two-antigen (CA) formulation to achieve increased VE. Although the ChAd63-CAT vaccine was associated with increased frequencies of systemic AEs compared to the CA vaccine and, historically, compared to the HuAd5 vectored malaria vaccine encoding CSP and AMA1, they were transient and associated with increased vector dosing.
Background: Studies of chromosome 15 abnormality have implicated over‐expression of paternally imprinted genes in the 15q11–13 region in the aetiology of autism. To test this hypothesis we compared ...individuals with Prader‐Willi syndrome (PWS) due to uniparental disomy (UPD – where paternally imprinted genes are over‐expressed) to individuals with the 15q11–13 deletion form of the syndrome (where paternally imprinted genes are not over‐expressed). We also tested reports that PWS cases due to the larger type I (TI) form of deletion show differences to cases with the smaller type II (TII) deletion.
Method: Ninety‐six individuals with PWS were recruited from genetic centres and the PWS association. Forty‐nine individuals were confirmed as having maternal UPD of chromosome 15 and were age and sex matched to 47 individuals with a deletion involving 15q11–13 (32 had the shorter (T II) deletion, and 14 had the longer (TI) deletion). Behavioural assessments were carried out blind to genetic status, using the Autism Diagnostic Observation Schedule (ADOS), the Autism Diagnostic Interview (ADI), the Autism Screening Questionnaire (ASQ), the Children's Yale‐Brown Obsessive‐Compulsive Scale (CY‐BOCS), the Vineland Adaptive Behaviour Scales (VABS), and measurements of intellectual ability, including the Wechsler and Mullen Scales and Raven's Matrices.
Results: UPD cases exhibited significantly more autistic‐like impairments in reciprocal social interaction on questionnaire, interview and standardised observational measures. Comparison of TI and TII deletion cases revealed few differences, but ability levels tended to be lower in the TI deletion cases.
Conclusions: Findings from a large study comparing deletion and UPD forms of Prader‐Willi syndrome were consistent with other evidence in indicating that paternally imprinted genes in the 15q11–13 region constitute a genetic risk factor for aspects of autistic symptomatology. These genes may therefore play a role in the aetiology of autism. By contrast with another report, there was no clear‐cut relationship between the size of the deletion and the form of cognitive and behavioural phenotype.
Human genetic variation is an important determinant of the outcome of infection with Mycobacterium tuberculosis. We have conducted a two-stage genome-wide linkage study to search for regions of the ...human genome containing tuberculosis-susceptibility genes. This approach uses sibpair families that contain two full siblings who have both been affected by clinical tuberculosis. For any chromosomal region containing a major tuberculosis-susceptibility gene, affected sibpairs inherit the same parental alleles more often than expected by chance. In the first round of the screen, 299 highly informative genetic markers, spanning the entire human genome, were typed in 92 sibpairs from The Gambia and South Africa. Seven chromosomal regions that showed provisional evidence of coinheritance with clinical tuberculosis were identified. To identify whether any of these regions contained a potential tuberculosis-susceptibility gene, 22 markers from these regions were genotyped in a second set of 81 sibpairs from the same countries. Markers on chromosomes 15q and Xq showed suggestive evidence of linkage (lod = 2.00 and 1.77, respectively) to tuberculosis. The potential identification of susceptibility loci on both chromosomes 15q and Xq was supported by an independent analysis designated common ancestry using microsatellite mapping. These results indicate that genome-wide linkage analysis can contribute to the mapping and identification of major genes for multifactorial infectious diseases of humans. An X chromosome susceptibility gene may contribute to the excess of males with tuberculosis observed in many different populations.
Cardiovascular disease is the leading cause of mortality in renal transplant recipients. Although renal transplant recipients frequently undergo cardiac functional tests prior to surgery, coronary ...atherosclerosis can remain undetected. Coronary artery calcification (CAC), an early marker of atherosclerosis can be quantified using EBCT. The purpose of this study was to determine the extent and characteristics of CAC at the time of renal transplantation. We evaluated 79 consecutive incident asymptomatic renal transplant recipients. Patients were mostly White (62%), male (54%) and had a deceased donor renal transplant (61%). The mean age was 47 (12.1) years. Sixty‐five percentage of subjects had CAC. The mean CAC score was 331.5 (562.4) with a median of 43.3. Older age, presence of diabetes, not having a preemptive transplant, deceased donor transplantation and hypercholesterolemia were significantly associated with presence of CAC univariately. Median CAC scores were significantly increased in subjects with diabetes (127.8 vs. 28.9, p = 0.05), exposed to dialysis (102.9 vs. 3.7, p < 0.001) and deceased donor recipients (169.7 vs. 7.5, p = 0.02). Using multiple logistic regression, age and time on dialysis were significantly associated with the presence of CAC at the time of transplant. In summary, CAC is prevalent in patients undergoing kidney transplant. CAC may be a method to identify renal transplant recipients at increased risk for future cardiovascular events.
The effects of habitat gaps on breeding success and parental daily energy expenditure (DEE) were investigated in great tits (Parus major) and blue tits (Cyanistes caeruleus) in urban parkland ...(Cardiff, UK) compared with birds in deciduous woodland (eastern England, UK). Tree canopy height, the percentage of gap in the canopy and the percentage of oak (in the wood only) within a 30 m radius of nest boxes were obtained from airborne remote-sensed data. Breeding success was monitored and parental DEE (great tits: both habitats; blue tits: park only) was measured using doubly labelled water in birds feeding young. In the park, mean (±SD) tree height (7.5 ± 4.7 m) was less than in the wood (10.6 ± 4.5 m), but the incidence of gaps (32.7 ± 22.6%) was greater (9.2 ± 14.7%). Great tits and blue tits both reared fewer young in the park and chick body mass was also reduced in park-reared great tits. Park great tits had a higher DEE (86.3 ± 12.3 kJ day⁻¹) than those in the wood (78.0 ± 11.7 kJ day⁻¹) and, because of smaller brood sizes, worked about 64% harder for each chick reared. Tits in the park with more than about 35% gap around their boxes had higher DEEs than the average for the habitat. In the wood, great tits with less oak around their boxes worked harder than average. Thus structural gaps, and functional gaps generated by variation in the quality of foraging habitat, increased the costs of rearing young.
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