Construction and biochemical characterization of recombinant cytoplasmic forms of the IucD protein (lysine:N6-hydroxylase) encoded by the pColV-K30 aerobactin gene cluster

The aerobactin gene cluster in pColV-K30 consists of five genes (iucABCD iutA); four of these (iucABCD) are involved in aerobactin biosynthesis, whereas the fifth one (iutA) encodes the ferriaerobactin outer membrane receptor. iucD encodes lysine:N6-hydroxylase, which catalyzes the first step in aerobactin biosynthesis. Regardless of the method used for cell rupture, we have consistently found that IucD remains membrane bound, and repeated efforts to achieve a purified and active soluble form of the enzyme have been unsuccessful. To circumvent this problem, we have constructed recombinant IucD proteins with modified amino termini by creating three in-frame gene fusions of IucD to the amino-terminal amino acids of the cytoplasmic enzyme beta-galactosidase. Two of these constructs resulted in the addition to the iucD coding region of a hydrophilic leader sequence of 13 and 30 amino acids. The other construct involved the deletion of the first 47 amino acids of the IucD amino terminus and the addition of 19 amino acids of the amino terminus of beta-galactosidase. Cells expressing any of the three recombinant IucD forms were found to produce soluble N6-hydroxylysine. One of these proteins, IucD439, was purified to homogeneity from the soluble fraction of the cell lysates, and it was capable of participating in the biosynthesis of aerobactin, as determined in vitro by a cell-free system and in vivo by a cross-feeding bioassay. A medium ionic strength of 0.25 (250 mM NaCl) or higher was required to maintain the protein in a catalytically functional, tetrameric state.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification, expression, and DNA sequence of the GDP-mannose biosynthesis genes encoded by the O7 rfb gene cluster of strain VW187 (Escherichia coli O7:K1)

The O7-specific lipopolysaccharide (LPS) in strains of Escherichia coli consists of a repeating unit made of galactose, mannose, rhamnose, 4-acetamido-2,6-dideoxyglucose, and N-acetylglucosamine. We have recently cloned and characterized genetically the O7-specific LPS biosynthesis region (rfbEcO7) of the E. coli O7:K1 strain VW187 (C. L. Marolda, J. Welsh, L. Dafoe, and M. A. Valvano, J. Bacteriol. 172:3590-3599, 1990). In this study, we localized the gnd gene encoding gluconate-6-phosphate dehydrogenase at one end of the rfbEcO7 gene cluster and sequenced that end of the cluster. Three open reading frames (ORF) encoding polypeptides of 275, 464, and 453 amino acids were identified upstream of gndEcO7, all transcribed toward the gnd gene. ORF275 had 45% similarity at the protein level with ORF16.5, which occupies a similar position in the Salmonella enterica LT2 rfb region, and presumably encodes a nucleotide sugar transferase. The polypeptides encoded by ORFs 464 and 453 were expressed under the control of the ptac promoter and visualized in Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels and by maxicell analysis. ORF464 expressed GDP-mannose pyrophosphorylase and ORF453 encoded a phosphomannomutase, the enzymes for the biosynthesis pathway of GDP-mannose, one of the nucleotide sugar precursors for the formation of the O7 repeating unit. They were designated rfbMEcO7 and rfbKEcO7, respectively. The RfbMEcO7 polypeptide was homologous to the corresponding protein in S. enterica LT2, XanB of Xanthomonas campestris, and AlgA of Pseudomonas aeruginosa, all GDP-mannose pyrophosphorylases. RfbKEcO7 was very similar to CpsG of S. enterica LT2, an enzyme presumably involved in the biosynthesis of the capsular polysaccharide colanic acid, but quite different from the corresponding RfbK protein of S. enterica LT2.

Effect of a multifaceted intervention on adherence to hand hygiene among healthcare workers:A cluster-randomized trial

OBJECTIVES. Adherence to hand hygiene among healthcare workers (HCWs) is widely believed to be a key factor in reducing the spread of healthcare-associated infection. The objective of this study was to evaluate the impact of a multifaceted intervention to increase rates of adherence to hand hygiene among HCWs and to assess the effect on the incidence of hospital-acquired methicillin-resistant Staphylococcus aureus (MRSA) colonization. DESIGN. Cluster-randomized controlled trial. SETTING. Thirty hospital units in 3 tertiary care hospitals in Hamilton, Ontario, Canada. INTERVENTION. After a 3-month baseline period of data collection, 15 units were randomly assigned to the intervention arm (with performance feedback, small-group teaching seminars, and posters) and 15 units to usual practice. Hand hygiene was observed during randomly selected 15-minute periods on each unit, and the incidence of MRSA colonization was measured using weekly surveillance specimens from June 2007 through May 2008. RESULTS. We found that 3,812 (48.2%) of 7,901 opportunities for hand hygiene in the intervention group resulted in adherence, compared with 3,205 (42.6%) of 7,526 opportunities in the control group (P <.001; independent t test). There was no reduction in the incidence of hospital-acquired MRSA colonization in the intervention group. CONCLUSION. Among HCWs in Ontario tertiary care hospitals, the rate of adherence to hand hygiene had a statistically significant increase of 6% with a multifaceted intervention, but the incidence of MRSA colonization was not reduced.

Do obesity and sleep problems cluster in the workplace?:A multivariate, multilevel study

OBJECTIVES: The aim of this study was to examine the co-occurrence of obesity and sleep problems among employees and workplaces. METHODS: We obtained data from 39 873 men and women working in 3040 workplaces in 2000-2002 (the Finnish Public Sector Study). Individual- and workplace-level characteristics were considered as correlates of obesity and sleep problems, which were modelled simultaneously using a multivariate, multilevel approach. RESULTS: Of the participants, 11% were obese and 23% reported sleep problems. We found a correlation between obesity and sleep problems at both the individual [correlation coefficient 0.048, covariance 0.047, standard error (SE) 0.005) and workplace (correlation coefficient 0.619, covariance 0.068, SE 0.011) level. The latter, but not the former, correlation remained after adjustment for individual- and workplace-level confounders, such as age, sex, socioeconomic status, shift work, alcohol consumption, job strain, and proportion of temporary employees and manual workers at the workplace. CONCLUSIONS: Obese employees and those with sleep problems tend to cluster in the same workplaces, suggesting that, in addition to targeting individuals at risk, interventions to reduce obesity and sleep problems might benefit from identifying "risky" workplaces.

Characterization and biological role of the O-polysaccharide gene cluster of Yersinia enterocolitica serotype O:9

Yersinia enterocolitica serotype O:9 is a gram-negative enteropathogen that infects animals and humans. The role of lipopolysaccharide (LPS) in Y. enterocolitica O:9 pathogenesis, however, remains unclear. The O:9 LPS consists of lipid A to which is linked the inner core oligosaccharide, serving as an attachment site for both the outer core (OC) hexasaccharide and the O-polysaccharide (OPS; a homopolymer of N-formylperosamine). In this work, we cloned the OPS gene cluster of O:9 and identified 12 genes organized into four operons upstream of the gnd gene. Ten genes were predicted to encode glycosyltransferases, the ATP-binding cassette polysaccharide translocators, or enzymes required for the biosynthesis of GDP-N-formylperosamine. The two remaining genes within the OPS gene cluster, galF and galU, were not ascribed a clear function in OPS biosynthesis; however, the latter gene appeared to be essential for O:9. The biological functions of O:9 OPS and OC were studied using isogenic mutants lacking one or both of these LPS parts. We showed that OPS and OC confer resistance to human complement and polymyxin B; the OPS effect on polymyxin B resistance could be observed only in the absence of OC.

Effect of a multifaceted intervention on number of antimicrobial prescriptions for suspected urinary tract infections in residents of nursing homes:Cluster randomised controlled trial

Objective: To assess whether a multifaceted intervention can reduce the number of prescriptions for antimicrobials for suspected urinary tract infections in residents of nursing homes. Design: Cluster randomised controlled trial. Setting: 24 nursing homes in Ontario, Canada, and Idaho, United States. Participants: 12 nursing homes allocated to a multifaceted intervention and 12 allocated to usual care. Outcomes were measured in 4217 residents. Interventions: Diagnostic and treatment algorithm for urinary tract infections implemented at the nursing home level using a multifaceted approach-small group interactive sessions for nurses, videotapes, written material, outreach visits, and one on one interviews with physicians. Main outcome measures: Number of antimicrobials prescribed for suspected urinary tract infections, total use of antimicrobials, admissions to hospital, and deaths. Results: Fewer courses of antimicrobials for suspected urinary tract infections per 1000 resident days were prescribed in the intervention nursing homes than in the usual care homes (1.17 v 1.59 courses; weighted mean difference -0.49, 95% confidence intervals -0.93 to -0.06). Antimicrobials for suspected urinary tract infection represented 28.4% of all courses of drugs prescribed in the intervention nursing homes compared with 38.6% prescribed in the usual care homes (weighted mean difference -9.6%, -16.9% to -2.4%). The difference in total antimicrobial use per 1000 resident days between intervention and usual care groups was not significantly different (3.52 v 3.93; weighed mean difference -0.37, -1.17 to 0.44). No significant difference was found in admissions to hospital or mortality between the study arms. Conclusion: A multifaceted intervention using algorithms can reduce the number of antimicrobial prescriptions for suspected urinary tract infections in residents of nursing homes.

Effectiveness of medicines review with web-based pharmaceutical treatment algorithms in reducing potentially inappropriate prescribing in older people in primary care:a cluster randomized trial (OPTI-SCRIPT study protocol)

Potentially inappropriate prescribing in older people is common in primary care and can result in increased morbidity, adverse drug events, hospitalizations and mortality. In Ireland, 36% of those aged 70 years or over received at least one potentially inappropriate medication, with an associated expenditure of over €45 million.The main objective of this study is to determine the effectiveness and acceptability of a complex, multifaceted intervention in reducing the level of potentially inappropriate prescribing in primary care.

PS1-12sk is a Peculiar Supernova From a He-rich Progenitor System in a Brightest Cluster Galaxy Environment

We report on our discovery and observations of the Pan-STARRS1 supernova (SN) PS1-12sk, a transient with properties that indicate atypical star formation in its host galaxy cluster or pose a challenge to popular progenitor system models for this class of explosion. The optical spectra of PS1-12sk classify it as a Type Ibn SN (c.f. SN 2006jc), dominated by intermediate-width (3x10^3 km/s) and time variable He I emission. Our multi-wavelength monitoring establishes the rise time dt = 9-23 days and shows an NUV-NIR SED with temperature > 17x10^3 K and a peak rise magnitude of Mz = -18.9 mag. SN Ibn spectroscopic properties are commonly interpreted as the signature of a massive star (17 - 100 M_sun) explosion within a He-enriched circumstellar medium. However, unlike previous Type Ibn supernovae, PS1-12sk is associated with an elliptical brightest cluster galaxy, CGCG 208-042 (z = 0.054) in cluster RXC J0844.9+4258. The expected probability of an event like PS1-12sk in such environments is low given the measured infrequency of core-collapse SNe in red sequence galaxies compounded by the low volumetric rate of SN Ibn. Furthermore, we find no evidence of star formation at the explosion site to sensitive limits (Sigma Halpha

Evaluation of a worksite cervical screening initiative to increase Pap smear uptake in Malaysia:a cluster randomized controlled trial

Despite the significant burden of cervical cancer, Malaysia like many middle-income countries relies on opportunistic cervical screening as opposed to a more organized population-based program. The aim of this study was to ascertain the effectiveness of a worksite screening initiative upon Papanicolaou smear test (Pap test) uptake among educated working women in Malaysia.

A cluster randomised controlled trial of “Bookstart+”: a book gifting programme

Purpose – The purpose of this paper is to evaluate the effectiveness of a free book gifting programme, called “Bookstart+”, in improving family reading outcomes.

Design/methodology/approach – Bookstart+ consists of a pack of books and reading materials provided to families at their two-year-old child's statutory health visit. The pack is accompanied by a short priming demonstration, delivered by the health visitor, on shared reading. The evaluation took the form of a randomised controlled trial (RCT) with 460 families from the client lists of 115 health visitors.

Findings – The study found evidence of: a positive significant effect on parents’ attitudes to reading and books (Cohen's d=+0.192, p=0.034); no significant effect on parental attitudes to their child reading (d=+0.085, p=0.279); and a negative effect, approaching significance, on public library usage (d=-0.160, p=0.055).

Research limitations/implications – The attrition rate was high, with only 43.9 per cent of the target families completing all of the research. However, this level of attrition did not lead to any significant differences between the control and intervention groups on their pre-test measures.

Practical implications – The study provides recommendations for free book gifting service provision in relation to pack contents and delivery.

Originality/value – This paper contributes to the limited international RCT evidence on free book gifting programmes.

Electron-induced hydrogen loss in uracil in a water cluster environment

Low-energy electron-impact hydrogen loss due to dissociative electron attachment (DEA) to the uracil and thymine molecules in a water cluster environment is investigated theoretically. Only the A'-resonance contribution, describing the near-threshold behavior of DEA, is incorporated. Calculations are based on the nonlocal complex potential theory and the multiple scattering theory, and are performed for a model target with basic properties of uracil and thymine, surrounded by five water molecules. The DEA cross section is strongly enhanced when the attaching molecule is embedded in a water cluster. This growth is due to two effects: the increase of the resonance lifetime and the negative shift in the resonance position due to interaction of the intermediate negative ion with the surrounding water molecules. A similar effect was earlier found in DEA to chlorofluorocarbons. 

Use of the child health utility and strengths and difficulties outcome measures in economic evaluations of school-based interventions: data from a cluster-randomised controlled trial in Northern Ireland

Background There is growing evidence linking early social and emotional wellbeing to later academic performance and various health outcomes including mental health. An economic evaluation was designed alongside the Roots of Empathy cluster-randomised trial evaluation, which is a school-based intervention for improving pupils’ social and emotional wellbeing. Exploration of the relevance of the Strengths and Diffi culties Questionnaire (SDQ) and Child Health Utility 9D (CHU9D) in school-based health economic evaluations is warranted. The SDQ is a behavioural screening questionnaire for 4–17-year-old children, consisting of a total diffi culties score, and also prosocial behaviour,
which aims to identify positive aspects of behaviour. The CHU9D is a generic preference-based health-related quality of life instrument for 7–17-year-old children.