Acinetobacter baumannii has emerged as an important and problematic human pathogen as it is the causative agent of several types of infections including pneumonia, meningitis, septicemia, and urinary tract infections. We explored the pathogenic content of this harmful pathogen using a combination of DNA sequencing and insertional mutagenesis. The genome of this organism was sequenced using a strategy involving high-density pyrosequencing, a novel, rapid method of high-throughput sequencing. Excluding the rDNA repeats, the assembled genome is 3,976,746 base pairs (bp) and has 3830 ORFs. A significant fraction of ORFs (17.2%) are located in 28 putative alien islands, indicating that the genome has acquired a large amount of foreign DNA. Consistent with its role in pathogenesis, a remarkable number of the islands (16) contain genes implicated in virulence, indicating the organism devotes a considerable portion of its genes to pathogenesis. The largest island contains elements homologous to the Legionella/Coxiella Type IV secretion apparatus. Type IV secretion systems have been demonstrated to be important for virulence in other organisms and thus are likely to help mediate pathogenesis of A. baumannii. Insertional mutagenesis generated avirulent isolates of A. baumannii and verified that six of the islands contain virulence genes, including two novel islands containing genes that lacked homology with others in the databases. The DNA sequencing approach described in this study allows the rapid elucidation of the DNA sequence of any microbe and, when combined with genetic screens, can identify many novel genes important for microbial pathogenesis.

This article investigates the relationship between democratic practices and the design of institutions operating in collaborative spaces, those policy and spatial domains where multiple public, private and non-profit actors join together to shape, make and implement public policy. Partnerships are organizational manifestations of institutional design for collaboration. They offer flexibility and stakeholder engagement, but are loosely coupled to representative democratic systems. A multimethod research strategy examines the impact of discourses of managerialism, consociationalism and participation on the design of partnerships in two UK localities. Analysing objective measures of democratic performance in partnerships and interpreting the discursive transition from earlier practices in representative democratic institutions we find that institutional designs for collaboration reflect different settlements between discourses, captured in the distinction between club, agency and polity-forming partnership types. The results show how the governance of collaborative spaces is mediated through a dominant set of discursively defined institutional practices.

We have discovered a microbial interaction between yeast, bacteria, and nematodes. Upon coculturing, Saccharomyces cerevisiae stimulated the growth of several species of Acinetobacter, including, A. baumannii, A. haemolyticus, A. johnsonii, and A. radioresistens, as well as several natural isolates of Acinetobacter. This enhanced growth was due to a diffusible factor that was shown to be ethanol by chemical assays and evaluation of strains lacking ADH1, ADH3, and ADH5, as all three genes are involved in ethanol production by yeast. This effect is specific to ethanol: methanol, butanol, and dimethyl sulfoxide were unable to stimulate growth to any appreciable level. Low doses of ethanol not only stimulated growth to a higher cell density but also served as a signaling molecule: in the presence of ethanol, Acinetobacter species were able to withstand the toxic effects of salt, indicating that ethanol alters cell physiology. Furthermore, ethanol-fed A. baumannii displayed increased pathogenicity when confronted with a predator, Caenorhabditis elegans. Our results are consistent with the concept that ethanol can serve as a signaling molecule which can affect bacterial physiology and survival.

To further understand the roles of protein glycosylation in eukaryotes, we globally identified glycancontaining proteins in yeast. A fluorescent lectin binding assay was developed and used to screen protein microarrays containing over 5000 proteins purified from yeast. A total of 534 yeast proteins were identified that bound either Concanavalin A (ConA) or Wheat-Germ Agglutinin (WGA); 406 of them were novel. Among the novel glycoproteins, 45 were validated by mobility shift upon treatment with EndoH and PNGase F, thereby extending the number of validated yeast glycoproteins to 350. In addition to many components of the secretory pathway, we identified other types of proteins, such as transcription factors and mitochondrial proteins. To further explore the role of glycosylation in mitochondrial function, the localization of four mitochondrial proteins was examined in the presence and absence of tunicamycin, an inhibitor of N-linked protein glycosylation. For two proteins, localization to the mitochondria is diminished upon tunicamycin treatment, indicating that protein glycosylation is important for protein function. Overall, our studies greatly extend our understanding of protein glycosylation in eukaryotes through the cataloguing of glycoproteins, and describe a novel role for protein glycosylation in mitochondrial protein function and localization.

Protein microarrays are similar to DNA microarrays; both enabling the parallel interrogation of thousands of probes immobilized on a surface. Consequently, they have benefited from technologies previously developed for DNA microarrays. However, assumptions for the analysis of DNA microarrays do not always translate to protein arrays, especially in the case of normalization. Hence, we have developed an experimental and computational framework to assess normalization procedures for protein microarrays. Specifically, we profiled two sera with markedly different autoantibody compositions. To analyze intra- and interarray variability, we compared a set of control proteins across subarrays and the corresponding spots across multiple arrays, respectively. To estimate the degree to which the normalization could help reveal true biological separability, we tested the difference in the signal between the sera relative to the variability within replicates. Next, by mixing the sera in different proportions (titrations), we correlated the reactivity of proteins with serum concentration. Finally, we analyzed the effect of normalization procedures on the list of reactive proteins. We compared global and quantile normalization, techniques that have traditionally been employed for DNA microarrays, with a novel normalization approach based on a robust linear model (RLM) making explicit use of control proteins. We show that RLM normalization is able to reduce both intra- and interarray technical variability while maintaining biological differences. Moreover, in titration experiments, RLM normalization enhances the correlation of protein signals with serum concentration. Conversely, while quantile and global normalization can reduce interarray technical variability, neither is as effective as RLM normalization in maintaining biological differences. Most importantly, both introduce artifacts that distort the signals and affect the correct identification of reactive proteins, impairing their use for biomarker discovery. Hence, we show RLM normalization is better suited to protein arrays than approaches used for DNA microarrays.

BackgroundQuestionnaires are valuable data collection instruments in public health research, and can serve to pre-screen respondents for suitability in future studies. Survey non-response leads to reduced effective sample sizes and can decrease representativeness of the study population, so high response rates are needed to minimize the risk of bias. Here we present results on the success of different postal questionnaire strategies at effecting response, and the effectiveness of these strategies at recruiting participants for a field study on the effects of aircraft noise on sleep.MethodsIn total, we mailed 17 rounds of 240 questionnaires (total n = 4080) to randomly selected households around Atlanta International Airport. Different mailing rounds were varied in the length of the questionnaire (11, 26 or 55 questions), survey incentive (gift card or $2 cash), number of follow-up waves (0, 2 or 3), incentive for participating in a 5-night in-home sleep study ($100, $150 or $200), and address personalization.ResultsWe received completed questionnaires from 407 respondents (response rate 11.4%). Personalizing the address, enclosing a $2 cash incentive with the initial questionnaire mailing and repeated follow-up mailings were effective at increasing response rate. Despite the increased expense of these approaches in terms of each household mailed, the higher response rates meant that they were more cost-effective overall for obtaining an equivalent number of responses. Interest in participating in the field study decreased with age, but was unaffected by the mailing strategies or cash incentives for field study participation. The likelihood that a respondent would participate in the field study was unaffected by survey incentive, survey length, number of follow-up waves, field study incentive, age or sex.ConclusionsPre-issued cash incentives and sending follow-up waves could maximize the representativeness and numbers of people from which to recruit, and may be an effective strategy for improving recruitment into field studies.

ObjectivesTransportation of goods on railways is increasing and the majority of the increased numbers of freight trains run during the night. Transportation noise has adverse effects on sleep structure, affects the heart rate (HR) during sleep and may be linked to cardiovascular disease. Freight trains also generate vibration and little is known regarding the impact of vibration on human sleep. A laboratory study was conducted to examine how a realistic nocturnal railway traffic scenario influences HR during sleep.DesignCase–control.SettingHealthy participants.Participants24 healthy volunteers (11 men, 13 women, 19–28 years) spent six consecutive nights in the sleep laboratory.InterventionsAll participants slept during one habituation night, one control and four experimental nights in which train noise and vibration were reproduced. In the experimental nights, 20 or 36 trains with low-vibration or high-vibration characteristics were presented.Primary and secondary outcome measuresPolysomnographical data and ECG were recorded.ResultsThe train exposure led to a significant change of HR within 1 min of exposure onset (p=0.002), characterised by an initial and a delayed increase of HR. The high-vibration condition provoked an average increase of at least 3 bpm per train in 79% of the participants. Cardiac responses were in general higher in the high-vibration condition than in the low-vibration condition (p=0.006). No significant effect of noise sensitivity and gender was revealed, although there was a tendency for men to exhibit stronger HR acceleration than women.ConclusionsFreight trains provoke HR accelerations during sleep, and the vibration characteristics of the trains are of special importance. In the long term, this may affect cardiovascular functioning of persons living close to railways.

There was a steady and marked increase in the number of medical helicopter accidents in the United States during the 10-year period (1993-2002). These findings are worrisome in light of recent research that has indicated use of medical helicopters may be excessive and nonbeneficial for most patients.