bluuebunny/crossref_metadata_2025 huggingface dataset:
Public Satisfaction with the Health System and Popular Support for State Involvement in an East Asian Welfare Regime: Health Policy Legitimacy of Hong Kong
Authors: He Alex Jingwei
Published: November 2016

Abstract

The legitimacy of social policies has gained increasing attention in the past decade, against the backdrop of fiscal austerity and retrenchment in many nations. Policy legitimacy encompasses public preferences for the underlying principles of policies and the actual outcomes as perceived by citizens. Scholarly knowledge concerning the legitimacy of health policy – a major element of modern social policy architecture – is, unfortunately, limited. This article seeks to extend the scholarly debates on health policy legitimacy from the West to Hong Kong, a member of the East Asian welfare state cluster. A bi‐dimensional definition of health policy legitimacy – encompassing both public satisfaction with the health system and the normative expectation as to the extent of state involvement in health care – is adopted. Based on analysis of data collected from a telephone survey of adult Hong Kong citizens between late 2014 and early 2015, the findings of this study demonstrate a fairly high level of satisfaction with the territory's health system, but popular support for government responsibility presents a clear residual characteristic. The study also tests the self‐interest thesis and the ideology thesis – major theoretical frameworks for explaining social policy legitimacy – in the Hong Kong context. Egalitarian ideology and trust in government are closely related to both public satisfaction with the system and popular support for governmental provision of care. However, the self‐interest thesis receives partial support. The findings are interpreted in the context of Hong Kong's health system arrangements, while implications for the territory's ongoing health policy reform are discussed.
Improving transfusion education for junior doctors; exploring UK experiences
Authors: Graham J.E. , Narayan S. , Pendry K.
Published: November 2016

Objectives

To provide evidence‐based guidance on how transfusion education should be delivered to junior doctors by employing established qualitative research methodology.

Background

There is a global call for increased transfusion education for doctors to support the delivery of evidence‐based practice. Education is reported as an effective measure to improve transfusion practice, although there is a paucity of research evaluating how this should be effectively delivered.

Methods

Serial focus groups with junior doctors and relevant healthcare professionals explored experiences of, and reactions to, education and competency assessments in transfusion, which were audio‐recorded and transcribed. Temporal and final analysis, performed by two independent assessors, informed subsequent recruitment, analysis and challenging of emerging theories – until saturation was reached.

Results

Eight focus groups were held involving 53 personnel, 77% of whom were junior doctors. Current transfusion education for doctors in the UK is reliant on e‐learning and ‘cascade training’ (on‐the‐job from senior clinicians/nursing staff). E‐learning is viewed as a ‘tick box exercise’. There is a call for relevant and practical continuing education delivered face to face by good educators in an environment away from clinical practice. Preferred methods include small group and simulation learning based on real‐life cases. In contrast to practical competency, the assessment of clinical competency is deemed unfeasible.

Conclusion

Current methods of transfusion education employed in the UK are unsatisfactory to ensure safe transfusion practice. Ongoing education is deemed necessary throughout career progression, and suggested improvements include increased emphasis on face‐to‐face teaching and simulation training. Employed educational methods and decision support tools require appropriate evaluation.
Negative experiences and pre‐donation blood pressure: the role of attitude and anxiety
Authors: Hoogerwerf M. D. , van Dongen A. , Middelburg R. A. , Merz E.‐M. , de Kort W. L. A. M. , Frings‐Dresen M. H. W. , Sluiter J. K. , Veldhuizen I. J. T.
Published: November 2016

Background/objectives

Negative experiences (NEs) have been shown to result in an increased stress response, as indicated by blood pressure, at the subsequent donation. This response might be influenced by how the donor rates the donation in terms of importance and pleasantness [affective attitude (AA)/cognitive attitude (CA)] or by anxiety about donating blood. We investigated the effect of AA/CA/anxiety on the impact of NEs on pre‐donation blood pressure (pd‐BP) in the subsequent donation.

Materials/methods

pd‐BP at visit 3 was compared between donors with and without a history of NEs during or after their first two visits (visit 1: medical check, visit 2: first donation). The effect of AA/CA/anxiety (measured 1 month prior to visit 1 on a 7‐point scale) on visit 3 pd‐BP was explored using linear regression and interaction analyses. Analyses were stratified for gender, age and pd‐BP at visit 1, which were taken into account as confounders.

Results

In 1106 first‐time blood donors (70% female), 632 donors (57% of total) indicated an NE at their first donation. Mean scores for AA/CA/anxiety were 5·2/6·5/2·2 (men without NE), 4·8/6·3/3·0 (men with NE), 5·2/6·6/2·6 (women without NE) and 4·8/6·6/3·2 (women with NE). No significant associations were found for NE and pd‐BP at visit 3 after adjusting for confounding. Of 48 interaction effects, four were significant, but effects were small and inconsistent.

Conclusion

In donors who had had negative experiences during their first donation, anxiety and attitude to donation did not influence their pre‐donation blood pressure at their subsequent visit.
Trends and risk factors of stillbirths and neonatal deaths in Eastern Uganda (1982–2011): a cross‐sectional, population‐based study
Authors: Kujala Sanni , Waiswa Peter , Kadobera Daniel , Akuze Joseph , Pariyo George , Hanson Claudia
Published: November 2016

Objectives

To identify mortality trends and risk factors associated with stillbirths and neonatal deaths 1982–2011.

Methods

Population‐based cross‐sectional study based on reported pregnancy history in Iganga‐Mayuge Health and Demographic Surveillance Site (HDSS) in Uganda. A pregnancy history survey was conducted among women aged 15–49 years living in the HDSS during May–July 2011 (n = 10 540). Time trends were analysed with cubic splines and linear regression. Potential risk factors were examined with multilevel logistic regression with adjusted odds ratios (AOR) and 95% confidence intervals (CI).

Results

34 073 births from 1982 to 2011 were analysed. The annual rate of decrease was 0.9% for stillbirths and 1.8% for neonatal mortality. Stillbirths were associated with several risk factors: multiple births (AOR 2.57, CI 1.66–3.99), previous adverse outcome (AOR 6.16, CI 4.26–8.88) and grand multiparity among 35‐ to 49‐year‐olds (AOR 1.97, CI 1.32–2.89). Neonatal deaths were associated with multiple births (AOR 6.16, CI 4.80–7.92) and advanced maternal age linked with parity of 1–4 (AOR 2.34, CI 1.28–4.25) and grand multiparity (AOR 1.44, CI 1.09–1.90). Education, marital status and household wealth were not associated with the outcomes.

Conclusions

The slow decline in mortality rates and easily identifiable risk factors calls for improving quality of care at birth and a rethinking of how to address obstetric risks, potentially a revival of the risk approach in antenatal care.
Costs of diarrhoea and acute respiratory infection attributable to not handwashing: the cases of India and China
Authors: Townsend Joy , Greenland Katie , Curtis Val
Published: November 2016

Objective

To estimate the national costs relating to diarrhoea and acute respiratory infections from not handwashing with soap after contact with excreta and the costs and benefits of handwashing behaviour change programmes in India and China.

Methods

Data on the reduction in risk of diarrhoea and acute respiratory infection attributable to handwashing with soap were used, together with World Health Organization (WHO) estimates of disability‐adjusted life years (DALYs) due to diarrhoea and acute respiratory infection, to estimate DALYs due to not handwashing in India and China. Costs and benefits of behaviour change handwashing programmes and the potential returns to investment are estimated valuing DALYs at per capita GDP for each country.

Results

Annual net costs to India from not handwashing are estimated at US$ 23 billion (16–35) and to China at US$ 12 billion (7–23). Expected net returns to national behaviour change handwashing programmes would be US$ 5.6 billion (3.4–8.6) for India at US$ 23 (16–35) per DALY avoided, which represents a 92‐fold return to investment, and US$ 2.64 billion (2.08–5.57) for China at US$ 22 (14–31) per DALY avoided – a 35‐fold return on investment.

Conclusion

Our results suggest large economic gains relating to decreases in diarrhoea and acute respiratory infection for both India and China from behaviour change programmes to increase handwashing with soap in households.
Monocyte‐mediated activation of endothelial cells occurs only after binding to extracellular vesicles from red blood cell products, a process mediated by β‐integrin
Authors: Straat Marleen , van Hezel Maike E. , Böing Anita , Tuip‐De Boer Anita , Weber Nina , Nieuwland Rienk , van Bruggen Robin , Juffermans Nicole P.
Published: November 2016

BACKGROUND

Red blood cell (RBC) transfusion is associated with organ failure. The mechanism remains unknown, but may include adherence of blood cells to the microvasculature. We hypothesized that RBC‐derived extracellular vesicles (EVs) interact with monocytes to activate endothelial cells.

STUDY DESIGN AND METHODS

Human umbilical vein endothelial cells were incubated with supernatant from fresh and stored RBC units either containing EVs or depleted from EVs, with or without the addition of immune cells. We measured expression of adhesion markers by flow cytometry and markers of coagulation and inflammation in the culture medium. We studied phagocytosis of EVs by monocytes by using confocal microscopy and flow cytometry.

RESULTS

Incubation of endothelial cells with monocytes alone did not induce up regulation of adhesion markers. The addition of both monocytes and supernatant from RBCs containing EVs resulted in up regulation of endothelial expression of intercellular adhesion molecule 1 and E‐selectin when compared to baseline. Up regulation was absent when stimulated with RBC supernatant depleted from EVs. EVs are phagocytosed by monocytes, which was partly abrogated after coincubation with two different complement receptor 3 (CR3)‐blocking antibodies. Addition of RBC‐derived EVs also increased levels of von Willebrand factor (VWF). There were no differences between groups related to storage time.

CONCLUSION

EVs from RBC transfusion bags activate monocytes with subsequent up regulation of endothelial cell adhesion markers. EVs are phagocytosed by monocytes through CR3. Furthermore, these EVs proved to be a source of VWF. These effects are unrelated to storage time. Thereby, EVs from RBC transfusion bags induce a proinflammatory and procoagulant endothelial cell response.
Contaminated particle characteristics influence on membrane fouling
Authors: Abdelrasoul Amira , Doan Huu , Lohi Ali , Cheng Chil‐Hung
Published: November 2016

The goal of the current research study was to critically examine the role of the shape of contaminated particles for an accurate prediction of membrane fouling phenomenon. Polycarbonate flat membranes with uniform pore sizes of 0.05 µm and 0.1 µm, in addition to Polysulfone membranes with molecular weight cut of 60,000, were used under a constant feed flow rate and a cross‐flow mode in ultrafiltration of a latex paint solution featuring a wide range of particle size distribution. The current mathematical model was developed to illustrate the effect of irregularity and polydispersity of latex particles on the mass of fouling and irreversible fouling on membranes. The results obtained indicate that the sphericity of contaminated particles had a significant influence on fouling potential at different aggregation levels.
Reuse of waste activated sludge for textile dyeing wastewater treatment by biosorption: performance optimization and comparison
Authors: Djafer A. , Djafer L. , Maimoun B. , Iddou A. , Kouadri Mostefai S. , Ayral A.
Published: November 2016

The aim of this study is to assess the ability of local low‐cost biomaterials to remove reactive dyes from aqueous solutions. Granules prepared from dried activated sludge (DAS) were used as a sorbent for the removal of red bimacid dye (E5R) chosen as model dye. The study involves batch type experiments to investigate the effects of initial dye concentration, adsorbent dose, contact time, temperature and pH of solution on biosorption process. Optimal experimental conditions were ascertained. The pseudo‐second‐order kinetic model fits very well with the experimental results. The thermodynamic parameters for the biosorption process have also been calculated and found the sorption process as exothermic. The sorption performance of this DAS is finally compared with that of different reference sorbents and of other low‐cost materials.
The effect of hypoxia on free and encapsulated adult porcine islets—an in vitro study
Authors: Muthyala Sudhakar , Safley Susan , Gordan Kereen , Barber Graham , Weber Collin , Sambanis Athanassios
Published: November 2016

Background

Adult porcine islets (APIs) constitute a promising alternative to human islets in treating type 1 diabetes. The intrahepatic site has been used in preclinical primate studies of API xenografts; however, an estimated two‐thirds of donor islets are destroyed after intraportal infusion due to a number of factors, including the instant blood‐mediated inflammatory reaction (IBMIR), immunosuppressant toxicity, and poor reestablishment of extracellular matrix connections. Intraperitoneal (ip) transplantation of non‐vascularized encapsulated islets offers several advantages over intrahepatic transplantation of free islets, including avoidance of IBMIR, immunoprotection, accommodation of a larger graft volume, and reduced risk of hemorrhage. However, there exists evidence that the peritoneal site is hypoxic, which likely impedes islet function.

Methods

We tested the effect of hypoxia (2%‐5% oxygen or pO2: 15.2‐38.0 mm Hg) on free and encapsulated APIs over a period of 6 days in culture. Free and encapsulated APIs under normoxia served as controls. Islet viability was evaluated with a viability/cytotoxicity assay using calcein AM and ethidium bromide on days 1, 3, and 6 of culture. Alamar blue assay was used to measure the metabolic activity on days 1 and 6. Insulin in spent medium was assayed by ELISA on days 1 and 6.

Results

Viability staining indicated that free islet clusters lost their integrity and underwent severe necrosis under hypoxia; encapsulated islets remained intact, even when they began to undergo necrosis. Under hypoxia, the metabolic activity and insulin secretion (normalized to metabolic activity) of both free and encapsulated islets decreased relative to islets cultured under normoxic conditions.

Conclusions

Hypoxia (2%‐5% oxygen or pO2: 15.2‐38.0 mm Hg) affects the viability, metabolic activity, and insulin secretion of both free and encapsulated APIs over a six‐day culture period. Encapsulation augments islet integrity under hypoxia, but it does not prevent loss of viability, metabolic activity, or insulin secretion.
Effect of Target Wall Curvature on Heat Transfer and Pressure Loss From Jet Array Impingement
Authors: Harrington John , Hossain Jahed , Wang Wenping , Kapat Jayanta , Maurer Michael , Thorpe Steven
Published: November 2016

Experiments to investigate the effect of target wall curvature on heat transfer and pressure loss from jet array impingement are performed. A jet plate configuration is studied with constant hole diameters and spacings. The geometry of the jet plate has streamwise jet spacings of 5.79 jet diameters, spanwise jet spacings of 4.49 jet diameters, and a jet-to-target plate distance of 3 jet diameters. For the curved case, the radius of the target plate is r/D = 31.57. A flat target wall setup with identical geometric spacing is also tested for direct comparison. Jet spacings were chosen such that validation and comparison can be made with open literature. For all configurations, spent air is drawn out in a single direction, which is tangential to the target plate curvature. Average jet Reynolds numbers ranging from 55,000 to 125,000 are tested. A steady-state measurement technique utilizing temperature-sensitive paint (TSP) is used on the target surface to obtain Nusselt number distributions. Pressure taps placed on the sidewall of the channel are used to evaluate the flow distribution in the impingement channel. Alongside the experimental work, CFD was performed utilizing the v2 − f turbulence model to better understand the relationship between the flow field and the heat transfer on the target surface. The main target of the current study is to quantify the impact of target wall radius and the decay of heat transfer after the impingement section, and to check the open literature correlations. It was found that the target wall curvature did not cause any significant changes in either the flow distribution or the heat transfer level. Comparisons with established correlations show similar level but different trends in heat transfer, potentially caused by differences in L/D. CFD results were able to show agreement in streamwise pitch-averaged Nusselt number levels with experimental results for the curved target plate at higher Re numbers.
Effect of Chemical Reactions of H2/O2 Combustion Gas on Wall Heat Flux in a Turbulent Channel Flow
Authors: Kitano Tomoaki , Iida Hiroaki , Kurose Ryoichi
Published: November 2016

The effect of chemical reactions of burnt gas on heat transfer on a cooled wall in a turbulent channel flow is investigated by direct numerical simulations. Burnt gas from a H2/O2 mixture is used as a fluid and a detailed chemical reaction mechanism that considers eight chemical species and 19 elemental reactions is used in the reaction calculation. The initial gas temperature and pressure are 3173 K and 2.0 MPa, respectively. The Reynolds number based on the channel width and mean streamwise velocity is approximately 6400 and that based on the channel half width and friction velocity is approximately 200. The results show that heat release because of consumption of radicals such as OH and H near the wall increases the heat flux on the wall and that the heat flux is enhanced by the significant increase in the local heat flux at high-speed streaks where radicals are supplied by sweep events constituting bursting motions in the turbulent boundary layer.
Smart optical coherence tomography for ultra-deep imaging through highly scattering media
Authors: Badon Amaury , Li Dayan , Lerosey Geoffroy , Boccara A. Claude , Fink Mathias , Aubry Alexandre
Published: November 2016

Iterative time reversal overcomes multiple scattering and breaks the imaging-depth limit in optical coherence tomography.
StrigoQuant: A genetically encoded biosensor for quantifying strigolactone activity and specificity
Authors: Samodelov Sophia L. , Beyer Hannes M. , Guo Xiujie , Augustin Maximilian , Jia Kun-Peng , Baz Lina , Ebenhöh Oliver , Beyer Peter , Weber Wilfried , Al-Babili Salim , Zurbriggen Matias D.
Published: November 2016

A ratiometric, quantitative, and genetically encoded strigolactone sensor, StrigoQuant, opens new avenues in SL signaling studies.
Quantitative Metaproteomics Highlight the Metabolic Contributions of Uncultured Phylotypes in a Thermophilic Anaerobic Digester
Authors: Hagen Live H. , Frank Jeremy A. , Zamanzadeh Mirzaman , Eijsink Vincent G. H. , Pope Phillip B. , Horn Svein J. , Arntzen Magnus Ø.
Published: November 2016

In this study, we used multiple meta-omic approaches to characterize the microbial community and the active metabolic pathways of a stable industrial biogas reactor with food waste as the dominant feedstock, operating at thermophilic temperatures (60°C) and elevated levels of free ammonia (367 mg/liter NH
3
-N). The microbial community was strongly dominated (76% of all 16S rRNA amplicon sequences) by populations closely related to the proteolytic bacterium
Coprothermobacter proteolyticus.
Multiple
Coprothermobacter-
affiliated strains were detected, introducing an additional level of complexity seldom explored in biogas studies. Genome reconstructions provided metabolic insight into the microbes that performed biomass deconstruction and fermentation, including the deeply branching phyla
Dictyoglomi
and
Planctomycetes
and the candidate phylum “
Atribacteria
.” These biomass degraders were complemented by a synergistic network of microorganisms that convert key fermentation intermediates (fatty acids) via syntrophic interactions with hydrogenotrophic methanogens to ultimately produce methane. Interpretation of the proteomics data also suggested activity of a
Methanosaeta
phylotype acclimatized to high ammonia levels. In particular, we report multiple novel phylotypes proposed as syntrophic acetate oxidizers, which also exert expression of enzymes needed for both the Wood-Ljungdahl pathway and β-oxidation of fatty acids to acetyl coenzyme A. Such an arrangement differs from known syntrophic oxidizing bacteria and presents an interesting hypothesis for future studies. Collectively, these findings provide increased insight into active metabolic roles of uncultured phylotypes and presents new synergistic relationships, both of which may contribute to the stability of the biogas reactor.

IMPORTANCE
Biogas production through anaerobic digestion of organic waste provides an attractive source of renewable energy and a sustainable waste management strategy. A comprehensive understanding of the microbial community that drives anaerobic digesters is essential to ensure stable and efficient energy production. Here, we characterize the intricate microbial networks and metabolic pathways in a thermophilic biogas reactor. We discuss the impact of frequently encountered microbial populations as well as the metabolism of newly discovered novel phylotypes that seem to play distinct roles within key microbial stages of anaerobic digestion in this stable high-temperature system. In particular, we draft a metabolic scenario whereby multiple uncultured syntrophic acetate-oxidizing bacteria are capable of syntrophically oxidizing acetate as well as longer-chain fatty acids (via the β-oxidation and Wood-Ljundahl pathways) to hydrogen and carbon dioxide, which methanogens subsequently convert to methane.
An Inducible Operon Is Involved in Inulin Utilization in Lactobacillus plantarum Strains, as Revealed by Comparative Proteogenomics and Metabolic Profiling
Authors: Buntin Nirunya , Hongpattarakere Tipparat , Ritari Jarmo , Douillard François P. , Paulin Lars , Boeren Sjef , Shetty Sudarshan A. , de Vos Willem M.
Published: November 2016

The draft genomes of
Lactobacillus plantarum
strains isolated from Asian fermented foods, infant feces, and shrimp intestines were sequenced and compared to those of well-studied strains. Among 28 strains of
L. plantarum
, variations in the genomic features involved in ecological adaptation were elucidated. The genome sizes ranged from approximately 3.1 to 3.5 Mb, of which about 2,932 to 3,345 protein-coding sequences (CDS) were predicted. The food-derived isolates contained a higher number of carbohydrate metabolism-associated genes than those from infant feces. This observation correlated to their phenotypic carbohydrate metabolic profile, indicating their ability to metabolize the largest range of sugars. Surprisingly, two strains (P14 and P76) isolated from fermented fish utilized inulin. β-Fructosidase, the inulin-degrading enzyme, was detected in the supernatants and cell wall extracts of both strains. No activity was observed in the cytoplasmic fraction, indicating that this key enzyme was either membrane-bound or extracellularly secreted. From genomic mining analysis, a predicted inulin operon of
fosRABCDXE
, which encodes β-fructosidase and many fructose transporting proteins, was found within the genomes of strains P14 and P76. Moreover,
pts1BCA
genes, encoding sucrose-specific IIBCA components involved in sucrose transport, were also identified. The proteomic analysis revealed the mechanism and functional characteristic of the
fosRABCDXE
operon involved in the inulin utilization of
L. plantarum
. The expression levels of the
fos
operon and
pst
genes were upregulated at mid-log phase. FosE and the LPXTG-motif cell wall anchored β-fructosidase were induced to a high abundance when inulin was present as a carbon source.

IMPORTANCE
Inulin is a long-chain carbohydrate that may act as a prebiotic, which provides many health benefits to the host by selectively stimulating the growth and activity of beneficial bacteria in the colon. While certain lactobacilli can catabolize inulin, this has not yet been described for
Lactobacillus plantarum
, and an associated putative inulin operon has not been reported in this species. By using comparative and functional genomics, we showed that two
L. plantarum
strains utilized inulin and identified functional inulin operons in their genomes. The proteogenomic data revealed that inulin degradation and uptake routes, which related to the
fosRABCDXE
operon and
pstBCA
genes, were widely expressed among
L. plantarum
strains. The present work provides a novel understanding of gene regulation and mechanisms of inulin utilization in probiotic
L. plantarum
generating opportunities for synbiotic product development.
Impacts of Seasonal Housing and Teat Preparation on Raw Milk Microbiota: a High-Throughput Sequencing Study
Authors: Doyle Conor J. , Gleeson David , O'Toole Paul W. , Cotter Paul D.
Published: November 2016

In pasture-based systems, changes in dairy herd habitat due to seasonality results in the exposure of animals to different environmental niches. These niches contain distinct microbial communities that may be transferred to raw milk, with potentially important food quality and safety implications for milk producers. It is postulated that the extent to which these microorganisms are transferred could be limited by the inclusion of a teat preparation step prior to milking. High-throughput sequencing on a variety of microbial niches on farms was used to study the patterns of microbial movement through the dairy production chain and, in the process, to investigate the impact of seasonal housing and the inclusion/exclusion of a teat preparation regime on the raw milk microbiota from the same herd over two sampling periods, i.e., indoor and outdoor. Beta diversity and network analyses showed that environmental and milk microbiotas separated depending on whether they were sourced from an indoor or outdoor environment. Within these respective habitats, similarities between the milk microbiota and that of teat swab samples and, to a lesser extent, fecal samples were apparent. Indeed, SourceTracker identified the teat surface as the most significant source of contamination, with herd feces being the next most prevalent source of contamination. In milk from cows grazing outdoors, teat prep significantly increased the numbers of total bacteria present. In summary, sequence-based microbiota analysis identified possible sources of raw milk contamination and highlighted the influence of environment and farm management practices on the raw milk microbiota.

IMPORTANCEThe composition of the raw milk microbiota is an important consideration from both a spoilage perspective and a food safety perspective and has implications for milk targeted for direct consumption and for downstream processing. Factors that influence contamination have been examined previously, primarily through the use of culture-based techniques. We describe here the extensive application of high-throughput DNA sequencing technologies to study the relationship between the milk production environment and the raw milk microbiota. The results show that the environment in which the herd was kept was the primary driver of the composition of the milk microbiota composition.
Immunogenic Properties of Lactobacillus plantarum Producing Surface-Displayed Mycobacterium tuberculosis Antigens
Authors: Kuczkowska Katarzyna , Kleiveland Charlotte R. , Minic Rajna , Moen Lars F. , Øverland Lise , Tjåland Rannei , Carlsen Harald , Lea Tor , Mathiesen Geir , Eijsink Vincent G. H.
Published: November 2016

Tuberculosis (TB) remains among the most deadly diseases in the world. The only available vaccine against tuberculosis is the bacille Calmette-Guérin (BCG) vaccine, which does not ensure full protection in adults. There is a global urgency for the development of an effective vaccine for preventing disease transmission, and it requires novel approaches. We are exploring the use of lactic acid bacteria (LAB) as a vector for antigen delivery to mucosal sites. Here, we demonstrate the successful expression and surface display of a
Mycobacterium tuberculosis
fusion antigen (comprising Ag85B and ESAT-6, referred to as AgE6) on
Lactobacillus plantarum
. The AgE6 fusion antigen was targeted to the bacterial surface using two different anchors, a lipoprotein anchor directing the protein to the cell membrane and a covalent cell wall anchor. AgE6-producing
L. plantarum
strains using each of the two anchors induced antigen-specific proliferative responses in lymphocytes purified from TB-positive donors. Similarly, both strains induced immune responses in mice after nasal or oral immunization. The impact of the anchoring strategies was reflected in dissimilarities in the immune responses generated by the two
L. plantarum
strains
in vivo
. The present study comprises an initial step toward the development of
L. plantarum
as a vector for
M. tuberculosis
antigen delivery.

IMPORTANCE
This work presents the development of
Lactobacillus plantarum
as a candidate mucosal vaccine against tuberculosis. Tuberculosis remains one of the top infectious diseases worldwide, and the only available vaccine, bacille Calmette-Guérin (BCG), fails to protect adults and adolescents. Direct antigen delivery to mucosal sites is a promising strategy in tuberculosis vaccine development, and lactic acid bacteria potentially provide easy, safe, and low-cost delivery vehicles for mucosal immunization. We have engineered
L. plantarum
strains to produce a
Mycobacterium tuberculosis
fusion antigen and to anchor this antigen to the bacterial cell wall or to the cell membrane. The recombinant strains elicited proliferative antigen-specific T-cell responses in white blood cells from tuberculosis-positive humans and induced specific immune responses after nasal and oral administrations in mice.
The Enterobacterial Genotoxins: Cytolethal Distending Toxin and Colibactin
Authors: Taieb Frederic , Petit Claude , Nougayrède Jean-Philippe , Oswald Eric
Published: November 2016

While the DNA damage induced by ionizing radiation and by many chemical compounds and drugs is well characterized, the genotoxic insults inflicted by bacteria are only scarcely documented. However, accumulating evidence indicates that we are exposed to bacterial genotoxins. The prototypes of such bacterial genotoxins are the Cytolethal Distending Toxins (CDTs) produced by
Escherichia coli
and
Salmonella enterica
serovar Typhi. CDTs display the DNase structure fold and activity, and induce DNA strand breaks in the intoxicated host cell nuclei.
E. coli
and certain other
Enterobacteriaceae
species synthesize another genotoxin, colibactin. Colibactin is a secondary metabolite, a hybrid polyketide/nonribosomal peptide compound synthesized by a complex biosynthetic machinery. In this review, we summarize the current knowledge on CDT and colibactin produced by
E. coli
and/or
Salmonella
Typhi. We describe their prevalence, genetic determinants, modes of action, and impact in infectious diseases or gut colonization, and discuss the possible involvement of these genotoxigenic bacteria in cancer.
Transcription mapping of the Escherichia coli chromosome by electron microscopy
Authors: French S L , Miller O L
Published: November 2016

The distinctive double Christmas tree morphology of rRNA operons as visualized by electron microscopy makes them easy to recognize in chromatin spreads from Escherichia coli. On the basis of the pattern of nascent transcripts on nearby transcription units and the relative distances of the operons from one another and the replication origin, we are now able to specifically identify five of the seven rRNA operons in E. coli. The use of rRNA operons as markers of both position and distance has resulted in the morphological mapping of a significant portion of the E. coli chromosome; over 600 kilobase pairs in the 84- to 90-min and 72-min regions can now be recognized. Since individual rRNA operons could be identified, direct comparisons could be made of their transcriptional activities. As judged by the densities of RNA polymerases along the operons, rrnA, rrnB, rrnC, rrnD, and rrnE were all transcribed at similar levels, with one RNA polymerase every 85 base pairs. The ability to recognize individual operons and specific regions of the chromosome allows direct comparisons of various genetic parameters.
A component of the side tail fiber of Escherichia coli bacteriophage lambda can functionally replace the receptor-recognizing part of a long tail fiber protein of the unrelated bacteriophage T4
Authors: Montag D , Schwarz H , Henning U
Published: November 2016

The distal part of the long tail fiber of Escherichia coli bacteriophage T4 consists of a dimer of protein 37. Dimerization requires the catalytic action of protein 38, which is encoded by T4 and is not present in the virion. It had previously been shown that gene tfa of the otherwise entirely unrelated phage lambda can functionally replace gene 38. Open reading frame (ORF) 314, which encodes a protein that exhibits homology to a COOH-terminal area of protein 37, is located immediately upstream of tfa. The gene was cloned and expressed in E. coli. An antiserum against the corresponding polypeptide showed that it was present in phage lambda. The serum also reacted with the long tail fibers of phage T4 near their free ends. An area of the gene encoding a COOH-terminal region of ORF 314 was recombined, together with tfa, into the genome of T4, thus replacing gene 38 and a part of gene 37 that codes for a COOH-terminal part of protein 37. Such T4-lambda hybrids, unlike T4, required the presence of outer membrane protein OmpC for infection of E. coli B. An ompC missense mutant of E. coli K-12, which was still sensitive to T4, was resistant to these hybrids. We conclude that the ORF 314 protein represents a subunit of the side tail fibers of phage lambda which probably recognize the OmpC protein. ORF 314 was designated stf (side tail fiber). The results also offer an explanation for the very unusual fact that, despite identical genomic organizations, T4 and T2 produce totally different proteins 38. An ancestor of T4 from the T2 lineage may have picked up tfa and stf from a lambdoid phase, thus possibly demonstrating horizontal gene transfer between unrelated phage species.
A mutation in the amino terminus of a hybrid TrpC-TonB protein relieves overproduction lethality and results in cytoplasmic accumulation
Authors: Skare J T , Roof S K , Postle K
Published: November 2016

We have developed a selection for mutations in a trpC-tonB gene fusion that takes advantage of the properties of the plasmid-encoded TrpC-TonB hybrid protein. The TrpC-TonB hybrid protein consists of amino acids 1 through 25 of the normally cytoplasmic protein, TrpC, fused to amino acids 12 through 239 of TonB. It is expressed from the trp promoter and is regulated by the trpR gene and the presence or absence of tryptophan. Under repressing conditions in the presence of tryptophan, the trpC-tonB gene can restore phi 80 sensitivity to a tonB deletion mutant, which indicates that TrpC-TonB can be exported and is functional. High-level expression of TrpC-TonB protein in the absence of tryptophan results in virtually immediate cessation of growth for strains carrying the trpC-tonB plasmid. By selecting for survivors of the induced growth inhibition (overproduction lethality), we have isolated a variety of mutations. Many of the mutations decrease expression of the TrpC-TonB protein, as expected. In addition, three independently isolated mutants expressing normal levels of TrpC-TonB protein result in a Gly----Asp substitution within the hydrophobic amino terminus of TonB. The mutant proteins are designated TrpC-TonBG26D. The mutations are suppressed by prlA alleles, known to suppress export (signal sequence) mutations. TrpC-TonB proteins carrying the Gly----Asp substitution accumulate in the cytoplasm. We conclude that the Gly----Asp substitution is an export mutation. TrpC-TonBG26D protein has been purified and used to raise polyclonal antibodies that specifically recognize both TrpC-TonB protein and wild-type TonB protein.
Staphylococcus aureus chromosomal mutations that decrease efficiency of Rep utilization in replication of pT181 and related plasmids
Authors: Iordanescu S , Bargonetti J
Published: November 2016

Staphylococcus aureus chromosomal mutants which maintain pT181 and related plasmids at a much reduced copy number but which do not affect the replication of other plasmids have been isolated. The origin of replication and the initiator protein of the affected plasmids are the only elements required for the response to these mutations. The host mutations do not interfere with the pT181 replication control mechanism.
Sporulation of Myxococcus xanthus in liquid shake flask cultures
Authors: Rosenbluh A , Rosenberg E
Published: November 2016

When suspended in a liquid starvation medium, exponentially growing Myxococcus xanthus sporulated within 3 days. These myxospores were similar to spores developed within fruiting bodies, as determined by electron microscopy and the production of spore-specific protein S. This liquid sporulation system may be useful as a means of preparing large quantities of myxospores and extracellular fluid for biochemical studies, including isolation of chemical signals produced during the sporulation process.
Cloning and sequence analysis of the LPD-glc structural gene of Pseudomonas putida
Authors: Palmer J A , Hatter K , Sokatch J R
Published: November 2016

Pseudomonas putida is able to produce three lipoamide dehydrogenases: (i) LPD-glc, which is the E3 component of the pyruvate and 2-ketoglutarate dehydrogenase complexes and the L-factor for the glycine oxidation system; (ii) LPD-val, which is the specific E3 component of the branched-chain keto acid dehydrogenase complex and is induced by growth on leucine, isoleucine, or valine; and (iii) LPD-3, which was discovered in a lpdG mutant and whose role is unknown. Southern hybridization with an oligonucleotide probe encoding the highly conserved redox-active site produced three bands corresponding to the genes encoding these three lipoamide dehydrogenases. The complete structural gene for LPD-glc, lpdG, was isolated, and its nucleotide sequence was determined. The latter consists of 476 codons plus a stop codon, TAA. The structural gene for LPD-glc is preceded by a partial open reading frame with strong similarity to the E2 component of 2-ketoglutarate dehydrogenase of Escherichia coli. This suggests that lpdG is part of the 2-ketoglutarate dehydrogenase operon. LPD-glc was expressed in Pseudomonas putida JS348 from pHP4 which contains a partial open reading frame corresponding to the E2 component, 94 bases of noncoding DNA, and the structural gene for lpdG. This result indicates that lpdG can be expressed separately from the other genes of the operon.
Evidence for energy-dependent transposition of core lipopolysaccharide across the inner membrane of Salmonella typhimurium
Authors: McGrath B C , Osborn M J
Published: November 2016

The uncoupler 2,4-dinitrophenol blocks the final step of lipopolysaccharide assembly--transfer of O antigen from undecaprenyl pyrophosphate to core lipopolysaccharide--in intact Salmonella typhimurium but not in isolated membrane fractions. The O-antigen ligase enzyme is not inhibited by dinitrophenol in vitro, and core lipopolysaccharide synthesized in the presence of uncoupler in vivo is functional as acceptor of O antigen in vitro. The evidence strongly suggests that maintenance of proton motive force is required for transmembrane transposition of core lipopolysaccharide to the active site of O-antigen ligase at the periplasmic face of the inner membrane.
Coralline shape of the bacterial nucleoid after cryofixation
Authors: Bohrmann B , Villiger W , Johansen R , Kellenberger E
Published: November 2016

A new procedure of immunostaining sections of cryofixed and freeze-substituted Escherichia coli shows that DNA extends from its bulk into small ribosome-free spaces throughout the cytoplasm, resulting in a coralline-shaped nucleoid. Low-resolution imaging of a bacterium reconstructed from serial sections demonstrated that the small excrescencies are not resolved. The resulting photograph shows the same features as phase-contrast light micrographs.
endAFS, a novel family E endoglucanase gene from Fibrobacter succinogenes AR1
Authors: Cavicchioli R , East P D , Watson K
Published: November 2016

The complete nucleotide sequence of endAFS, an endoglucanase gene isolated from the ruminal anaerobe Fibrobacter succinogenes AR1, was determined. endAFS encodes two overlapping open reading frames (ORF1 and ORF2), and it was proposed that a -1 ribosomal frameshift was required to allow contiguous synthesis of a 453-amino-acid endoglucanase. A proline- and threonine-rich region at the C terminus of ORF1 and rare codons for arginine and threonine were coincident with the proposed frameshift site. ENDAFS is proposed to be a member of subgroup 1 of family E endoglucanases, of which endoglucanases from Thermomonospora fusca and Persea americana (avocado) are also members. Endoglucanases from Clostridium thermocellum and Pseudomonas fluorescens form subgroup 2.
Expression of Escherichia coli pabA
Authors: Tran P V , Nichols B P
Published: November 2016

Escherichia coli pabA encodes the glutamine amidotransferase subunit of p-aminobenzoate synthase. p-Aminobenzoate synthase catalyzes the conversion of chorismate and glutamine to 4-amino-4-deoxychorismate, which is then converted to p-aminobenzoate by a 4-amino-4-deoxychorismate lyase. The 5'-terminal segment of pabA was previously shown to be transcribed from two different promoters, one near the pabA coding sequence (P1) and one preceding fic (P2). However, a pabA-lacZ translational fusion was expressed only from the mRNA originating at P1. We have determined that expression of a pabA-lacZ chromosomal fusion is not changed by p-aminobenzoate limitation, growth rate, catabolite repression, overexpression of either p-aminobenzoate synthase subunit, or gene dosage of pabA and pabB. The lack of pabA expression from P2 appears to be the result of a stable secondary structure in the intergenic space preceding pabA that sequesters the pabA ribosome binding site. Disruption of the secondary structure by mutation allowed expression of pabA from P2, as did translation of ribosomes into the fic-pabA intergenic region.
Effect of ermC leader region mutations on induced mRNA stability
Authors: Hue K K , Bechhofer D H
Published: November 2016

Induction of translation of the ermC gene product in Bacillus subtilis occurs upon exposure to erythromycin and is a result of ribosome stalling in the ermC leader peptide coding sequence. Another result of ribosome stalling is stabilization of ermC mRNA. The effect of leader RNA secondary structure, methylase translation, and leader peptide translation on induced ermC mRNA stability was examined by constructing various mutations in the ermC leader region. Analysis of deletion mutations showed that ribosome stalling causes induction of ermC mRNA stability in the absence of methylase translation and ermC leader RNA secondary structure. Furthermore, deletions that removed much of the leader peptide coding sequence had no effect on induced ermC mRNA stability. A leader region mutation was constructed such that ribosome stalling occurred in a position upstream of the natural stall site, resulting in induced mRNA stability without induction of translation. This mutation was used to measure the effect of mRNA stabilization on ermC gene expression.
Rhizobium meliloti exoG and exoJ mutations affect the exoX-exoY system for modulation of exopolysaccharide production
Authors: Reed J W , Capage M , Walker G C
Published: November 2016

R. meliloti Rm1021 normally produces an acidic Calcofluor-binding exopolysaccharide, called succinoglycan or EPS I, which is required for successful nodulation of alfalfa by this strain. At least 13 loci affecting production of EPS I were previously mapped to a cluster on the second of two symbiotic megaplasmids in Rm1021, pRmeSU47b. A putative regulatory region was originally defined by the exoG and exoJ mutations. exoG and exoJ mutants produced less exopolysaccharide than wild-type strains and induced nitrogen-fixing nodules on alfalfa with reduced efficiency compared with the wild type. These mutants appeared to produce only a low-molecular-weight form of EPS I. Mutations called exoX cause an increase in exopolysaccharide production and map in the same region as the exoG and exoJ mutations. The DNA sequence of this region reveals that it contains two open reading frames, called exoX and exoY, which have homologs in other Rhizobium species. Interestingly, the exoG insertion mutations fall in an intergenic region and may affect the expression of exoX or exoY. The exoJ mutation falls in the 3' portion of the exoX open reading frame and is probably an allele of exoX that results in altered function. exoG and exoJ mutations limit EPS I production in the presence of exoR95 or exoS96 mutations, which cause overproduction of EPS I. Gene regulation studies suggest that ExoX and ExoY constitute a system that modulates exopolysaccharide synthesis at a posttranslational level. The deduced sequence of ExoY is homologous to a protein required for an early step in xanthan gum biosynthesis, further suggesting that the modulatory system may affect the exopolysaccharide biosynthetic apparatus.
Growth rate regulation of translation initiation factor IF3 biosynthesis in Escherichia coli
Authors: Liveris D , Klotsky R A , Schwartz I
Published: November 2016

infC, the gene encoding translation initiation factor IF3 in Escherichia coli, can be transcribed from three promoters. Two of these promoters, PI1 and PI2, are located in the upstream thrS sequence which codes for threonyl-tRNA synthetase. Previous studies had shown that PI2 was the major promoter for infC. In the present study, the extent of transcription from PI1 and/or PI2 at a variety of steady-state growth rates was analyzed by promoter fusion studies. PI2 was the more active promoter (two- to threefold stronger than PI1) at all growth rates tested. A fusion plasmid containing both PI1 and PI2 exhibited a transcription level approximately equal to the sum of those observed with the fusion plasmids containing the individual promoters. The transcriptional activities of PI1 and PI2 did not change as the growth rate was varied from 0.3 to 1.7 doublings per h. In contrast, a fusion plasmid carrying the rrnB P1 promoter displayed the expected growth rate response. The steady-state concentrations of infC mRNA in cells grown at different rates were measured and found not to vary. These results indicate that the previously reported growth rate regulation of IF3 biosynthesis neither is accomplished by transcriptional control nor is a result of differential mRNA stability. In view of these results, the steady-state levels of IF3 in cells grown at a number of different growth rates were determined by quantitative immunoblotting. IF3 levels were found to vary with growth rate in a manner essentially identical to that observed for ribosomes. A model accounting for these results and describing a mechanism for coordinate growth rate-regulated expression of ribosomes and IF3 is presented.