Construction of a multiple fluorescence labelling system for use in co-invasion studies of Listeria monocytogenes

BACKGROUND: Existing virulence models are often difficult to apply for quantitative comparison of invasion potentials of Listeria monocytogenes. Well-to-well variation between cell-line based in vitro assays is practically unavoidable, and variation between individual animals is the cause of large deviations in the observed capacity for infection when animal models are used. One way to circumvent this problem is to carry out virulence studies as competition assays between 2 or more strains. This, however, requires invasion-neutral markers that enable easy discrimination between the different strains. RESULTS: A fluorescent marker system, allowing visualization and identification of single L. monocytogenes cells as well as colonies in a non-destructive manner, was developed. Five different fluorescent labels are available, and allowed simultaneous visual discrimination between three differently labelled strains at the single cell level by use of fluorescence microscopy. More than 90% of the L. monocytogenes host cells maintained the fluorescence tags for 40 generations. The fluorescence tags did not alter the invasive capacity of the L. monocytogenes cells in a traditional Caco-2 cell invasion assay, and visual discrimination between invaded bacteria carrying different fluorescent labels inside the cells was possible. CONCLUSION: The constructed fluorescent marker system is stable, easy to use, does not affect the virulence of L. monocytogenes in Caco-2 cell assays, and allows discrimination between differently labelled bacteria after internalization in these cells

Phase variable expression of capsular polysaccharide modifications allows <em>Campylobacter jejuni</em> to avoid bacteriophage infection in chickens

Bacteriophages are estimated to be the most abundant entities on earth and can be found in every niche where their bacterial hosts reside. The initial interaction between phages and Campylobacter jejuni, a common colonizer of poultry intestines and a major source of foodborne bacterial gastroenteritis in humans, is not well understood. Recently, we isolated and characterized a phage F336 resistant variant of C. jejuni NCTC11168 called 11168R. Comparisons of 11168R with the wildtype lead to the identification of a novel phage receptor, the phase variable O-methyl phosphoramidate (MeOPN) moiety of the C. jejuni capsular polysaccharide (CPS). In this study we demonstrate that the 11168R strain has gained cross-resistance to four other phages in our collection (F198, F287, F303, and F326). The reduced plaquing efficiencies suggested that MeOPN is recognized as a receptor by several phages infecting C. jejuni. To further explore the role of CPS modifications in C. jejuni phage recognition and infectivity, we tested the ability of F198, F287, F303, F326, and F336 to infect different CPS variants of NCTC11168, including defined CPS mutants. These strains were characterized by high-resolution magic angle spinning NMR spectroscopy. We found that in addition to MeOPN, the phase variable 3-O-Me and 6-O-Me groups of the NCTC11168 CPS structure may influence the plaquing efficiencies of the phages. Furthermore, co-infection of chickens with both C. jejuni NCTC11168 and phage F336 resulted in selection of resistant C. jejuni bacteria, which either lack MeOPN or gain 6-O-Me groups on their surface, demonstrating that resistance can be acquired in vivo. In summary, we have shown that phase variable CPS structures modulate phage infectivity in C. jejuni and suggest that the constant phage predation in the avian gut selects for changes in these structures leading to a continuing phage–host co-evolution

Settling velocity of microplastic particles having regular and irregular shapes

The settling velocities of 66 microplastic particle groups, having both regular (58) and irregular (eight) shapes, are measured experimentally. Regular shapes considered include: spheres, cylinders, disks, square plates, cubes, other cuboids (square and rectangular prisms), tetrahedrons, and fibers. The experiments generally consider Reynolds numbers greater than 102, extending the predominant range covered by previous studies. The present data is combined with an extensive data set from the literature, and the settling velocities are systematically analyzed on a shape-by-shape basis. Novel parameterizations and predictive drag coefficient formulations are developed for both regular and irregular particle shapes, properly accounting for preferential settling orientation. These are shown to be more accurate than the best existing predictive formulation from the literature. The developed method for predicting the settling velocity of irregularly-shaped microplastic particles is demonstrated to be equally well suited for natural sediments in the Appendix

Experimental investigation on the nearshore transport of buoyant microplastic particles

This paper presents experimental measurements of beaching times for buoyant microplastic particles released, both in the pre-breaking region and within the surf zone. The beaching times are used to quantify cross-shore Lagrangian transport velocities of the microplastics. Prior to breaking the particles travel onshore with a velocity close to the Lagrangian fluid particle velocity, regardless of particle characteristics. In the surf zone the Lagrangian velocities of the microplastics increase and become closer to the wave celerity. Furthermore, it is demonstrated that particles having low Dean numbers (dimensionless fall velocity) are transported at higher mean velocities, as they have a larger tendency to be at the free-surface relative to particles with higher Dean numbers. An empirical relation is formulated for predicting the cross-shore Lagrangian transport velocities of buoyant microplastic particles, valid for both non-breaking and breaking irregular waves. The expression matches the present experiments well, in addition to two prior studies

CNV-association meta-analysis in 191,161 European adults reveals new loci associated with anthropometric traits

Funding Information: This research has been conducted using the UK Biobank Resource. This research has been conducted using the Danish National Biobank resource. The authors are grateful to the Raine Study participants and their families, and to the Raine Study research staff for cohort co-ordination and data collection. QIMR is grateful to the twins and their families for their generous participation in these studies. We would like to thank staff at the Queensland Institute of Medical Research: Anjali Henders, Dixie Statham, Lisa Bowdler, Ann Eldridge, and Marlene Grace for sample collection, processing and genotyping, Scott Gordon, Brian McEvoy, Belinda Cornes and Beben Benyamin for data QC and preparation, and David Smyth and Harry Beeby for IT support. HBCS Acknowledgements: We thank all study participants as well as everybody involved in the Helsinki Birth Cohort Study. Helsinki Birth Cohort Study has been supported by grants from the Academy of Finland, the Finnish Diabetes Research Society, Folkhälsan Research Foundation, Novo Nordisk Foundation, Finska Läkaresällskapet, Juho Vainio Foundation, Signe and Ane Gyllenberg Foundation, University of Helsinki, Ministry of Education, Ahokas Foundation, Emil Aaltonen Foundation. Finrisk study is grateful for the THL DNA laboratory for its skillful work to produce the DNA samples used in this study and thanks the Sanger Institute and FIMM genotyping facilities for genotyping the samples. We thank the MOLGENIS team and Genomics Coordination Center of the University Medical Center Groningen for software development and data management, in particular Marieke Bijlsma and Edith Adriaanse. This work was supported by the Leenards Foundation (to Z.K.), the Swiss National Science Foundation (31003A_169929 to Z.K., Sinergia grant CRSII33-133044 to AR), Simons Foundation (SFARI274424 to AR) and SystemsX.ch (51RTP0_151019 to Z.K.). A.R.W., H.Y. and T.M.F. are supported by the European Research Council grant: 323195:SZ-245. M.A.T., M.N.W. and An.M. are supported by the Wellcome Trust Institutional Strategic Support Award (WT097835MF). For full funding information of all participating cohorts see Supplementary Note 2. Publisher Copyright: © 2017 The Author(s).There are few examples of robust associations between rare copy number variants (CNVs) and complex continuous human traits. Here we present a large-scale CNV association meta-analysis on anthropometric traits in up to 191,161 adult samples from 26 cohorts. The study reveals five CNV associations at 1q21.1, 3q29, 7q11.23, 11p14.2, and 18q21.32 and confirms two known loci at 16p11.2 and 22q11.21, implicating at least one anthropometric trait. The discovered CNVs are recurrent and rare (0.01-0.2%), with large effects on height (> 2.4 cm), weight ( 5 kg), and body mass index (BMI) (> 3.5 kg/m(2)). Burden analysis shows a 0.41 cm decrease in height, a 0.003 increase in waist-to-hip ratio and increase in BMI by 0.14 kg/m2 for each Mb of total deletion burden (P = 2.5 x 10(-10), 6.0 x 10(-5), and 2.9 x 10(-3)). Our study provides evidence that the same genes (e.g., MC4R, FIBIN, and FMO5) harbor both common and rare variants affecting body size and that anthropometric traits share genetic loci with developmental and psychiatric disorders.Peer reviewe

Effect of bacterial distribution and activity on conjugal gene transfer on the phylloplane of bush bean (Phaseolus vulgaris). Appl Environ Microbiol 64

2 , corresponding to about one-third of the recipient population. At 25% RH, numbers of transconjugants were below the detection limit. Immediately after inoculation onto the leaves, the per-cell metabolic activity of the inocula increased by up to eight times (100% RH), followed by a decrease to the initial level after 96 h. The metabolic activity of the bacteria was not rate limiting for conjugation, and no correlation between the two parameters was observed. Apparently, leaf exudates insured that the activity of the bacteria was above a threshold value for transfer to occur. Transconjugants were primarily observed in junctures between epidermal cells and in substomatal cavities. The distribution of the transconjugants was similar to the distribution of indigenous bacteria on nonsterile leaves. Compared to polycarbonate filters, with cell densities equal to the overall density on the leaves, transfer ratios on leaves were up to 30 times higher. Thus, aggregation of the bacteria into microhabitats on the phylloplane had a great stimulatory effect on transfer