Development of primary invasive pneumococcal disease caused by serotype 1 pneumococci is driven by early increased type I interferon response in the lung

The pneumococcus is the world's foremost respiratory pathogen, but the mechanisms allowing this pathogen to proceed from initial asymptomatic colonization to invasive disease are poorly understood. We have examined the early stages of invasive pneumococcal disease (IPD) by comparing host transcriptional responses to an invasive strain and a noninvasive strain of serotype 1 Streptococcus pneumoniae in the mouse lung. While the two strains were present in equal numbers in the lung 6 h after intranasal challenge, only the invasive strain (strain 1861) had invaded the pleural cavity at that time point; this correlated with subsequent development of bacteremia in mice challenged with strain 1861 but not the noninvasive strain (strain 1). Progression beyond the lung was associated with stronger induction of the type I interferon (IFN-I) response in the lung at 6 h. Suppression of the IFN-I response through administration of neutralizing antibody to IFNAR1 (the receptor for type I interferons) led to significantly reduced invasion of the pleural cavity by strain 1861 at 6 h postchallenge. Our data suggest that strong induction of the IFN-I response is a key factor in early progression of invasive serotype 1 strain 1861 beyond the lung during development of IPD

Overlapping functionality of the Pht proteins in zinc homeostasis of streptococcus pneumoniae

Streptococcus pneumoniae is a globally significant pathogen that causes a range of diseases, including pneumonia, sepsis, meningitis, and otitis media. Its ability to cause disease depends upon the acquisition of nutrients from its environment, including transition metal ions such as zinc. The pneumococcus employs a number of surface proteins to achieve this, among which are four highly similar polyhistidine triad (Pht) proteins. It has previously been established that these proteins collectively aid in the delivery of zinc to the ABC transporter substrate-binding protein AdcAII. Here we have investigated the contribution of each individual Pht protein to pneumococcal zinc homeostasis by analyzing mutant strains expressing only one of the four pht genes. Under conditions of low zinc availability, each of these mutants showed superior growth and zinc accumulation profiles relative to a mutant strain lacking all four genes, indicating that any of the four Pht proteins are able to facilitate delivery of zinc to AdcAII. However, optimal growth and zinc accumulation in vitro and pneumococcal survival and proliferation in vivo required production of all four Pht proteins, indicating that, despite their overlapping functionality, the proteins are not dispensable without incurring a fitness cost. We also show that surface-attached forms of the Pht proteins are required for zinc recruitment and that they do not contribute to defense against extracellular zinc stress

Precision metering of microliter volumes of biological fluids in micro-gravity

Concepts were demonstrated and investigated for transferring accurately known and reproducible microliter volumes of biological fluids from sample container onto dry chemistry slides in microgravity environment. Specific liquid transfer tip designs were compared. Information was obtained for design of a liquid sample handling system to enable clinical chemical analysis in microgravity. Disposable pipet tips and pipet devices that were designed to transfer microliter volumes of biological fluid from a (test tube) sample container in 1-G environment were used during microgravity periods of parabolic trajectories of the KC-135 aircraft. The transfer process was recorded using charge coupled device camera and video cassette equipment. Metering behavior of water, a synthetic aqueous protein solution, and anticoagulated human blood was compared. Transfer of these liquids to 2 substrate materials representative of rapidly wettable and slowly wettable dry chemistry slide surface was compared

The first histidine triad motif of phtd is critical for zinc homeostasis in Streptococcus pneumoniae

Streptococcus pneumoniae is the world's foremost human pathogen. Acquisition of the first row transition metal ion zinc is essential for pneumococcal colonization and disease. Zinc is acquired via the ATP-binding cassette transporter AdcCB and two zinc-binding proteins, AdcA and AdcAII. We have previously shown that AdcAII is reliant upon the polyhistidine triad (Pht) proteins to aid in zinc recruitment. Pht proteins generally contain five histidine (His) triad motifs that are believed to facilitate zinc binding and therefore play a significant role in pneumococcal metal ion homeostasis. However, the importance and potential redundancy of these motifs have not been addressed. We examined the effects of mutating each of the five His triad motifs of PhtD. The combination of in vitro growth assays, active zinc uptake, and PhtD expression studies show that the His triad closest to the protein's amino terminus is the most important for zinc acquisition. Intriguingly, in vivo competitive infection studies investigating the amino- and carboxyl-terminal His triad mutants indicate that the motifs have similar importance in colonization. Collectively, our new insights into the contributions of the individual His triad motifs of PhtD, and by extension the other Pht proteins, highlight the crucial role of the first His triad site in zinc acquisition. This study also suggests that the Pht proteins likely play a role beyond zinc acquisition in pneumococcal virulence

CompGO: an R package for comparing and visualizing gene ontology enrichment differences between DNA binding experiments

Background: Gene ontology (GO) enrichment is commonly used for inferring biological meaning from systems biology experiments. However, determining differential GO and pathway enrichment between DNA-binding experiments or using the GO structure to classify experiments has received little attention. Results: Herein, we present a bioinformatics tool, CompGO, for identifying Differentially Enriched Gene Ontologies, called DiEGOs, and pathways, through the use of a z-score derivation of log odds ratios, and visualizing these differences at GO and pathway level. Through public experimental data focused on the cardiac transcription factor NKX2-5, we illustrate the problems associated with comparing GO enrichments between experiments using a simple overlap approach. Conclusions: We have developed an R/Bioconductor package, CompGO, which implements a new statistic normally used in epidemiological studies for performing comparative GO analyses and visualizing comparisons from .BED data containing genomic coordinates as well as gene lists as inputs. We justify the statistic through inclusion of experimental data and compare to the commonly used overlap method. CompGO is freely available as a R/Bioconductor package enabling easy integration into existing pipelines and is available at: http://www.bioconductor.org/packages/release/bioc/html/CompGO.html packages/release/bioc/html/CompGO.htm

On the cross-section of Dark Matter using substructure infall into galaxy clusters

We develop a statistical method to measure the interaction cross-section of Dark Matter, exploiting the continuous minor merger events in which small substructures fall into galaxy clusters. We find that by taking the ratio of the distances between the galaxies and Dark Matter, and galaxies and gas in accreting sub-halos, we form a quantity that can be statistically averaged over a large sample of systems whilst removing any inherent line-of-sight projections. In order to interpret this ratio as a cross-section of Dark Matter we derive an analytical description of sub-halo infall which encompasses; the force of the main cluster potential, the drag on a gas sub-halo, a model for Dark Matter self-interactions and the resulting sub-halo drag, the force on the gas and galaxies due to the Dark Matter sub-halo potential, and finally the buoyancy on the gas and Dark Matter. We create mock observations from cosmological simulations of structure formation and find that collisionless Dark Matter becomes physically separated from X-ray gas by up to 20h^-1 kpc. Adding realistic levels of noise, we are able to predict achievable constraints from observational data. Current archival data should be able to detect a difference in the dynamical behaviour of Dark Matter and standard model particles at 6 sigma, and measure the total interaction cross-section sigma/m with 68% confidence limits of +/- 1cm2g^-1. We note that this method is not restricted by the limited number of major merging events and is easily extended to large samples of clusters from future surveys which could potentially push statistical errors to 0.1cm^2g^-1.Comment: 14 pages, 11 figure

The pH-dependence of lipid-mediated antimicrobial peptide resistance in a model Staphylococcal plasma membrane: a two-for-one mechanism of epithelial defence circumvention.

The mechanisms of membrane defence by lysylphosphatidylglycerol (LPG), were investigated using synthetic biomimetic mono- and bilayer models of methicillin resistant S. aureus ST239 TW, based on its lipid composition in both pH 7.4 (28% LPG) and pH 5.5 (51% LPG) cultures. These models incorporated a stable synthetic analogue of LPG (3adLPG) to facilitate long-duration biophysical studies, which were previously limited by the lability native LPG. Both increased 3adLPG content and full headgroup ionization at pH 5.5, increased bilayer order and dampened overall charge, via the formation of neutral ion pairs with anionic lipids. Ion pair formation in air/liquid interface lipid monolayers elicited a significant condensing effect, which correlated with the inhibition of subphase-injected magainin 2 F5W partitioning. In fluid phase lipid vesicles, increasing the proportion of 3adLPG from 28 to 51 mol% completely inhibited the adoption of the membrane-active ?-helical conformation of the peptide, without the need for full headgroup ionization. Neutron reflectivity measurements performed on biomimetic PG/3adLPG fluid floating bilayers, showed a significant ordering effect of mild acidity on a bilayer containing 30 mol% 3adLPG, whilst peptide binding/partitioning was only fully inhibited in a bilayer with 55 mol% 3adLPG at pH 5.5. These findings are discussed with respect to the roles of LPG in resistance to human epithelial defences in S. aureus and the continued evolution of this opportunistic pathogen’s virulence

The Hlx homeobox transcription factor is required early in enteric nervous system development

BACKGROUND: Development of the enteric nervous system (ENS) requires interactions between migrating neural crest cells and the nascent gastrointestinal tract that are dependent upon genes expressed by both cell compartments. Hlx, a homeobox transcription factor gene that is expressed in mouse intestinal and hepatic mesenchyme, is required for normal embryonic growth of intestine and liver, and the Hlx(-/- )genotype is embryonic lethal. We hypothesized that Hlx is required for ENS development. RESULTS: Enteric neurons were identified in Hlx(+/+ )and Hlx(-/- )mouse embryos by immunostaining of embryo sections for the neural markers PGP9.5 and Phox2b, or by staining for β-galactosidase in whole-mount embryos containing the dopamine β-hydroxylase-nLacZ transgene. In Hlx(+/+ )embryos, neural crest cells/enteric neurons have moved from the stomach into the intestine by E10.5. By contrast, neural crest cells/enteric neurons remain largely restricted to the lateral stomach mesenchyme of Hlx(-/- )embryos, with only a few scattered neural crest cells/enteric neurons in the intestine between E10.5–16.5. CONCLUSION: The Hlx homeobox transcription factor is required for early aspects of ENS development