RNA-Seq Differentiates Tumour and Host mRNA Expression Changes Induced by Treatment of Human Tumour Xenografts with the VEGFR Tyrosine Kinase Inhibitor Cediranib.

Pre-clinical models of tumour biology often rely on propagating human tumour cells in a mouse. In order to gain insight into the alignment of these models to human disease segments or investigate the effects of different therapeutics, approaches such as PCR or array based expression profiling are often employed despite suffering from biased transcript coverage, and a requirement for specialist experimental protocols to separate tumour and host signals. Here, we describe a computational strategy to profile transcript expression in both the tumour and host compartments of pre-clinical xenograft models from the same RNA sample using RNA-Seq. Key to this strategy is a species-specific mapping approach that removes the need for manipulation of the RNA population, customised sequencing protocols, or prior knowledge of the species component ratio. The method demonstrates comparable performance to species-specific RT-qPCR and a standard microarray platform, and allowed us to quantify gene expression changes in both the tumour and host tissue following treatment with cediranib, a potent vascular endothelial growth factor receptor tyrosine kinase inhibitor, including the reduction of multiple murine transcripts associated with endothelium or vessels, and an increase in genes associated with the inflammatory response in response to cediranib. In the human compartment, we observed a robust induction of hypoxia genes and a reduction in cell cycle associated transcripts. In conclusion, the study establishes that RNA-Seq can be applied to pre-clinical models to gain deeper understanding of model characteristics and compound mechanism of action, and to identify both tumour and host biomarkers

Nasopharyngeal Colonization and Invasive Disease Are Enhanced by the Cell Wall Hydrolases LytB and LytC of Streptococcus pneumoniae

Background: Streptococcus pneumoniae is a common colonizer of the human nasopharynx and one of the major pathogens causing invasive disease worldwide. Dissection of the molecular pathways responsible for colonization, invasion, and evasion of the immune system will provide new targets for antimicrobial or vaccine therapies for this common pathogen. Methodology/Principal Findings: We have constructed mutants lacking the pneumococcal cell wall hydrolases (CWHs) LytB and LytC to investigate the role of these proteins in different phases of the pneumococcal pathogenesis. Our results show that LytB and LytC are involved in the attachment of S. pneumoniae to human nasopharyngeal cells both in vitro and in vivo. The interaction of both proteins with phagocytic cells demonstrated that LytB and LytC act in concert avoiding pneumococcal phagocytosis mediated by neutrophils and alveolar macrophages. Furthermore, C3b deposition was increased on the lytC mutant confirming that LytC is involved in complement evasion. As a result, the lytC mutant showed a reduced ability to successfully cause pneumococcal pneumonia and sepsis. Bacterial mutants lacking both LytB and LytC showed a dramatically impaired attachment to nasopharyngeal cells as well as a marked degree of attenuation in a mouse model of colonization. In addition, C3b deposition and phagocytosis was more efficient for the double lytB lytC mutant and its virulence was greatly impaired in both systemic and pulmonary models of infection. Conclusions/Significance: This study confirms that the CWHs LytB and LytC of S. pneumoniae are essential virulence factor

Step-Wise Loss of Bacterial Flagellar Torsion Confers Progressive Phagocytic Evasion

Phagocytosis of bacteria by innate immune cells is a primary method of bacterial clearance during infection. However, the mechanisms by which the host cell recognizes bacteria and consequentially initiates phagocytosis are largely unclear. Previous studies of the bacterium Pseudomonas aeruginosa have indicated that bacterial flagella and flagellar motility play an important role in colonization of the host and, importantly, that loss of flagellar motility enables phagocytic evasion. Here we use molecular, cellular, and genetic methods to provide the first formal evidence that phagocytic cells recognize bacterial motility rather than flagella and initiate phagocytosis in response to this motility. We demonstrate that deletion of genes coding for the flagellar stator complex, which results in non-swimming bacteria that retain an initial flagellar structure, confers resistance to phagocytic binding and ingestion in several species of the gamma proteobacterial group of Gram-negative bacteria, indicative of a shared strategy for phagocytic evasion. Furthermore, we show for the first time that susceptibility to phagocytosis in swimming bacteria is proportional to mot gene function and, consequently, flagellar rotation since complementary genetically- and biochemically-modulated incremental decreases in flagellar motility result in corresponding and proportional phagocytic evasion. These findings identify that phagocytic cells respond to flagellar movement, which represents a novel mechanism for non-opsonized phagocytic recognition of pathogenic bacteria

Streptococcus pneumoniae in Biofilms Are Unable to Cause Invasive Disease Due to Altered Virulence Determinant Production

It is unclear whether Streptococcus pneumoniae in biofilms are virulent and contribute to development of invasive pneumococcal disease (IPD). Using electron microscopy we confirmed the development of mature pneumococcal biofilms in a continuous-flow-through line model and determined that biofilm formation occurred in discrete stages with mature biofilms composed primarily of dead pneumococci. Challenge of mice with equal colony forming units of biofilm and planktonic pneumococci determined that biofilm bacteria were highly attenuated for invasive disease but not nasopharyngeal colonization. Biofilm pneumococci of numerous serotypes were hyper-adhesive and bound to A549 type II pneumocytes and Detroit 562 pharyngeal epithelial cells at levels 2 to 11-fold greater than planktonic counterparts. Using genomic microarrays we examined the pneumococcal transcriptome and determined that during biofilm formation S. pneumoniae down-regulated genes involved in protein synthesis, energy production, metabolism, capsular polysaccharide (CPS) production, and virulence. We confirmed these changes by measuring CPS by ELISA and immunoblotting for the toxin pneumolysin and the bacterial adhesins phosphorylcholine (ChoP), choline-binding protein A (CbpA), and Pneumococcal serine-rich repeat protein (PsrP). We conclude that biofilm pneumococci were avirulent due to reduced CPS and pneumolysin production along with increased ChoP, which is known to bind C-reactive protein and is opsonizing. Likewise, biofilm pneumococci were hyper-adhesive due to selection for the transparent phase variant, reduced CPS, and enhanced production of PsrP, CbpA, and ChoP. These studies suggest that biofilms do not directly contribute to development of IPD and may instead confer a quiescent mode of growth during colonization

Nucleotide sequence of the alpha ribosomal protein operon of Escherichia coli.

In Escherichia coli some 19 transcription units encoding the 52 ribosomal proteins are scattered throughout the genome. One of the units, the alpha operon, encodes genes for the ribosomal proteins S13, S11, S4 and L17 as well as the alpha subunit of RNA polymerase. We report here the complete 3.0 kb nucleotide sequence of the alpha operon. In addition, we have determined by S1 nuclease mapping the site of transcription termination in this operon

Interleukin-1 regulates CXCL8 release and influences disease outcome in response to streptococcus pneumoniae, defining intercellular cooperation between pulmonary epithelial cells and macrophages

The success of Streptococcus pneumoniae (the pneumococcus) as a pulmonary pathogen is related to its restriction of innate immune responses by respiratory epithelial cells. The mechanisms used to overcome this restriction are incompletely elucidated. Pulmonary chemokine expression involves complex cellular and molecular networks, involving the pulmonary epithelium, but the specific cellular interactions and the cytokines that control them are incompletely defined. We show that serotype 2 or 4 pneumococci induce only modest levels of CXCL8 expression from epithelial cell lines, even in the absence of a polysaccharide capsule. In contrast, coculture of A549 cells with the macrophage-like THP-1 cell line, differentiated with vitamin D, or monocyte-derived macrophages enhanced CXCL8 release. Supernatants from the THP-1 cell line prime A549 cells to release CXCL8 at levels similar to cocultures. Interleukin-1Ra (IL-1Ra) inhibits CXCL8 release from cocultures and reduces the activity of macrophage-conditioned media, but inhibition of tumor necrosis factor alpha (TNF-alpha) had only a minimal effect on CXCL8 release. Release of IL-1 beta but not TNF-alpha was upregulated in cocultures. IL-1 type 1 receptor knockout C57BL/6 and BALB/c mice confirmed the importance of IL-1 signaling in CXC chemokine expression and neutrophil recruitment in vivo. In fulminant disease, increased IL-1 signaling resulted in increased neutrophils in the airway and more invasive disease. These results demonstrate that IL-1 is an important component of the cellular network involving macrophages and epithelial cells, which facilitates CXC chemokine expression and aids neutrophil recruitment during pneumococcal pneumonia. They also highlight a potential clinical role for anti-IL-1 treatment to limit excessive neutrophilic inflammation in the lung

Transport of Snow by the Wind: A Comparison Between Observations in Adélie Land, Antarctica, and Simulations Made with the Regional Climate Model MAR

For the first time a simulation of blowing snow events was validated in detail using one-month long observations (January 2010) made in Adélie Land, Antarctica. A regional climate model featuring a coupled atmosphere/blowing snow/snowpack model is forced laterally by meteorological re-analyses. The vertical grid spacing was 2 m from 2 to 20 m above the surface and the horizontal grid spacing was 5 km. The simulation was validated by comparing the occurrence of blowing snow events and other meteorological parameters at two automatic weather stations. The Nash test allowed us to compute effi- ciencies of the simulation. The regional climate model simulated the observed wind speed with a positive efficiency (0.69). Wind speeds higher than 12 m s−1 were underestimated. Positive efficiency of the simulated wind speed was a prerequisite for validating the blowing snow model. Temperatures were simulated with a slightly negative efficiency (−0.16) due to overestimation of the amplitude of the diurnal cycle during one week, probably because the cloud cover was underestimated at that location during the period concerned. Snowfall events were correctly simulated by our model, as confirmed by field reports. Because observations suggested that our instrument (an acoustic sounder) tends to overestimate the blowing snow flux, data were not sufficiently accurate to allow the complete validation of snow drift val- ues. However, the simulation of blowing snow occurrence was in good agreement with the observations made during the first 20 days of January 2010, despite the fact that the blowing snow flux may be underestimated by the regional climate model during pure blowing snow events. We found that blowing snow occurs in Adélie Land only when the 30-min wind speed value at 2 m a.g.l. is >10 m s−1. The validation for the last 10 days of January 2010 was less satisfactory because of complications introduced by surface melting and refreezing