On the filling factor of emitting material in the upper atmosphere of ε Eri (K2 V)

The emission measure distribution in the upper transition region and corona of e Eri is derived from observed emission-line fluxes. Theoretical emission measure distributions are calculated assuming that the radiation losses are balanced by the net conductive flux. We discuss how the area factor of the emitting regions as a function of temperature can be derived from a comparison between these emission measure distributions. It is found that the filling factor varies from ~0.2 in the mid-transition region to ~1.0 in the inner corona. The sensitivity of these results to the adopted ion fractions, the iron abundance and other parameters is discussed. The area factors found are qualitatively similar to the observed structure of the solar atmosphere, and can be used to constrain two-component models of the chromosphere. Given further observations, the method could be applied to investigate the trends in filling factors with indicators of stellar activity.

Differences in Mucosal Gene Expression in the Colon of Two Inbred Mouse Strains after Colonization with Commensal Gut Bacteria

The host genotype has been proposed to contribute to individually composed bacterial communities in the gut. To provide deeper insight into interactions between gut bacteria and host, we associated germ-free C3H and C57BL/10 mice with intestinal bacteria from a C57BL/10 donor mouse. Analysis of microbiota similarity between the animals with denaturing gradient gel electrophoresis revealed the development of a mouse strain-specific microbiota. Microarray-based gene expression analysis in the colonic mucosa identified 202 genes whose expression differed significantly by a factor of more than 2. Application of bioinformatics tools demonstrated that functional terms including signaling/secretion, lipid degradation/catabolism, guanine nucleotide/guanylate binding and immune response were significantly enriched in differentially expressed genes. We had a closer look at the 56 genes with expression differences of more than 4 and observed a higher expression in C57BL/10 mice of the genes coding for Tlr1 and Ang4 which are involved in the recognition and response to gut bacteria. A higher expression of Pla2g2a was detected in C3H mice. In addition, a number of interferon-inducible genes were higher expressed in C3H than in C57BL/10 mice including Gbp1, Mal, Oasl2, Ifi202b, Rtp4, Ly6g6c, Ifi27l2a, Usp18, Ifit1, Ifi44, and Ly6g indicating that interferons may play an essential role in microbiota regulation. However, genes coding for interferons, their receptors, factors involved in interferon expression regulation or signaling pathways were not differentially expressed between the two mouse strains. Taken together, our study confirms that the host genotype is involved in the establishment of host-specific bacterial communities in the gut. Based on expression differences after colonization with the same bacterial inoculum, we propose that Pla2g2a and interferon-dependent genes may contribute to this phenomenon.

Application of factor analysis and geostatistics to identify processes controlling the distribution and bioaccessibility of trace elements in Northern Ireland soils

Geologic and environmental factors acting over varying spatial scales can control
trace element distribution and mobility in soils. In turn, the mobility of an element in soil will affect its oral bioaccessibility. Geostatistics, kriging and principal component analysis (PCA) were used to explore factors and spatial ranges of influence over a suite of 8 element oxides, soil organic carbon (SOC), pH, and the trace elements nickel (Ni), vanadium (V) and zinc (Zn). Bioaccessibility testing was carried out previously using the Unified BARGE Method on a sub-set of 91 soil samples from the Northern Ireland Tellus1 soil archive. Initial spatial mapping of total Ni, V and Zn concentrations shows their distributions are correlated spatially with local geologic formations, and prior correlation analyses showed that statistically significant controls were exerted over trace element bioaccessibility by the 8 oxides, SOC and pH. PCA applied to the geochemistry parameters of the bioaccessibility sample set yielded three principal components accounting for 77% of cumulative variance in the data
set. Geostatistical analysis of oxide, trace element, SOC and pH distributions using 6862 sample locations also identified distinct spatial ranges of influence for these variables, concluded to arise from geologic forming processes, weathering processes, and localised soil chemistry factors. Kriging was used to conduct a spatial PCA of Ni, V and Zn distributions which identified two factors comprising the majority of distribution variance. This was spatially accounted for firstly by basalt rock types, with the second component associated with sandstone and limestone in the region. The results suggest trace element bioaccessibility and distribution is controlled by chemical and geologic processes which occur over variable spatial ranges of influence.

Tumour necrosis factor-alpha (TNF-alpha) increases nuclear factor kappaB (NFkappaB) activity in and interleukin-8 (IL-8) release from bovine mammary epithelial cells

Epithelia play important immunological roles at a variety of mucosal sites. We examined NFkappaB activity in control and TNF-alpha treated bovine mammary epithelial monolayers (BME-UV cells). A region of the bovine IL-8 (bIL-8) promoter was sequenced and a putative kappaB consensus sequence was identified bioinformatically. We used this sequence to analyse nuclear extracts for IL-8 specific NFkappaB activity. As a surrogate marker of NFkappaB activation, we investigated IL-8 release in two models. Firstly in BME-UV monolayers, IL-8 release in the presence of pro- and anti-inflammatory agents was determined by enzyme-linked immunosorbent assay (ELISA). Secondly, we measured IL-8 secretion from a novel model of intact mucosal sheets of bovine teat sinus. IL-8 release into bathing solutions was assessed following treatment with pro- and anti-inflammatory agents. TNF-alpha enhanced NFkappaB activity in bovine mammary epithelial monolayers. p65 NFkappaB homodimer was identified in both control and TNF-alpha treated cells. Novel sequencing of the bovine IL-8 promoter identified a putative kappaB consensus sequence, which specifically bound TNF-alpha inducible p50/p65 heterodimer. TNF-alpha induced primarily serosal IL-8 release in the cell culture model. Pre-treatment with anti-TNF or dexamethasone inhibited TNF-alpha induced IL-8 release. High dose interleukin-1beta (IL-1beta) induced IL-8 release, however significantly less potently than TNF-alpha. Bovine mammary mucosal tissue released high basal levels of IL-8 which were unaffected by TNF-alpha or IL-1beta but inhibited by both dexamethasone and anti-TNF. These data support a role for TNF-alpha in activation of NFkappaB and release of IL-8 from bovine mammary epithelial cells.

The Opportunistic Pathogen Propionibacterium acnes: Insights into Typing, Human Disease, Clonal Diversification and CAMP Factor Evolution

We previously described a Multilocus Sequence Typing (MLST) scheme based on eight genes that facilitates population genetic and evolutionary analysis of P. acnes. While MLST is a portable method for unambiguous typing of bacteria, it is expensive and labour intensive. Against this background, we now describe a refined version of this scheme based on two housekeeping (aroE; guaA) and two putative virulence (tly; camp2) genes (MLST) that correctly predicted the phylogroup (IA, IA, IB, IC, II, III), clonal complex (CC) and sequence type (ST) (novel or described) status for 91% isolates (n = 372) via cross-referencing of the four gene allelic profiles to the full eight gene versions available in the MLST database (http://pubmlst.org/pacnes/). Even in the small number of cases where specific STs were not completely resolved, the MLST method still correctly determined phylogroup and CC membership. Examination of nucleotide changes within all the MLST loci provides evidence that point mutations generate new alleles approximately 1.5 times as frequently as recombination; although the latter still plays an important role in the bacterium's evolution. The secreted/cell-associated 'virulence' factors tly and camp2 show no clear evidence of episodic or pervasive positive selection and have diversified at a rate similar to housekeeping loci. The co-evolution of these genes with the core genome might also indicate a role in commensal/normal existence constraining their diversity and preventing their loss from the P. acnes population. The possibility that members of the expanded CAMP factor protein family, including camp2, may have been lost from other propionibacteria, but not P. acnes, would further argue for a possible role in niche/host adaption leading to their retention within the genome. These evolutionary insights may prove important for discussions surrounding camp2 as an immunotherapy target for acne, and the effect such treatments may have on commensal lineages. © 2013 McDowell et al.

Fluid Shear Induces Conformation Change in Human Blood Protein von Willebrand Factor in Solution

Many of the physiological functions of von Willebrand Factor (VWF), including its binding interaction with blood platelets, are regulated by the magnitude of applied fluid/hydrodynamic stress. We applied two complementary strategies to study the effect of fluid forces on the solution structure of VWF. First, small-angle neutron scattering was used to measure protein conformation changes in response to laminar shear rates (G) up to 3000/s. Here, purified VWF was sheared in a quartz Couette cell and protein conformation was measured in real time over length scales from 2-140 nm. Second, changes in VWF structure up to 9600/s were quantified by measuring the binding of a fluorescent probe 1,1'-bis(anilino)-4-,4'-bis(naphtalene)-8,8'-disulfonate (bis-ANS) to hydrophobic pockets exposed in the sheared protein. Small angle neutron scattering studies, coupled with quantitative modeling, showed that VWF undergoes structural changes at G < 3000/s. These changes were most prominent at length scales <10 nm (scattering vector (q) range >0.6/nm). A mathematical model attributes these changes to the rearrangement of domain level features within the globular section of the protein. Studies with bis-ANS demonstrated marked increase in bis-ANS binding at G > 2300/s. Together, the data suggest that local rearrangements at the domain level may precede changes at larger-length scales that accompany exposure of protein hydrophobic pockets. Changes in VWF conformation reported here likely regulate protein function in response to fluid shear.

Mutations within subdomain II of the extracellular region of epidermal growth factor receptor selectively alter TGF alpha binding

The interactions of epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) with the epidermal growth factor receptor (EGFR) were examined by insertion mutagenesis of the receptor. Seventeen insertions were made throughout a construct containing only the extracellular domain. This truncated receptor (sEGFR) was secreted and had a dissociation constant similar to that of the full-length solubilized receptor. Receptors with insertions within subdomain III were not secreted. Two receptors with insertions at positions 291 and 474, which border subdomain III, have significantly decreased binding to both EGF and TGF alpha relative to wild type. This confirms previous work demonstrating that subdomain III forms the primary binding site for EGF and TGF alpha. Four of the mutants within subdomain II had a decreased binding to TGF alpha relative to wild type, but had wild type binding to EGF. These results suggest that a region within subdomain II may selectively regulate the binding of TGF alpha. Two receptors which contained insertions within subdomains II and IV, approximately equidistant from the center of subdomain III, bound twofold more ligand molecules than wild type receptor, with an affinity similar to that of wild type receptor. These findings suggest that insertion at these positions allows the access of more than one ligand molecule to the binding site.

An inherited duplication at the gene p21 Protein-Activated Kinase 7 (PAK7) is a risk factor for psychosis

Identifying rare, highly penetrant risk mutations may be an important step in dissecting the molecular etiology of schizophrenia. We conducted a gene-based analysis of large (>100kb), rare copy number variants (CNVs) in the Wellcome Trust Case Control Consortium 2 (WTCCC2) schizophrenia sample of 1,564 cases and 1,748 controls all from Ireland, and further extended the analysis to include an additional 5,196 UK controls. We found association with duplications at chr20p12.2 (P=0.007) and evidence of replication in large independent European schizophrenia (P=0.052) and UK bipolar disorder case-control cohorts (P=0.047). A combined analysis of Irish/UK subjects including additional psychosis cases (schizophrenia and bipolar disorder) identified 22 carriers in 11,707 cases and 10 carriers in 21,204 controls (meta-analysis CMH P value=2x10(-4) (odds ratio (OR)=11.3, 95% CI=3.7, ∞)). Nineteen of the 22 cases and 8 of the 10 controls carried duplications starting at 9.68Mb with similar breakpoints across samples. By haplotype analysis and sequencing we identified a tandem ∼149kb duplication overlapping the gene p21 Protein-Activated Kinase 7 (PAK7, also called PAK5) which was in linkage disequilibrium with local haplotypes (P=2.5x10(-21)), indicative of a single ancestral duplication event. We confirmed the breakpoints in 8/8 carriers tested and found co-segregation of the duplication with illness in two additional family members of one of the affected probands. We demonstrate that PAK7 is developmentally co-expressed with another known psychosis risk gene (DISC1) suggesting a potential molecular mechanism involving aberrant synapse development and plasticity.

Anthrax Lethal Factor as an Immune Target in Humans and Transgenic Mice and the Impact of HLA Polymorphism on CD4+ T Cell Immunity

Bacillus anthracis produces a binary toxin composed of protective antigen (PA) and one of two subunits, lethal factor (LF) or edema factor (EF). Most studies have concentrated on induction of toxin-specific antibodies as the correlate of protective immunity, in contrast to which understanding of cellular immunity to these toxins and its impact on infection is limited. We characterized CD4+ T cell immunity to LF in a panel of humanized HLA-DR and DQ transgenic mice and in naturally exposed patients. As the variation in antigen presentation governed by HLA polymorphism has a major impact on protective immunity to specific epitopes, we examined relative binding affinities of LF peptides to purified HLA class II molecules, identifying those regions likely to be of broad applicability to human immune studies through their ability to bind multiple alleles. Transgenics differing only in their expression of human HLA class II alleles showed a marked hierarchy of immunity to LF. Immunogenicity in HLA transgenics was primarily restricted to epitopes from domains II and IV of LF and promiscuous, dominant epitopes, common to all HLA types, were identified in domain II. The relevance of this model was further demonstrated by the fact that a number of the immunodominant epitopes identified in mice were recognized by T cells from humans previously infected with cutaneous anthrax and from vaccinated individuals. The ability of the identified epitopes to confer protective immunity was demonstrated by lethal anthrax challenge of HLA transgenic mice immunized with a peptide subunit vaccine comprising the immunodominant epitopes that we identified.

The Influence of the User in Body-Centric Antennas and Propagation at 3–6 GHz—A Rician K-Factor Approach

We investigate whether the presence of a human body in wearable communications should be considered as part of the radiating structure or as part of the local radio environment. The Rician $K$ -factor was employed as a quantitative measure of the effect of the user's body for five environments and two mounting locations. Presented empirical results indicated that the environment had a greater impact on the $K$-factor values than the position of the transmit antenna for the ultrawideband signals used, confirming that the human body should be considered primarily as part of the overall radiating system when the antenna is worn on the body. Furthermore, independent variations also existed in the $K$-factor values for the differing antenna-body mounting positions, indicating that as the position changed, then the radiating effects and the contribution from the body changed. This is significant for ensuring body-antenna systems are accurately modeled in system-level simulations.

Functional Genomic Screen Identifies Klebsiella pneumoniae Factors Implicated in Blocking Nuclear Factor κB (NF-κB) Signaling

Klebsiella pneumoniae is etiologic agent of community-acquired and nosocomial pneumonia. It has been shown that K. pneumoniae infections are characterized by reduced early inflammatory response. Recently our group have shown that K. pneumoniae dampens the activation of inflammatory responses by antagonizing the activation of the NF-κB canonical pathway. Our results revealed that K. pneumoniae capsule (CPS) was necessary but not sufficient to attenuate inflammation. To identify additional Klebsiella factors required to dampen inflammation, we standardized and applied a high-throughput gain-on-function screen to examine a Klebsiella transposon mutant library. We identified 114 mutants that triggered the activation of NF-κB. Two gene ontology categories accounted for half of the loci identified in the screening, that of metabolism and transport (32% of the mutants), and of enveloperelated genes (17%). Characterization of the mutants revealed that the lack of the enterobactin siderophore was linked to a reduced CPS expression which in turn underlined the NF- κB activation induced by the mutant. The lipopolysaccharide (LPS) O-polysaccharide and the pullulanase (PulA) type 2 secretion system (T2SS) are required for full effectiveness of immune evasion. Importantly, these factors do not play a redundant role. The fact that LPS Opolysaccharide and T2SS mutants-induced responses were dependent on TLR2-TLR4- MyD88 activation suggested that LPS Opolysaccharide and PulA perturbed TLRdependent recognition of K. pneumoniae. Finally, we demonstrate that LPS O-polysaccharide and pulA mutants are attenuated in the pneumonia mouse model. We propose that LPS Opolysaccharide and PulA T2SS could be new targets for designing new antimicrobials. Increasing TLR-governed defence responses might provide also selective alternatives for the management of K. pneumoniae pneumonia.

Comparative analysis of Klebsiella pneumoniae genomes identifies a phospholipase D family protein as a novel virulence factor

BACKGROUND: Klebsiella pneumoniae strains are pathogenic to animals and humans, in which they are both a frequent cause of nosocomial infections and a re-emerging cause of severe community-acquired infections. K. pneumoniae isolates of the capsular serotype K2 are among the most virulent. In order to identify novel putative virulence factors that may account for the severity of K2 infections, the genome sequence of the K2 reference strain Kp52.145 was determined and compared to two K1 and K2 strains of low virulence and to the reference strains MGH 78578 and NTUH-K2044.

RESULTS: In addition to diverse functions related to host colonization and virulence encoded in genomic regions common to the four strains, four genomic islands specific for Kp52.145 were identified. These regions encoded genes for the synthesis of colibactin toxin, a putative cytotoxin outer membrane protein, secretion systems, nucleases and eukaryotic-like proteins. In addition, an insertion within a type VI secretion system locus included sel1 domain containing proteins and a phospholipase D family protein (PLD1). The pld1 mutant was avirulent in a pneumonia model in mouse. The pld1 mRNA was expressed in vivo and the pld1 gene was associated with K. pneumoniae isolates from severe infections. Analysis of lipid composition of a defective E. coli strain complemented with pld1 suggests an involvement of PLD1 in cardiolipin metabolism.

CONCLUSIONS: Determination of the complete genome of the K2 reference strain identified several genomic islands comprising putative elements of pathogenicity. The role of PLD1 in pathogenesis was demonstrated for the first time and suggests that lipid metabolism is a novel virulence mechanism of K. pneumoniae.

Insulin and insulin-like growth factor signaling increases proliferation and hyperplasia of the ovarian surface epithelium and decreases follicular integrity through upregulation of the PI3-kinase pathway

BACKGROUND: The ovarian surface epithelium responds to cytokines and hormonal cues to initiate proliferation and migration following ovulation. Although insulin and IGF are potent proliferative factors for the ovarian surface epithelium and IGF is required for follicle development, increased insulin and IGF activity are correlated with at least two gynecologic conditions: polycystic ovary syndrome and epithelial ovarian cancer. Although insulin and IGF are often components of in vitro culture media, little is known about the effects that these growth factors may have on the ovarian surface epithelium morphology or how signaling in the ovarian surface may affect follicular health and development.

METHODS: Ovaries from CD1 mice were cultured in alginate hydrogels in the presence or absence of 5 μg/ml insulin or IGF-I, as well as small molecule inhibitors of IR/IGF1R, PI 3-kinase signaling, or MAPK signaling. Tissues were analyzed by immunohistochemistry for expression of cytokeratin 8 to mark the ovarian surface epithelium, Müllerian inhibiting substance to mark secondary follicles, and BrdU incorporation to assess proliferation. Changes in gene expression in the ovarian surface epithelium in response to insulin or IGF-I were analyzed by transcription array. Extracellular matrix organization was evaluated by expression and localization of collagen IV.

RESULTS: Culture of ovarian organoids with insulin or IGF-I resulted in formation of hyperplastic OSE approximately 4-6 cell layers thick with a high rate of proliferation, as well as decreased MIS expression in secondary follicles. Inhibition of the MAPK pathway restored MIS expression reduced by insulin but only partially restored normal OSE growth and morphology. Inhibition of the PI 3-kinase pathway restored MIS expression reduced by IGF-I and restored OSE growth to a single cell layer. Insulin and IGF-I altered organization of collagen IV, which was restored by inhibition of PI 3-kinase signaling.

CONCLUSIONS: While insulin and IGF are often required for propagation of primary cells, these cytokines may act as potent mitogens to disrupt cell growth, resulting in formation of hyperplastic OSE and decreased follicular integrity as measured by MIS expression and collagen deposition. This may be due partly to altered collagen IV deposition and organization in the ovary in response to insulin and IGF signaling mediated by PI 3-kinase.

On the diversity of superluminous supernovae: ejected mass as the dominant factor

We assemble a sample of 24 hydrogen-poor superluminous supernovae(SLSNe). Parameterizing the light-curve shape through rise and declinetime-scales shows that the two are highly correlated. Magnetar-poweredmodels can reproduce the correlation, with the diversity in rise anddecline rates driven by the diffusion time-scale. Circumstellarinteraction models can exhibit a similar rise-decline relation, but onlyfor a narrow range of densities, which may be problematic for thesemodels. We find that SLSNe are approximately 3.5 mag brighter and havelight curves three times broader than SNe Ibc, but that the intrinsicshapes are similar. There are a number of SLSNe with particularly broadlight curves, possibly indicating two progenitor channels, butstatistical tests do not cleanly separate two populations. The generalspectral evolution is also presented. Velocities measured from Fe II aresimilar for SLSNe and SNe Ibc, suggesting that diffusion timedifferences are dominated by mass or opacity. Flat velocity evolution inmost SLSNe suggests a dense shell of ejecta. If opacities in SLSNe aresimilar to other SNe Ibc, the average ejected mass is higher by a factor2-3. Assuming κ = 0.1 cm2 g-1, we estimate amean (median) SLSN ejecta mass of 10 M⊙ (6M⊙), with a range of 3-30 M⊙. Doubling theassumed opacity brings the masses closer to normal SNe Ibc, but with ahigh-mass tail. The most probable mechanism for generating SLSNe seemsto be the core collapse of a very massive hydrogen-poor star, forming amillisecond magnetar.

Activated protein C inhibits tumor necrosis factor and macrophage migration inhibitory factor production in monocytes

The precise regulatory mechanisms of amplification and downregulation of the pro- and anti-inflammatory cytokines in the inflammatory response have not been fully delineated. Although activated protein C (APC) and its precursor protein C (PC) have recently been reported to be promising therapeutic agents in the management of meningococcal sepsis, direct evidence for the anti-inflammatory effect remains scarce. We report that APC inhibits in vitro the release of tumor necrosis factor (TNF) and macrophage migration inhibitory factor (MIF), two known cytokine mediators of bacterial septic shock, from lipopolysaccharide (LPS)-stimulated human monocytes. The THP-1 monocytic cell line, when stimulated with LPS and concomitant APC, exhibited a marked reduction in the release of TNF and MIF protein in a concentration-dependent manner compared to cells stimulated with LPS alone. This effect was observed only when incubations were performed in serum-free media, but not in the presence of 1-10% serum. Serum-mediated inhibition could only be overcome by increasing APC concentrations to far beyond physiological levels, suggesting the presence of endogenous serum-derived APC inhibitors. Inhibition of MIF release by APC was found to be independent of TNF, as stimulation of MIF release by LPS was unaltered in the presence of anti-TNF antibodies. Our data confirm that the suggested anti-inflammatory properties of APC are due to direct inhibition of the release of the pro-inflammatory monokine TNF, and imply that the anti-inflammatory action of APC is also mediated via inhibition of MIF release.