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In order to promote infection, the blood-borne parasite Trypanosoma brucei releases factors that upregulate arginase expression and activity in myeloid cells.
Methodology/Principal findings
By screening a cDNA library of T. brucei with an antibody neutralizing the arginase-inducing activity of parasite released factors, we identified a Kinesin Heavy Chain isoform, termed TbKHC1, as responsible for this effect. Following interaction with mouse myeloid cells, natural or recombinant TbKHC1 triggered SIGN-R1 receptor-dependent induction of IL-10 production, resulting in arginase-1 activation concomitant with reduction of nitric oxide (NO) synthase activity. This TbKHC1 activity was IL-4Rα-independent and did not mirror M2 activation of myeloid cells. As compared to wild-type T. brucei, infection by TbKHC1 KO parasites was characterized by strongly reduced parasitaemia and prolonged host survival time. By treating infected mice with ornithine or with NO synthase inhibitor, we observed that during the first wave of parasitaemia the parasite growth-promoting effect of TbKHC1-mediated arginase activation resulted more from increased polyamine production than from reduction of NO synthesis. In late stage infection, TbKHC1-mediated reduction of NO synthesis appeared to contribute to liver damage linked to shortening of host survival time.
A kinesin heavy chain released by T. brucei induces IL-10 and arginase-1 through SIGN-R1 signaling in myeloid cells, which promotes early trypanosome growth and favors parasite settlement in the host. Moreover, in the late stage of infection, the inhibition of NO synthesis by TbKHC1 contributes to liver pathogenicity.
Author Summary
From the first invasive step onwards, African trypanosomes can efficiently undermine the protective immune response of their mammalian host to favor their survival within the host and successful transmission by its vector, the tsetse fly. Identifying the parasite factors affecting the protective immune response is thus critical to detail the immune evasion mechanisms of these organisms. We report here that during acute infection, a Trypanosoma brucei protein named Kinesin Heavy Chain 1 (TbKHC1) sustains the development of the first (most prominent) peak of parasitaemia in the blood and its control by the host. Mechanistically, TbKHC1 was found to interact with the SIGN-R1 molecule at the surface of immune cells. Hereby, TbKHC1 modulates arginine/NO metabolism in immune cells towards the production by the host of nutrients (polyamines) required for parasite growth via an IL-10-dependent induction of arginase 1 and down-regulation of iNOS activities. Consequently, IL-10/arginase 1 producing immune cells are impaired in their capacity to destroy the parasite, favouring parasite settlement. Moreover, in the late stage of infection, the inhibition of NO synthesis by TbKHC1 increases liver pathogenicity that contributes to compromised host survival. Thus, targeting TbKHC1 may benefit the host protective immunity against T. brucei parasite.
Citation: De Muylder G, Daulouède S, Lecordier L, Uzureau P, Morias Y, Van Den Abbeele J, et al. (2013) A Trypanosoma brucei Kinesin Heavy Chain Promotes Parasite Growth by Triggering Host Arginase Activity. PLoS Pathog 9(10): e1003731. doi:10.1371/journal.ppat.1003731
Editor: L. David Sibley, Washington University School of Medicine, United States of America
Received: January 28, 2013; Accepted: September 11, 2013; Published: October 31, 2013
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: The work was supported by the Belgian National Fund for Scientific Research (FRSM, http://www2.frs-fnrs.be/), the J. Brachet Fund (http://www.ulb.ac.be/brachet/actions.htm), the Interuniversity Attraction Poles Programme – Belgian Science Policy (http://www.belspo.be) and the Conseil Régional d'Aquitaine (France, www.aquitaine.fr/institution/conseil-regional/). GDM is Research Fellow at the FRIA (http://www2.frs-fnrs.be/). YM is funded by the IWT-Vlaanderen (www.iwt.be/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The protozoan flagellate parasite Trypanosoma brucei is responsible for the diseases human sleeping sickness and nagana in cattle. In experimental murine models, the host immune response to this blood-borne pathogen involves antibody production against the Variant Surface Glycoprotein (VSG), as well as interferon-γ (IFN-γ)-mediated activation of macrophages/myeloid cells into cells of the M1 phenotype. These engulf opsonized parasites and synthesize factors that interfere with trypanosome growth including tumor necrosis factor-α (TNF). However, uncontrolled IFN-γ-induced immune responses including TNF and NO production as the infection persists induce tissue pathogenicity and death of the host [1]–[5]. Induction of IL-10 can attenuate the IFN-γ/M1 response and hereby enables prolonged survival of T. brucei-infected mice [6]–[8].
Only few T. brucei-derived immunomodulatory factors have been identified. These include the glycosylphosphatidylinositol anchor of VSG, CpG oligodeoxynucleotides and the trypanosome suppressive immunomodulating factor (TSIF) which have been found to induce TNF- and NO-secreting M1 cells [9]–[12]. Factors inducing M1 cells favor the control of parasite development in the early stage of infection, but their continuous release in the late stage of infection can sustain inflammation responsible for tissue pathogenicity. Components found in the culture medium of T. brucei have also been shown to affect immune cells of the host. In particular, factors released by the parasite promote the degradation of L-arginine through increase of arginase activity in macrophages/myeloid cells, and antagonize NO synthases (NOS)-mediated conversion of L-arginine into NO in infected mice. Arginase induction appears to attenuate the innate response at the early stage of infection, and likely contributes to the synthesis of polyamines and the trypanosome anti-oxidant trypanothione, known to promote trypanosome growth and colonization of the host [5], [13]–[15]. We have identified TbKHC1, a kinesin heavy chain isoform, as a factor released by T. brucei to trigger host arginase-1 activity for promotion of its own growth in the host. Although arginase-1 expression is commonly associated with alternatively activated myeloid cell (M2) functions, TbKHC1-induced arginase-1 was independent of IL-4Rα signaling but relied on a SIGN-R1 receptor-dependent IL-10 secretion.
A T. brucei kinesin heavy chain induces arginase activity in myeloid cells
T. brucei parasites were found to induce arginase activity in myeloid cells from non-infected mice (Fig. 1A). This induction was maintained when myeloid cells and trypanosomes were separated by a cell-retaining insert, indicating that soluble components from trypanosomes were involved (Fig. 1A). Parasite-released factors (PRF) were prepared under conditions leading to no detectable trypanosome death. PRF induced arginase activity, and this effect was abolished by heat-treatment (Fig. 1A). Monoclonal antibodies raised against T. brucei PRF (2C12) inhibited arginase activity induced by PRF, whereas other antibodies from the same hybridoma fusion (2A3, 2C6, 4B5) or of irrelevant specificity (5F6, 4J5) had no effect (Fig. 1B). The PRF fraction eluted after binding to a 2C12 antibody-based affinity column retained full arginase-inducing activity, confirming that this activity was directly targeted by 2C12 (Fig. 1C).
Arginase activity was determined in myeloid cells from non-infected mice incubated with (A) WT parasites separated or not by an insert, or WT parasite-released factors (PRF), (B) PRF from WT parasites in presence of indicated antibodies and (C) PRF from WT or TbKHC1 KO parasites, or material purified on anti-PRF antibody affinity column, in presence of indicated antibodies. Heat treatment was at 45°C for 30 min. Data are means ± SEM of 3–4 individual mice of one representative from 3 independent experiments (A, B), or of triplicate cultures of one representative from 2 independent experiments (C). * p<0.05 compared to non-stimulated (-) cells.
The 2C12 antibody was used to screen a cDNA expression library and identify the T. brucei arginase-inducing protein. Among the 6 positive clones identified, 5 encoded fragments of the same putative kinesin heavy chain isoform (geneDB accession number Tb927.6.4390), which we termed TbKHC1 for T. brucei kinesin heavy chain 1 (Fig. S1in Text S1). The protein N-terminal domain contained the highly conserved kinesin motor domain with a typical ATP binding site. The region targeted by 2C12 antibody located downstream from the motor domain and was characterized by a predicted coiled structure (Fig. S1 in Text S1).
TbKHC1 knock-out (KO) parasites were generated from wild-type (WT) T. brucei. In contrast to PRF from WT parasites, PRF from TbKHC1 KO parasites did not trigger arginase activity in myeloid cells from non-infected mice (Fig. 1C).
TbKHC1 partially localizes in the endocytic compartment and is released from the parasite
Pleomorphic trypanosomes, differentiating in the bloodstream from proliferative slender forms into quiescent stumpy forms, can induce long-standing infection in mammals and perform cyclical transmission in tsetse flies while differentiating into procyclic forms. In contrast, monomorphic trypanosomes, resulting from prolonged cultivation in vitro, are unable to develop long-lasting infection and cyclical transmission. The TbKHC1 gene was found to be expressed preferentially in slender bloodstream forms (Fig. 2A,B). Parasite immunolabeling with both monoclonal 2C12 and polyclonal antibodies generated against recombinant TbKHC1 (rTbKHC1) vanished in TbKHC1 KO parasites or RNAi-mediated TbKHC1 knock-down (KD) parasites, while it increased in trypanosomes over-expressing TbKHC1 (Fig. 2A–D), confirming that TbKHC1 is the genuine target of the anti-PRF 2C12 antibody.
(A) Detection of TbKHC1 transcripts by Northern blotting of RNA from procyclic forms (PF), bloodstream (BF) slender (SL) and stumpy (ST) forms, BF TbKHC1 knock-out (KO), overexpressor (OV) and TbKHC1 knock-down (RNAi) trypanosomes (d: days after doxycyclin induction; ds RNA: double-stranded RNA; histone H2B mRNA: loading control). (B) Detection of TbKHC1 with anti-rTbKHC1 polyclonal antibody in whole extracts from the indicated trypanosome forms (Tub: Tubulin loading control). (C) Parasite staining with anti-PRF monoclonal 2C12 antibody. Nucleus (N) and kinetoplast (K) are stained in blue with 4′,6-Diamidino-2-phenylindole (DAPI). (D) Localization of TbKHC1 on WT, KO and OV trypanosomes with anti-rTbKHC1 polyclonal antibody. (E) Immunoprecipitation of TbKHC1, actin, enolase and PDI-2 from 35S-metabolically labelled WT parasite total extracts (1) or supernatants (2).
Both antibodies revealed a scattered protein distribution primarily between the kinetoplast and the nucleus (Fig. 2C,D). TbKHC1 partially co-localized with tomato lectin, which detects linear chains of poly-N-acetyllactosamine typical of the endocytic compartment [16] (Fig. S2A in Text S1). However, TbKHC1 did not bind to tomato lectin in affinity chromatography assay (Fig. S2B in Text S1), suggesting that TbKHC1 is not an endocytic component. Moreover, a fraction of TbKHC1 was reproducibly found in the culture medium of parasites, as revealed by immunoprecipitation of detergent-free supernatants from metabolically 35S-labelled T. brucei (Fig. 2E). In these experiments, trypanosome lysis was unlikely as the supernatants did not contain any evidence of the cytoplasmic markers actin [17] or protein disulfide isomerase 2 [18], but did contain enolase, a protein released from trypanosome-related parasites such as Leishmania [19] (Fig. 2E). TbKHC1 immunoprecipitated from the trypanosome supernatant did not associate with other 35S-labelled proteins (Fig. 2E), as could have been expected in case of release as a complex.
IL-10 and mannose regulate TbKHC1-induced arginase activity
We screened for pathways altering the levels of PRF-induced arginase activity in myeloid cells. Co-incident with the induction of arginase activity, PRF induced myeloid cells to express the regulatory cytokine IL-10 (Fig. 3A). The arginase activity induced by PRF was inhibited by neutralizing anti-IL-10 antibody and by D-mannose, but not by D-galactose (Fig. 3B).
Myeloid cells from non-infected WT (A-D,F, G) or IL-4Rα KO (E) mice were incubated with PRF or rTbKHC1. (A) IL-10 production induced by PRF. (B) Arginase activity induced by PRF incubated with indicated antibodies or sugars. (C) Arginase activity and IL-10 secretion induced by rTbKHC1 incubated with indicated antibodies or sugars. (D–G) Relative expression level of M2 genes after incubation with rTbKHC1. In F, M2 IL-10-dependent genes are indicated, and in G, rTbKHC1 was incubated with the indicated antibodies. Data are means ± SEM of 3 individual mice of one representative from 3 independent experiments. * p<0.05 compared to non-stimulated (-) cells.
rTbKHC1 mimicked the arginase-inducing effect of PRF, including its inhibition by 2C12, anti-IL-10R antibodies and D-mannose (Fig. 3C). The increased arginase activity induced by rTbKHC1 associated with increased expression of the Arg1, but not Arg2 gene, as well as impaired expression of the iNOS gene (Fig. 3D). Activation of myeloid cells by rTbKHC1 also resulted in increased expression of the IL-10 gene (Fig. 3D) and protein (Fig. 3C), the latter being inhibited by the 2C12 antibody and D-mannose. Thus, TbKHC1 interaction with myeloid cells involved a mannose-sensitive event triggering the synthesis of IL-10, resulting in enhanced Arg1 gene expression and activity, and inhibition of iNOS expression.
Although arginase-1 expression is commonly associated with alternatively activated myeloid cell (M2) functions, rTbKHC1 did not trigger Il4 and Il13 gene expression in myeloid cells from WT mice (Fig. 3D). Moreover, the induction of Arg1 and Il10 genes by rTbKHC1 was conserved in IL-4Rα KO mice (Fig. 3E), excluding that IL-10 synergistically enhanced IL-4Rα-induced Arg1 expression as observed in M2 cells [20], [21]. Accordingly, rTbKHC1 did not trigger the expression of M2-associated genes characteristic of African trypanosome infection [22] (Fig. 3F). We also addressed whether the induction of Arg1 by rTbKHC1 required IL-6 and G-CSF in combination with IL-10 [23]. rTbKHC1 only induced Il6 gene expression (Fig. 3D). This expression was not affected following addition of anti-IL-10R antibody that inhibited the induction of arginase activity by rTbKHC1 (Fig. 3G), indicating that IL-10 induced by TbKHC1 is the main trigger of Arg1 expression in myeloid cells.
TbKHC1 promotes parasite growth in mice by increasing ornithine availability
In vitro, the parasite growth rate appeared to be identical between WT, TbKHC1 KD, TbKHC1 KO and TbKHC1-rescued parasites obtained by reinsertion of the TbKHC1 gene in one allele of its original locus in the TbKHC1 KO genome (Fig. 4A,B), indicating that TbKHC1 is not required for parasite growth. Moreover, TbKHC1 KO parasites motility or flagellum movement addressed by microscopic examination and sedimentation analysis [24] appeared normal (not shown). TbKHC1 KO parasites were able to perform the full parasite life-cycle, since procyclic transformants could cycle through tsetse flies to produce infective metacyclic forms.
(A,B) In vitro parasite growth (A: monomorphic trypanosomes, either WT or induced and non-induced TbKHC1 KD; B: pleomorphic trypanosomes, either WT or TbKHC1 KO and rescued TbKHC1 KO). Data are means ± SEM of one representative from 3 independent experiments (C,D) Parasitemia in C57Bl/6 mice infected with pleomorphic WT and TbKHC1 KO parasites (C: intraperitoneal injection; D: injection through the bite of infected tsetse flies). Data are means ± SEM of 4 individual mice of one representative from 6 independent experiments. * p<0.05 comparing TbKHC1 KO- and WT-infected mice. (E–G) TbKHC1-mediated myeloid cell activation. At day 6 p.i. with WT and TbKHC1 KO parasites, mice were treated with anti-IL-10R (anti-IL-10R Ab) or control antibody (Ctrl Ab), and at day 7 spleen myeloid cells were analyzed for arginase activity (E), spontaneous NO secretion (F) and IL-10 secretion (G). Data are means ± SEM of 3 individual mice of one representative from 3 independent experiments. ∇ p<0.05 comparing WT- or TbKHC1 KO-infected to non-infected mice; £ p<0.05 comparing WT- or TbKHC1 KO-infected mice treated with anti-IL-10R antibody to WT- or TbKHC1 KO-infected control antibody-treated mice; # p<0.05 comparing TbKHC1 KO- and WT-infected mice treated with control antibody.
In C57Bl/6 mice, the cumulative parasite load in the first peak of parasitaemia by TbKHC1 KO trypanosomes was reduced by >70% as compared to WT parasites (Fig. 4C). This reduced TbKHC1 KO parasite load was also observed under natural infection conditions in which infected tsetse flies were allowed to feed on mice (Fig. 4D). Reinsertion of TbKHC1 in TbKHC1 KO parasites reverted early parasitaemia to WT levels (Fig. S3 in Text S1).
As compared to infection with WT parasites, at day 7 post-infection (p.i.) with TbKHC1 KO parasites, spleen myeloid cells showed a reduced induction of arginase activity that coincided with increased NO2 accumulation, likely reflecting a shift towards NOS activity (Fig. 4E,F). Moreover, the induction of arginase activity was not observed in infected mice treated with a neutralizing anti-IL-10R antibody (Fig. 4E). Furthermore, mice infected with TbKHC1 KO parasites exhibited lower IL-10 serum levels than mice infected with WT parasites (Fig. 4G). The reduction of IL-10 secretion in TbKHC1 KO-infected mice could account for the higher inhibition of arginase activity observed in the presence of anti-IL-10R antibodies in these mice (Fig. 4E).
In TbKHC1 KO-infected mice, it is unlikely that the reduction of the first peak of parasitaemia resulted from increased NO production. Indeed, inhibition of NOS activity by N-(G)-nitro-L-arginine methyl ester (L-NAME) or absence of iNOS activity in iNOS KO mice did not affect TbKHC1 KO parasitaemia, while it strongly reduced WT parasitaemia (Fig. 5A,B). The lower parasitaemia in TbKHC1 KO-infected mice might rather result from reduced arginase activity, which would limit nutrient availability for the parasite. Indeed, this enzyme converts L-arginine to L-ornithine, a precursor of polyamines that are required for trypanosome growth [13]. Accordingly, in mice lacking arginase-1 in myeloid cells/macrophages following a cross between Arg1 loxP-targeted mice and LysMCre or Tie2Cre deleter mice [21], WT parasitaemia dropped to that of TbKHC1 KO parasitaemia (Fig. 5C,D). Moreover, treatment of mice with L-ornithine increased the cumulative parasite load to a greater extent in TbKHC1 KO- than in WT-infected mice (∼94% vs ∼43%, respectively; Fig. 5E). In addition, spermine levels tended to increase although without reaching statistical significance in spleen myeloid cells and blood from mice infected with WT but not TbKHC1 KO parasites (Fig. S4A in Text S1). Furthermore, an increase in L-ornithine production, coinciding with the consumption of L-arginine and the induction of N-acetylputrescine production, was observed in the supernatant of myeloid cells from non-infected mice activated in vitro with rTbKHC1 (Fig. S4B in Text S1).
WT and TbKHC1 KO parasitaemias were monitored in various mice and conditions: (A) WT mice treated with L-NAME or D-NAME; (B) iNOS KO and WT mice; (C,D) myeloid cell Arg1 KO mice (KO1 = LysMcreArg1-/lox; KO2 = Tie2creArg1-/lox) and controls (Arg1lox/lox); (E,F) WT mice treated with L-ornithine, D-mannose or D-galactose; (G) MMR KO and WT mice; (H) SIGN-R1 KO and control (Ctrl) mice. Data are means ± SEM of 4 individual mice of one representative from 3 independent experiments. * p<0.05 comparing D-NAME and L-NAME treated mice (A) or WT and iNOS KO mice (B) infected with WT parasites; ∇ p<0.05 comparing Arg1lox/lox and LysMcreArg1-/lox or Tie2creArg1-/lox mice infected with WT parasites; # p<0.05 comparing L-ornithine treated and non treated mice infected with TbKHC1 KO parasites; ¥ p<0.05 comparing D-mannose and D-galactose-treated mice infected with WT parasites; £ p<0.05 comparing SIGN-R1 KO and control mice infected with WT parasites.
The effect of TbKHC1 on parasite growth involves the myeloid cell receptor SIGN-R1
Since the in vitro induction of arginase activity by PRF and rTbKHC1 was inhibited by D-mannose (Fig. 3B,C), we treated infected mice with D-mannose. This treatment reduced WT parasitaemia to the level of TbKHC1 KO parasitaemia, but did not significantly affect TbKHC1 KO parasitaemia (Fig. 5F). To follow up on this in vivo finding and the in vitro observation that arginase activation by PRF and rTbKHC1 was inhibited by D-mannose as well as by an antibody recognizing coils of TbKHC1 (Fig. 1B, 3C), we monitored the course of WT- and TbKHC1 KO-infection in mice deficient for myeloid cell receptors able to bind both mannose and peptidic coils, namely MMR (CD206) and SIGN-R1 (CD209b), using appropriate congenic control mice [25]–[27]. In MMR KO mice, WT and TbKHC1 KO parasitaemias were not affected as compared to infection in WT mice (Fig. 5G). In SIGN-R1 KO animals the differential control of the first peak of parasitaemia between WT and TbKHC1 KO parasites vanished, due to the reduction of the WT parasite level to that of TbKHC1 KO parasites (Fig. 5H). Thus, either the absence of SIGN-R1 in infected mice or the absence of TbKHC1 in the parasite decreased T. brucei parasitaemia similarly. These data suggested that TbKHC1 interacts with the SIGN-R1 receptor. To evaluate this hypothesis, we tested the in vitro activity of rTbKHC1 on myeloid cells from SIGN-R1 KO mice. In contrast to results obtained with myeloid cells from control mice, in myeloid cells from SIGN-RI KO mice rTbKHC1 did not stimulate IL-10 and Arg1 gene expression (Fig. 6A) and did not increase arginase activity (Fig. 6B). Conversely, in myeloid cells from MMR KO mice rTbKHC1 induced expression of Il10 and Arg1 genes as in WT mouse myeloid cells (Fig. S5 in Text S1). Therefore, TbKHC1 appeared to trigger arginase activity through SIGN-R1-mediated signaling.
(A,B) Effects of rTbKHC1 on myeloid cells from non-infected control (Ctrl) or SIGN-R1 KO mice (A: relative expression of Arg1, Arg2, Il10 and iNOS genes; B: arginase activity in presence of indicated antibodies or sugars). Data are means ± SEM of 3 individual mice of one representative from 3 independent experiments. * p<0.05 compared to non-stimulated (-) cells. (C) Effects of rTbKHC1 on liver injury: microscopic analysis (hematoxylin-eosin staining, magnification 20×) of sections from WT- and TbKHC1 KO-infected mice at day 30 p.i. Anoxic infarcts (ai), periportal (>>) and lobular (>) mononuclear cell infiltrates were representative of 8 animals tested in 2 independent experiments. (D) Spontaneous NO and IL-10 secretions in spleen myeloid cell supernatants from WT- and TbKHC1 KO-infected mice treated with D-NAME or L-NAME (day 30 p.i.). (E) Idem as D in SIGN-R1 KO and control (Ctrl) mice. Data are means ± SEM of 3–4 individual mice of one representative from 3 independent experiments. ∇ p<0.05 comparing WT or TbKHC1 KO- infected mice to non-infected mice; £ p<0.05 comparing L-NAME and D-NAME treatment in WT- or TbKHC1 KO-infected mice; # p<0.05 comparing WT- and TbKHC1 KO-infected mice.
TbKHC1 increases liver pathogenicity and reduces host survival by NO-dependent mechanisms
The survival time of TbKHC1 KO-infected mice was significantly prolonged as compared to WT-infected animals (Fig. S6 in Text S1, Table 1). Death of mice infected with African trypanosomes is related to systemic inflammatory response syndrome (SIRS) that causes multiple organ failure [28]. Liver myeloid cells are critical to the clearance of trypanosomes from the bloodstream [29], hence maintaining liver integrity is fundamental to the host's ability to clear trypanosomes. At day 30 p.i. several indicators of liver damage differed between WT- and TbKHC1 KO-infected mice: (i) the serum levels of the liver damage marker alanine aminotransferase (ALT) were lower in TbKHC1 KO- than in WT-infected mice; (ii) anoxic infarcts were observed in none of the TbKHC1 KO-infected mice, but in 6 out of 8 WT-infected mice; (iii) lobular and portal mononuclear cell infiltrates were milder in all TbKHC1 KO- than in WT-infected mice (Table 1, Fig. 6C). Concomitantly, NO production was higher in TbKHC1 KO- than in WT-infected mice, while IL-10 production was not induced and similar in all infected experimental groups (Fig. 6D). A moderate increase in iNOS mRNA expression level was also observed in myeloid cells from mice infected with TbKHC1 KO parasites (4.2±1.5 vs 1.32±0.33 fold in mice infected with WT parasites, n = 3; p = 0.08). These data suggested that increased NO production protected liver integrity in TbKHC1 KO-infected mice. Accordingly, L-NAME treatment or knock-out of iNOS activity in TbKHC1 KO-infected mice reduced the survival time and increased ALT levels to those found during WT parasite infection (Table 1). L-NAME treatment or absence of iNOS activity in WT-infected mice did not affect the survival time, although it reduced the serum levels of ALT (Table 1). Therefore, a threshold of NOS activity, achieved in TbKHC1 KO- but not in WT-infected mice, might be required during T. brucei infection to protect liver integrity, thereby contributing to extended host survival. This hepatoprotective effect of NO was not due to difference in parasite load in mice infected with WT and TbKHC1 KO parasites (Fig. S7 in Text S1).
The survival times and ALT levels in WT and TbKHC1 KO-infected mice were similar in Arg1-deficient mice and control mice (Table 1). However, treatment of WT-infected mice with L-ornithine slightly increased the serum level of ALT, while in TbKHC1 KO-infected mice L-ornithine treatment reduced the survival time and increased ALT levels to those found during WT parasite infection (Table 1). Therefore, L-ornithine as a polyamine precursor favored the development of liver injury and negatively affected the survival of T. brucei-infected mice.
As in the liver, brain injury characteristic of African trypanosomiasis was also lower in TbKHC1 KO- than in WT-infected mice. Indeed, no parasites could be detected in the choroid plexus of TbKHC1 KO-infected mice, sharply contrasting with the presence of numerous parasites, associated with immune infiltrates, in the choroid plexus of WT-infected mice (Fig. S8 in Text S1).
The survival times as well as ALT, NO and IL-10 levels in WT- and TbKHC1 KO-infected mice were similar in SIGN-R1 KO and control mice (Table 1, Fig. 6E), suggesting that in late infection TbKHC1 signaling no longer operates primarily through SIGN-R1.
We report that a particular isoform of the large KHC family of T. brucei is released by the trypanosome and influences both parasite growth and host pathogenicity.
T. brucei contains 51 sequences encoding kinesin-like proteins. The T. brucei isoform described here appears to be an orphan member without predicted function [30]. That a KHC isoform could be released in the extracellular environment is not unprecedented, since a KHC-like protein has been reported as secreted from the coagulating gland in rat [31]. TbKHC1 did not appear to be contained in high molecular weight complexes released from the parasite, for instance in exosomes as occurs in T. cruzi [32] and Leishmania [33]. Our immunoprecipitation data, rather, suggested that TbKHC1 is free in the medium, but the mechanism of release remains unclear.
TbKHC1 did not appear to be essential for T. brucei cellular proliferation or differentiation.
In vitro, TbKHC1 triggered arginase activity and, in contrast to the other documented T. brucei immunomodulators VSG, DNA or TSIF [9], [10], [12], inhibited NO synthesis by myeloid cells. These effects involved a region rich in coils in the TbKHC1 C-terminal domain, since they were inhibited by an antibody specifically recognizing the coiled region of TbKHC1.
Arg1 is often recognized as a prototypic M2 myeloid cell marker induced following STAT6 activation by IL-4/IL-13/IL-4Rα signaling. However, Arg1 can be expressed by M1 cells in a STAT3-dependent pathway triggered by IL-10, IL-6 and G-CSF [21], [23]. In agreement, rTbKHC1 induced the expression of Il10 and Il6 genes in myeloid cells, and this neither involved IL-4Rα nor induced the expression of genes associated with M2 activation. Since Il6 expression was still induced in the presence of the Arg1 expression inhibitor anti-IL-10R antibody, we conclude that rTbKHC1-induced Arg1 expression primarily depended on IL-10 production.
In mice, TbKHC1 activity appeared to promote parasite growth in the early stage of infection. Reduced parasitaemia induced by TbKHC1 KO trypanosomes was associated with increased NO production, but did not directly result from this increase. Indeed, inhibition of NO production by L-NAME treatment or in iNOS KO mice did not improve TbKHC1 KO growth. Rather, in agreement with previous studies [34] L-NAME treatment or absence of iNOS activity in mice actually increased the control of WT parasitaemia. Together, these experiments suggested that the negative/braking effect of NO on the control of WT parasitaemia requires the expression/activity of TbKHC1. We propose that TbKHC1-mediated induction of host arginase activity fuels L-ornithine production and hereby the synthesis of polyamines, which are essential nutrients for growth of extracellular trypanosomes in the host. In bloodstream forms of T. brucei the arginase gene and activity are absent, but when exogenous L-ornithine is lacking in body niches these parasites can use L-arginine to produce L-ornithine through an arginase-independent mechanism [35], [36]. However, the differential growth-promoting effect of exogenous L-ornithine during infection by WT or TbKHC1 KO parasites suggests that this arginase-independent mechanism is insufficient to provide all the amines necessary for the polyamine pathway. Similarly, the related but intracellular parasite Leishmania also requires host arginase induction for optimal growth, despite the presence of an arginase gene [37]. Therefore, optimal production of L-ornithine/polyamines for T. brucei growth would rely on host arginase activity induced by TbKHC1. In accordance, in two models of mice deficient for arginase-1 in myeloid cells/macrophages, WT T. brucei growth was reduced to the level observed with TbKHC1 KO parasites.
In T. brucei-infected mice, the TbKHC1-mediated effect on parasitaemia appeared to involve the myeloid cell receptor SIGN-R1. Indeed, improved parasite growth control was observed after either removal of TbKHC1 from the parasite or removal of SIGN-R1 from the host. Moreover, rTbKHC1-mediated induction of arginase activity was lost in myeloid cells from SIGN-R1 KO mice. The involvement of SIGN-R1 in TbKHC1-mediated IL-10 and arginase production is in keeping with previous data demonstrating the anti-inflammatory role of SIGN-R1 in various diseases [38]–[40]. Although LPS and intracellular mycobacteria infection can induce Arg1 in a MyD88/IL-10 dependent manner [21], [23], we can exclude that TbKHC1 is simultaneously a ligand for SIGN-R1 and a trigger for MyD88 signaling in our extracellular infection model since the differential infection phenotype between WT and TbKHC1 KO T. brucei was conserved in Myd88 KO mice (data not shown).
D-mannose was found to decrease parasite growth during the first peak of parasitaemia by WT but not TbKHC1 KO parasites, and it inhibited rTbKHC1-mediated induction of IL-10 and arginase by myeloid cells. Similarly, the 2C12 antibody, which is specific to the coiled region of TbKHC1, inhibited IL-10 production and arginase activity triggered by rTbKHC1. Mannose-specific receptors such as SIGN-R1 and MMR are known to interact with coiled proteins, the complement protein C1q and collagen respectively [26], [27]. Therefore, it is possible that mannose binding to SIGN-R1 could affect the binding of TbKHC1 peptidic coils to a second binding site of this receptor.
After the parasite growth-promoting effect occurring during the first peak of parasitaemia, a distinct effect of TbKHC1 consisted in reducing the survival time of infected mice while contributing to liver injury. Given the similar in vitro growth properties between parasites expressing or not TbKHC1, the mechanism by which this protein influenced the host survival time is likely to be independent from differences in parasite virulence. Parasite load throughout the infection, survival and pathogenicity are independent traits in experimental and natural infection with African trypanosomes [2], [4], [6]–[8], [28]. Therefore, the survival time difference between WT- and TbKHC1 KO-infected mice is primarily due to differences in tissue damage inflicted by inflammatory immune responses, rather than differences of parasite load in the first peak of parasitaemia. NO contributes to peripheral pathogenicity induced by African trypanosomes, while IL-10 exerts counter-pathogenic activity [2], [3]. In late stages of infection, IL-10 production was unaffected by the absence of TbKHC1, while NO production (and iNOS expression) was lower when TbKHC1 was present. Moreover, the reduction of liver injury in TbKHC1 KO-infected mice was erased by inhibition of NO synthesis upon L-NAME treatment or by infecting iNOS KO mice. Thus, TbKHC1-mediated depletion of NO might contribute to the liver damage associated with WT parasite infection. Accordingly, NO plays a pivotal role in vascular tone, and decreased NO bioavailability contributes to pathogenesis in chronic infectious diseases like malaria, and in liver ischemia reperfusion injury [41], [42]. Our observation that after the first peak of parasitaemia parasite load was similar in WT- and TbKHC1 KO -infected mice, even upon inhibition of NO production, supports the notion that blood parasite load is not a major contributor to liver pathogenicity.
Besides NO, polyamines contribute to the pathogenicity of T. brucei infection [43]. Since our data suggest that induction of arginase activity by TbKHC1 could sustain the production of polyamines, these molecules could contribute to increase liver injury in WT- as well as in TbKHC1 KO-infected mice fed with L-ornithine.
In the late stage of infection, the effects of TbKHC1 did not appear to depend on SIGN-R1 any longer, since the absence of SIGN-R1 did not affect the survival time and liver injury in WT- and TbKHC1 KO-infected mice. A major difference between early and late infection is the evolution of the immune response driven by the continuous release of immunomodulatory/inflammatory factors by the parasite, such as VSG and TSIF, which induce pathogenic TNF-α and IFN-γ [1], [4], [9] while switching off the IL-10 production [7]. The evolving context of host-parasite interaction could affect TbKHC1 signaling, and indeed the myeloid cell activation state and corresponding expression of receptors is known to vary during the time course of T. brucei infection [44]. Therefore, we cannot exclude that in infected SIGN-R1 KO mice, compensation by other myeloid cell receptors influences the immune response and outcome of pathogenicity in the late stage of the disease.
In conclusion, we have identified an unexpected function for a KHC of extracellular T. brucei, which is SIGN-R1-mediated activation of myeloid cells to produce IL-10 leading to increase in arginase activity, resulting in promotion of parasite growth in the host.
Ethic statement
Experiments, maintenance and care of mice complied with guidelines of the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (CETS n°123) and were approved by the Ethical Committee for Animal Experiments of the Université Libre de Bruxelles, Brussels, Belgium (laboratory accreditation number LA2500482).
Mice, trypanosomes and infection protocol
WT C57Bl/6 mice were from Harlan, iNOS KO C57Bl/6 mice from Jackson Laboratory. MMR KO, IL4Rα KO, control Arg1lox/lox, LysMcreArg1-/lox and Tie2creArg1-/lox mice (C57BL/6 background), SIGN-R1 KO and congenic control mice (BALB/c background) were bred in-house.
T. brucei bloodstream monomorphic "single-marker" [45] and pleomorphic AnTat 1.1E (EATRO1125 strain) were used.
Mice were infected i.p. with 2,000 AnTat1.1E parasites [6]. Parasitaemia was monitored by tail blood puncture. When required, mice were injected i.p. 2 h before infection with 2 mg L-NAME, then 1 mg/ml L-NAME was added to drinking water. The less active enantiomer D-NAME was used as control. For L-ornithine treatment, mice received 2 mg i.p. 2 h before infection, and then were given 5 mg/ml in drinking water. For D-mannose or D-galactose treatment, mice received 50 mg i.p. 2 h before infection, then every 2 days. Sugar treatment caused massive inflammation and the animals were euthanized 12 days p.i. To neutralise IL-10 activity, mice were treated once with 200 µg control antibody (rat IgG1, BD Biosciences) or 1B1.3a anti-IL-10R antibody at day 6 p.i. Injections of anti-IL-10R antibody at day 2, 4 and 6 p.i. killed WT- and TbKHC1 KO- infected mice within 48 h, as reported [6].
Parasite released factors, antibodies and PRF immunoaffinity column
PRF was prepared as described [15]. Monoclonal antibodies were obtained following an immunoenzymatic screening that selected antibodies directed against PRF, followed by a test based on inhibition of arginase activity induction. rTbKHC1 C-terminal fragment was used for production of rabbit polyclonal antibodies. PRF was incubated overnight with 2C12 monoclonal antibody coupled to Sepharose 4B CNBr resin at 4°C in carbonate buffer (v/v). After loading on a column and washings, bound material was eluted with glycine-HCl (pH 3), then glycine-NaOH (pH 10), dialysed for 24 h against milliQ water, lyophilised and solubilized at 1 mg/ml in 50 mM phosphate buffer, 1 M NaCl (pH 6.2). The residual level of endotoxins was below 2 U/µg (LAL assay, Biowhittaker).
cDNA expression library screening
Approximately 105 plaques from a bloodstream T. b. gambiense LiTat1.3 cDNA expression library (lambda ZAP express, Stratagene) were screened by incubation with the mouse 2C12 monoclonal antibody. Clones targeted by an anti-mouse antibody/alkaline phosphatase conjugate were identified by incubation at pH 9.5 with the chromogenic substrate NBT/BCIP. Reactive clones were plaque purified by 2 additional rounds of screening. The positive clones were processed to obtain the circular pBK CMV plasmid version of the phage, according to the manufacturer instructions.
TbKHC1 KD and KO parasites
For TbKHC1 KD, a 500 bp fragment from the TbKHC1 open reading frame (ORF) was amplified by polymerase chain reaction (PCR) using the KD primers (Table S1 in Text S1), digested with XhoI and BamHI and ligated into the p2T7-177TiTA plasmid [46]. The vector was linearized with NotI and transfected into monomorphic bloodstream forms. Stable transformants were selected with 1 µg/ml hygromycin. Double stranded RNA production was induced by addition of 1 µg/ml doxycyclin. For TbKHC1 KO parasites, both alleles of the complete TbKHC1 ORF were replaced in procyclic forms by genes encoding resistance to neomycin and bleomycin, following transfection of these genes flanked by 75 bp sequences from the TbKHC1 UTRs. Primers used for PCR amplification of the resistance genes were KOneo and KObleo (Table S1 in Text S1). Stable transformants were selected with 15 µg/ml geneticin and 2.5 µg/ml phleomycin. Pleomorphic TbKHC1 KO parasites were obtained by cyclical transmission of TbKHC1 KO procyclic forms in tsetse flies.
Rescue of TbKHC1 KO parasites
TbKHC1 gene and UTRs were PCR-amplified (Rescue primers, Table S1 in Text S1) and cloned into pCR-XL-TOPO vector (Invitrogen). The neomycin resistance cassette was extracted from pLew114hyg5′ [45] with AflII and StuI restriction enzymes and cloned into the AflII and EcoRV digestion of the pCR-XL-TOPO-TbKHC1 clones presenting the 5′ sequence of TbKHC1 gene next to the pUC origin. pCR-XL-TOPO-TbKHC1-Neo was linearized at AvrII and transfected in vivo into TbKHC1 KO pleomorphic parasites as described [47].
TbKHC1 overexpressing trypanosomes
TbKHC1 ORF was amplified by PCR from the initial pBK-CMV plasmid clone 4 (primers OV, Table S1 in Text S1) digested with EcoRV and HindIII, and cloned in the pTSARib vector [48]. The construct was linearized with BglII before transfection.
The full length TbKHC1 and a 2,235 bp TbKHC1 fragment were amplified by PCR from the initial pBK-CMV plasmid clone 4 using primers FL and C-term (Table S1 in Text S1). The products were digested with SalI and NotI and cloned in-frame in pET 21d (Novagen). rTbKHC1 was extracted from IPTG-induced E. coli and purified on Ni-NTA agarose (Qiagen) using 60% isopropanol in the washing steps to remove endotoxins [49]. The protein was additionally purified by HPLC Superdex 200 (10/300 GL, Pharmacia) in 50 mM phosphate buffer, 1 M NaCl (pH 6.2). The residual level of endotoxins was below 2 U/µg (LAL assay, Biowhittaker).
Immunofluorescence assay
Fixed parasites (4% formaldehyde in PBS, 10 min, 20°C) spread on poly(L-lysine)-coated slides were permeabilized (0.1% Triton X-100 in Tris-buffered saline 5 min, 20°C) and incubated with 5% BSA. TbKHC1 was detected with the 2C12 monoclonal (dilution 1/200) or rabbit polyclonal (dilution 1/1000) antibodies and Alexa 488-conjugated anti-mouse or anti-rabbit IgG (dilution 1/1000).
Metabolic labelling
Washed trypanosomes were incubated 15 min at 37°C at a density of 2×107 cells/ml in IMDM methionine-free medium supplemented with 10% FCS, 0.07 mM L-arginine, 0.2 mM L-glutamine, 0.04 mM L-leucine, 0.1 µM inositol, 0.05 mM D-glucose, 30 mM Hepes, 1 mg/ml BSA, 0.5 mM adenosine and 5 µg/ml catalase. Then, a mix of 35S-labelled methionine and cysteine was added (100 µCi/ml). After 1 h labelling, cells were washed and incubated for 2 h at 37°C at a density of 2×106/ml. Trypanosomes were centrifuged and lysed in 50 mM Tris, 150 mM NaCl, 2 mM EDTA, 1% NP40, complete protease inhibitors (Roche).
Tomato lectin chromatography
Fractionation of trypanosome extracts on tomato lectin affinity column was performed as described [16].
Preparation of myeloid cells
Non elicited peritoneal cells were collected in 0.34 M sucrose. Spleen homogenates were incubated 10 min in cold 0.83% NH4Cl/0.01 M Tris-HCl, pH 7.2 to lyse erythrocytes. Peritoneal and spleen cells were washed twice in complete medium (RPMI 1640 supplemented with 10% heat-inactivated FCS, 5×10−5 M 2-mercaptoethanol, 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 0.1 mM non essential amino acids; all from Invitrogen Life Technologies) and counted by trypan blue exclusion. To prepare myeloid cells, peritoneal cells adjusted to 5×106 cells/ml in complete medium were dispensed by 1 ml in 6-well plates and spleen cells adjusted to 107 cells/ml were dispensed by 10 ml in 10-cm tissue culture dishes. After 3 h at 37°C in 5% CO2, cells were washed five times with warm complete medium to remove non adherent cells. Adherent cells were collected in cold complete medium using a scraper, washed and viability was determined by trypan blue exclusion (usually >95%). Cells (CD11b+ for >90% as determined by flow cytometry, data not shown) were suspended in complete medium at 106 cells/ml.
In vitro culture of myeloid cells
Peritoneal myeloid cells from non infected mice (5×105/ml) were cultured in 24-well plates in presence of 5×105/ml trypanosomes, 80 µg/ml PRF, 3 µg/ml eluate of anti-PRF antibody affinity column or 3 µg/ml rTbKHC1. When required, antibodies (monoclonal anti-PRF, JES5-2A5 anti-IL-10 (BD Biosciences), 1B1.3a anti-IL-10R or appropriate isotype-matched antibodies (3 µg/ml), D-mannose or D-galactose (50 mM) were added to PRF- or rTbKHC1-stimulated cells. Unless specified, gene expression, arginase activity and NO2 and IL-10 secretions were determined after 24 h of culture. Alternatively, spleen myeloid cells from infected mice (106/ml) were collected at day 7 or 30 p.i. and stored for arginase activity determination or cultured for 24 h before determining spontaneous NO2 and IL-10 production in culture supernatants.
Myeloid cell metabolite extraction, HPLC-MS analysis and data processing
Spleen myeloid cells prepared as described above were cultured in modified complete medium (2% FCS, no 2-mercaptoethanol, no antibiotic) in 24-well plates in presence of 3 µg/ml rTbKHC1. After 12 h culture, supernatants were collected, rapidly cooled to 4°C in dry ice ethanol bath and centrifuged (3 min, 4°C, 13,000 g). Protein denaturation was achieved by adding 200 µl of 4°C chloroform∶methanol∶water (1∶3∶1) to 5 µl of cell supernatants. Samples were shaken for 1 h at 4°C and centrifuged again. The supernatants were extracted and frozen at −80°C until the samples were submitted to High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS) analysis at the Scottish Metabolomics Facility using High Performance Liquid Chromatography (Dionex UltiMate 3000 RSLC system) with a ZIC-HILIC 150×4.6 mm, 5 µm columns (Merck Sequant). The mobile phase comprised 0.1% formic acid in water (phase A) and 0.08% formic acid in acetonitrile (phase B). A linear gradient was applied to B from 80-20% over 30 minutes, followed by an 8 minute wash with 5% B and concluded with 8 minute equilibration with 80% B. The flow rate was 300 µL/min and 10 µL was injected into the column. The column temperature was 20°C and the autosampler temperature was 4°C.
For data processing, LC-MS raw data were processed using IDEOM software (version 17) [50] which is a composite of XCMS [51], mzMatch [52], R and Microsoft Excel packages. Firstly, XCMS detected peaks with a peak width between 10 and 100 seconds; signal to noise ratio of 5 and mass deviation between scans of 2 parts per million (ppm). MzMatch then matched the peaks between the two samples, comparing their retention times (RT) and ion densities before calculating any deviations. Peaks were identified according to their mass-to-charge ratio and RT.
Arginase activity, NO2 and IL-10 secretion, spermine levels
Arginase activity was determined as described [53]. NO2 quantification (reflecting NO production) was assayed by a Griess reaction. IL-10 was quantified with a specific sandwich ELISA (PharMingen) in accordance to the manufacturers' protocols. Spermine levels (Spermine ELISA Kit, Antibodies-online) were determined in blood serum and myeloid cell homogenates following the manufacturers' protocols.
Gene expression analysis
Quantitative real time PCR was performed as described [6]. Results of the PCR analyses were normalized against the house-keeping gene S12. Primers used are described in Table S2 in Text S1.
Microscopy, ALT levels
Brains and livers were fixed in 4% formaldehyde. Histological sections embedded in paraffin were stained with hematoxylin-eosin-saffron for microscopic evaluations. ALT was measured in serum samples using commercially available kits (Boehringer Mannheim).
Cumulative parasitaemia load, statistical analysis
GraphPad Prism 4.0 software was used to determine the cumulative parasitemia load (Area Under Curve function) and the statistical significance (Two-way ANOVA).
Supporting Information
Text S1.
Contains Table S1 (Primers used to generate genetically modified parasites) and Table S2 (Primers used for gene expression analysis in myeloid cells) as supplement to Experimental procedures. The Text S1 contains also Fig. S1 (Schematic primary structure of T. brucei TbKHC1), Fig. S2 (Localization of T. brucei TbKHC1), Fig. S3 (Characterization and growth pattern of TbKHC1-rescued T. brucei), Fig. S4 (Effect of T. brucei infection on metabolites involved in L-arginine pathway), Fig. S5 (Effect of rTbKHC1 on Arg1 and Il10 gene expression in MMR KO mice), Fig. S6 (Effect of TbKHC1 on the survival of T. brucei infected mice), Fig. S7 (Effects of absence of iNOS gene activity on T. brucei parasitaemia) and Fig. S8 (Effects of TbKHC1 on cerebral injury in T. brucei infected mice), as well as their legends.
We thank P. Poelvoorde, B. Vanhollebeke, L. Brys, M.A. Laute and P. Loi for help; G. Van Walleghem for statistical analyses; L. Martinez-Pomares (Nottingham, UK) for suggestions; M. Moser, O. Leo, C. Coquerelle (ULB, Belgium), C. Carnoy (IBL, France), K. Gull (Oxford, UK), S.M. Levitz (UMASS, USA), K. Benlagha (Paris, France), M.A. McDowell (Notre-Dame, USA), and P. Michels (Louvain, Belgium) for transgenic mice or reagents; Glasgow Polyomics, College of Medical, Veterinary and Life Sciences, University of Glasgow' for metabolomics and bioinformatics analyses.
Author Contributions
Conceived and designed the experiments: GDM AB EP PV. Performed the experiments: GDM AB SD LL PU YM JVDA GC MH PH SS PC BS MPB. Analyzed the data: GDM AB PV EP MH PH PDB LV MPB. Contributed reagents/materials/analysis tools: JVDA JLB ANJM LB TAW. Wrote the paper: AB EP GDM PV.
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- 40. Zhou Y, Kawasaki H, Hsu SC, Lee RT, Yao X, et al. (2010) Oral tolerance to food-induced systemic anaphylaxis mediated by the C-type lectin SIGNR1. Nat Med 16: 1128–1133. doi: 10.1038/nm.2201
- 41. Cabrales P, Zanini GM, Meays D, Frangos JA, Carvalho LJ (2011) Nitric oxide protection against murine cerebral malaria is associated with improved cerebral microcirculatory physiology. J Infect Dis 203: 1454–1463. doi: 10.1093/infdis/jir058
- 42. Abu-Amara M, Yang SY, Seifalian A, Davidson B, Fuller B (2012) The nitric oxide pathway–evidence and mechanisms for protection against liver ischaemia reperfusion injury. Liver Int 32: 531–543. doi: 10.1111/j.1478-3231.2012.02755.x
- 43. Nishimura K, Yanase T, Araki N, Ohnishi Y, Kozaki S, et al. (2006) Effects of polyamines on two strains of Trypanosoma brucei in infected rats and in vitro culture. J Parasitol 92: 211–217. doi: 10.1645/ge-633r.1
- 44. Grosskinsky CM, Ezekowitz RA, Berton G, Gordon S, Askonas BA (1983) Macrophage activation in murine African trypanosomiasis. Infect Immun 39: 1080–1086.
- 45. Wirtz E, Leal S, Ochatt C, Cross GA (1999) A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei. Mol Biochem Parasitol 99: 89–101. doi: 10.1016/s0166-6851(99)00002-x
- 46. Wickstead B, Ersfeld K, Gull K (2002) Targeting of a tetracycline-inducible expression system to the transcriptionally silent minichromosomes of Trypanosoma brucei. Mol Biochem Parasitol 125: 211–216. doi: 10.1016/s0166-6851(02)00238-4
- 47. Uzureau P, Felu C, De Muylder G, Pays E, Vanhamme L (2007) G418, phleomycin and hygromycin selection of recombinant Trypanosoma brucei parasites refractory to long-term in vitro culture. Mol Biochem Parasitol 154: 90–94. doi: 10.1016/j.molbiopara.2007.03.007
- 48. Xong HV, Vanhamme L, Chamekh M, Chimfwembe CE, Van Den Abbeele J, et al. (1998) A VSG expression site-associated gene confers resistance to human serum in Trypanosoma rhodesiense. Cell 95: 839–846. doi: 10.1016/s0092-8674(00)81706-7
- 49. Franken KL, Hiemstra HS, van Meijgaarden KE, Subronto Y, den Hartigh J, et al. (2000) Purification of his-tagged proteins by immobilized chelate affinity chromatography: the benefits from the use of organic solvent. Protein Expr Purif 18: 95–99. doi: 10.1006/prep.1999.1162
- 50. Creek DJ, Jankevics A, Burgess KE, Breitling R, Barrett MP (2012) IDEOM: an Excel interface for analysis of LC-MS-based metabolomics data. Bioinformatics 28: 1048–1049. doi: 10.1093/bioinformatics/bts069
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- 52. Scheltema RA, Jankevics A, Jansen RC, Swertz MA, Breitling R (2011) PeakML/mzMatch: a file format, Java library, R library, and tool-chain for mass spectrometry data analysis. Anal Chem 83: 2786–2793. doi: 10.1021/ac2000994
- 53. Corraliza IM, Campo ML, Soler G, Modolell M (1994) Determination of arginase activity in macrophages: a micromethod. J Immunol Methods 174: 231–235. doi: 10.1016/0022-1759(94)90027-2


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Body In California Cabin Is Positively Identified As Ex-Cop Christopher Dorner
The San Bernardino sheriff said today that the charred body found in a cabin at the Big Bear mountain resort was that of alleged cop killer Christopher Dorner, the AP reports.
USA Today adds that the sheriff said the remains were positively identified using dental records.
Dorner, who was fired from the Los Angeles Police Department in 2009, had threatened many current police officers in an online manifesto. He's suspected of killing four people — two of them police officers — during a crime spree that led to a massive multi-state manhunt.
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subset: commoncrawl


text:
Video Industry News Video Company News Technology Industry News Technology Company News Novelty Industry News Novelty Company News Gay Industry News Gay Company News Legal Industry News All Company News
Released: Aug 22nd, 2011
Running Time: 213 Min.
Company: Jules Jordan Video
DVD Extras: Behind the Scenes, Cumshot Recap, Still Gallery(ies)
Cast: Lexi Belle, Gracie Glam, Kristina Rose, Chanel Preston, Jana Jordan, Angelina Valentine, Skin Diamond, Sophia Santi, Asa Akira, Adriana Sephora
Genre: All-Sex
Only a woman knows how to please a woman! Rough and ready to show who's the top bitch. Playing dress-up was never this much fun. Pretty young things try a girlfriend experience. Class turns to ass in this classy ass scene. Gracie and Lexi give the term "play date" a whole new meaning.


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A teenager suffered life-threatening injuries Friday night after a man stabbed him, Amarillo Police Sgt. Dustin Ponder said.
About 10:15 p.m., police responded to the parking lot of Westminster Apartments, 2415 S. Coulter St., where they found the teen suffering from stab wounds. He was transported to a local hospital, police said.
"The way that I understood (the stabbing), it started as a road rage incident, and it progressed into a physical fight," Ponder said.
Police found the alleged stabber near Interstate 40 and South Georgia Street. He was arrested on an aggravated assault charge and booked into Potter County jail. Police and jail authorities did not identify him Saturday.
Yolanda Miller, 45, lives at the apartment complex. She said police found the teen in front of her apartment, but she wasn't sure what had happened. She thought he might have been injured while working underneath a vehicle.
Police taped off the area, she said. A woman — whom Miller thought knew the victim — was crying while police investigated.
"I was hugging another person, trying to make her feel all right, because she was just going nuts," she said.
Ponder said police would release more information at a later time.


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The 4 Slice Egg And Muffin Toaster is the easy way to make great egg-and-muffin sandwiches, poached eggs, scrambled eggs, hardboiled eggs, softboiled eggs, Eggs Benedict, Eggs Florentine and more. The toaster adjusts for lighter or darker toast, and the four wide slots accommodate bread, English muffins, croissants and bagels. The included meat tray lets you heat pre-cooked meats and bacon. Cleans up easily.
See more details
Item # 202055
The 4 Slice Egg And Muffin Toaster lets you create delicious egg-and-muffin sandwiches in minutes.
• Easy to use and clean
• Holds up to 8 eggs and 4 slices of bread
• Egg cooker timing is controlled by the amount of water placed in the heating tray — adjust water level to change the consistency of boiled or poached eggs
• Variable toast settings
• Toaster accommodates bread, English muffins and bagels
• Hard or soft boil up to 8 eggs at one time
• Poach or scramble 2 eggs at one time
• Included meat tray lets you heat up pre-cooked meats and bacon
• Instruction book includes tips and six easy recipes
• Plugs into an AC outlet
Length: 19.6
Width: 12.5
Height: 10
Weight: 8 Lbs
Our Price: $99.99
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text:
NAIROBI, Kenya - Secretary of State Colin Powell said Saturday that the United States strongly supports reconciliation of Sudan's warring factions so its people can "find a way to live in peace together in one nation."
Powell discussed the 18-year conflict in a meeting with Kenyan President Daniel arap Moi, a leader in efforts to end the strife.
At a joint news conference with Moi at Kenya's State House, Powell said President Bush soon will appoint a special envoy to Sudan.
He also attended a wreath-laying ceremony to honor victims of the 1998 bombing at the U.S. embassy in Nairobi, and met with opposition-party leaders.
Powell, making the third stop on his four-nation African tour, also urged Kenya to pass anti-corruption, anti-economics-crime and ethics reforms so the International Monetary Fund and World Bank can restore suspended loans.
He sidestepped giving a direct opinion on whether Moi, who has served as president since Aug. 22, 1978, should step down. But Powell expressed strong faith in Kenya's constitution, which limits presidents to two five-year terms.
Moi, serving his second term under that constitution, is required to step down in elections scheduled for 2002.
Moi has not disclosed his plans, and efforts have begun to write a new constitution.
Asked directly whether he would step down in 2002, Moi said, "Those who will decide the destiny of Kenya ... or other countries will be the people themselves." He criticized those who wanted to "undermine the intelligence of the African people," adding, "I don't know what the worry is."
Mwai Kibaki, leader of the official opposition party, told reporters after meeting Powell, "Our constitution is very clear: One should serve two terms and that's the end, finished."
He said Powell wanted to get an idea of the situation "as we see it in Kenya ... so he understands the problems Kenya faces, hopefully so that he may help us."
While in South Africa, Powell made a point of suggesting that Democratic leaders on the continent must know when to retire or step aside.
Powell said he and Moi spent much time discussing the situation in Sudan.
He planned to meet today with those engaged in the relief operation - but said he would not see any Sudanese leaders.
The United States is in the process of trying to develop a new approach to the humanitarian effort in Sudan.
"We have completed our policy review and the Bush administration will be starting to take action," Powell said.
"We are anxious to see reconciliation in Sudan," Powell said. "We want to see these peoples find a way to live in peace together in one nation in a system that they design."
Powell also met Saturday with some of the Kenyan survivors of the 1998 embassy bombing in Nairobi. 224 died and 5,000 were injured in the explosion, the vast majority of them Kenyans.
Powell declined comment on whether the United States should provide more than the $43 million it has already paid in compensation, noting that the issue is the subject of several pending lawsuits brought on behalf of the victims.
Moi, 76, head of the regional Intergovernmental Authority on Development, has led African efforts to end the war.
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text:
David Forrest
5 Magazines | 8,421 Articles | 821 Followers | 16 Likes | @innovationwatch | David Forrest is a Canadian writer and strategy consultant Twitter: @innovationwatch
Paralyzed Man Walks After Nose Cells Transplanted Into Spinal Cord
By Carl Engelking | October 22, 2014 12:22 pm
The same cells that give Darek Fidyka his sense of smell are also helping him walk again.
The man, who …University College London
Macy's Makes Rapid Advances In Technology
There is much to be excited about over IT at Macy's. Management recognizes that the future of the stores is dependent on customer interest and that technological innovation may create renewed interest in shopping in stores. It could be the Magic Mirror, Smart Fitting Rooms, Hointer robotic …IBM Watson
SingularityU Goes International
Announcing Our First Innovation Hub Outside of the US
In 1965, Gordon Moore, well known as co-founder of Intel, was a director of R&D at Fairchild …Innovation
How an 18-year-old from a Kolkata slum cracked the IIT exam this year — Quartz
By the time the motley group of teenagers—sons of farmers, daily labourers and shopkeepers—woke up on June 18, the tripods, microphones and television cameras had already started arriving for their annual ritual at Anand Kumar's house.
In 2002, Kumar established the Super 30 programme, a free …Kolkata
Cancer-fighting gene-editing tool to begin human trials in China
It may be banned in the UK, but DNA editing technology will begin trials on humans in China next month in the hope of curing lung cancer. The …China
The race is on to start drilling on the moon
The race for the Moon is on. Nobody is really sure exactly what resources are located on the Moon, or how vast they are, but a growing number of companies believe the Moon could hold large quantities of natural resources for extraction.
This concept of the Moon as a source of natural resources for …South Pole
Predicting the Future of Robotics
In 1994, Dante II, a 1,700 pound robot built by Carnegie Mellon and NASA, rappelled into the volcano of Mt. Spurr at the rate of one centimeter per …The Future
Walmart has a smart new ploy to reduce rampant food waste: Selling ugly produce
After decades of US lawmakers passing up opportunities to fight food waste on a national level, a concerted effort is beginning to emerge from parts of the private sector instead.
The front lines: Expiration-date labels and lumpy potatoes.
That Walmart, the world's largest retailer, is involved in …Food waste
The assimilation of robots into the workforce as peers, not replacements
One might ask why we would ever want to create robots that can do human work when we have so many people who need jobs. The goal of robotics should not be to replace humans with robots, but rather to improve productivity and safety, removing humans from harm's way and enabling them to focus on …Artificial Intelligence
It has the potential to change millions of lives - so what is the 'digital grid'?
When I was first asked to write about 'green energy' ahead of President Obama's Global Entrepreneurship Summit (GES) 2016, I was debating whether I …Environment
Six Ways Drones Are Revolutionizing Agriculture
Unmanned aerial vehicles (UAVs)—better known as drones—have been used commercially since the early 1980s. Today, however, practical applications for …Agriculture
Cheap and clean: Australian company creates hydrogen with near-zero emissions
An Australian company is using "cheap as dirt" iron ore to convert methane in natural gas into hydrogen. Importantly, their process generates near-zero emissions, as the carbon content of the gas is captured in the form of high-quality graphite.
As a clean-burning fuel, hydrogen could play a key …Energy
China seeks top-10 automation ranking by 2020: robot industry group
FRANKFURT (Reuters) - China is aiming for a top-10 ranking in automation for its industries by 2020 by putting more robots in its factories, the International Federation of Robotics (IFR) said.
China's push to modernize its manufacturing with robotics is partly a response to labor shortages and …Asia
4 Ways Your Office Is Crushing Your Creativity
Two-thirds of employees believe that they work in spaces that stifle creativity and innovation, a Gensler survey finds.
Is your office stifling your creativity? A recent survey, conducted by the global architecture firm Gensler, suggests as much. The good news: You can do something about it.
The …Human Resources
Facebook's terrifying internet drone has made its maiden voyage
Facebook wants to bring internet to the most remote parts of the world, and the first part of its plan to do so has taken flight. A massive, foreboding wing-shaped drone, nicknamed Project Aquila, recently completed its first test flight in Arizona, Facebook announced today.
Nearly a year ago, …Drones
This floating underwater tunnel in Norway could save half a day in travel time
Norwegians treasure their beloved fjords — and at 1,190 in total, there are plenty to love — but the giant bodies of water pose serious hurdles to traveling across the country, especially when roads are icy in the winter. Journeys that should take hours end up taking upwards of a day, all because …Travel
The Coal Industry Collapsed—Is Oil Next?
The major oil companies don't seem at all prepared for what's about to hit them. Investors should be worried.
Five years ago, if you had asked someone in the coal industry about the future, they would have predicted growth—and ignored a nonprofit report arguing that the industry was likely to …Energy
Macy's has launched an in-store AI assistant powered by IBM's Watson
The new assistant is called "Macy's On Call" and comes in the form of a mobile web app users can access on their smartphone. The assistant, which is powered by IBM's Watson AI, allows shoppers in specific Macy's stores to input natural language questions regarding the store's product assortment, …IBM
Plan to Bury Greenhouse Gases in North Sea May Be Europe's 'Best Hope'
Energy companies aren't sure what to do
A scheme to collect millions of tons of greenhouse gases and bury them under the North Sea off the coast of Rotterdam is Europe's best hope of showing it can make carbon capture and storage (CCS) technology work.
Rotterdam, Europe's largest port, is the home to …Global Perspectives
Google's DeepMind trains AI to cut its energy bills by 40%
The AI firm used machine learning to reduce electricity use in its data centres
Google has created artificial intelligence that's able to save the amount of electricity it uses to power its data centres.
Using machine learning developed by the firm's AI research company, DeepMind, it was possible to …Google
Design Thinking Comes of Age
There's a shift under way in large organizations, one that puts design much closer to the center of the enterprise. But the shift isn't about aesthetics. It's about applying the principles of design to the way people work.
This new approach is in large part a response to the increasing complexity of …Design thinking
2016 Is Breaking All Kinds of Climate Records, and It's Only Half Over | Motherboard
2016 is only half over, and already two big indicators of climate change (global surface temperatures, and the diminishing extent of Arctic sea ice) are smashing record after record, NASA scientists said on Tuesday in a press conference.
It's a troubling trend that's hitting home in many parts of …Climate
China's One Belt, One Road: Will it reshape global trade?
One of the biggest stories in Asian business is China's One Belt, One Road initiative, an economic and diplomatic program that could transform …Asia
3 Things That Pokémon Go Says About the Future of Technology
It's a worldwide phenomenon
It is hard to think of another app that has reached such high levels of awareness only a little over a week after its release. Pokémon Go is all the rage. All you need to do is spend any time in a public location to see herds of people walking around hunting for Pokémon. …Augmented Reality
Norway Just Banned Deforestation
Norway is the first country to adopt a ban on deforestation, a policy that will likely have global effects. The Norwegian parliament recently committed to a deforestation-free supply chain of goods coming into the country. Norway is not, however, new to putting large sums of money to stop …Deforestation
Social innovation in property taking off
The rise of B Corporations, themed community hubs, maker spaces, affordable housing funds and even showers for the homeless are some of the trends …Finance
As Pokémon Go Evolves, Businesses Are Squeezing Themselves In
Pokémon Go has taken over everything, everywhere. At least it feels like it has. People are scrambling to make the most of the mobile game's extraordinary popularity.
It's only been out a week, but Pokémon Go is making more money than a Meowth using Pay Day.
According to Fortune, as of Thursday the …Mobile
Robot factories could threaten jobs of millions of garment workers
The jobs of nearly 90% of garment and footwear workers in Cambodia and Vietnam are at risk from automated assembly lines – or "sewbots" – according to a new report from the International Labour Organisation (ILO).
There are 9 million people, mostly young women, dependent upon jobs in textiles, …Clothing
How Will Pokémon Go Affect the Future of App Development?
Pokémon Go is a bona fide phenomenon. If someone would have told me six months ago that Pokémon Go would become a breakout sensation that fundamentally changes how we use technology to interact with our environments, I would have laughed. Today, I'm starting to believe it.
Despite a handful of …Augmented Reality
Immersion is going to be immense
Pokémon; Macbeth; the Illuminati. Those may not sound like they have a lot in common, but they exemplify the three whole new forms of technology-driven entertainment that have erupted in recent years. We'll soon combine all three–and, eventually, use them to create whole new multi-faceted immersive …Augmented Reality


text:
You are here
Tomato Vermicelli Soup
|2 , homemade or any desired store-bought kind
|2 Tablespoon
|Ripe tomatoes
|3 , sliced
|Consomme/Potato cooking water
|5 Cup (80 tbs)
|1⁄4 Teaspoon
|Bay leaf
|Parsley sprigs
|1 Teaspoon
|1⁄4 Cup (4 tbs)
1) Take a pan and melt the butter.
2) Brown the onions in it by stirring it often with a wooden spoon.
3) Add tomatoes to it and cook over medium flame for 10 to 15 minutes until it gets reduced.
4) Add thyme, parsley, bay leaf and sugar to the liquid.
5) Boil the covered pan with all the ingredients for 15 to 20 minutes by placing it on medium heat.
6) Mash the ingredients with the back of a wooden spoon while forcing the liquid through a sieve.
7) Boil the resultant puree by transferring it into a saucepan.
8) Add some vermicelli along with the salt and pepper.
9) Simmer for an additional 10 minutes.
10) Serve immediately.
Recipe Summary
Rate It
Nutrition Rank
Nutrition Facts
Serving size
Calories 176 Calories from Fat 57
% Daily Value*
Total Fat 7 g10%
Saturated Fat 3.9 g19.6%
Trans Fat 0 g
Cholesterol 16.1 mg5.4%
Sodium 849.7 mg35.4%
Total Carbohydrates 20 g6.8%
Dietary Fiber 2.7 g10.9%
Sugars 6.9 g
Protein 10 g19.7%
Vitamin A 20.2% Vitamin C 32.3%
Calcium 4.4% Iron 8.1%
*Based on a 2000 Calorie diet


text:
Adam Woods is a physicist. His research interests include building software to run and build mathematical models. Adam got interested in personal finance in the great recession when it became obvious an interest was necessary.
After harassing his friends and family (and a short intervention) he took to the web where he blogs about finance, investment, politics, and economics.
Adam is currently located in Boulder, Colorado where he can generally be found hiking, biking, or running a D&D campaign. He can also be contacted at email@example.com.


text:
5 Simple but Effective SEO/Social Tactics
1 November 2011
Some great examples of simple linkbait or visibility tactics that I've seen in the past few weeks.
Search Marketers Get Alerted Most Through Twitter, Then Facebook
31 October 2011
At State of Search we are working hard on improving the site to fit the needs of the people who visit the site. So every week we ask them...
Has Google+ missed the boat?
27 October 2011
I was involved in the State of Search radio show this Tuesday night with Bas and Roy, something I have been fortunate to have been involved with on a...
Google vs Facebook: the Privacy Battle
14 October 2011
We probably don't know half of all the stuff Google and Facebook know of us. And we probably even don't want to know how much they know, because it...
Facebook pulls review functionality from pages – next steps for SMBs
10 October 2011
Last week admins for Facebook pages that were utilizing the Reviews application were warned that this functionality (along with discussions) would cease to exist from the 31st October, with Facebook taking...
When To Time Your Posts on Social Networks (infographic)
28 September 2011
If you are simply using Facebook or Twitter as a consumer you probably don't think about when you post something. It just comes to you and you do it....
Facebook Getting into the Data: Read, Listened, Watched, And Want Buttons
20 September 2011
This week Facebook has its famous F8 Conference. And the expectations are high as they always are. This year they might even be a bit higher. There are rumors...
Facebook and Google – who influences who?
30 August 2011
This is a guest post by Marcus Tober, co-founder and CTO of Searchmetrics, the world leader in search analytics software. As the company's CTO, he is responsible for advanced technology...
10 Real Ways to Get More Facebook Likes
29 August 2011
This post is a collection of real methods for driving likes. Take away at least one of the Facebook tips presented here.
Who is Giving Facebook the Most Referrals? Google!
23 August 2011
Google and Facebook have become huge competitors in the past few years. Where they started of as two completely different services on the web they now are connected like...


text:
Bypass the box office line at many theaters with guaranteed advance tickets. If something comes up, you can return or exchange them up until the posted showtime.
Earn rewards points from our partners, including AMC Stubs, Regal Crown Club and more.
Save your favorite theaters and movies to experience a customized Fandango just for you.
Get the VIP treatment, free screenings, digital downloads, discounts and more.
Zachary Gordon began his career at the tender age of nine, playing a bratty kid at a barbecue in the 2007 comedy Sex and Death 101. The California native subsequently wasted no time building up his momentum as an actor, amassing a résumé over the coming years that included roles in National Treasure: Book of Secrets, The Brothers Bloom, and Madagascar: Escape 2 Africa, as well as on shows like How...


text: Christopher Eccleston 10 adult jokes in Doctor Who you might have missed News post details 8:21 AM, 17 July 2016 Carry on Up the Tardis Meet the stars of Doctor Who before they were famous News post details 10:40 AM, 23 June 2016 From a teenage Tennant to Peter Capaldi's stint in The Beatles, here are some of our favourite clips of Doctor Who stars before they stepped aboard the TARDIS… 11 OMG moments from Doctor Who News post details 7:30 AM, 18 June 2016 Here's a gobsmacker for each year of New Who's life Christopher Eccleston drama Safe House to be re-written with a new cast and location News post details 8:22 AM, 11 June 2016 Former Doctor Who star will no longer be in the ITV drama that has been "stood down" for "confidential" reasons, say ITV sources This new Doctor Who montage perfectly sums up NuWho News post details 12:16 PM, 10 June 2016 You're bound to have a tear in your eye and a smile on your face Can you match the Doctor to their first line? News post details 11:20 AM, 01 June 2016 Are you a Whovian who pays attention? The A Word gets a second series News post details 10:00 AM, 26 May 2016 Pete Bowker's drama starring Christopher Eccleston gets a second run with two more seasons for BBC2 drama Peaky Blinders This wonderful fan video unites modern and classic Doctor Who in the most magical mash-up News post details 10:10 AM, 30 April 2016 The Doctor's story is seamlessly told through both the classic and modern tales ITV to screen Jimmy McGovern's Hillsborough this Sunday News post details 4:30 PM, 27 April 2016 In light of yesterday's inquest verdict, the broadcaster will re-show the award-winning film that tells the story of the 1989 disaster and its aftermath Which Doctor Who series has the most deaths? News post details 10:38 AM, 27 April 2016 Turns out David Tennant has a lot on his conscience


text:
Catalog 2015-2016 > College of Social Sciences, Mathematics and Education > Department of Psychology > Four-Year Degree Plan for Major in Psychology, B.S.
Catalog 2015-2016
Note that this is a sample four-year plan. There are other course sequences that will allow a student to graduate within four years as long as prerequisite courses are taken in the proper sequence. This sample plan does not guarantee course availability, and adjustments to students' plans may be necessary if they are unable to take specific courses during specific semesters. Students who are placed into lower level FYW, MAT or other prerequisite courses will need to adjust their four-year plans accordingly. Similarly, students who bring in Advanced Placement, Dual Enrollment or transfer credit for courses will need to adjust their four-year plans. A minimum of a 2.0 GPA both overall and in the major is required for graduation. In addition to major requirements, all components of the Baccalaureate Experience must be completed in order to successfully graduate.
Total Credit Hours: 124
In addition to the required courses listed above, students must take a minimum of 24 hours in psychology that includes 20 hours of 300-400 level courses (at least one course at the 400 level, but not 499). The specific courses chosen for the major must fill the emphasis requirements as described in the UT catalog. Many courses have prerequisites, which are listed in the Course Descriptions chapter of this catalog.


text:
Hello, Hello. Sign In
Please enter a valid city, state or zip code
2 reviews | 4.5 out of 5
I sent a dozen stargazers to my aunt, she reported that they were beautiful and lasted over a week. They arrived on the date they were promised. So, no complaints from me.
Flowers arrived on time in beautiful condition...tks.


text:
HOUSTON - Houston Airport System spokeswoman Marlene McClinton said that someone Wednesday reported a passenger at George Bush Intercontinental Airport as a potential breach. The passenger had been going down the escalator out of the secured area before running back up.
The checkpoints in three of five terminals were closed.
McClinton said the passenger, who was changing planes in the airport, never actually crossed into the unsecured area, which begins just after passengers exit the escalator.
Checkpoints were reopened at 5:30 p.m., and McClinton said the brief closure caused "minimal disruption to operations."
- The Associated Press
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text:
Which sex is the worst about washing up? Why is it so important? We've got the dirty truth on how and when to wash your hands.
Altitude Sickness
Lipman GS, et al. (2012). Ibuprofen prevents altitude illness: A randomized controlled trial for prevention of altitude illness with nonsteroidal anti-inflammatories. Annals of Emergency Medicine, 59(6): 484-490.
Murdoch D (2010). Altitude sickness, search date October 2009. Online version of BMJ Clinical Evidence: http://www.clinicalevidence.com.
Other Works Consulted
Hackett PH, Roach RC (2012). High-altitude medicine and physiology. In PS Auerbach, ed., Wilderness Medicine, 6th ed., pp. 2-33. Philadelphia: Mosby Elsevier.
Luks AM, et al. (2010). In R Mason et al., eds., Murray and Nadel's Textbook of Respiratory Medicine, 5th ed., vol. 2, pp. 1651-1673. Philadelphia: Saunders Elsevier.
Luks AM, et al. (2010). Wilderness Medical Society consensus guidelines for the prevention and treatment of acute altitude sickness. Wilderness and Environmental Medicine, 21(2): 146-155.
WebMD Medical Reference from Healthwise
Altitude Sickness Topics
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Patricia A. Houston, RN, MPH, MEd, coordinator of the bariatric program at Beebe Medical Center in Lewes, Del., has published an article in the January/February 2013 issue of the peer-reviewed Journal of Infusion Nursing, "Obtaining Vascular Access in the Obese Patient Population." According to a news release, the article compiles the research that has been done on safe and effective techniques when using IVs on obese patients.
"We chose Pattys article for publication because of its timeliness and unique focus," Mary Alexander, RN, MA, CRNI, CAE, FAAN, editor-in-chief of the Journal of Infusion Nursing, said in the release.
"Not only does this article address a real need for information for our readers, but Pattys research is also significant as we add to the science of the infusion nursing specialty."
According to the release, Houston became interested in the subject in 2010 after taking over the coordinators role for Beebes bariatric program.
"When I started doing research, I couldnt find comprehensive information on obtaining IV access for our patient population," Houston said in the release. "Instead, each journal article I found dealt with a single aspect of IV insertion. I decided to put all pertinent research into one article so infusion staff at any hospital can use it as a guideline to improve practice."
The several techniques Houston discusses in her article include: using ultrasound to find the vein; using longer IV devices; angling the needle differently; and measuring the arm to allow for the extra tissue in cases of obtaining peripherally inserted central lines.
"Research shows that using these specialized techniques leads to fewer needle sticks, faster time starting the IV, a reduced risk of infection and improved patient satisfaction," Houston said in the release. Anesthesiologists and vascular access team members already use these techniques at Beebe.
Houston presented her research in November 2011 at the Infusion Nurses Societys National Academy of Infusion Therapy in Los Angeles.
"Speaking to nurses from all over the country about bariatric surgery patients was a delightful experience," Houston said in the release. "They came with vast experiences from small community hospitals to large teaching facilities. I feel very proud to have the privilege of being able to represent Beebe Medical Center on the national level."
Paul Minnick, RN, MSN, NEA-BC, executive vice president and chief operating officer of Beebe Medical Center, said in the release that Houstons research reflects her commitment to patient safety and to quality care.
"I applaud Patty for her commitment to patient safety and to advanced education," Minnick said in the release. "Patient safety is our No. 1 goal at Beebe Medical Center, and it is critical that our staff members stay up-to-date with the latest nursing practices."


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Salinas city leaders said Sunday's Ciclovia bicycle event will not be a repeat of the embarrassing failure that happened at Criterium.
KIRO; Chris Harris via CNN
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Hey guys, does anyone live in Toronto?  If so whereabouts do you drive your RC cars???
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Type: Posts; User: SumoRcNoob
Hey guys, does anyone live in Toronto?  If so whereabouts do you drive your RC cars???
Ok, just checked the batteries and nothing has come loose. Any other possibilities anyone knows of?? Also my air filter was lost in the crash and the buggy is covered in dirt, including the open...
I was driving my Tornado S30 earlier today when all of a sudden I couldn't control it and it took off full throttle and crashed while still throttling at top speed. It was pretty bad. No idea why it...
im thinking about replacing the 2.67cc engine in my Redcat Lightening STR with a 3.0cc engine from my Tornado S30. Has anyone done this?  aslo what othr mods have people done to their Redcat...
oops sorry forgot to paste it! check it out
http://amazingrcstore.com/

found an RC store that is a GOLDMINE for Redcat parts & accessories today!! This store has everything!!! :)
hey guys, do u know if these tires will fit my Tornado S30 ??
http://toronto.kijiji.ca/c-buy-and-sell-hobbies-crafts-KYOSHO-tires-for-RC-models-scale-1-10-W0QQAdIdZ451852352

hey guys i have a Tornado S30, i was wondering if anyone knows if these tires would fit on my car. ?? ...
alright fair enough. I went to my local hobby shop and discussed the wheels. Getting some ordered. Also contemplating dropping a Traxxas 3.3cc engine and header into my Tornado. Im told this will...
what wheels would you suggest that are compatible with my S30?
alright thanks "Supertib"
Hey all, any approximate on how long a fully charged "HSP" glow plug igniter (1.2 volt 1800mah) will last ?
Ok so i'll pass on buying the Hop up kit. I'll look into putting the <span style="font-family: Verdana, Arial; font-size: 13px; -webkit-border-horizontal-spacing: 1px;...
alright thank you for the advice "Dads like RC too"! i have a hobby shop just up the street that has Redcat Racing parts so im in luck if i break anything. And i have already bought a few back up...
Hey all ,
im new to the RC hobby and this forum. I have just recently purchased a Tornado S30 and love it so far. what mods are worthwhile? any advic e or tips would be much appreciated thx.
...
i was also wondering,  when i change the motor if i should change the pinion?
Aalright thanks Nitro, sounds like im on the right track. Ive got to read up on this battery alternative you speak of as i dont know too much about it.

i just purchased a Sumo Rc truck and am new to this hobby. i am upgrading the bearings, 180 Motor and heat sink this week. Can anyone advise me on what is worthwhile to modify next? thanks