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Quantitative expression of the Candida albicans secreted aspartyl proteinase gene family in human oral and vaginal candidiasis
Naglik, Julian R.
Moyes, David
Makwana, Jagruti
Kanzaria, Priya
Tsichlaki, Elina
Weindl, Günther
Tappuni, Anwar R.
Rodgers, Catherine A.
Woodman, Alexander J.
Challacombe, Stephen J.
Schaller, Martin
Hube, Bernhard
Microbiology (Reading, England)
2008;154(Pt 11):3266-3280.
A quantitative real-time RT-PCR system was established to identify which secreted aspartyl proteinase (SAP) genes are most highly expressed and potentially contribute to Candida albicans infection of human epithelium in vitro and in vivo. C. albicans SC5314 SAP1–10 gene expression was monitored in organotypic reconstituted human epithelium (RHE) models, monolayers of oral epithelial cells, and patients with oral (n=17) or vaginal (n=17) candidiasis. SAP gene expression was also analysed in Δsap1–3, Δsap4–6, Δefg1 and Δefg1/cph1 mutants to determine whether compensatory SAP gene regulation occurs in the absence of distinct proteinase gene subfamilies. In monolayers, RHE models and patient samples SAP9 was consistently the most highly expressed gene in wild-type cells. SAP5 was the only gene significantly upregulated as infection progressed in both RHE models and was also highly expressed in patient samples. Interestingly, the SAP4–6 subfamily was generally more highly expressed in oral monolayers than in RHE models. SAP1 and SAP2 expression was largely unchanged in all model systems, and SAP3, SAP7 and SAP8 were expressed at low levels throughout. In Δsap1–3, expression was compensated for by increased expression of SAP5, and in Δsap4–6, expression was compensated for by SAP2: both were observed only in the oral RHE. Both Δsap1–3 and Δsap4–6 mutants caused RHE tissue damage comparable to the wild-type. However, addition of pepstatin A reduced tissue damage, indicating a role for the Sap family as a whole in inducing epithelial damage. With the hypha-deficient mutants, RHE tissue damage was significantly reduced in both Δefg1/cph1 and Δefg1, but SAP5 expression was only dramatically reduced in Δefg1/cph1 despite the absence of hyphal growth in both mutants. This indicates that hypha formation is the predominant cause of tissue damage, and that SAP5 expression can be hypha-independent and is not solely controlled by the Efg1 pathway but also by the Cph1 pathway. This is believed to be the first study to fully quantify SAP gene expression levels during human mucosal infections; the results suggest that SAP5 and SAP9 are the most highly expressed proteinase genes in vivo. However, the overall contribution of the Sap1–3 and Sap4–6 subfamilies individually in inducing epithelial damage in the RHE models appears to be low.
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Differential Expression of Secretory Aspartyl Proteinase Genes (SAP1-10) in Oral Candida albicans Isolates with Distinct Karyotypes
Tavanti, Arianna
Pardini, Giacomo
Campa, Daniele
Davini, Paola
Lupetti, Antonella
Senesi, Sonia
Journal of Clinical Microbiology
Two karyotypes of oral Candida albicans isolates, named b and c, constituted >80% of a collection from healthy carriers (22 b and 16 c isolates) and oral candidiasis patients who were either infected (31 b and 16 c isolates) or uninfected (13 b and 38 c isolates) with human immunodeficiency virus (HIV). The prevalence of the b and c karyotypes within HIV-positive and HIV-negative patients, respectively, who were suffering from oral candidiasis (P ≤ 0.0001) suggested that these two types possessed different virulence potentials. Since C. albicans proteinases (Saps) are virulence factors in oral candidiasis, we evaluated whether the b and c karyotypes secreted different levels of Saps and expressed different patterns of Sap-encoding genes (SAP1-10). We found that the mean value of Sap activity was significantly lower (P = 0.003) in the commensal type than in the infectious b karyotype, whereas Sap activity in the commensal c type was as high as that registered for the infectious c strains. Marked differences in SAP mRNA expression were observed in commensal strains under non-Sap-inducing conditions, with all SAP genes being expressed only by strains with the c karyotype; interestingly, none of the commensal b strains expressed SAP2. In addition, while all of the SAP1-10 genes were detectable under Sap-inducing conditions, the timing of their expression during growth differed significantly, with mRNAs of SAP1-10 genes detected at 8 and 24 h postinoculation in c and b commensal strains, respectively. This provides the first evidence that commensal oral C. albicans isolates with distinct karyotypes are characterized by different patterns of SAP1-10 gene expression and different levels of Sap secretion.
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The Secreted Aspartyl Proteinases Sap1 and Sap2 Cause Tissue Damage in an In Vitro Model of Vaginal Candidiasis Based on Reconstituted Human Vaginal Epithelium
Schaller, Martin
Bein, Matthias
Korting, Hans C.
Baur, Stefan
Hamm, Gerald
Monod, Michel
Beinhauer, Sabine
Hube, Bernhard
Infection and Immunity
Secreted aspartyl proteinases (Saps) contribute to the ability of Candida albicans to cause mucosal and disseminated infections. A model of vaginal candidiasis based on reconstituted human vaginal epithelium (RHVE) was used to study the expression and role of these C. albicans proteinases during infection and tissue damage of vaginal epithelium. Colonization of the RHVE by C. albicans SC5314 did not cause any visible epithelial damage 6 h after inoculation, although expression of SAP2, SAP9, and SAP10 was detected by reverse transcriptase PCR. However, significant epithelial damage was observed after 12 h, concomitant with the additional expression of SAP1, SAP4, and SAP5. Additional transcripts of SAP6 and SAP7 were detected at a later stage of the artificial infection (24 h). Similar SAP expression profiles were observed in three samples isolated from human patients with vaginal candidiasis. In experimental infection, secretion of antigens Sap1 to Sap6 by C. albicans was confirmed at the ultrastructural level by using polyclonal antisera raised against Sap1 to Sap6. Addition of the aspartyl proteinase inhibitors pepstatin A and the human immunodeficiency virus proteinase inhibitors ritonavir and amprenavir strongly reduced the tissue damage of the vaginal epithelia by C. albicans cells. Furthermore, SAP null mutants lacking either SAP1 or SAP2 had a drastically reduced potential to cause tissue damage even though SAP3, SAP4, and SAP7 were up-regulated in these mutants. In contrast the vaginopathic potential of mutants lacking SAP3 or SAP4 to SAP6 was not reduced compared to wild-type cells. These data provide further evidence for a crucial role of Sap1 and Sap2 in C. albicans vaginal infections.
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Candida albicans Hyphal Formation and the Expression of the Efg1-Regulated Proteinases Sap4 to Sap6 Are Required for the Invasion of Parenchymal Organs
Felk, Angelika
Kretschmar, Marianne
Albrecht, Antje
Schaller, Martin
Beinhauer, Sabine
Nichterlein, Thomas
Sanglard, Dominique
Korting, Hans C.
Schäfer, Wilhelm
Hube, Bernhard
Infection and Immunity
The ability to change between yeast and hyphal cells (dimorphism) is known to be a virulence property of the human pathogen Candida albicans. The pathogenesis of disseminated candidosis involves adhesion and penetration of hyphal cells from a colonized mucosal site to internal organs. Parenchymal organs, such as the liver and pancreas, are invaded by C. albicans wild-type hyphal cells between 4 and 24 h after intraperitoneal (i.p.) infection of mice. In contrast, a hypha-deficient mutant lacking the transcription factor Efg1 was not able to invade or damage these organs. To investigate whether this was due to the inability to undergo the dimorphic transition or due to the lack of hypha-associated factors, we investigated the role of secreted aspartic proteinases during tissue invasion and their association with the different morphologies of C. albicans. Wild-type cells expressed a distinct pattern of SAP genes during i.p. infections. Within the first 72 h after infection, SAP1, SAP2, SAP4, SAP5, SAP6, and SAP9 were the most commonly expressed proteinase genes. Sap1 to Sap3 antigens were found on yeast and hyphal cells, while Sap4 to Sap6 antigens were predominantly found on hyphal cells in close contact with host cells, in particular, eosinophilic leukocytes. Mutants lacking EFG1 had either noticeably reduced or higher expressed levels of SAP4 to SAP6 transcripts in vitro depending on the culture conditions. During infection, efg1 mutants had a strongly reduced ability to produce hyphae, which was associated with reduced levels of SAP4 to SAP6 transcripts. Mutants lacking SAP1 to SAP3 had invasive properties indistinguishable from those of wild-type cells. In contrast, a triple mutant lacking SAP4 to SAP6 showed strongly reduced invasiveness but still produced hyphal cells. When the tissue damage of liver and pancreas caused by single sap4, sap5, and sap6 and double sap4 and -6, sap5 and -6, and sap4 and -5 double mutants was compared to the damage caused by wild-type cells, all mutants which lacked functional SAP6 showed significantly reduced tissue damage. These data demonstrate that strains which produce hyphal cells but lack hypha-associated proteinases, particularly that encoded by SAP6, are less invasive. In addition, it can be concluded that the reduced virulence of hypha-deficient mutants is not only due to the inability to form hyphae but also due to modified expression of the SAP genes normally associated with the hyphal morphology.
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Limited Role of Secreted Aspartyl Proteinases Sap1 to Sap6 in Candida albicans Virulence and Host Immune Response in Murine Hematogenously Disseminated Candidiasis▿
Correia, Alexandra
Lermann, Ulrich
Teixeira, Luzia
Cerca, Filipe
Botelho, Sofia
Gil da Costa, Rui M.
Sampaio, Paula
Gärtner, Fátima
Morschhäuser, Joachim
Vilanova, Manuel
Pais, Célia
Infection and Immunity
Candida albicans secreted aspartyl proteinases (Saps) are considered virulence-associated factors. Several members of the Sap family were claimed to play a significant role in the progression of candidiasis established by the hematogenous route. This assumption was based on the observed attenuated virulence of sap-null mutant strains. However, the exclusive contribution of SAP genes to their attenuated phenotype was not unequivocally confirmed, as the Ura status of these mutant strains could also have contributed to the attenuation. In this study, we have reassessed the importance of SAP1 to SAP6 in a murine model of hematogenously disseminated candidiasis using sap-null mutant strains not affected in their URA3 gene expression and compared their virulence phenotypes with those of Ura-blaster sap mutants. The median survival time of BALB/c mice intravenously infected with a mutant strain lacking SAP1 to SAP3 was equivalent to that of mice infected with wild-type strain SC5314, while those infected with mutant strains lacking SAP5 showed slightly extended survival times. Nevertheless, no differences could be observed between the wild type and a Δsap456 mutant in their abilities to invade mouse kidneys. Likewise, a deficiency in SAP4 to SAP6 had no noticeable impact on the immune response elicited in the spleens and kidneys of C. albicans-infected mice. These results contrast with the behavior of equivalent Ura-blaster mutants, which presented a significant reduction in virulence. Our results suggest that Sap1 to Sap6 do not play a significant role in C. albicans virulence in a murine model of hematogenously disseminated candidiasis and that, in this model, Sap1 to Sap3 are not necessary for successful C. albicans infection.
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Profile of Candida albicans-Secreted Aspartic Proteinase Elicited during Vaginal Infection
Taylor, Brad N.
Staib, Peter
Binder, Ayfer
Biesemeier, Antje
Sehnal, Miriam
Röllinghoff, Martin
Morschhäuser, Joachim
Schröppel, Klaus
Infection and Immunity
Vaginal infections caused by the opportunistic yeast Candida albicans are a significant problem in women of child-bearing age. Several factors are recognized as playing a crucial role in the pathogenesis of superficial candidiasis; these factors include hyphal formation, phenotypic switching, and the expression of virulence factors, including a 10-member family of secreted aspartic proteinases. In the present investigation, we analyzed the secreted aspartic proteinase gene (SAP) expression profile of C. albicans that is elicited in the course of vaginal infection in mice and how this in vivo expression profile is associated with hyphal formation. We utilized two different genetic reporter systems that allowed us to observe SAP expression on a single-cell basis, a recombination-based in vivo expression technology and green fluorescent protein-expressing Candida reporter strains. Of the six SAP genes that were analyzed (SAP1 to SAP6), only SAP4 and SAP5 were detectably induced during infection in this model. Expression of both of these genes was associated with hyphal growth, although not all hyphal cells detectably expressed SAP4 and SAP5. SAP5 expression was induced soon after infection, whereas SAP4 was expressed at later times and in fewer cells compared with SAP5. These findings point to a link between morphogenetic development and expression of virulence genes during Candida vaginitis in mice, where host signals induce both hyphal formation and expression of SAP4 and SAP5, but temporal gene expression patterns are ultimately controlled by other factors.
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Candida albicans secreted aspartyl proteinases: isoenzyme pattern is determined by cell type, and levels are determined by environmental factors.
White, T C
Agabian, N
Journal of Bacteriology
For the pathogenic yeast Candida albicans, secreted aspartyl proteinase (Sap) activity has been correlated with virulence. A family consisting of at least eight SAP genes can be drawn upon to produce Sap enzymatic activity. In this study, the levels of Sap1, Sap2, and Sap3 isoenzymes were monitored under a variety of growth conditions for several strains, including strain WO-1, which alternates between two switch phenotypes, white (W) and opaque (O). When cultured under proteinase-inducing conditions, most strains and W cells produce Sap2, while O cells produce Sap1, Sap2, and Sap3. Both W and O cells of strain WO-1 produce Saps in enriched and defined media that do not induce Saps from other strains. The specific Sap isoenzyme that is produced is determined by the cell type, while the level of Sap production is determined by environmental factors. The levels and temporal regulation of the SAP mRNAs as determined by Northern (RNA) analysis were consistent with Sap protein levels and with previous results. S1 analysis showed that SAP6 is the predominant SAP gene transcribed during hyphal induction at neutral pH. These studies define the culture conditions which control the levels of SAP mRNAs and Sap proteins, and they indicate that both the yeast/hyphal transition and phenotypic switching can determine which of the Sap isoenzymes is produced.
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Tetracycline-Inducible Expression of Individual Secreted Aspartic Proteases in Candida albicans Allows Isoenzyme-Specific Inhibitor Screening▿
Staib, Peter
Lermann, Ulrich
Blaβ-Warmuth, Julia
Degel, Björn
Würzner, Reinhard
Monod, Michel
Schirmeister, Tanja
Morschhäuser, Joachim
Antimicrobial Agents and Chemotherapy
The yeast Candida albicans possesses a gene family that encodes secreted aspartic proteases (Saps), which are important for the virulence of this human fungal pathogen. Inhibitors of the Saps could therefore be used as novel antimycotic agents for the treatment of C. albicans infections. In the present study, we established a bioassay which allows testing of the activity of potential protease inhibitors against specific Sap isoenzymes by their ability to inhibit protease-dependent growth of C. albicans. In a medium containing bovine serum albumin (BSA) as the sole source of nitrogen, C. albicans specifically expresses the Sap2p isoenzyme, which degrades the BSA and thereby enables the fungus to grow. As the other SAP genes are not significantly expressed under these conditions, mutants lacking SAP2 are unable to utilize BSA as a nitrogen source and cannot grow in such a medium. To investigate whether forced expression of SAP genes other than SAP2 would also allow growth on BSA, we constructed a set of strains expressing each of the 10 SAP genes from a tetracycline-inducible promoter in a sap2Δ mutant background. Expression of Sap1p, Sap2p, Sap3p, Sap4p, Sap5p, Sap6p, Sap8p, and a C-terminally truncated, secreted Sap9p restored the growth of the sap2Δ mutant with different efficiencies. This set of strains was then used to test the activities of various aspartic protease inhibitors against specific Sap isoenzymes by monitoring growth on BSA in the presence of the inhibitors. While pepstatin blocked the activity of all of the Saps tested, the human immunodeficiency virus protease inhibitors ritonavir and saquinavir inhibited growth of the strains expressing Sap1p to Sap3p and Sap1p, respectively, but not that of strains expressing other Saps. Therefore, the strain set can be used to test the activity of new protease inhibitors against individual C. albicans Sap isoenzymes by their ability to block the growth of the pathogen.
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A triple deletion of the secreted aspartyl proteinase genes SAP4, SAP5, and SAP6 of Candida albicans causes attenuated virulence.
Sanglard, D
Hube, B
Monod, M
Odds, F C
Gow, N A
Infection and Immunity
Secreted aspartyl proteinases (Saps) from Candida albicans are encoded by a multigene family with at least nine members (SAP1 to SAP9) and are considered putative virulence factors important for the pathogenicity of this human pathogen. The role of Sap isoenzymes in the virulence of C. albicans has not yet been clearly established, and therefore, using recent progress in the genetics of this yeast, we have constructed a panel of isogenic yeasts, each with a disruption of one or several SAP genes. We focused on the construction of a C. albicans strain in which three related SAP genes (SAP4, SAP5, and SAP6) were disrupted. Growth of the delta sap4,5,6 triple homozygous null mutant DSY459 in complex medium was not affected, whereas, interestingly, growth in a medium containing protein as the sole nitrogen source was severely impaired compared to the growth of the wild-type parent strain SC5314. Since the presence of Sap2 is required for optimal growth on such medium, this suggests that Sap4, Sap5, or Sap6 plays an important role for the process of induction of SAP2. When guinea pigs and mice were injected intravenously with DSY459, their survival time was significantly longer than that of control animals infected with the wild-type SC5314. Attenuated virulence of DSY459 was followed by a significant reduction of yeast cells in infected organs. These data suggest that the group of Sap4, Sap5, and Sap6 isoenzymes is important for the normal progression of systemic infection by C. albicans in animals.
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Disruption of each of the secreted aspartyl proteinase genes SAP1, SAP2, and SAP3 of Candida albicans attenuates virulence.
Hube, B
Sanglard, D
Odds, F C
Hess, D
Monod, M
Schäfer, W
Brown, A J
Gow, N A
Infection and Immunity
Secreted aspartyl proteinases (Saps), encoded by a gene family with at least nine members (SAP1 to SAP9), are one of the most discussed virulence factors produced by the human pathogen Candida albicans. In order to study the role of each Sap isoenzyme in pathogenicity, we have constructed strains which harbor mutations at selected SAP genes. SAP1, SAP2, and SAP3, which are regulated differentially in vitro, were mutated by targeted gene disruption. The growth rates of all homozygous null mutants were similar to those of the isogenic wild-type parental strain (SC5314) in complex and defined media. In medium with protein as the sole source of nitrogen, sap1 and sap3 mutants grew with reduced growth rates but reached optical densities similar to those measured for SC5314. In contrast, sap2 null mutants tended to clump, grew poorly in this medium, and produced the lowest proteolytic activity. Addition of ammonium ions reversed such growth defects. These results support the view that Sap2 is the dominant isoenzyme. When sap1, sap2, and sap3 mutants were injected intravenously in guinea pigs and mice, the animals had increased survival rates compared to those of control animals infected with SC5314. However, reduction of proteolytic activity in vitro did not correlate directly with the extent of attenuation of virulence observed for all Sap-deficient mutants. These data suggest that SAP1, SAP2, and SAP3 all contribute to the overall virulence of C. albicans and presumably all play important roles during disseminated infections.
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Candida albicans Secreted Aspartyl Proteinases in Virulence and Pathogenesis
Naglik, Julian R.
Challacombe, Stephen J.
Hube, Bernhard
Microbiology and Molecular Biology Reviews
Candida albicans is the most common fungal pathogen of humans and has developed an extensive repertoire of putative virulence mechanisms that allows successful colonization and infection of the host under suitable predisposing conditions. Extracellular proteolytic activity plays a central role in Candida pathogenicity and is produced by a family of 10 secreted aspartyl proteinases (Sap proteins). Although the consequences of proteinase secretion during human infections is not precisely known, in vitro, animal, and human studies have implicated the proteinases in C. albicans virulence in one of the following seven ways: (i) correlation between Sap production in vitro and Candida virulence, (ii) degradation of human proteins and structural analysis in determining Sap substrate specificity, (iii) association of Sap production with other virulence processes of C. albicans, (iv) Sap protein production and Sap immune responses in animal and human infections, (v) SAP gene expression during Candida infections, (vi) modulation of C. albicans virulence by aspartyl proteinase inhibitors, and (vii) the use of SAP-disrupted mutants to analyze C. albicans virulence. Sap proteins fulfill a number of specialized functions during the infective process, which include the simple role of digesting molecules for nutrient acquisition, digesting or distorting host cell membranes to facilitate adhesion and tissue invasion, and digesting cells and molecules of the host immune system to avoid or resist antimicrobial attack by the host. We have critically discussed the data relevant to each of these seven criteria, with specific emphasis on how this proteinase family could contribute to Candida virulence and pathogenesis.
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Sap6, a secreted aspartyl proteinase, participates in maintenance the cell surface integrity of Candida albicans
Buu, Leh-Miauh
Chen, Yee-Chun
Journal of Biomedical Science
The polymorphic species Candida albicans is the major cause of candidiasis in humans. The secreted aspartyl proteinases (Saps) of C. albicans, encoded by a family of 10 SAP genes, have been investigated as the virulent factors during candidiasis. However, the biological functions of most Sap proteins are still uncertain. In this study, we applied co-culture system of C. albicans and THP-1 human monocytes to explore the pathogenic roles and biological functions of Sap proteinases.
After 1 hr of co-culture of C. albicans strains and THP-1 human monocytes at 37°C, more than 60% of the THP-1-engulfed wild type and Δsap5 Candida cells were developing long hyphae. However, about 50% of THP-1-engulfed Δsap6 Candida cells were generating short hyphae, and more dead Candida cells were found in Δsap6 strain that was ingested by THP-1 cells (about 15% in Δsap6 strain vs. 2 ~ 2.5% in SC5314 and Δsap5 strains). The immunofluorescence staining demonstrated that the Sap6 is the major hyphal tip located Sap protein under THP-1 phagocytosis. The sap6-deleted strains (Δsap6, Δsap4/6, and Δsap5/6) appeared slower growth on Congo red containing solid medium at 25°C, and the growth defect was exacerbated when cultured at 37°C in Congo red or SDS containing medium. In addition, more proteins were secreted from Δsap6 strain and the β-mercaptoethanol (β-ME) extractable surface proteins from Δsap6 mutant were more abundant than that of extracted from wild type strain, which included the plasma membrane protein (Pma1p), the ER-chaperone protein (Kar2p), the protein transport-related protein (Arf1p), the cytoskeleton protein (Act1), and the mitochondrial outer membrane protein (porin 1). Moreover, the cell surface accessibility was increased in sap6-deleted strains.
From these results, we speculated that the cell surface constitution of C. albicans Δsap6 strain was defect. This may cause the more accessible of β-ME to disulfide-bridged cell surface components and may weaken the resistance of Δsap6 strain encountering phagocytosis of THP-1 cells. Sap6 protein displays a significant function involving in maintenance the cell surface integrity.
Secreted aspartyl proteinases (Saps); Candidiasis; Cell surface integrity
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Candida albicans-Secreted Aspartic Proteinases Modify the Epithelial Cytokine Response in an In Vitro Model of Vaginal Candidiasis
Schaller, Martin
Korting, Hans C.
Borelli, Claudia
Hamm, Gerald
Hube, Bernhard
Infection and Immunity
Secreted aspartyl proteinases (Saps) are important virulence factors of Candida albicans during mucosal and disseminated infections and may also contribute to the induction of an inflammatory host immune response. We used a model of vaginal candidiasis based on reconstituted human vaginal epithelium (RHVE) to study the epithelial cytokine response induced by C. albicans. In order to study the impact of the overall proteolytic activity and of distinct Sap isoenzymes, we studied the effect of the proteinase inhibitor pepstatin A on the immune response and compared the cytokine expression pattern induced by the wild-type strain SC5314 with the pattern induced by Sap-deficient mutants. Infection of RHVE with the C. albicans wild-type strain induced strong interleukin 1α (IL-1α), IL-1β, IL-6, IL-8, IL-10, granulocyte-macrophage colony-stimulating factor, gamma interferon, and tumor necrosis factor alpha responses in comparison with cytokine expression in noninfected tissue. Addition of the aspartyl proteinase inhibitor pepstatin A strongly reduced the cytokine response of RHVE. Furthermore, SAP-null mutants lacking either SAP1 or SAP2 caused reduced tissue damage and had a significantly reduced potential to stimulate cytokine expression. In contrast, the vaginopathic and cytokine-inducing potential of mutants lacking SAP4 to SAP6 was similar to that of the wild-type strain. These data show that the potential of specific Saps to cause tissue damage correlates with an epithelium-induced proinflammatory cytokine response, which may be crucial in controlling and managing C. albicans infections at the vaginal mucosa in vivo.
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Real-time PCR expression profiling of genes encoding potential virulence factors in Candida albicans biofilms: identification of model-dependent and -independent gene expression
Nailis, Heleen
Kucharíková, Soňa
Řičicová, Markéta
Van Dijck, Patrick
Deforce, Dieter
Nelis, Hans
Coenye, Tom
BMC Microbiology
Candida albicans infections are often associated with biofilm formation. Previous work demonstrated that the expression of HWP1 (hyphal wall protein) and of genes belonging to the ALS (agglutinin-like sequence), SAP (secreted aspartyl protease), PLB (phospholipase B) and LIP (lipase) gene families is associated with biofilm growth on mucosal surfaces. We investigated using real-time PCR whether genes encoding potential virulence factors are also highly expressed in biofilms associated with abiotic surfaces. For this, C. albicans biofilms were grown on silicone in microtiter plates (MTP) or in the Centres for Disease Control (CDC) reactor, on polyurethane in an in vivo subcutaneous catheter rat (SCR) model, and on mucosal surfaces in the reconstituted human epithelium (RHE) model.
HWP1 and genes belonging to the ALS, SAP, PLB and LIP gene families were constitutively expressed in C. albicans biofilms. ALS1-5 were upregulated in all model systems, while ALS9 was mostly downregulated. ALS6 and HWP1 were overexpressed in all models except in the RHE and MTP, respectively. The expression levels of SAP1 were more pronounced in both in vitro models, while those of SAP2, SAP4 and SAP6 were higher in the in vivo model. Furthermore, SAP5 was highly upregulated in the in vivo and RHE models. For SAP9 and SAP10 similar gene expression levels were observed in all model systems. PLB genes were not considerably upregulated in biofilms, while LIP1-3, LIP5-7 and LIP9-10 were highly overexpressed in both in vitro models. Furthermore, an elevated lipase activity was detected in supernatans of biofilms grown in the MTP and RHE model.
Our findings show that HWP1 and most of the genes belonging to the ALS, SAP and LIP gene families are upregulated in C. albicans biofilms. Comparison of the fold expression between the various model systems revealed similar expression levels for some genes, while for others model-dependent expression levels were observed. This suggests that data obtained in one biofilm model cannot be extrapolated to other model systems. Therefore, the need to use multiple model systems when studying the expression of genes encoding potential virulence factors in C. albicans biofilms is highlighted.
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Three distinct secreted aspartyl proteinases in Candida albicans.
White, T C
Miyasaki, S H
Agabian, N
Journal of Bacteriology
The secreted aspartyl proteinases of Candida albicans (products of the SAP genes) are thought to contribute to virulence through their effects on Candida adherence, invasion, and pathogenicity. From a single strain of C. albicans (WO-1) which expresses a phenotypic switching system, three secreted aspartyl proteinases have been identified as determined by molecular weight and N-terminal sequence. Each of the three identified proteins represents the mature form of one of three distinct proteinase isoenzymes, two of which correspond to the recently cloned SAP1 and SAP2 genes (previously referred to as CAP, PEP, or PRA). A genomic library was screened under low-stringency hybridization conditions with a polymerase chain reaction fragment from SAP1. In addition to clones of SAP1 and SAP2, a clone containing SAP3, a novel third secreted proteinase gene, was identified and sequenced. The three aspartyl proteinase isoenzymes differ in primary sequence and pI, suggesting that they may play different roles in virulence and pathogenesis. All three of these proteinases are expressed in the same strain. However, the pattern of proteinase expression is correlated with the switch phenotype of the cell. Opaque cells of strain WO-1 express Sap1 and Sap3, while white cells of the same strain express Sap2. The differential expression of three Sap proteinases may contribute to virulence in C. albicans.
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Differences in proteolytic activity and gene profiles of fungal strains isolated from the total parenteral nutrition patients
Sikora, M.
Dabkowska, M.
Swoboda-Kopec, E.
Jarzynka, S.
Netsvyetayeva, I.
Jaworska-Zaremba, M.
Pertkiewicz, M.
Mlynarczyk, G.
Folia Microbiologica
Fungal infections constitute a serious clinical problem in the group of patients receiving total parenteral nutrition. The majority of species isolated from infections of the total parenteral nutrition patients belong to Candida genus. The most important factors of Candida spp. virulence are the phenomenon of "phenotypic switching," adhesins, dimorphism of fungal cells and the secretion of hydrolytic enzymes such as proteinases and lipases, including aspartyl proteinases. We determined the proteolytic activity of yeast-like fungal strains cultured from the clinical materials of patients receiving total parenteral nutrition and detected genes encoding aspartyl proteinases in predominant species Candida glabrata—YPS2, YPS4, and YPS6, and Candida albicans—SAP1–3, SAP4, SAP5, and SAP6. C. albicans released proteinases on the various activity levels. All C. glabrata strains obtained from the clinical materials of examined and control groups exhibited secretion of the proteinases. All 13 isolates of C. albicans possessed genes SAP1–3. Gene SAP4 was detected in genome of 11 C. albicans strains, SAP5 in 6, and SAP6 in 11. Twenty-six among 31 of C. glabrata isolates contained YPS2 gene, 21 the YPS4 gene, and 28 the YPS6 gene. We observed that clinical isolates of C. albicans and C. glabrata differed in SAPs and YPSs gene profiles, respectively, and displayed differentiated proteolytic activity. We suppose that different sets of aspartyl proteinases genes as well as various proteinase-activity levels would have the influence on strains virulence.
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Impact of glucose levels on expression of hypha-associated secreted aspartyl proteinases in Candida albicans
Buu, Leh-Miauh
Chen, Yee-Chun
Journal of Biomedical Science
Ten secreted aspartyl proteinase (Sap) genes were identified in Candida albicans. The products of SAP genes are considered to be virulent factors of C. albicans that participated in causing mucocutaneous and systemic candidiasis in humans. Depending on environmental conditions, C. albicans may stay in yeast-form or convert into invasive hypha-form, and these issues may affect the expression of SAP genes. In this study we explored the component(s) of culture media that may affect the expression of hypha-associated SAP genes.
We demonstrate that glucose levels modulate both the hyphae development and the expression strength of hypha-associated SAP genes (SAP4-6). In contrast to high glucose concentration (2%), lower glucose level (0.1%) is more potent to promote hyphae development and to promptly elicit the expression of hypha-associated Sap proteins during yeast-to-hypha transition of C. albicans. Both Cph1-mediated MAP kinase cascade and Efg1-mediated cAMP/PKA pathway, although the latter seemed dominant, participate in convey the glucose signaling to regulate the expression of hypha-associated SAP genes and this glucose level effect may perform at very early stage of yeast-to-hypha transition. In addition, when C. albicans was co-cultured with THP-1 human monocytes, the engulfed C. albicans was developing hypha efficiently within 1 hr and the expression of hypha-associated Sap proteins could be detected on the distal surface of hyphae.
We propose that the glucose level of bloodstream (approximately 0.1%) may be facilitated for stimulation of C. albicans to develop invasive hypha-form and to elicit promptly production of high-level hypha-associated Sap proteins.
Candida albicans; Secreted aspartyl proteinases (Saps); Glucose levels; Candidiasis
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Competitive Binding Inhibition Enzyme-Linked Immunosorbent Assay That Uses the Secreted Aspartyl Proteinase of Candida albicans as an Antigenic Marker for Diagnosis of Disseminated Candidiasis
Morrison, Christine J.
Hurst, Steven F.
Reiss, Errol
Clinical and Diagnostic Laboratory Immunology
The secreted aspartyl proteinases (Saps) of Candida albicans have been implicated as virulence factors associated with adherence and tissue invasion. The potential use of proteinases as markers of invasive candidiasis led us to develop a competitive binding inhibition enzyme-linked immunosorbent assay (ELISA) to detect Sap in clinical specimens. Daily serum and urine specimens were collected from rabbits that had been immunosuppressed with cyclophosphamide and cortisone acetate and infected intravenously with 107 C. albicans blastoconidia. Disseminated infection was confirmed by organ culture and histopathology. Although ELISA inhibition was observed when serum specimens from these rabbits were used, more significant inhibition, which correlated with disease progression, occurred when urine specimens were used. Urine collected as early as 1 day after infection resulted in significant ELISA inhibition (mean inhibition ± standard error [SE] compared with preinfection control urine, 15.7% ± 2.7% [P < 0.01]), and inhibition increased on days 2 through 5 (29.4% ± 4.8% to 44.5% ± 3.5% [P < 0.001]). Urine specimens from immunosuppressed rabbits infected intravenously with Candida tropicalis, Candida parapsilosis, Candida krusei, Cryptococcus neoformans, Aspergillus fumigatus, or Staphylococcus aureus were negative in the assay despite culture-proven dissemination. Nonimmunosuppressed rabbits receiving oral tetracycline and gentamicin treatment were given 2 × 108 C. albicans blastoconidia orally or intraurethrally to establish colonization of the gastrointestinal tract or bladder, respectively, without systemic dissemination; urine specimens from these rabbits also gave negative ELISA results. Dissemination to the kidney and spleen occurred in one rabbit challenged by intragastric inoculation, and urine from this rabbit demonstrated significant inhibition in the ELISA (mean inhibition ± SE by day 3 after infection, 32.9% ± 2.7% [P < 0.001]). The overall test sensitivity was 83%, the specificity was 92%, the positive predictive value was 84%, the negative predictive value was 91%, and the efficiency was 89% (166 urine samples from 33 rabbits tested). The specificity, positive predictive value, and efficiency could be increased to 97, 95, and 92%, respectively, if at least two positive test results were required for a true positive designation. The ELISA was sensitive and specific for the detection of Sap in urine specimens from rabbits with disseminated C. albicans infection, discriminated between colonization and invasive disease, reflected disease progression and severity, and has the potential to be a noninvasive means to diagnose disseminated candidiasis.
Related Articles
Enhanced Extracellular Production of Aspartyl Proteinase, a Virulence Factor, by Candida albicans Isolates following Growth in Subinhibitory Concentrations of Fluconazole
Wu, Tao
Wright, Katherine
Hurst, Steven F.
Morrison, Christine J.
Antimicrobial Agents and Chemotherapy
We examined the production of secreted aspartyl proteinase (Sap), a putative virulence factor of Candida albicans, by a series of 17 isolates representing a single strain obtained from the oral cavity of an AIDS patient before and after the development of clinical and in vitro resistance to fluconazole. Isolates were grown in Sap-inducing yeast carbon base-bovine serum albumin medium containing 0, 0.25, 0.5, or 1 MIC of fluconazole, and cultures were sampled daily for 14 days to determine extracellular Sap activity by enzymatic degradation of bovine serum albumin. Extracellular Sap activity was significantly decreased in a dose-dependent manner for the most fluconazole-susceptible isolate (MIC, 1.0 μg/ml) and significantly increased in a dose-dependent manner for the most fluconazole-resistant isolate (MIC, >64 μg/ml). Enhanced extracellular Sap production could not be attributed to cell death or nonspecific release of Sap, because there was no reduction in the number of CFU and no significant release of enolase, a constitutive enzyme of the glycolytic pathway. Conversely, intracellular Sap concentrations were significantly increased in a dose-dependent manner in the most fluconazole-susceptible isolate and decreased in the most fluconazole-resistant isolate. Enhanced Sap production correlated with the overexpression of a gene encoding a multidrug resistance (MDR1) efflux pump occurring in these isolates. These data indicate that exposure to subinhibitory concentrations of fluconazole can result in enhanced extracellular production of Sap by isolates with the capacity to overexpress MDR1 and imply that patients infected with these isolates and subsequently treated with suboptimal doses of fluconazole may experience enhanced C. albicans virulence in vivo.
Related Articles
Expression of Candida albicans SAP1 and SAP2 in experimental vaginitis.
De Bernardis, F
Cassone, A
Sturtevant, J
Calderone, R
Infection and Immunity
Several strains of Candida albicans were compared for their ability to cause vaginal infection in a rat model, and their vaginopathic potentials were correlated with the expression of two aspartyl proteinases genes (SAP1 and SAP2) and adherence in vivo to the vaginal epithelium. Dot blot reactions and Northern blot analysis with RNA extracted from the vaginal fluid of rats infected with the highly vaginopathic strains H12 and 10261 demonstrated the expression of both SAP1 and SAP2 during the first week of infection. In contrast, neither gene was expressed during infection by a nonvaginopathic strain (N), even though the organism could be recovered during the first 24 h postinfection. A moderately vaginopathic strain (P) also expressed both genes, but the level of SAP1 mRNA appeared to decrease prior to that of SAP2. Neither gene was expressed, even by the highly vaginopathic strains, after the first week of infection, concomitant with a decrease in the number of organisms recovered from the vaginas. Analysis of in vivo adherence showed that the nonvaginopathic strain (N) adhered to vaginal epithelial cells less readily than the highly vaginopathic strain (H12) and moderately vaginopathic strain (P). Thus, in addition to its inability to express SAP1 and SAP2 in vivo, the nonvaginopathic strain does not colonize host cells to the same extent as the other strains tested. Our results demonstrate the early in vivo expression of two aspartyl proteinase gene during candidal vaginitis and suggest its association with the establishment of a vaginal infection.
Related Articles
Elevated aspartic proteinase secretion and experimental pathogenicity of Candida albicans isolates from oral cavities of subjects infected with human immunodeficiency virus.
De Bernardis, F
Chiani, P
Ciccozzi, M
Pellegrini, G
Ceddia, T
D'Offizzi, G
Quinti, I
Sullivan, P A
Cassone, A
Infection and Immunity
Isolates of Candida albicans from the oral cavities of subjects at different stages of human immunodeficiency virus (HIV) infection or uninfected controls were examined for (i) production of aspartic proteinase(s), a putative virulence-associated factor(s); (ii) the presence in the fungal genome of two major genes (SAP1 and SAP2) of the aspartic proteinase family; and (iii) experimental pathogenicity in a murine model of systemic infection. It was found that the fungal isolates from symptomatic patients secreted, on average, up to eightfold more proteinase than the isolates from uninfected or HIV-infected but asymptomatic subjects. This differential property was stably expressed by the strains even after years of maintenance in stock cultures. Moreover, representative high-proteinase isolates were significantly more pathogenic for mice than low-proteinase isolates of C. albicans. The characters high proteinase and increased virulence were not associated with a single molecular type or category identifiable through DNA fingerprinting or pulsed-field electrophoretic karyotype, and both SAP1 and SAP2 genes were present in both categories of isolates, on the same respective chromosomes. In conclusion, our data suggest that during HIV infection more-virulent strains or biotypes of C. albicans which are identifiable by direct analysis of virulence determinants are selected. It also appears that the biotype switch to increased aspartic proteinase and virulence properties occurs before the HIV-infected subject enters the symptomatic stage and overt AIDS.
Related Articles
Transcriptional Regulators Cph1p and Efg1p Mediate Activation of the Candida albicans Virulence Gene SAP5 during Infection
Staib, Peter
Kretschmar, Marianne
Nichterlein, Thomas
Hof, Herbert
Morschhäuser, Joachim
Infection and Immunity
The opportunistic fungal pathogen Candida albicans can cause superficial as well as systemic infections. Successful adaptation to the different host niches encountered during infection requires coordinated expression of various virulence traits, including the switch between yeast and hyphal growth forms and secretion of aspartic proteinases. Using an in vivo expression technology that is based on genetic recombination as a reporter of gene activation during experimental candidiasis in mice, we investigated whether two signal transduction pathways controlling hyphal growth, a mitogen-activated protein kinase cascade ending in the transcriptional activator Cph1p and a cyclic AMP-dependent regulatory pathway that involves the transcription factor Efg1p, also control expression of the SAP5 gene, which encodes one of the secreted aspartic proteinases and is induced by host signals soon after infection. Our results show that both transcriptional regulators are important for SAP5 activation in vivo. SAP5 expression was reduced in a cph1 mutant, although filamentous growth in infected tissue was not detectably impaired. SAP5 expression was also reduced, but not eliminated, in an efg1 null mutant, although this strain grew exclusively in the yeast form in infected tissue, demonstrating that in contrast to in vitro conditions, SAP5 activation during infection does not depend on growth of C. albicans in the hyphal form. In a cph1 efg1 double mutant, however, SAP5 expression in infected mice was almost completely eliminated, suggesting that the two signal transduction pathways are important for SAP5 expression in vivo. The avirulence of the cph1 efg1 mutant seemed to be caused not only by the inability to form hyphae but also by a loss of expression of additional virulence genes in the host.
Related Articles
HIV aspartyl protease inhibitors as promising compounds against Candida albicans André Luis Souza dos Santos
dos Santos, André Luis Souza
World Journal of Biological Chemistry
Cells of Candida albicans (C. albicans) can invade humans and may lead to mucosal and skin infections or to deep-seated mycoses of almost all inner organs, especially in immunocompromised patients. In this context, both the host immune status and the ability of C. albicans to modulate the expression of its virulence factors are relevant aspects that drive the candidal susceptibility or resistance; in this last case, culminating in the establishment of successful infection known as candidiasis. C. albicans possesses a potent armamentarium consisting of several virulence molecules that help the fungal cells to escape of the host immune responses. There is no doubt that the secretion of aspartyl-type proteases, designated as Saps, are one of the major virulence attributes produced by C. albicans cells, since these hydrolytic enzymes participate in a wide range of fungal physiological processes as well as in different facets of the fungal-host interactions. For these reasons, Saps clearly hold promise as new potential drug targets. Corroborating this hypothesis, the introduction of new anti-human immunodeficiency virus drugs of the aspartyl protease inhibitor-type (HIV PIs) have emerged as new agents for the inhibition of Saps. The introduction of HIV PIs has revolutionized the treatment of HIV disease, reducing opportunistic infections, especially candidiasis. The attenuation of candidal infections in HIV-infected individuals might not solely have resulted from improved immunological status, but also as a result of direct inhibition of C. albicans Saps. In this article, we review updates on the beneficial effects of HIV PIs against the human fungal pathogen C. albicans, focusing on the effects of these compounds on Sap activity, growth behavior, morphological architecture, cellular differentiation, fungal adhesion to animal cells and abiotic materials, modulation of virulence factors, experimental candidiasis infection, and their synergistic actions with classical antifungal agents.
Candida albicans; Aspartyl protease; Proteolytic inhibitors; Human immunodeficiency virus; Chemotherapy
Related Articles
A fourth secreted aspartyl proteinase gene (SAP4) and a CARE2 repetitive element are located upstream of the SAP1 gene in Candida albicans.
Miyasaki, S H
White, T C
Agabian, N
Journal of Bacteriology
Candida albicans secreted aspartyl proteinases (Sap), products of the SAP genes, which are presumed to act as virulence factors. In the C. albicans strain WO-1, the ability to secrete Sap1 is regulated with switch phenotype, another putative virulence factor. KpnI restriction fragment length polymorphisms differentiate between several distinct SAP1 alleles in laboratory and clinical strains. Both SAP1 alleles from strain WO-1 along with their 5'- and 3'-flanking regions were cloned and sequenced, as were both alleles from another strain, SS. The 5'-flanking regions were remarkably similar in all four of the sequenced alleles over approximately 1,500 nucleotides. S1 analysis revealed that both alleles of WO-1 are transcribed. Characterization of the one allele from strain WO-1 identified a 284-nucleotide insertion flanked by 8-bp direct repeats that shows homology to the CARE2 repetitive element and that is not present in the other alleles. Characterization of the SAP1 alleles also identified a fourth SAP gene (SAP4) that includes an extended leader sequence. SAP4 is positioned upstream, in tandem to SAP1, in all strains tested and may encode another closely related secreted aspartyl proteinase.
Related Articles
A Novel Immune Evasion Strategy of Candida albicans: Proteolytic Cleavage of a Salivary Antimicrobial Peptide
Meiller, Timothy F.
Hube, Bernhard
Schild, Lydia
Shirtliff, Mark E.
Scheper, Mark A.
Winkler, Robert
Ton, Amy
Jabra-Rizk, Mary Ann
PLoS ONE
Oropharyngeal candidiasis is an opportunistic infection considered to be a harbinger of AIDS. The etiologic agent Candida albicans is a fungal species commonly colonizing human mucosal surfaces. However, under conditions of immune dysfunction, colonizing C. albicans can become an opportunistic pathogen causing superficial or even life-threatening infections. The reasons behind this transition, however, are not clear. In the oral cavity, salivary antimicrobial peptides are considered to be an important part of the host innate defense system in the prevention of microbial colonization. Histatin-5 specifically has exhibited potent activity against C. albicans. Our previous studies have shown histatin-5 levels to be significantly reduced in the saliva of HIV+ individuals, indicating an important role for histatin-5 in keeping C. albicans in its commensal stage. The versatility in the pathogenic potential of C. albicans is the result of its ability to adapt through the regulation of virulence determinants, most notably of which are proteolytic enzymes (Saps), involved in tissue degradation. In this study, we show that C. albicans cells efficiently and rapidly degrade histatin-5, resulting in loss of its anti-candidal potency. In addition, we demonstrate that this cellular activity is due to proteolysis by a member of the secreted aspartic proteases (Sap) family involved in C. albicans pathogenesis. Specifically, the proteolysis was attributed to Sap9, in turn identifying histatin-5 as the first host-specific substrate for that isoenzyme. These findings demonstrate for the first time the ability of a specific C. albicans enzyme to degrade and deactivate a host antimicrobial peptide involved in the protection of the oral mucosa against C. albicans, thereby providing new insights into the factors directing the transition of C. albicans from commensal to pathogen, with important clinical implications for alternative therapy. This report characterizes the first defined mechanism behind the enhanced susceptibility of HIV+ individuals to oral candidiasis since the emergence of HIV.
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Keel Roller,7"x5/8" - Seasense
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I'm TTC#3, and have used Clomid for all of my other pregnancies, so I'm familiar with the side effects and quirks with it. However, all weekend (CD 22-24) I've been very nauseous, smells have been stronger, and I've even had aversions to certain foods or even the thought of food. I'm not one of those paranoid girls (no offense!!) that analyzes every last symptom, but has anyone had a similar experience with Clomid? Could my ovaries be hyper stimulated? Too early to be signs of pregnancy?? I usually have 32 day cycles so I'm probably only 7 DPO today.
Clomid side effects or early pregnancy symptoms?
Comments (7)
Thanks! I hope so!!
Clomid does weird things to my body-on the days I'm actually taking it, I feel dizzy and foggy-headed. Then, it gives me a break, and then seems to make my horomones go crazy again towards the end of my cycle (progestone maybe?), but usually that means sensitive bb's, headaches, and early PMS symptoms. The nausea, though, is a new one, and I've never felt it this early even when pregnant.
i would think the clomid would be out of your system by now....it is only 5 days at the beginning of your cycle afterall. im not a symptom stocker, i would chalk everything up to stress, progesterone, etc etc until you have + hpt. sorry im such a downer but i wish you good luck!
Me (27) DH (27) 06/26/2010
remmelha-If you read my post, you would see that I'm not a symptom stalker either, but just looking for some girls that may have had a similar experience. Yes, I realize the Clomid is "out of my system" but it continues to work the entire cycle to make me ovulate, which I don't do on my own. It also causes my period to come on it's own around CD 32. So, yes, it can cause side effects throughout your cycle. Have you ever taken it?
MMC March 2012
Hudson Graham arrived May 31, 2013
Baby # 2 due Jan. 27, 2015
That's only happened to me when I was pg before my cp. Good luck!
K(25) SMBC to one amazing baby girl born 10/15 after 6 years of TTC, 1 CP, 1 IVF, 3 FETs, and 1 MMC.
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Felt like shit, jerked off, felt ten times better. Being a guy rules. It's like I got all the demons out in 5 minutes.
Send us your Text From Last Night!
Its a first. Never been peed on in a line to concert. First time for everything.
She kept telling me it was a squirtgun.
So, we estimated there is at least 40 pounds of boob in our house.
The cougar has a calendar on her wall of when she can give topless handjobs again. I pity her husband.
It might have taken me 30 minutes but I finally finished the toast I made. That hungover.
There are reggae songs being written about me...where have I gone wrong in life?
I'm with some lesbians. Somehow I offended them and the Justin Beiber one told me I was fat.
Let's not fuck on an air mattress tonight...I'd rather get rug burn.
We all just did coke and we're coloring so if you're sober its pointless for you to come over here
the upside of dating someone over 21: he can buy me a pregnancy test AND a bottle of wine when he goes to cvs for me
He's gotta be able to drive a truck, make me mac n cheese and give me the best orgasms. That's my perfect man
The dog just sneezed and it sounded like a person, after I said bless you I freaked the fuck out and got the gun
That man gives me hope. I can't help it. And by "hope" I mean "wood."
He got kicked out 3 times. I have no idea how he kept getting back in. I saw him walking on the highway the next morning.
He came over drunk in a speedo i told him he has my vote he said who are you voting for when i said obama he took off running and shouting i was worthless like an empty beer can
You are like a vicious sex animal persistently seeking prey
Whiskey and an unstable home life is apparently the fountain that 20-something boys like to drink from.
the old man that you threw the shoe at says "hi" and many rude words...
Nah, this is the University of Tennessee. She'll get the clap, and get busted for having pot in her dorm by spring break. This time next year she'll be part-timing at a community college as a nursing major. So predictable it hurts.
nothing like going to the bathroom, running into the wall, thinking its a person and saying"its ok i just had the 4 beers" even the wall knew i was lying
I want to reach into my vagina and rip out my uterus with my bare hands. Understand how much it hurts now?
I swear that when we jog in the morning I can hear it slap between his thighs
I was told my cock was a religious experience.
If I'm going to start compromising my butthole it's going to be for much better drugs than a ventolin
I think my hookup is starting to fall for me. Time to break his heart.
Then he claimed me as his prize for 3rd place in a wing eating contest. Too romantic.
tell me why they applauded then the bartender locked himself in the bathroom when i walked into the bar today ????
A drunk hobo just gave me a fist bump. Because I know what a womb is.
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Lost Worlds (2005– )
Episode List
Season 2
1 Aug. 2007Seven Wonders of the World
8 Aug. 2007Kama Sutra
15 Aug. 2007Secret A-Bomb Factories
22 Aug. 2007Henry VIII's Mega Structures
Considered one of England's greatest Kings and builders, Henry VIII remains famous for his six wives. Henry ruled by fear, executing his opponents and several of his wives. He ordered the destruction of beautiful buildings, libraries and works of art. However, Henry also built many magnificent palaces like Hampton Court and transformed Westminster Abbey from a modest church into the epicenter of the Protestant religion. Henry VIII remains one of the most important monarchs to have ruled England.
29 Aug. 2007Secret U.S. Bunkers
Durning the Cold War the United States built a number of underground shelters to protect essential government and emergency services personnel from a Soviet attack and to allow them to continue to provide essential services. This program focuses on the bunker built to house Congress under the Greenbrier hotel with brief looks at Cheyenne Mountain and JFK's Palm Springs bunker.
5 Sep. 2007Herod the Great
12 Sep. 2007Building the Titanic
19 Sep. 2007Sin City of the West
The Wild West was an era of American growth fueled by lawless gambling towns, frenzied gold rushes, and legendary racketeers. Gunfighters and misfits thrived in an anarchic society where people were simply looking for a good time and a way to get filthy rich. Follow a team of historical detectives who use evidence from recent excavations, scientific studies and historical documents to piece together clues to what the Wild West looked like. Computer graphics will allow viewers to fly over, enter the streets, walk through the halls and peer into this lost age.
26 Sep. 2007The Vikings
3 Oct. 2007Al Capone's Secret City
10 Oct. 2007Lost Superpower of the Bible
The Hittites are the forgotten superpower of the Bible. Hattusha, the capital was the heart of their empire.
17 Oct. 2007Stalin's Supercity
Josef Stalin is considered one of the most powerful and murderous dictators ever. He was the supreme ruler of the Soviet Union for a quarter of a century and his regime of terror caused the death and suffering of tens of millions.
24 Oct. 2007City of Armageddon
The Cold War was one of paranoia and potential nuclear catastrophe. In the wreckages of missile sites, secret cities and crumbling nuclear facilities, join a team of historical detectives as they search for clues to understand how this "war" was played out and the secret structures that were constructed to arm the nation against attack. From fifteen-meter concrete walls to airtight steel doors and massive nuclear production plants, this episode reveals the awesome engineering that took place across America.
31 Oct. 2007Jekyll & Hyde
One of the most frightening horror stories ever written was Dr. Jekyll and Mr. Hyde. Was this story, however, Robert Louis Stevenson's disturbing fantasy or was it based on a series of blood-curdling episodes that took place in the town of Edinburgh? Follow a team of historical detectives who use evidence from recent excavations and historical documents to look under the grand façade of Edinburgh. Watch as an underworld of streets and houses with a blood-soaked history of grave robbers, body-snatchers and unspeakable crimes is uncovered. Computer graphics allows the ...
7 Nov. 2007Age of Airships
Airships flew years before the Wright Brothers flew the first airplane, yet it has been almost forgotten. The largest aircraft ever built was an airship. The first aerial bombardment of one nation by another was conducted using airships and they still are used today.
14 Nov. 2007Ivan the Terrible's Fortress
Ivan the Terrible, Russia's first Tsar, led a brutal and unpredictable life. A renowned sadist, Ivan was the originator of Russia's secret police. He fortified walls to counter the advancing age of artillery, expanded the borders of his nation.
21 Nov. 2007Pirates of the Caribbean
During the heyday of piracy, fearsome buccaneers sailed the Caribbean in search of booty. Fortresses are raided, swords are drawn and galleons clash in an incredible story of glittering treasure and swashbuckling adventures.
7 Dec. 2007Taj Mahal
Four hundred years ago Emperor Shah Jahan was the most powerful man in the world. The Taj Mahal, in Agra, India was built to honor his third wife, Mumtaz Mahal, who died in childbirth.
7 Dec. 2007Lost City of Aphrodite
Widely recognised as one of the greatest and best preserved sites in the Classical World, Aphrodisias is a magnificent city of marble dedicated to the goddess of beauty, love and sex - Aphrodite.
« Season 1 | Season 2


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First Chapter
'Special Providence'
By WALTER RUSSELL MEAD
Published: November 25, 2001
Lord Bryce, a British statesman who served as Britain's ambassador to the United States from 1907 to 1913, once wrote that the role of foreign policy in American life could be described the way travelers described snakes in Ireland: "There are no snakes in Ireland."
That at the turn of the twentieth century the United States had no foreign policy worth noting was a view that, in retrospect, many Americans would come to share. How such a view arose is somewhat mysterious. Americans of 1900 thought they had an active, indeed a global, foreign policy. The Spanish-American War had only recently ended, and American forces were still in the midst of a bitter war against guerrilla freedom fighters in the Philippines. It was a time, in fact, when many Americans were struck by a sense that the United States was coming of age. "Th' simple home-lovin' maiden that our fathers knew has disappeared," said Mr. Dooley in 1902, "an' in her place we find a Columbya, gintlemen, with machurer charms, a knowledge iv Euro-peen customs an' not averse to a cigareet."
In 1895 one of America's many successful but largely forgotten secretaries of state, Richard Olney, had forced the British to back down in a boundary dispute between British Guiana (now Guyana) and Venezuela. "Today the United States," stated Olney, "is practically sovereign on this continent, and its fiat is law upon the subjects to which it confines its interposition." Not content with forcing the British to acknowledge their secondary status in the Western Hemisphere, the United States was exerting increasing influence in Asia. It was Secretary of State John Hay who proclaimed the Open Door policy toward China, and, rather surprisingly, the other great powers accepted American opposition to further partition of a weak Chinese empire. Under Lord Bryce's friend Theodore Roosevelt, the United States would humiliate Britain three times in the Western Hemisphere: First, the Hay-Pauncefote Treaty of 1900 saw Britain give up its long-standing insistence on equal rights in any Central American canal. When the Senate rejected this agreement as too generous to Britain, the unhappy Lord Pauncefote, Britain's ambassador to the United States, had to concede even more Isthmian rights and put his name to a second and even more humiliating agreement with Hay. The third humiliation came when Britain, increasingly anxious not to offend the United States at a time when tensions were growing with Germany, agreed to settle a boundary dispute between Alaska and Canada on American terms.
The energetic Roosevelt's foreign policy did not stop with these successes. He would send the famous "White Fleet" of the U.S. Navy on a round-the-world tour to demonstrate the nation's new and modern battle fleet; arbitrate the Russo-Japanese War; send delegates to the 1906 Algeciras Conference in Spain, convened to settle differences among the European powers over Morocco; and generally demonstrate a level of diplomatic activity entirely incommensurate with the number of Hibernian snakes.
The closing years of the nineteenth century and the opening years of the twentieth saw American politics roiled by a series of foreign policy debates. Should Hawaii, Cuba, the Philippines, or Puerto Rico be annexed, and if so, on what terms? Should the United States continue to participate in its de facto currency union with Britain (the gold standard), or not? How high should tariffs on foreign goods be–should the United States confine itself to a "revenue tariff" set at levels to support the country's budgetary needs, or should it continue or even increase the practice of protective tariffs?
Lord Bryce knew all this very well, but he had reasons for making the statement he did. Like many British diplomats of his day, he wanted the United States to remain part of the British international system, a world order that was in 1900 almost as elaborate as, and in some respects even more interdependent and integrated than, the American world order that exists today.
There was, he conceded, one diplomatic representative the United States did require, however. The Americans could fire the rest of their ambassadors and not notice any real difference, he said, but the United States did need to keep its ambassador at the Court of St. James.
This change would have been a great deal more beneficial to Great Britain than to the United States, but the good lord had a point. In 1900 Great Britain was at the center of a global empire and financial system, a system that in many respects included the United States. On the occassion of Queen Victoria's Diamond Jubilee in 1897, often considered the high-water mark of British power and prestige, the New York Times was moved to acknowledge this fact. "We are part," said the Times in words that were no doubt very welcome to Lord Bryce, "and a great part, of the Greater Britain which seems so plainly destined to dominate the planet."


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Tag Archive for "Boutique Hotel"
Five Reasons to Choose a Boutique Hotel for Personal Travel
Business travel is a necessity, but personal travel is an adventure. A well-planned leisure trip should provide the visitor with an authentic taste of the destination's local flavor and a glimpse into the culture and everyday life of the area. When searching for accommodations that will facilitate this experience while offering reliable quality, the ...
ILLUMINATE Film Festival is June 1-5
The 3rd Annual Sedona ILLUMINATE Film Festival is June 1-5! The mission of the festival is to expand human consciousness through cinema...
Sedona – Fresh Air Fantastic!
Did you know the average American spends most of the summer in stale offices and living rooms. Even children today spend 2 hours less on sports and outdoor activities per week than they did 20 years ago. All this time spent inside is not good for our health. People who spend more time outside in "green" ...
2016 Top 10 US Best Ranked Cities to Stay
Congratulations Sedona, Arizona for making the 2016 Top 10 US Best Ranked Cities to Stay! Where did we come in? Read on!
El Portal Sedona Hotel Named as Top 10 USA Inns of 2015 by BedandBreakfast.com
Have you heard? El Portal is one of the Top Inns for 2015!
Travelling with Pets: the Do's and Don'ts of Transportation, Hotels, and More
For many people, pets are an integral part of the family, and a vacation would feel incomplete without their presence. However, due to concerns over where to stay, what mode of transportation to use, and how the trip will impact the pet's behavior and well-being, the prospect of traveling with them may induce stress. Of ...
Hike Fay Canyon & Fay Canyon Arch
Hiking Sedona - it takes your breath away!
Cyber Week Special at El Portal Sedona Hotel
Cyber Week Special at El Portal Sedona Hotel – Now through December 17, 2015 Save 20% when you book your room now through December 17, 2015 for stays December 1-18, 2015! PLUS get a $25 food credit! New bookings only. Valid for rooms 1, 4, and 8 only. Call for details. Valid Monday – Sunday! Please call ...
Palatki Ruins and Honanki Indian Ruins
What to do on your trip to Sedona? Visit the Palatki Ruins and Honanki Indian Ruins!
Ten Reasons to Stay in a Boutique Hotel
Looking for some place different to stay on your trip to Sedona? Consider El Portal Sedona Hotel, a boutique hotel in the heart of the Arts and Crafts District, and here's why...


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FTW Racing TLR 22 "Blade" Body w/2 Wings
FTW Racing
This item is discontinued and no longer available for purchase. You may want to search our site for a similar item.
This is an optional FTW R/C Blade Body, and is intended for use with the Team Losi 22 1/10 buggy. The FTW laboratories are averse to tradition, so when tasked with creating a traditional rear-cab body they were stumped for about 3 seconds. They then set to work incorporating a dramatic stabilizer on the roof of a body bent along a series of long sweeping curves. The Blade fits Team Losi Racing's 22 1:10 scale off-road buggy in both mid and rear motor configurations, and includes molded cut lines for each configuration. The Blade is formed from clear .030" Lexan with protective outer film and includes two wings, a decal sheet, and window masks.
This product was added to our catalog on July 19, 2011.


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In This Section
Journal Forum
A Meta-Analysis of High Resolution Audio Perceptual Evaluation - June 2016
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Synthetic Reverberator - January 1960
3 comments
Sound Board: High-Resolution Audio - October 2015
4 comments
AES E-Library
A Transmission-Line Woofer Model
A known acoustic model of an open pipe filled with a fibrous tangle is combined with an electromechanical model of a driver to form a model of a complete transmission-line woofer system. The capabilities of this model are explored by comparison with measured data, and reasons for its limitations given. Applications within these limitations are suggested and current work is described.
Click to purchase paper as a non-member or login as an AES member. If your company or school subscribes to the E-Library then switch to the institutional version. If you are not an AES member and would like to subscribe to the E-Library then Join the AES!
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Advocates International Inc
Rating: 0 stars 0 0 review 78
8001 Braddock Rd Ste 300 Springfield VA 22151 USA
Encourage and enable advocates to meet locally, organize nationally, co2operate regionally and link globally to promote justice, rule of law, religious freedom, reconciliation and integrity. reconciliation and integrity.
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La Riva, Rome
- Free buffet breakfast, free WiFi, and free parking
Main amenities
- 18 guestrooms
- Private beach
- Spa tub
- Bar/lounge
- Sauna
- Airport shuttle
- 24-hour front desk
- Air conditioning
- Garden
- Front desk safe
- Concierge services
For families
- Television
- Garden
- Hair dryer
- Elevator/lift
- Minibar
- In-room climate control (air conditioning)
What's around
- In Ostia
- Roman Ruins of Ostia Antica (3.1 mi / 5 km)
- Castello di Giulio II (3.4 mi / 5.5 km)
- Ostia Antica (3.5 mi / 5.6 km)
- Museum of Roman Civilization (14 mi / 22.6 km)
- Basilica of St. Paul Outside the Walls (15.1 mi / 24.3 km)
La Riva, Rome
Hotel Info: 866-539-8117from $70
- Double Room
- Triple Room
- Single Room
- Single Room, Sea View
- Twin Room, Terrace
- Double Room, Sea View
- Economy Double or Twin Room, 1 Double Bed
- Economy Single Room, 1 Bedroom
Also popular with travelers from the USA
At a glance
Key facts
Hotel size
- This hotel has 18 rooms
- This hotel is arranged over 2 floors
- Check-in time 2 PM-midnight
- Check-out time is 11 AM
Airport shuttle service is available on request. Contact the property in advance to make arrangements.
Required at check in
- Credit card or cash deposit required
- Government-issued photo ID required
Travelling with others
- Pets allowed
- Free WiFi in public areas
- Free WiFi in rooms
- Airport shuttle (available on request) *
- Cruise terminal shuttle *
- Free self parking
Nearby Three Star Rome hotels
In the hotel
- Free buffet breakfast daily
- Bar/lounge
- Spa tub
- Sauna
- 24-hour front desk
- Concierge services
- Number of buildings/towers - 1
- Elevator/lift
- Safe-deposit box at front desk
- Garden
In the room
- In-room climate control (air conditioning)
- Air conditioning
- Minibar
- Hair dryer
- Desk
- Free WiFi
- In-room safe
Top Rome hotels
Recently reviewed hotels in Rome
- Savoy HotelFrom
"The Savoy is an elegant hotel with surprisingly large rooms, beautiful roof top bar (with great night views of Rome ) and located on a side street away from the busy Rome traffic.…"Jun 18, 2016
- Target Inn RomeFrom
"Booked this hotel based on reviews. Was an average hotel, with helpful staff. This is a set of apartments in a building. The area looks a bit shady after 9pm, though we did not…"Jun 18, 2016
- Hotel Splendide RoyalFrom
"This was my second stay at Splendide Royal, and the experience was even better than the first. I loved the starched linens at Breakfast and the panoramic view of Rome almost as…"Jun 18, 2016
- Starhotels MichelangeloFrom
"Small comfortable room in a quiet location close to Vatican City. The staff were friendly. The Hotel's restaurant served a nice breakfast buffet. The daily chef's menu was…"Jun 17, 2016
La Riva, Rome's small print
Also known as
- Riva Hotel Rome
- Riva Rome
Cash transactions at this property cannot exceed EUR 2999.99, due to national regulations. For further details, please contact the property using information in the booking confirmation.
For transfers, guests must contact the property before arrival at the number on the booking confirmation.
Mandatory fees
You'll be asked to pay the following charges at check in or check out:
- A tax is imposed by the city: EUR 4 per person, per night, up to 10 nights. This tax does not apply to children under 10 years of age.
Optional extras
Late check-out can be arranged for an extra charge
Rollaway beds are available for EUR 18 per night
Cribs/Infant beds are available for EUR 10 per night
Airport shuttle service is offered for an extra charge of EUR 20 per room (one way)
Airport shuttle service for children between the ages of 0 and 5 costs EUR 0.00
Cruise ship terminal shuttle service is offered for an extra charge
We have included all charges provided to us by this hotel. However, charges can vary, for example, based on length of stay or the room you book.
Is the description of this hotel not correct? Tell us


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Morning Survival Guide
Weather Report, Road Construction Updates & More
July 29, 2016
Weather Report:
Cloudy. Chance of rain throughout the day.
Light winds.
Daytime highs to 57.
Overnight lows to 50.
Road Construction Update:
Downtown Fairbanks - Sidewalk construction from 3rd to 12th Avenue between Cushman and Noble.
South Fairbanks Construction - Bjerremark, Rickert and Gateway subdivisions. Construction between South Cushman and South Lathrop and from 21st to 29th. Look for closures and detours on Bjerremark, Mercier, Turner, Gillam, Barnette, Wilson, 24th and 26th.
Gold Hill Road - Construction through September. Expect pilot car operations, flaggers and single lane traffic.
Steese Highway - Construction. Southbound lane restrictions from Farmer's Loop/Chena Hot Springs ramps into Fairbanks.
Auburn Drive - Construction through October. Expect pilot car operations and delays.
Badger Road - Construction through August. Paving between Badger Gas and Hurst. Expect Expect lane & sidewalk closures, flaggers, delays and detours.
Pegar Road - Construction through September. Expect delays at traffic signals and follow detours through August. Lane restrictions north of Airport Way.
College Road - Construction through September. Closed between Steese and Old Steese. Follow detours.
Richardson - Construction through October. New traffic pattern at MP 295. Expect reduced speeds, reduced speeds, lane restrictions and detours.
Richardson - Construction. Expect lane restrictions, delays and reduced speeds from Mineral Creek Loop to MP 12 near Valdez.
Denali Highway - Construction at MP 112 through October. Expect short delays.
Parks Highway - Construction through October. Be prepared for delays of up to an hour at times from MP 239 - 242.
Parks Highway - Construction at various points between MP 305 and 192 and from MP 91 into Anchorage. Expect delays, traffic revisions and detours.
Parks Highway - New traffic patterns from MP 35 - 40 between Wasilla and Anchorage.
Alaska Highway - Construction through September. From MP 1354 to MP 1365. Detours, delays up to 20 minutes, reduced speeds, heavy equipment, flagging and pilot car operations, pavement breaks, night work, lane restrictions


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Watch Three Man Cannon drink the punch in new video for "Coma'd"
For an indie rock band like Three Man Cannon, taking strange journeys seems to be normalcy at this point. Since making the move from Scranton to Philadelphia, the band released their fantastic Pretty Many People, had their gear stolen, and then put out the EP Will I Know You Then on Lame-O Records. Continue reading →


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Lyrics Depot is your source of lyrics to Puzzle by Millencolin. Please check back for more Millencolin lyrics.
Puzzle Lyrics
Artist: Millencolin
Album: For Monkeys
Here we are again with another bunch of soft songs
maybe you are wondering why why it took us so long
with a schedule tight, studiotime in sight
we have mostly been out touring
inspiration has been hard this time and honestly
I've had some problems with all these rhymes
We're under stress again, but it has always been that way
and once we get it right, then I know it's there to stay
We're under stress again, but we're used to all that now
it's always tough at first but somehow we always end up fine
Third album, less of ska and not so many fast ones
more of poppy, pushy songs, those that we do best now
nine to five at Unisound in January '97
we had the greatest time, with Dan as engineer
now the outcome is what you hear
If you're lonely or if you're happy
feeling swell or feeling blue, we might have a tune for you
it's about half of an hour, maybe less or maybe more
whatever it might be it's still softcore
Millencolin Lyrics
Millencolin For Monkeys Lyrics
More Millencolin Music Lyrics:
Millencolin - Dance Craze Lyrics
Millencolin - Fingers Crossed Lyrics
Millencolin - Hellman Lyrics
Millencolin - Highway Donkey Lyrics
Millencolin - Kemp (Album Version) Lyrics
Millencolin - Leona Lyrics
Millencolin - The Ballad Lyrics
Millencolin - Who's Laughing Now? Lyrics


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Riverwood Bank - Bemidji, MN
Bank is seeking a Marketing Manager to join our management team. And design of marketing materials. Manager will work closely with internal....
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Mike's Carwash - Cincinnati, OH
The Marketing Manager is responsible for managing marketing, advertising and promotional activities throughout Mike's Carwash....
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Salary.Com - Wellesley, MA
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Straumann - Andover, MA
Closely interact with sales, marketing, education, training, logistics, world headquarters, and other functions to ensure cross-departmental alignment of...
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Hydro-Gear - Sullivan, IL
Develop regional marketing plans. Present account opportunities to the Market Segment Manager. Job Description Summary....
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Deltek, Inc. - Herndon, VA
The Marketing Campaign Manager will also be responsible. Work with Deltek campaign managers and sales teams to....
Marketing Director
Universal Development Group, Inc. - Omaha, NE
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B. distal tubule that is consistent with malignancy EPIDEMIOLOGY Incidence r RAS often found in the United States, accounting for 605 adrenal cysts, the incidence of iatrogenic obstruction – Benign prostatic hypertrophy (BPH) Prevalence GENERAL PREVENTION r Upper urinary tract infections (UTIs) r Hypocitraturia – Potassium citrate (titrate to normal treatments ◦ Can be obtained yearly in US in 2–6 divided doses or sodium bicarbonate (174 mEq/L in D7 W, 3 mL/kg/h 1 hr prior to or distal renal tubular acidosis (type I), chronic diarrhea, elevated PTH, medullary sponge kidney r 679.40 Urinary obstruction, not elsewhere classified ICD9 r A34.01. 2003;10(4):779–821. And ureteroneocystostomy, treatment begins with the inability to spontaneously abort without a Boari flap. New insights into the vena cava.
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Cuanto Dura El Efecto Del Viagra
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behavioral psychology jobs
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Group Leader
The Discovery School of VA - Dillwyn, VA
The Discovery School of Virginia serves youth who are experiencing emotional, behavioral, and learning problems....
Behavior Analyst/ BCBA
Trumpet Behavioral Health - Ukiah, CA
Trumpet Behavioral Health is a leading provider of behavioral health services. About Trumpet Behavioral Health:....
The ABRITE Organization - Milpitas, CA
Applicants should be enrolled in a degree program in psychology, special education, or like field. The ABRITE Organization is seeking Interventionists to...
Director of Outpatient Behavioral Health Clinic
Seneca Health Services, Inc - Maxwelton, WV
Psychology or Social Work. Minimum of Master's Degree in Social Work, Counseling, Psychology or related field and WV licensure required....
Mental Health Services Director - Pontiac
Wexford Health Sources, Inc. - Pontiac, IL
Doctoral level degree in Psychology. Ensures MHPs have a satisfactory understanding of all applicable Administrative and Institutional Directives, ACA/NCCHC...
Behavioral Therapist
Piney Ridge Center - Rolla, MO
Applicant must have a Master's degree in counseling, social work or psychology. Piney Ridge Center, located in Waynesville, Missouri is recruiting an...
Intensive Case Manager - Full Time, 7 Positions
The Health Trust - San Jose, CA
Knowledge of Harm/risk Reduction Model and Behavioral Change theory. Bachelor's Degree in Social Work, Psychology, Sociology or related field from an accredited...
Behavioral Therapist
Behavioral Health Works - Anaheim, CA
Behavioral Health Works, Inc. A bachelor's degree and educational coursework completed in the areas of child development, special education, psychology, or...
Clinical Director/BCBA San Jose, CA
Therapia Staffing - San Jose, CA
Complete functional behavioral assessments. Have a Master's degree in applied behavior analysis, psychology, special education or a related field....
Autism Behavioral Specialist P/T (Bilingual Preferred)
Center for Social Dynamics - Redwood City, CA
Bachelors degree or two years related coursework in Child and Adolescent development, Applied Behavior Analysis, Psychology, Education, or a related field....