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Sample records for flt3 ligand elicits

  1. The nasal dendritic cell-targeting Flt3 ligand as a safe adjuvant elicits effective protection against fatal pneumococcal pneumonia.

    PubMed

    Kataoka, Kosuke; Fujihashi, Kohtaro; Oma, Keita; Fukuyama, Yoshiko; Hollingshead, Susan K; Sekine, Shinichi; Kawabata, Shigetada; Ito, Hiro-O; Briles, David E; Oishi, Kazunori

    2011-07-01

    We have previously shown that a pneumococcal surface protein A (PspA)-based vaccine containing DNA plasmid encoding the Flt3 ligand (FL) gene (pFL) as a nasal adjuvant prevented nasal carriage of Streptococcus pneumoniae. In this study, we further investigated the safety and efficacy of this nasal vaccine for the induction of PspA-specific antibody (Ab) responses against lung infection with S. pneumoniae. C57BL/6 mice were nasally immunized with recombinant PspA/Rx1 (rPspA) plus pFL three times at weekly intervals. When dynamic translocation of pFL was initially examined, nasal pFL was taken up by nasal dendritic cells (DCs) and epithelial cells (nECs) but not in the central nervous systems, including olfactory nerve and epithelium. Of importance, nasal pFL induced FL protein synthesis with minimum levels of inflammatory cytokines in the nasal washes (NWs) and bronchoalveolar lavage fluid (BALF). NWs and BALF as well as plasma of mice given nasal rPspA plus pFL contained increased levels of rPspA-specific secretory IgA and IgG Ab responses that were correlated with elevated numbers of CD8(+) and CD11b(+) DCs and interleukin 2 (IL-2)- and IL-4-producing CD4(+) T cells in the nasal mucosa-associated lymphoid tissues (NALT) and cervical lymph nodes (CLNs). The in vivo protection by rPspA-specific Abs was evident in markedly reduced numbers of CFU in the lungs, airway secretions, and blood when mice were nasally challenged with Streptococcus pneumoniae WU2. Our findings show that nasal pFL is a safe and effective mucosal adjuvant for the enhancement of bacterial antigen (Ag) (rPspA)-specific protective immunity through DC-induced Th2-type and IL-2 cytokine responses.

  2. Dual inhibition of AKT/FLT3-ITD by A674563 overcomes FLT3 ligand-induced drug resistance in FLT3-ITD positive AML

    PubMed Central

    Wang, Wenchao; Yu, Kailin; Liu, Xiaochuan; Zou, Fengming; Zhao, Zheng; Wu, Jiaxin; Liu, Juan; Liu, Feiyang; Wang, Li; Stone, Richard M.; Galinksy, Ilene A.; Griffin, James D.; Zhang, Shanchun; Weisberg, Ellen L.; Liu, Jing; Liu, Qingsong

    2016-01-01

    The FLT3-ITD mutation is one of the most prevalent oncogenic mutations in AML. Several FLT3 kinase inhibitors have shown impressive activity in clinical evaluation, however clinical responses are usually transient and clinical effects are rapidly lost due to drug resistance. One of the resistance mechanisms in the AML refractory patients involves FLT3-ligand induced reactivation of AKT and/or ERK signaling via FLT3 wt kinase. Via a screen of numerous AKT kinase inhibitors, we identified the well-established orally available AKT inhibitor, A674563, as a dual suppressor of AKT and FLT3-ITD. A674563 suppressed FLT3-ITD positive AML both in vitro and in vivo. More importantly, compared to other FLT3 inhibitors, A674563 is able to overcome FLT3 ligand-induced drug resistance through simultaneous inhibition of FLT3-ITD- and AKT-mediated signaling. Our findings suggest that A674563 might be a potential drug candidate for overcoming FLT3 ligand-mediated drug resistance in FLT3-ITD positive AML. PMID:27074558

  3. Ectodomain shedding of FLT3 ligand is mediated by TACE 1

    PubMed Central

    Horiuchi, Keisuke; Morioka, Hideo; Takaishi, Hironari; Akiyama, Haruhiko; Blobel, Carl P.; Toyama, Yoshiaki

    2010-01-01

    FLT3 ligand (FLT3L) has diverse roles in the hematopoietic system, which include stimulating proliferation of hematopoietic precursors and development of natural killer cells and dendritic cells. FLT3L is initially synthesized as a membrane-bound protein, which must be cleaved to become a soluble growth factor. However, little is known about the enzyme involved in the proteolytic release of FLT3L. In the current study, we show that shedding of FLT3L is metalloprotease-dependent, and that this proteolytic activity was abolished in fibroblasts lacking TNFα converting enzyme (TACE) and could be rescued by reintroducing wildtype TACE in these cells. Moreover, we found that cells derived from the thymus of conditional TACE-deficient mice produce less FLT3L, and that serum FLT3L levels in these TACE mutant mice are significantly lower, both after LPS treatment and in the absence of such a challenge, further corroborating the relevance of TACE as FLT3L sheddase in vivo. Considering the involvements of FLT3 and FLT3L in hematopoietic malignancies and stem cell mobilization, the identification of the enzyme involved in FLT3L shedding may have important clinical implications. PMID:19494263

  4. Barley as a green factory for the production of functional Flt3 ligand.

    PubMed

    Erlendsson, Lýdur S; Muench, Marcus O; Hellman, Ulf; Hrafnkelsdóttir, Soffía M; Jonsson, Anders; Balmer, Yves; Mäntylä, Einar; Orvar, Björn L

    2010-02-01

    Biologically active recombinant human Flt3 ligand was expressed and isolated from transgenic barley seeds. Its expression is controlled by a tissue specific promoter that confines accumulation of the recombinant protein to the endosperm tissue of the seed. The recombinant Flt3 ligand variant expressed in the seeds contains an HQ-tag for affinity purification on immobilized metal ion affinity chromatography (IMAC) resin. The tagged protein was purified from seed extracts to near homogeneity using sequential chromatography on IMAC affinity resin and cation exchange resin. We also show that the recombinant Flt3 ligand protein undergoes posttranslational modifications: it is a glycoprotein containing alpha-1,3-fucose and alpha-1,2-xylose. The HQ-tagged Flt3 ligand variant exhibits comparable biological activity to commercial Flt3 ligand. This is the first report showing expression and accumulation of recombinant human growth factor in barley seeds with a yield of active protein similar to a bacterial expression system. The present results demonstrate that plant molecular farming is a viable approach for the bioproduction of human-derived growth factors.

  5. FLT3 Ligand as a Molecular Adjuvant for Naked RNA Vaccines.

    PubMed

    Kreiter, Sebastian; Diken, Mustafa; Selmi, Abderraouf; Petschenka, Jutta; Türeci, Özlem; Sahin, Ugur

    2016-01-01

    Intranodal immunization with antigen-encoding naked mRNA has proven to be an efficacious and safe approach to induce antitumor immunity. Thanks to its unique characteristics, mRNA can act not only as a source for antigen but also as an adjuvant for activation of the immune system. The search for additional adjuvants that can be combined with mRNA to further improve the potency of the immunization revealed Fms-like tyrosine kinase 3 (FLT3ligand as a potent candidate. Systemic administration of the dendritic cell-activating FLT3 ligand prior to or along with mRNA immunization-enhanced priming and expansion of antigen-specific CD8(+) T cells in lymphoid organs, T-cell homing into melanoma tumors, and therapeutic activity of the intranodally administered mRNA. Both compounds demonstrate a successful combination in terms of boosting the immune response. This chapter describes methods for intranodal immunization with naked mRNA by co-administration of FLT3 ligand, which leads to strong synergistic effects.

  6. A type I IFN–Flt3 ligand axis augments plasmacytoid dendritic cell development from common lymphoid progenitors

    PubMed Central

    Chen, Yi-Ling; Chen, Ting-Ting; Pai, Li-Mei; Wesoly, Joanna; Bluyssen, Hans A.R.

    2013-01-01

    During infections and inflammation, plasmacytoid dendritic cells (pDCs) are the most potent type I interferon (IFN-I)–producing cells. However, the developmental origin of pDCs and the signals dictating pDC generation remain incompletely understood. Here, we report a synergistic role for IFN-I and Flt3 ligand (FL) in pDC development from common lymphoid progenitors (CLPs). Both conventional DCs (cDCs) and pDCs were generated from CLPs in response to FL, whereas pDC generation required higher concentrations of FL and concurrent IFN-I signaling. An absence of IFN-I receptor, impairment of IFN-I signaling, or neutralization of IFN-I significantly impeded pDC development from CLPs. Furthermore, FL induced IFN-I expression in CLPs, which in turn induced Flt3 up-regulation that facilitated survival and proliferation of CLPs, as well as their differentiation into pDCs. Collectively, these results define a critical role for the FL/IFN-I/Flt3 axis in pDC differentiation from CLPs. PMID:24145513

  7. HIV gag protein is efficiently cross-presented when targeted with an antibody towards the DEC-205 receptor in Flt3 ligand-mobilized murine dendritic cells

    PubMed Central

    Bozzacco, Leonia; Trumpfheller, Christine; Huang, Yaoxing; Longhi, Maria Paula; Shimeliovich, Irina; Schauer, Joseph D.; Park, Chae Gyu; Steinman, Ralph M.

    2010-01-01

    Summary Dendritic cells present exogenous proteins to MHC class I restricted CD8+ T cells. This function does not require endogenous antigen synthesis within DC, providing the potential to elicit CD8+ T cell responses to immune complexes, inactivated microbes, dying cells and proteins like ovalbumin. In mice, the CD8+ or DEC-205+ DC are specialized for cross-presentation, and this subset can be increased 10 fold in numbers following Flt3L treatment in vivo. Therefore we studied cross-presentation by abundant Flt3L DC using HIV gag protein. When enriched by positive selection with anti-CD11c beads, cells from Flt3L mice are not only more abundant but are more highly enriched in CD11c high DC, particularly the DEC-205+ subset. DC cross-present HIV gag to primed CD8+ T cells, but when the antigen is delivered within an antibody to DEC-205 receptor, cross-presentation becomes 100 fold more efficient than non-targeted antigen. This finding requires gag to be engineered into anti-DEC antibody, not just mixed with antibody. Flt3L DC are a valuable tool to study cross-presentation, since their use overcomes the obstacle posed by the low number of cross-presenting DC in the steady state. These findings support future experiments to use Flt3L to enhance presentation of DC-targeted vaccines. PMID:19830741

  8. Flt3 ligand improves the innate response to respiratory syncytial virus and limits lung disease upon RSV reexposure in neonate mice.

    PubMed

    Remot, Aude; Descamps, Delphyne; Jouneau, Luc; Laubreton, Daphné; Dubuquoy, Catherine; Bouet, Stephan; Lecardonnel, Jérôme; Rebours, Emmanuelle; Petit-Camurdan, Agnès; Riffault, Sabine

    2016-04-01

    Respiratory syncytial virus (RSV) causes severe bronchiolitis in infants worldwide. The immunological factors responsible for RSV susceptibility in infants are poorly understood. Here, we used the BALB/c mouse model of neonatal RSV infection to study the mechanisms leading to severe disease upon reexposure to the virus when adults. Two major deficiencies in neonatal lung innate responses were found: a poor DCs mobilization, and a weak engagement of the IFNI pathway. The administration of Flt3 ligand (Flt3-L), a growth factor that stimulates the proliferation of hematopoietic cells, to neonates before RSV-infection, resulted in increased lung DC number, and reconditioned the IFNI pathway upon RSV neonatal infection. Besides, neonates treated with Flt3-L were protected against exacerbated airway disease upon adult reexposure to RSV. This was associated with a reorientation of RSV-specific responses toward Th1-mediated immunity. Thus, the poor lung DCs and IFNI responses to RSV in neonates may be partly responsible for the deleterious long-term consequences revealed upon adult reexposure to RSV, which could be prevented by Flt3-L treatment. These results open new perspectives for developing neonatal immuno-modulating strategies to reduce the burden of bronchiolitis.

  9. Flt3L is a novel regulator of skeletal myogenesis

    PubMed Central

    Ge, Yejing; Waldemer, Rachel J.; Nalluri, Ramakrishna; Nuzzi, Paul D.; Chen, Jie

    2013-01-01

    Summary Various cues initiate multiple signaling pathways to regulate the highly coordinated process of skeletal myogenesis. Myoblast differentiation comprises a series of ordered events starting with cell cycle withdrawal and ending with myocyte fusion, with each step probably controlled by multiple extracellular signals and intracellular signaling pathways. Here we report the identification of Fms-like tyrokine kinase 3 ligand (Flt3L) signaling as a novel regulator of skeletal myogenesis. Flt3L is a multifunctional cytokine in immune cells, but its involvement in skeletal muscle formation has not been reported. We found that Flt3L is expressed in C2C12 myoblasts, with levels increasing throughout differentiation. Knockdown of Flt3L, or its receptor Flt3, suppresses myoblast differentiation, which is rescued by recombinant Flt3L or Flt3, respectively. Differentiation is not rescued, however, by recombinant ligand when the receptor is knocked down, or vice versa, suggesting that Flt3L and Flt3 function together. Flt3L knockdown also inhibits differentiation in mouse primary myoblasts. Both Flt3L and Flt3 are highly expressed in nascent myofibers during muscle regeneration in vivo, and Flt3L siRNA impairs muscle regeneration, validating the physiological significance of Flt3L function in myogenesis. We have identified a cellular mechanism for the myogenic function of Flt3L, as we show that Flt3L promotes cell cycle exit that is necessary for myogenic differentiation. Furthermore, we identify Erk as a relevant target of Flt3L signaling during myogenesis, and demonstrate that Flt3L suppresses Erk signaling through p120RasGAP. In summary, our work reveals an unexpected role for an immunoregulatory cytokine in skeletal myogenesis and a new myogenic pathway. PMID:23704355

  10. Flt3 ligand expands CD103+ dendritic cells, FoxP3+ T regulatory cells and attenuates Crohn’s-like murine ileitis

    PubMed Central

    Collins, Colm B.; Aherne, Carol M.; McNamee, Eóin N.; Lebsack, Matthew D. P.; Eltzschig, Holger; Jedlicka, Paul; Rivera-Nieves, Jesús

    2013-01-01

    Background Imprinting an effector or regulatory phenotype on naïve T cells requires education at induction sites by dendritic cells (DC). In the current studies we analyzed the effect of inflammation on the frequency of mononuclear phagocytes (MP) and the effect of altering their frequency by administration of Flt3-L in chronic ileitis. Design Using a TNF-driven model of ileitis (i.e. TNFΔARE) that recapitulates many features of Crohn’s disease (CD), we assessed dynamic changes in the frequency and functional state of MP within the inflamed ileum by flow cytometry, immunofluorescence and real-time reverse-transcription polymerase chain reaction and by generating CX3CR1 GFP-reporter TNFΔARE mice. Finally, we assessed the effect of Flt3-L supplementation on the severity of ileitis, the frequency of CD103+ DC and of FoxP3+ Tregs in TNFΔARE mice. Results CD11cHi/MHCII+ MP accumulated in inflamed ilea, predominantly mediated by expansion of the CX3CR1+ MP subpopulation. This coincided with a decreased pro-regulatory CD103+ DC. The phenotype of these MP was that of activated cells, as they expressed increased CD80 and CD86 on their surface. Flt3-ligand administration resulted in a preferential expansion of CD103+ DC that attenuated the severity of ileitis in 20-week-old TNFΔARE mice, mediated by increased CD4+/CD25+/FoxP3+ Tregs. Conclusions Our findings support a role for Flt3-L as a potential therapeutic in Crohn’s-like ileitis. PMID:22068168

  11. An autologous in situ tumor vaccination approach for hepatocellular carcinoma. 2. Tumor-specific immunity and cure after radio-inducible suicide gene therapy and systemic CD40-ligand and Flt3-ligand gene therapy in an orthotopic tumor model.

    PubMed

    Kawashita, Yujo; Deb, Niloy J; Garg, Madhur K; Kabarriti, Rafi; Fan, Zuoheng; Alfieri, Alan A; Roy-Chowdhury, Jayanta; Guha, Chandan

    2014-08-01

    Diffuse hepatocellular carcinoma (HCC) is a lethal disease that radiation therapy (RT) currently has a limited role in treating because of the potential for developing fatal radiation-induced liver disease. However, recently diffuse HCC, "radio-inducible suicide gene therapy" has been shown to enhance local tumor control and residual microscopic disease within the liver for diffuse HCC, by using a combination of chemoactivation and molecular radiosensitization. We have demonstrated that the addition of recombinant adenovirus-expressing human Flt3 ligand (Adeno-Flt3L) after radio-inducible suicide gene therapy induced a Th1-biased, immune response and enhanced tumor control in an ectopic model of HCC. We hypothesized that sequential administration of recombinant adenovirus-expressing CD40L (Adeno-CD40L) could further potentiate the efficacy of our trimodal therapy with RT + HSV-TK + Adeno-Flt3L. We examined our hypothesis in an orthotopic model of diffuse HCC using BNL1ME A.7R.1 (BNL) cells in Balb/c mice. BNL murine hepatoma cells (5 × 10(4)) transfected with an expression vector of HSV-TK under the control of a radiation-inducible promoter were injected intraportally into BALB/cJ mice. Fourteen days after the HCC injection, mice were treated with a 25 Gy dose of radiation to the whole liver, followed by ganciclovir (GCV) treatment and systemic adenoviral cytokine gene therapy (Flt3L or CD40L or both). Untreated mice died in 27 ± 4 days. Radiation therapy alone had a marginal effect on survival (median = 35 ± 7 days) and the addition of HSV-TK/GCV gene therapy improved the median survival to 47 ± 6 days. However, the addition of Adeno-Flt3L to radiation therapy and HSV-TK/GCV therapy significantly (P = 0.0005) increased survival to a median of 63 ± 20 days with 44% (7/16) of the animals still alive 116 days after tumor implantation. The curative effect of Flt3L was completely abolished when using immunodeficient nude mice or mice depleted for CD4, CD8 and

  12. Revisiting emergency anti-apoptotic cytokinotherapy: erythropoietin synergizes with stem cell factor, FLT-3 ligand, trombopoietin and interleukin-3 to rescue lethally-irradiated mice.

    PubMed

    Drouet, Michel; Grenier, Nancy; Hérodin, Francis

    2012-06-01

    We have re-evaluated the benefit of using erythropoietin (Epo) as a pleiotropic cytokine to counteract hematological and extra-hematological toxicity following lethal irradiation. B6D2F1 mice were exposed to a dose of 9 Gy gamma radiation resulting in 90% mortality at 30 days, and then injected with stem cell factor, FLT-3 ligand, thrombopoietin and interleukin-3 [i.e. SFT3] at two and 24 hours with or without Epo (1,000 IU/kg) at 2 hours and day 8. As controls, two groups of irradiated mice were given only Epo or Phosphate-buffered saline. Epo synergized with SFT3 to rescue lethally-irradiated mice from radiation-induced death (survival: 60%, 95% and 5% respectively for SFT3, SFT3+Epo and controls at 30 days, p<0.05), whereas Epo alone exhibited no protective effect. Hematopoietic parameters did not differ significantly between SFT3 and SFT3+Epo groups during the animal death period. Some beneficial effects on gastro-intestinal toxicity were noticed following administration of Epo, although lung, liver and kidney were not protected. Further studies are necessary to understand fully the mechanisms involved in these effects of Epo in order to optimize treatment with cytokines following high-dose irradiation.

  13. The new and recurrent FLT3 juxtamembrane deletion mutation shows a dominant negative effect on the wild-type FLT3 receptor

    PubMed Central

    Sandhöfer, Nadine; Bauer, Julia; Reiter, Katrin; Dufour, Annika; Rothenberg, Maja; Konstandin, Nikola P.; Zellmeier, Evelyn; Tizazu, Belay; Greif, Philipp A.; Metzeler, Klaus H.; Hiddemann, Wolfgang; Polzer, Harald; Spiekermann, Karsten

    2016-01-01

    In acute myeloid leukemia (AML), the Fms-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes. Recently, a new and recurrent juxtamembrane deletion mutation (p.Q569Vfs*2) resulting in a truncated receptor was identified. The mutated receptor is expressed on the cell surface and still binds its ligand but loses the ability to activate ERK signaling. FLT3 p.Q569fs-expressing Ba/F3 cells show no proliferation after ligand stimulation. Furthermore, coexpressed with the FLT3 wild-type (WT) receptor, the truncated receptor suppresses stimulation and activation of the WT receptor. Thus, FLT3 p.Q569Vfs*2, to our knowledge, is the first FLT3 mutation with a dominant negative effect on the WT receptor. PMID:27346558

  14. Activation of protein phosphatase 2A in FLT3+ acute myeloid leukemia cells enhances the cytotoxicity of FLT3 tyrosine kinase inhibitors

    PubMed Central

    Lee, Erwin M.; Harrison, Celeste; Kahl, Richard; Flanagan, Hayley; Panicker, Nikita; Mashkani, Baratali; Don, Anthony S.; Morris, Jonathan; Toop, Hamish; Lock, Richard B.; Powell, Jason A.; Thomas, Daniel; Guthridge, Mark A.; Moore, Andrew; Ashman, Leonie K.; Skelding, Kathryn A.; Enjeti, Anoop; Verrills, Nicole M.

    2016-01-01

    Constitutive activation of the receptor tyrosine kinase Fms-like tyrosine kinase 3 (FLT3), via co-expression of its ligand or by genetic mutation, is common in acute myeloid leukemia (AML). In this study we show that FLT3 activation inhibits the activity of the tumor suppressor, protein phosphatase 2A (PP2A). Using BaF3 cells transduced with wildtype or mutant FLT3, we show that FLT3-induced PP2A inhibition sensitizes cells to the pharmacological PP2A activators, FTY720 and AAL(S). FTY720 and AAL(S) induced cell death and inhibited colony formation of FLT3 activated cells. Furthermore, PP2A activators reduced the phosphorylation of ERK and AKT, downstream targets shared by both FLT3 and PP2A, in FLT3/ITD+ BaF3 and MV4-11 cell lines. PP2A activity was lower in primary human bone marrow derived AML blasts compared to normal bone marrow, with blasts from FLT3-ITD patients displaying lower PP2A activity than WT-FLT3 blasts. Reduced PP2A activity was associated with hyperphosphorylation of the PP2A catalytic subunit, and reduced expression of PP2A structural and regulatory subunits. AML patient blasts were also sensitive to cell death induced by FTY720 and AAL(S), but these compounds had minimal effect on normal CD34+ bone marrow derived monocytes. Finally, PP2A activating compounds displayed synergistic effects when used in combination with tyrosine kinase inhibitors in FLT3-ITD+ cells. A combination of Sorafenib and FTY720 was also synergistic in the presence of a protective stromal microenvironment. Thus combining a PP2A activating compound and a FLT3 inhibitor may be a novel therapeutic approach for treating AML. PMID:27329844

  15. G-749, a novel FLT3 kinase inhibitor, can overcome drug resistance for the treatment of acute myeloid leukemia

    PubMed Central

    Lee, Hee Kyu; Kim, Hong Woo; Lee, In Yong; Lee, Jungmi; Lee, Jaekyoo; Jung, Dong Sik; Lee, Sang Yeop; Park, Sung Ho; Hwang, Haejun; Choi, Jang-Sik; Kim, Jung-Ho; Kim, Se Won; Kim, Jung Keun; Cools, Jan; Koh, Jong Sung

    2014-01-01

    Aberrant activations of Fms-like tyrosine receptor kinase (FLT) 3 are implicated in the pathogenesis of 20% to 30% of patients with acute myeloid leukemia (AML). G-749 is a novel FLT3 inhibitor that showed potent and sustained inhibition of the FLT3 wild type and mutants including FLT3-ITD, FLT3-D835Y, FLT3-ITD/N676D, and FLT3-ITD/F691L in cellular assays. G-749 retained its inhibitory potency in various drug-resistance milieus such as patient plasma, FLT3 ligand surge, and stromal protection. Furthermore, it displayed potent antileukemic activity in bone marrow blasts from AML patients regardless of FLT3 mutation status, including those with little or only minor responses to AC220 or PKC412. Oral administration of G-749 yielded complete tumor regression and increased life span in animal models. Thus, G-749 appears to be a promising next-generation drug candidate for the treatment of relapsed and refractory AML patients with various FLT3-ITD/FLT3-TKD mutants and further shows the ability to overcome drug resistance. PMID:24532805

  16. [Analysis of Flt-3 expression and Flt-3/ITD mutation in acute myeloid leukemia cells].

    PubMed

    Wang, Ya-Lun; Wang, Tong; Xu, Feng; Gang, Yan; Wang, Jie

    2006-06-01

    This study was aimed to explore the relationship between Flt-3 expression, Flt-3/ITD mutation in acute leukemia (AL) cell line and pathogenesis of AL, especially AML. The Flt-3 expression and Flt-3/ITD mutation were detected by RT-PCR and sequencing method in 82 leukemia cell lines including 20 AML, 57 ALL and 5 CML cell lines. The results indicated that positive results of Flt-3 expression were obtained in 48 out of 77 AL cell line, the positive rate was 62%; 12 cell lines were positive in 20 AML cell lines, the positive rate was 60%; 33 cell lines was positive in 57 ALL cell lines, the positive rate was 58%; 3 cell lines were positive in 5 CML cell lines, the positive rate was 60%. There was abnormal gene product in 1 AMOL cell line out of 12 AML cell lines with Flt-3 positive expression (positive rate 8.3%). DNA sequencing of abnormal gene product showed two coding duplication sequence with 29 bp long. The positive rate of Flt-3 expression in undifferentiated cell line was prominently higher than that in mature B cell ALL (P < 0.05). It is concluded that the Flt-3 expression is different in various leukemia cells. Flt-3/ITD duplication was found in one AML cell line. The detection of Flt-3 gene and Flt-3/ITD mutation may contribute to the diagnosis of ALL, especially to AML.

  17. The stromal cell-derived factor-1alpha dependent migration of human cord blood CD34 haematopoietic stem and progenitor cells switches from protein kinase C (PKC)-alpha dependence to PKC-alpha independence upon prolonged culture in the presence of Flt3-ligand and interleukin-6.

    PubMed

    Kasenda, Benjamin; Kassmer, Susannah H; Niggemann, Bernd; Schiermeier, Sven; Hatzmann, Wolfgang; Zänker, Kurt S; Dittmar, Thomas

    2008-09-01

    Addition of the inflammatory cytokine interleukin (IL)-6 to the culture medium of human cord blood haematopoietic stem and progenitor cells (HSPCs) has been shown to lead to an altered stromal cell-derived factor-1alpha-dependent migratory phenotype. This study investigated whether this effect was attributed to a differential engagement of protein kinase C (PKC) isotypes. The migratory activity of both Flt3-ligand and Flt3-ligand/IL-6 cultured cord blood HSPCs was PKC-alpha dependent on day 1, but PKC-alpha independent after 5 d of cultivation. PKC-alpha expression was not down-regulated in cells cultured for 5 d indicating a switch of signalling molecules directing cell migration.

  18. How I treat FLT3-mutated AML

    PubMed Central

    Pratz, Keith W.

    2017-01-01

    FLT3-mutated acute myeloid leukemia (AML), despite not being recognized as a distinct entity in the World Health Organization (WHO) classification system, is readily recognized as a particular challenge by clinical specialists who treat acute leukemia. This is especially true with regards to the patients harboring the most common type of FLT3 mutation, the internal tandem duplication (FLT3-ITD) mutation. Here we present 4 patient cases from our institution and discuss how our management reflects what we have learned about this subtype of the disease. We also reflect on how we anticipate the management might change in the near future, with the emergence of clinically useful tyrosine kinase inhibitors. PMID:27872057

  19. FLT3 kinase inhibitor TTT-3002 overcomes both activating and drug resistance mutations in FLT3 in acute myeloid leukemia

    PubMed Central

    Ma, Hayley S.; Nguyen, Bao; Duffield, Amy S.; Li, Li; Galanis, Allison; Williams, Allen B.; Brown, Patrick A.; Levis, Mark J.; Leahy, Daniel J.; Small, Donald

    2014-01-01

    There have been a number of clinical trials testing the efficacy of FLT3 tyrosine kinase inhibitors (TKIs) in acute myeloid leukemia (AML). patients harboring a constitutively activating mutation in FLT3 However, there has been limited efficacy, most often due to inadequate achievement of FLT3 inhibition through a variety of mechanisms In a previous study, TTT-3002 was identified as a novel FLT3 inhibitor with the most potent activity to date against FLT3 internal tandem duplication (FLT3/ITD) mutations Here the activity of TTT-3002 is demonstrated against a broad spectrum of FLT3 activating point mutations (FLT3/PMs), including the most frequently occurring D835 mutations The compound is also active against a number of point mutations selected for in FLT3/ITD alleles that confer resistance to other TKIs, including the F691L gatekeeper mutation TTT-3002 maintains activity against relapsed AML patient samples that are resistant to sorafenib and AC220 Studies utilizing human plasma samples from healthy donors and AML patients indicate that TTT-3002 is only moderately protein bound compared to several other TKIs currently in clinical trials Tumor burden of mice in a FLT3 TKI-resistant transplant model is significantly improved by oral dosing of TTT-3002 Therefore, TTT-3002 has demonstrated preclinical potential as a promising new FLT3 TKI that may overcome some of the limitations of other TKIs in the treatment of FLT3-mutant AML PMID:25060518

  20. FLT3LG — EDRN Public Portal

    Cancer.gov

    From NCBI Gene: Dendritic cells (DCs) provide the key link between innate and adaptive immunity by recognizing pathogens and priming pathogen-specific immune responses. FLT3LG controls the development of DCs and is particularly important for plasmacytoid DCs and CD8 (see MIM 186910)-positive classical DCs and their CD103 (ITGAE; MIM 604682)-positive tissue counterparts (summary by Sathaliyawala et al., 2010 [PubMed 20933441]).[supplied by OMIM, Jan 2011

  1. Microenvironmental hypoxia regulates FLT3 expression and biology in AML.

    PubMed

    Sironi, Silvia; Wagner, Michaela; Kuett, Alexander; Drolle, Heidrun; Polzer, Harald; Spiekermann, Karsten; Rieger, Christina; Fiegl, Michael

    2015-11-30

    Fms-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase constitutively expressed by acute myeloid leukaemia (AML) blasts. In addition, 25% of AML patients harbour a FLT3-ITD mutation, associated with inferior outcome due to increased relapse rate. Relapse might be propagated by interactions between AML blasts and the bone marrow microenvironment. Besides cellular elements of the microenvironment (e.g. mesenchymal stromal cells), bone marrow hypoxia has emerged as an additional crucial component. Hence, effects of hypoxia on FLT3 expression and biology could provide novel insight into AML biology. Here we show that 25% of AML patients down-regulate FLT3 expression on blasts in response to in vitro hypoxia (1% O2), which was independent of its mutational state. While virtually no AML cell lines regulate FLT3 in response to hypoxia, the down-regulation could be observed in Ba/F3 cells stably transfected with different FLT3 mutants. Hypoxia-mediated down-regulation was specific for FLT3, reversible and proteasome-dependent; with FLT3 half-life being significantly shorter at hypoxia. Also, PI-3K inhibition could partially abrogate down-regulation of FLT3. Hypoxia-mediated down-regulation of FLT3 conferred resistance against cytarabine in vitro. In conclusion, FLT3 expression in AML is dependent on the oxygen partial pressure, but response to hypoxia differs.

  2. Pim kinases modulate resistance to FLT3 tyrosine kinase inhibitors in FLT3-ITD acute myeloid leukemia

    PubMed Central

    Green, Alexa S.; Maciel, Thiago T.; Hospital, Marie-Anne; Yin, Chae; Mazed, Fetta; Townsend, Elizabeth C.; Pilorge, Sylvain; Lambert, Mireille; Paubelle, Etienne; Jacquel, Arnaud; Zylbersztejn, Florence; Decroocq, Justine; Poulain, Laury; Sujobert, Pierre; Jacque, Nathalie; Adam, Kevin; So, Jason C. C.; Kosmider, Olivier; Auberger, Patrick; Hermine, Olivier; Weinstock, David M.; Lacombe, Catherine; Mayeux, Patrick; Vanasse, Gary J.; Leung, Anskar Y.; Moura, Ivan C.; Bouscary, Didier; Tamburini, Jerome

    2015-01-01

    ABSTRACT Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is frequently detected in acute myeloid leukemia (AML) patients and is associated with a dismal long-term prognosis. FLT3 tyrosine kinase inhibitors provide short-term disease control, but relapse invariably occurs within months. Pim protein kinases are oncogenic FLT3-ITD targets expressed in AML cells. We show that increased Pim kinase expression is found in relapse samples from AML patients treated with FLT3 inhibitors. Ectopic Pim-2 expression induces resistance to FLT3 inhibition in both FLT3-ITD–induced myeloproliferative neoplasm and AML models in mice. Strikingly, we found that Pim kinases govern FLT3-ITD signaling and that their pharmacological or genetic inhibition restores cell sensitivity to FLT3 inhibitors. Finally, dual inhibition of FLT3 and Pim kinases eradicates FLT3-ITD+ cells including primary AML cells. Concomitant Pim and FLT3 inhibition represents a promising new avenue for AML therapy. PMID:26601252

  3. The Protein Tyrosine Phosphatase, Shp2, Positively Contributes to FLT3-ITD-Induced Hematopoietic Progenitor Hyperproliferation and Malignant Disease In Vivo

    PubMed Central

    Nabinger, Sarah C.; Li, XingJun; Ramdas, Baskar; He, Yantao; Zhang, Xian; Zeng, Lifan; Richine, Briana; Bowling, Joshua D.; Fukuda, Seiji; Goenka, Shreevrat; Liu, Ziyue; Feng, Gen-Sheng; Yu, Menggang; Sandusky, George E.; Boswell, H. Scott; Zhang, Zhong-Yin; Kapur, Reuben; Chan, Rebecca J.

    2014-01-01

    Internal tandem duplications in the fms-like tyrosine kinase receptor (FLT3-ITDs) confer a poor prognosis in acute myeloid leukemia. We hypothesized that increased recruitment of the protein tyrosine phosphatase, Shp2, to FLT3-ITDs contributes to FLT3 ligand (FL)-independent hyperproliferation and STAT5 activation. Co-immunoprecipitation demonstrated constitutive association of Shp2 with the FLT3-ITD, N51-FLT3, as well as with STAT5. Knock-down of Shp2 in Baf3/N51-FLT3 cells significantly reduced proliferation while having little effect on WT-FLT3-expressing cells. Consistently, mutation of N51-FLT3 tyrosine 599 to phenylalanine or genetic disruption of Shp2 in N51-FLT3-expressing bone marrow low density mononuclear cells reduced proliferation and STAT5 activation. In transplants, genetic disruption of Shp2 in vivo yielded increased latency to and reduced severity of FLT3-ITD-induced malignancy. Mechanistically, Shp2 co-localizes with nuclear phospho-STAT5, is present at functional interferon-γ activation sites (GAS) within the BCL2L1 promoter, and positively activates the human BCL2L1 promoter, suggesting that Shp2 works with STAT5 to promote pro-leukemogenic gene expression. Further, using a small molecule Shp2 inhibitor, the proliferation of N51-FLT3-expressing bone marrow progenitors and primary AML samples was reduced in a dose-dependent manner. These findings demonstrate that Shp2 positively contributes to FLT3-ITD-induced leukemia and suggest that Shp2 inhibition may provide a novel therapeutic approach to acute myeloid leukemia. PMID:23103841

  4. Incorporating FLT3 inhibitors into acute myeloid leukemia treatment regimens

    PubMed Central

    Pratz, Keith; Levis, Mark

    2011-01-01

    FMS-Like-Tyrosine kinase-3 (FLT3) mutations are found in about 30% of cases of acute myeloid leukemia and confer an increased relapse rate and reduced overall survival. Targeting of this tyrosine kinase by direction inhibition is the focus of both preclinical and clinical research in AML. Several molecules in clinical development inhibit FLT3 with varying degrees of specificity. Preclinical models suggest that these compounds enhance the cytotoxicity of conventional chemotherapeutics against FLT3 mutant leukemia cells. The pharmacodynamic interactions between FLT3 inhibitors and chemotherapy appear to be sequence dependent. When the FLT3 inhibitor is used prior to chemotherapy, antagonism is displayed, while if FLT3 inhibition is instituted after to exposure to chemotherapy, synergistic cytotoxicity is seen. The combination of FLT3 inhibitors with chemotherapy is also complicated by potential pharmacokinetic obstacles, such as plasma protein binding and p-glycoprotein interactions. Ongoing and future studies are aimed at incorporating FLT3 inhibitors into conventional induction and consolidation therapy specifically for patients with FLT3 mutant AML. PMID:18452067

  5. FLT3 inhibitors: clinical potential in acute myeloid leukemia

    PubMed Central

    Hospital, Marie-Anne; Green, Alexa S; Maciel, Thiago T; Moura, Ivan C; Leung, Anskar Y; Bouscary, Didier; Tamburini, Jerome

    2017-01-01

    Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy that is cured in as few as 15%–40% of cases. Tremendous improvements in AML prognostication arose from a comprehensive analysis of leukemia cell genomes. Among normal karyotype AML cases, mutations in the FLT3 gene are the ones most commonly detected as having a deleterious prognostic impact. FLT3 is a transmembrane tyrosine kinase receptor, and alterations of the FLT3 gene such as internal tandem duplications (FLT3-ITD) deregulate FLT3 downstream signaling pathways in favor of increased cell proliferation and survival. FLT3 tyrosine kinase inhibitors (TKI) emerged as a new therapeutic option in FLT3-ITD AML, and clinical trials are ongoing with a variety of TKI either alone, combined with chemotherapy, or even as maintenance after allogenic stem cell transplantation. However, a wide range of molecular resistance mechanisms are activated upon TKI therapy, thus limiting their clinical impact. Massive research efforts are now ongoing to develop more efficient FLT3 TKI and/or new therapies targeting these resistance mechanisms to improve the prognosis of FLT3-ITD AML patients in the future. PMID:28223820

  6. FLT-3 Expression and Function on Microglia in Multiple Sclerosis

    PubMed Central

    DeBoy, Cynthia A.; Rus, Horea; Tegla, Cosmin; Cudrici, Cornelia; Jones, Melina V.; Pardo, Carlos A.; Small, Donald; Whartenby, Katharine A.; Calabresi, Peter A.

    2010-01-01

    Inflammatory cell infiltration and resident microglial activation within the central nervous system (CNS) are pathological events in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). While MS therapies target the peripheral immune system, no treatment is currently known to also modulate microglia. FMS-like tyrosine-3 (FLT-3) is expressed on hematopoietic and dendritic cells. We reported that FLT-3 inhibition ameliorates early actively induced EAE by predominantly modulating dendritic cell function as compared to microglia. We demonstrate in this report that FLT-3 is expressed in perivascular cuffs, brain parenchyma and in non-lesioned gray and white matter within MS brain but not in these regions within control brain. Furthermore, we demonstrate that FLT-3 is expressed on two populations of cells within MS brain; one which expresses the dendritic cell marker CD209, and the other which does not, suggesting that FLT-3 within MS brain is expressed on infiltrating dendritic cells and a non-dendritic cell such as microglia. Additionally, we report that FLT-3 inhibition in murine microglia blocks, in a dose dependent manner, IFN-γ-induced expression of MHC class II and CD86, and LPS-induced secretion of IL-6. These data suggest that FLT-3 is involved in microglial cell’s capacity to respond to environmental cues to function as antigen presenting cells and mediate CNS inflammation. Furthermore these data suggest that FLT-3 may be a therapeutic target on microglia to mitigate CNS inflammation. PMID:20566414

  7. Prolonged exposure to FLT3 inhibitors leads to resistance via activation of parallel signaling pathways

    PubMed Central

    Piloto, Obdulio; Wright, Melissa; Brown, Patrick; Kim, Kyu-Tae; Levis, Mark; Small, Donald

    2007-01-01

    Continuous treatment of malignancies with tyrosine kinase inhibitors (TKIs) may select for resistant clones (ie, imatinib mesylate). To study resistance to TKIs targeting FLT3, a receptor tyrosine kinase that is frequently mutated in acute myelogenous leukemia (AML), we developed resistant human cell lines through prolonged coculture with FLT3 TKIs. FLT3 TKI-resistant cell lines and primary samples still exhibit inhibition of FLT3 phosphorylation on FLT3 TKI treatment. However, FLT3 TKI-resistant cell lines and primary samples often show continued activation of downstream PI3K/Akt and/or Ras/MEK/MAPK signaling pathways as well as continued expression of genes involved in FLT3-mediated cellular transformation. Inhibition of these signaling pathways restores partial sensitivity to FLT3 TKIs. Mutational screening of FLT3 TKI-resistant cell lines revealed activating N-Ras mutations in 2 cell lines that were not present in the parental FLT3 TKI-sensitive cell line. Taken together, these data indicate that FLT3 TKI-resistant cells most frequently become FLT3 independent because of activation of parallel signaling pathways that provide compensatory survival/proliferation signals when FLT3 is inhibited. Anti-FLT3 mAb treatment was still cytotoxic to FLT3 TKI-resistant clones. An approach combining FLT3 TKIs with anti-FLT3 antibodies and/or inhibitors of important pathways downstream of FLT3 may reduce the chances of developing resistance. PMID:17047150

  8. Arsenic trioxide and all-trans-retinoic acid selectively exert synergistic cytotoxicity against FLT3-ITD AML cells via co-inhibition of FLT3 signaling pathways.

    PubMed

    Wang, Li-Na; Tang, Yan-Lai; Zhang, Yin-Chuan; Zhang, Zu-Han; Liu, Xiao-Jian; Ke, Zhi-Yong; Li, Yu; Tan, Hui-Zhen; Huang, Li-Bin; Luo, Xue-Qun

    2017-03-09

    FLT3-ITD mutations occur in approximately 30% of acute myeloid leukemia (AML) and are associated with a poor outcome. Currently available FLT3 inhibitors have in vitro but limited clinical activity in FLT3-ITD AML. Reports have shown that an arsenic trioxide (ATO)/all-trans-retinoic acid (ATRA) combination improves prognosis in acute promyelocytic leukemia, especially with FLT3-ITD, and ATO or ATRA alone enhances apoptosis in FLT3-ITD AML cells treated with FLT3 inhibitors, providing a rationale to investigate the role of ATO/ATRA in FLT3-ITD AML. Here, we demonstrate that an ATO/ATRA combination selectively exerts synergistic cytotoxicity against FLT3-ITD AML cell lines (MV4;11/MOLM-13). The signaling pathways affected by ATO/ATRA include FLT3/STAT5/MYC, FLT3/STAT5/E2F1, FLT3/ERK/ATF5 and FLT3/AKT/ATF5.ATF5 may function as an oncogene in FLT3-ITD AML. Our findings provide experimental evidence that supports further exploration of ATO/ATRA in FLT3-ITD AML in vivo and warrants a clinical evaluation of regimens comprising an ATO/ATRA combination.

  9. TALENs-mediated gene disruption of FLT3 in leukemia cells: Using genome-editing approach for exploring the molecular basis of gene abnormality.

    PubMed

    Wang, Jue; Li, Tongjuan; Zhou, Mi; Hu, Zheng; Zhou, Xiaoxi; Zhou, Shiqiu; Wang, Na; Huang, Liang; Zhao, Lei; Cao, Yang; Xiao, Min; Ma, Ding; Zhou, Pengfei; Shang, Zhen; Zhou, Jianfeng

    2015-12-16

    Novel analytic tools are needed to elucidate the molecular basis of leukemia-relevant gene mutations in the post-genome era. We generated isogenic leukemia cell clones in which the FLT3 gene was disrupted in a single allele using TALENs. Isogenic clones with mono-allelic disrupted FLT3 were compared to an isogenic wild-type control clone and parental leukemia cells for transcriptional expression, downstream FLT3 signaling and proliferation capacity. The global gene expression profiles of mutant K562 clones and corresponding wild-type controls were compared using RNA-seq. The transcriptional levels and the ligand-dependent autophosphorylation of FLT3 were decreased in the mutant clones. TALENs-mediated FLT3 haplo-insufficiency impaired cell proliferation and colony formation in vitro. These inhibitory effects were maintained in vivo, improving the survival of NOD/SCID mice transplanted with mutant K562 clones. Cluster analysis revealed that the gene expression pattern of isogenic clones was determined by the FLT3 mutant status rather than the deviation among individual isogenic clones. Differentially expressed genes between the mutant and wild-type clones revealed an activation of nonsense-mediated decay pathway in mutant K562 clones as well as an inhibited FLT3 signaling. Our data support that this genome-editing approach is a robust and generally applicable platform to explore the molecular bases of gene mutations.

  10. The MERTK/FLT3 inhibitor MRX-2843 overcomes resistance-conferring FLT3 mutations in acute myeloid leukemia.

    PubMed

    Minson, Katherine A; Smith, Catherine C; DeRyckere, Deborah; Libbrecht, Clara; Lee-Sherick, Alisa B; Huey, Madeline G; Lasater, Elisabeth A; Kirkpatrick, Gregory D; Stashko, Michael A; Zhang, Weihe; Jordan, Craig T; Kireev, Dmitri; Wang, Xiaodong; Frye, Stephen V; Earp, H Shelton; Shah, Neil P; Graham, Douglas K

    2016-03-01

    FMS-like tyrosine kinase 3-targeted (FLT3-targeted) therapies have shown initial promise for the treatment of acute myeloid leukemia (AML) expressing FLT3-activating mutations; however, resistance emerges rapidly. Furthermore, limited options exist for the treatment of FLT3-independent AML, demonstrating the need for novel therapies that reduce toxicity and improve survival. MERTK receptor tyrosine kinase is overexpressed in 80% to 90% of AMLs and contributes to leukemogenesis. Here, we describe MRX-2843, a type 1 small-molecule tyrosine kinase inhibitor that abrogates activation of both MERTK and FLT3 and their downstream effectors. MRX-2843 treatment induces apoptosis and inhibits colony formation in AML cell lines and primary patient samples expressing MERTK and/or FLT3-ITD, with a wide therapeutic window compared with that of normal human cord blood cells. In murine orthotopic xenograft models, once-daily oral therapy prolonged survival 2- to 3-fold over that of vehicle-treated controls. Additionally, MRX-2843 retained activity against quizartinib-resistant FLT3-ITD-mutant proteins with clinically relevant alterations at the D835 or F691 loci and prolonged survival in xenograft models of quizartinib-resistant AML. Together, these observations validate MRX-2843 as a translational agent and support its clinical development for the treatment of AML.

  11. The MERTK/FLT3 inhibitor MRX-2843 overcomes resistance-conferring FLT3 mutations in acute myeloid leukemia

    PubMed Central

    Minson, Katherine A.; Smith, Catherine C.; Libbrecht, Clara; Lee-Sherick, Alisa B.; Huey, Madeline G.; Lasater, Elisabeth A.; Kirkpatrick, Gregory D.; Stashko, Michael A.; Zhang, Weihe; Jordan, Craig T.; Kireev, Dmitri; Wang, Xiaodong; Frye, Stephen V.; Earp, H. Shelton; Shah, Neil P.; Graham, Douglas K.

    2016-01-01

    FMS-like tyrosine kinase 3–targeted (FLT3-targeted) therapies have shown initial promise for the treatment of acute myeloid leukemia (AML) expressing FLT3-activating mutations; however, resistance emerges rapidly. Furthermore, limited options exist for the treatment of FLT3-independent AML, demonstrating the need for novel therapies that reduce toxicity and improve survival. MERTK receptor tyrosine kinase is overexpressed in 80% to 90% of AMLs and contributes to leukemogenesis. Here, we describe MRX-2843, a type 1 small-molecule tyrosine kinase inhibitor that abrogates activation of both MERTK and FLT3 and their downstream effectors. MRX-2843 treatment induces apoptosis and inhibits colony formation in AML cell lines and primary patient samples expressing MERTK and/or FLT3-ITD, with a wide therapeutic window compared with that of normal human cord blood cells. In murine orthotopic xenograft models, once-daily oral therapy prolonged survival 2- to 3-fold over that of vehicle-treated controls. Additionally, MRX-2843 retained activity against quizartinib-resistant FLT3-ITD–mutant proteins with clinically relevant alterations at the D835 or F691 loci and prolonged survival in xenograft models of quizartinib-resistant AML. Together, these observations validate MRX-2843 as a translational agent and support its clinical development for the treatment of AML. PMID:27158668

  12. Rat bone marrow-derived dendritic cells generated with GM-CSF/IL-4 or FLT3L exhibit distinct phenotypical and functional characteristics.

    PubMed

    N'diaye, Marie; Warnecke, Andreas; Flytzani, Sevasti; Abdelmagid, Nada; Ruhrmann, Sabrina; Olsson, Tomas; Jagodic, Maja; Harris, Robert A; Guerreiro-Cacais, Andre Ortlieb

    2016-03-01

    Dendritic cells are professional APCs that play a central role in the initiation of immune responses. The limited ex vivo availability of dendritic cells inspires the widespread use of bone marrow-derived dendritic cells as an alternative in research. However, the functional characteristics of bone marrow-derived dendritic cells are incompletely understood. Therefore, we compared functional and phenotypic characteristics of rat bone marrow-derived dendritic cells generated with GM-CSF/IL-4 or FLT3 ligand bone marrow-derived dendritic cells. A comparison of surface markers revealed that FLT3 ligand-bone marrow-derived dendritic cells expressed signal regulatory protein α, CD103, and CD4 and baseline levels of MHC class II, CD40, and CD86, which were highly up-regulated upon stimulation. Conversely, GM-CSF/IL-4-bone marrow-derived dendritic cells constitutively expressed signal regulatory protein α, CD11c, and CD11b but only mildly up-regulated MHC class II, CD40, or CD86 following stimulation. Expression of dendritic cell-associated core transcripts was restricted to FLT3 ligand-bone marrow-derived dendritic cells . GM-CSF/IL-4-bone marrow-derived dendritic cells were superior at phagocytosis but were outperformed by FLT3 ligand-bone marrow-derived dendritic cells at antigen presentation and T cell stimulation in vitro. Stimulated GM-CSF/IL-4-bone marrow-derived dendritic cells secreted more TNF, CCL5, CCL20, and NO, whereas FLT3 ligand-bone marrow-derived dendritic cells secreted more IL-6 and IL-12. Finally, whereas GM-CSF/IL-4-bone marrow-derived dendritic cell culture supernatants added to resting T cell cultures promoted forkhead box p3(+) regulatory T cell populations, FLT3 ligand-bone marrow-derived dendritic cell culture supernatants drove Th17 differentiation. We conclude that rat GM-CSF/IL-4-bone marrow-derived dendritic cells and FLT3 ligand-bone marrow-derived dendritic cells are functionally distinct. Our data support the current rationale that FLT3

  13. Temporal Changes in FLT3-ITD Regulation of Stem Cell Self-Renewal and Leukemogenesis

    DTIC Science & Technology

    2015-09-01

    whether FLT3-ITD depletes HSCs, expands restricted progenitors and promotes a myeloproliferative neoplasm during the adult, but not fetal stage of...goals: Aim 1: To test whether FLT3-ITD depletes HSCs, expands restricted progenitors and promotes a myeloproliferative neoplasm during the adult, but...progenitor pool and myeloproliferative neoplasms (MPN) were also evident in 14 day old FLT3-ITD mice but not fetal mice (Fig. 1F and not shown). FLT3-ITD

  14. FLT3 inhibition: a moving and evolving target in acute myeloid leukaemia.

    PubMed

    Leung, A Y H; Man, C-H; Kwong, Y-L

    2013-02-01

    Internal tandem duplication (ITD) of the fms-like tyrosine kinase 3 (FLT3) gene is a gain-of-function mutation common in acute myeloid leukaemia (AML). It is associated with inferior prognosis and response to chemotherapy. Single base mutations at the FLT3 tyrosine kinase domain (TKD) also leads to a gain of function, although its prognostic significance is less well defined because of its rarity. The clinical benefits of FLT3 inhibition are generally limited to AML with FLT3-ITD. However, responses are transient and leukaemia progression invariably occurs. There is compelling evidence that leukaemia clones carrying both ITD and TKD mutations appear when resistance to FLT3 inhibitors occurs. Interestingly, the emergence of double ITD and TKD mutants can be recapitulated in vitro when FLT3-ITD+ leukaemia cell lines are treated with mutagens and FLT3 inhibitors. Furthermore, murine xenotransplantation models also suggest that, in some cases, the FTL3-ITD and TKD double mutants actually exist in minute amounts before treatment with FLT3 inhibitors, expand under the selection pressure of FLT3 inhibition and become the predominant resistant clone(s) during the drug-refractory phase. On the basis of this model of clonal evolution, a multipronged strategy using more potent FLT3 inhibitors, and a combinatorial approach targeting both FLT3-dependent and FLT3-independent pathways, will be needed to improve outcome.

  15. Development and Characterization of FLT3-Specific Curcumin-Loaded Polymeric Micelles as a Drug Delivery System for Treating FLT3-Overexpressing Leukemic Cells

    PubMed Central

    Tima, Singkome; Okonogi, Siriporn; Ampasavate, Chadarat; Pickens, Chad; Berkland, Cory; Anuchapreeda, Songyot

    2016-01-01

    This study aimed to develop a curcumin (CM) nanoparticle targeted to Feline McDonough Sarcoma (FMS)-like tyrosine kinase 3 (FLT3) protein on the surface of leukemic cells and to evaluate their properties, specificity, cytotoxicity, and inhibitory effect on FLT3 protein level in FLT3 overexpressing leukemic cells, EoL-1 and MV-4-11 cells. FLT3-specific peptides were conjugated onto modified poloxamer 407 (P407) using the copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC). The thin film hydration method was performed for FLT3-specific CM-loaded polymeric micelles (FLT3-CM-micelles) preparation. Flow cytometry and fluorescence microscopy were used to determine rate of cellular uptake. 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was used to test the cytotoxicity of the micelles on leukemic cells. FLT3-CM-micelles demonstrated a mean particle size less than 50 nm, high entrapment efficiency, and high rate of CM uptake by leukemic cells. The intracellular CM fluorescence is related to FLT3 protein levels on the leukemic cell surfaces. Moreover, FLT3-CM-micelles demonstrated an excellent cytotoxic effect and decreased FLT3 protein expression in the leukemic cells. The FLT3-CM-micelles could enhance both solubility and cytotoxicity of CM on FLT3 overexpressing leukemic cells. These promising nanoparticles may be used for enhancing anti-leukemic activity of CM and developed as a targeted drug delivery system in the future. PMID:27751588

  16. Expression of caveolin-1 and podocalyxin in rat lungs challenged with 2-kDa macrophage-activating lipopeptide and Flt3L.

    PubMed

    Tschernig, T; Pabst, R; Kasper, M; El-Hadi, Mustafa; Singh, B

    2014-04-01

    Caveolin-1 is one of the important regulators of vascular permeability in inflamed lungs. Podocalyxin is a CD34 protein expressed on vascular endothelium and has a role in podocyte development in the kidney. Few data are available on the expression of caveolin-1 and podocalyxin in lungs challenged with Toll-like receptor 2 (TLR2) agonists such as mycoplasma-derived macrophage activating lipopeptide or with immune modulators such as Fms-like tyrosine kinase receptor-3 ligand (Flt3L), which expands dendritic cell populations in the lung. Because of the significance of pathogen-derived molecules that act through TLR2 and of the role of immune modulators in lung physiology, we examine the immunohistochemical expression of caveolin-1 and podocalyxin in lungs from rats challenged with a 2-kDa macrophage-activating lipopeptide (MALP-2) and Flt3L. Normal rat lungs expressed caveolin-1 in alveolar septa, vascular endothelium and airway epithelium, especially along the lateral borders of epithelial cells but not in alveolar macrophages. MALP-2 and Flt3L decreased and increased, respectively, the expression of caveolin-1. Caveolin-1 expression seemed to increase in microvessels in bronchiole-associated lymphoid tissue (BALT) in Flt3L-challenged lungs but not in normal or MALP-2-treated lungs. Podocalyxin was absent in the epithelium and alveolar macrophages but was present in the vasculature of control, Flt3L- and MALP-2-treated rats. Compared with control and MALP-2-treated rats, Flt3L-treated lungs showed greater expression of podocalyxin in BALT vasculature and at the interface of monocytes and the endothelium. These immunohistochemical data describing the altered expression of caveolin-1 and podocalyxin in lungs treated with MALP-2 or Flt3L encourage further mechanistic studies on the role of podocalyxin and caveolin-1 in lung inflammation.

  17. EphrinA1-EphA2 interaction-mediated apoptosis and Flt3L-induced immunotherapy inhibits tumor growth in a breast cancer mouse model

    PubMed Central

    Tandon, Manish; Vemula, Sai V.; Sharma, Anurag; Ahi, Yadvinder S.; Mittal, Shalini; Bangari, Dinesh S.; Mittal, Suresh K.

    2014-01-01

    Background The receptor tyrosine kinase EphA2 is overexpressed in several types of cancers and is currently being pursued as a target for breast cancer therapeutics. The EphA2 ligand EphrinA1 induces EphA2 phosphorylation and intracellular internalization and degradation, thus inhibiting tumor progression. The hematopoietic growth factor, FMS-like tyrosine kinase receptor ligand (Flt3L), promotes expansion and mobilization of functional dendritic cells. Methods We tested the EphrinA1-EphA2 interaction in MDA-MB-231 breast cancer cells focusing on the receptor-ligand-mediated apoptosis of breast cancer cells. In order to determine whether the EphrinA1-EphA2 interaction-associated apoptosis and Flt3L-mediated immunotherapy would have an additive effect in inhibiting tumor growth, we used an immunocompetent mouse model of breast cancer to evaluate intratumoral (i.t.) inoculation strategies with human adenovirus (HAd) vectors expressing either EphrinA1 (HAd-EphrinA1-Fc), Flt3L (HAd-Flt3L) or a combination of EphrinA1-Fc + Flt3L (HAd-EphrinA1-Fc + HAd-Flt3L). Results In vitro analysis demonstrated that an EphrinA1-EphA2 interaction led to apoptosis-related changes in breast cancer cells. In vivo, three i.t. inoculations of HAd-EphrinA1-Fc showed potent inhibition of tumor growth. Furthermore, increased inhibition in tumor growth was observed with the combination of HAd-EphrinA1-Fc and HAd-Flt3L accompanied by the generation of an anti-tumor adaptive immune response. Conclusions The results indicating induction of apoptosis and inhibition of mammary tumor growth show the potential therapeutic benefits of HAd-EphrinA1-Fc. In combination with HAd-Flt3L, this represents a promising strategy to effectively induce mammary tumor regression by HAd vector-based therapy. PMID:22228563

  18. Palbociclib treatment of FLT3-ITD+ AML cells uncovers a kinase-dependent transcriptional regulation of FLT3 and PIM1 by CDK6

    PubMed Central

    Uras, Iris Z.; Walter, Gina J.; Scheicher, Ruth; Bellutti, Florian; Prchal-Murphy, Michaela; Tigan, Anca S.; Valent, Peter; Heidel, Florian H.; Kubicek, Stefan; Scholl, Claudia; Fröhling, Stefan

    2016-01-01

    Up to 30% of patients with acute myeloid leukemia have constitutively activating internal tandem duplications (ITDs) of the FLT3 receptor tyrosine kinase. Such mutations are associated with a poor prognosis and a high propensity to relapse after remission. FLT3 inhibitors are being developed as targeted therapy for FLT3-ITD+ acute myeloid leukemia; however, their use is complicated by rapid development of resistance, which illustrates the need for additional therapeutic targets. We show that the US Food and Drug Administration–approved CDK4/6 kinase inhibitor palbociclib induces apoptosis of FLT3-ITD leukemic cells. The effect is specific for FLT3-mutant cells and is ascribed to the transcriptional activity of CDK6: CDK6 but not its functional homolog CDK4 is found at the promoters of the FLT3 and PIM1 genes, another important leukemogenic driver. There CDK6 regulates transcription in a kinase-dependent manner. Of potential clinical relevance, combined treatment with palbociclib and FLT3 inhibitors results in synergistic cytotoxicity. Simultaneously targeting two critical signaling nodes in leukemogenesis could represent a therapeutic breakthrough, leading to complete remission and overcoming resistance to FLT3 inhibitors. PMID:27099147

  19. Using combination therapy to override stromal-mediated chemoresistance in mutant FLT3-positive AML: Synergism between FLT3 inhibitors, dasatinib/multi-targeted inhibitors, and JAK inhibitors

    PubMed Central

    Weisberg, Ellen; Liu, Qingsong; Nelson, Erik; Kung, Andrew L.; Christie, Amanda L.; Bronson, Rod; Sattler, Martin; Sanda, Takaomi; Zhao, Zheng; Hur, Wooyoung; Mitsiades, Constantine; Smith, Robert; Daley, John F.; Stone, Richard; Galinsky, Ilene; Griffin, James D.; Gray, Nathanael

    2014-01-01

    Acute myeloid leukemia (AML) progenitors are frequently characterized by activating mutations in the receptor tyrosine kinase FLT3. Protein tyrosine kinases are integral components of signaling cascades that play a role in both FLT3-mediated transformation as well as viability pathways that are advantageous to leukemic cell survival. The bone marrow microenvironment can diminish AML sensitivity to tyrosine kinase inhibitors (TKIs). We hypothesized that inhibition of protein kinases in addition to FLT3 may be effective in overriding drug resistance in AML. We used a cell-based model mimicking stromal protection as part of an unbiased high-throughput chemical screen to identify kinase inhibitors with the potential to override microenvironment-mediated drug resistance in mutant FLT3-positive AML. Several related multi-targeted kinase inhibitors, including dasatinib, with the capability of reversing microenvironment-induced resistance to FLT3 inhibition were identified and validated. We validated synergy in vitro and demonstrated effective combination potential in vivo. In particular Janus kinase (JAK) inhibitors were effective in overriding stromal protection and potentiating FLT3 inhibition in primary AML and cell lines. These results hint at a novel concept of using combination therapy to override drug resistance in mutant FLT3-positive AML in the bone marrow niche and suppress or eradicate residual disease. PMID:22469781

  20. Specific detection of Flt3 point mutations by highly sensitive real-time polymerase chain reaction in acute myeloid leukemia.

    PubMed

    Scholl, Sebastian; Krause, Claudia; Loncarevic, Ivan F; Müller, Rouven; Kunert, Christa; Wedding, Ulrich; Sayer, Herbert G; Clement, Joachim H; Höffken, Klaus

    2005-06-01

    Among activating class III receptor tyrosine kinase (Flt3) mutations, internal tandem duplications of Flt3 (Flt3-ITD) are detected in about 25% of patients with acute myeloid leukemia (AML). In contrast, mutations within the tyrosine kinase domain of Flt3 (Flt3-TKD mutations) are less frequent (approximately 7%), and there are only limited data on the frequency of recently demonstrated activating Flt3 point mutation at codon 592 (Flt3-V592A mutation). We evaluated a new approach for rapid screening of Flt3-TKD and Flt3-V592A mutations using the fluorescence resonance energy transfer (FRET) principle in a group of 122 patients. Based on individual Flt3-TKD mutations, we designed patient-specific primers to perform a highly sensitive polymerase chain reaction (PCR) assay for rapid detection of minimal residual disease (MRD). We also used a model system with MonoMac-6 cells carrying the Flt3-V592A mutation to establish a mutation-specific real-time PCR approach also for this molecular aberration. We identified 9 cases (8%) of Flt3-TKD mutations (5 cases of mutation D835Y, 3 cases of mutation D835H, and 1 case of mutation Del836), and no cases of Flt3-V592A mutation. Screening for Flt3-TKD mutations with fluorescent probes is equivalent to conventional screening using standard PCR followed by EcoRV restriction. We present a real-time PCR protocol that can be used for MRD analyses based on individual Flt3-TKD mutations. Examples of MRD analyses are presented for all 3 subtypes of Flt3-TKD mutation identified in this study. In summary, we demonstrate new methodological approaches for rapid screening of Flt3 point mutations and for detection of MRD based on patient-specific Flt3-TKD mutations.

  1. Fetal and neonatal hematopoietic progenitors are functionally and transcriptionally resistant to Flt3-ITD mutations

    PubMed Central

    Porter, Shaina N; Cluster, Andrew S; Yang, Wei; Busken, Kelsey A; Patel, Riddhi M; Ryoo, Jiyeon; Magee, Jeffrey A

    2016-01-01

    The FLT3 Internal Tandem Duplication (FLT3ITD) mutation is common in adult acute myeloid leukemia (AML) but rare in early childhood AML. It is not clear why this difference occurs. Here we show that Flt3ITD and cooperating Flt3ITD/Runx1 mutations cause hematopoietic stem cell depletion and myeloid progenitor expansion during adult but not fetal stages of murine development. In adult progenitors, FLT3ITD simultaneously induces self-renewal and myeloid commitment programs via STAT5-dependent and STAT5-independent mechanisms, respectively. While FLT3ITD can activate STAT5 signal transduction prior to birth, this signaling does not alter gene expression until hematopoietic progenitors transition from fetal to adult transcriptional states. Cooperative interactions between Flt3ITD and Runx1 mutations are also blunted in fetal/neonatal progenitors. Fetal/neonatal progenitors may therefore be protected from leukemic transformation because they are not competent to express FLT3ITD target genes. Changes in the transcriptional states of developing hematopoietic progenitors may generally shape the mutation spectra of human leukemias. DOI: http://dx.doi.org/10.7554/eLife.18882.001 PMID:27879203

  2. FLT3 tyrosine kinase inhibitors in acute myeloid leukemia: clinical implications and limitations

    PubMed Central

    Kayser, Sabine; Levis, Mark J.

    2015-01-01

    Internal tandem duplications of the FMS-like tyrosine kinase 3 (FLT3) gene are one of the most frequent gene mutations in acute myeloid leukemia (AML) and are associated with poor clinical outcome. The remission rate is high with intensive chemotherapy, but most patients eventually relapse. During the last decade, FLT3 mutations have emerged as an attractive target for a molecularly specific treatment strategy. Targeting FLT3 receptor tyrosine kinases in AML has shown encouraging results in the treatment of FLT3 mutated AML, but in most patients responses are incomplete and not sustained. Newer, more specific compounds seem to have a higher potency and selectivity against FLT3. During therapy with FLT3 tyrosine kinase inhibitors (TKIs) the induction of acquired resistance has emerged as a clinical problem. Therefore, optimization of the targeted therapy and potential treatment options to overcome resistance is currently the focus of clinical research. In this review we discuss the use and limitations of TKIs as a therapeutic strategy for the treatment of FLT3 mutated AML, including mechanisms of resistance to TKIs as well as possible novel strategies to improve FLT3 inhibitor therapy. PMID:23631653

  3. Crystal structure of the FLT3 kinase domain bound to the inhibitor quizartinib (AC220)

    SciTech Connect

    Zorn, Julie A.; Wang, Qi; Fujimura, Eric; Barros, Tiago; Kuriyan, John; Boggon, Titus J.

    2015-04-02

    More than 30% of acute myeloid leukemia (AML) patients possess activating mutations in the receptor tyrosine kinase FMS-like tyrosine kinase 3 or FLT3. A small-molecule inhibitor of FLT3 (known as quizartinib or AC220) that is currently in clinical trials appears promising for the treatment of AML. Here, we report the co-crystal structure of the kinase domain of FLT3 in complex with quizartinib. FLT3 with quizartinib bound adopts an “Abl-like” inactive conformation with the activation loop stabilized in the “DFG-out” orientation and folded back onto the kinase domain. This conformation is similar to that observed for the uncomplexed intracellular domain of FLT3 as well as for related receptor tyrosine kinases, except for a localized induced fit in the activation loop. The co-crystal structure reveals the interactions between quizartinib and the active site of FLT3 that are key for achieving its high potency against both wild-type FLT3 as well as a FLT3 variant observed in many AML patients. This co-complex further provides a structural rationale for quizartinib-resistance mutations.

  4. Crystal structure of the FLT3 kinase domain bound to the inhibitor quizartinib (AC220)

    DOE PAGES

    Zorn, Julie A.; Wang, Qi; Fujimura, Eric; ...

    2015-04-02

    More than 30% of acute myeloid leukemia (AML) patients possess activating mutations in the receptor tyrosine kinase FMS-like tyrosine kinase 3 or FLT3. A small-molecule inhibitor of FLT3 (known as quizartinib or AC220) that is currently in clinical trials appears promising for the treatment of AML. Here, we report the co-crystal structure of the kinase domain of FLT3 in complex with quizartinib. FLT3 with quizartinib bound adopts an “Abl-like” inactive conformation with the activation loop stabilized in the “DFG-out” orientation and folded back onto the kinase domain. This conformation is similar to that observed for the uncomplexed intracellular domain ofmore » FLT3 as well as for related receptor tyrosine kinases, except for a localized induced fit in the activation loop. The co-crystal structure reveals the interactions between quizartinib and the active site of FLT3 that are key for achieving its high potency against both wild-type FLT3 as well as a FLT3 variant observed in many AML patients. This co-complex further provides a structural rationale for quizartinib-resistance mutations.« less

  5. Spontaneous Remission in an Older Patient with Relapsed FLT3 ITD Mutant AML

    PubMed Central

    Mendler, Jason H.; Evans, Andrew; Deeb, George; Starostik, Petr; Wallace, Paul K.; Wang, Eunice S.

    2016-01-01

    Spontaneous remission (SR) of acute myeloid leukemia (AML) is a very rare phenomenon. AML characterized by FLT3 internal tandem duplication (FLT3 ITD) is typically associated with an aggressive clinical course with rapid progression, relapse, and short overall survival in the absence of transplantation. We report here the first case of SR of FLT3 ITD mutant AML in the literature. Our patient was an elderly woman with relapsed NPM1 and FLT3 ITD mutant AML whose disease underwent SR for a brief duration without precipitating cause. We review the potential immune mechanisms underlying SR in AML and discuss the implications for novel immunotherapeutic approaches for FLT3 mutant AML. PMID:28127477

  6. Midostaurin and Decitabine in Treating Older Patients With Newly Diagnosed Acute Myeloid Leukemia and FLT3 Mutation

    ClinicalTrials.gov

    2016-10-10

    Acute Myeloid Leukemia With FLT3/ITD Mutation; Acute Myeloid Leukemia With Gene Mutations; FLT3 Tyrosine Kinase Domain Point Mutation; Secondary Acute Myeloid Leukemia; Untreated Adult Acute Myeloid Leukemia

  7. IMC-EB10, an anti-FLT3 monoclonal antibody, prolongs survival and reduces nonobese diabetic/severe combined immunodeficient engraftment of some acute lymphoblastic leukemia cell lines and primary leukemic samples.

    PubMed

    Piloto, Obdulio; Nguyen, Bao; Huso, David; Kim, Kyu-Tae; Li, Yiwen; Witte, Larry; Hicklin, Daniel J; Brown, Patrick; Small, Donald

    2006-05-01

    The class III receptor tyrosine kinase FLT3 is expressed on the blasts of >90% of patients with B-lineage acute lymphoblastic leukemias (ALL). In addition, it is expressed at extremely high levels in ALL patients with mixed lineage leukemia rearrangements or hyperdiploidy and is sometimes mutated in these same patients. In this report, we investigate the effects of treating ALL cell lines and primary samples with human anti-FLT3 monoclonal antibodies (mAb) capable of preventing binding of FLT3 ligand. In vitro studies, examining the ability of two anti-FLT3 mAbs (IMC-EB10 and IMC-NC7) to affect FLT3 activation and downstream signaling in ALL cell lines and primary blasts, yielded variable results. FLT3 phosphorylation was consistently inhibited by IMC-NC7 treatment, but in some cell lines, IMC-EB10 actually stimulated FLT3 activation, possibly as a result of antibody-mediated receptor dimerization. Through antibody-dependent, cell-mediated cytotoxicity, such an antibody could still prove efficacious against leukemia cells in vivo. In fact, IMC-EB10 treatment significantly prolonged survival and/or reduced engraftment of several ALL cell lines and primary ALL samples in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. This occurred even when IMC-EB10 treatment resulted in FLT3 activation in vitro. Moreover, fluorescence-activated cell sorting and PCR analysis of IMC-EB10-treated NOD/SCID mice surviving 150 days post-leukemic cell injection revealed that FLT3 immunotherapy reduced leukemic engraftment below the level of detection in these assays (<0.001%). Furthermore, in vivo IMC-EB10 treatment did not select for resistant cells, because cells surviving IMC-EB10 treatment remain sensitive to IMC-EB10 cytotoxicity upon retransplantation. In vivo studies involving either partial depletion or activation of natural killer (NK) cells show that most of the cytotoxic effect of IMC-EB10 is mediated through NK cells. Therefore, such an antibody, either

  8. FLT3-ITD drives Ara-C resistance in leukemic cells via the induction of RUNX3.

    PubMed

    Damdinsuren, Anar; Matsushita, Hiromichi; Ito, Masatoshi; Tanaka, Masayuki; Jin, Guilan; Tsukamoto, Hideo; Asai, Satomi; Ando, Kiyoshi; Miyachi, Hayato

    2015-12-01

    Internal tandem duplication (ITD) mutations of the FLT3 gene (FLT3-ITD) are well known to correlate with a poor prognosis in acute myeloid leukemia (AML). We previously reported that FLT3-ITD confers resistance to cytosine arabinoside (Ara-C), a key cytotoxic agent in AML treatments. In order to elucidate the detailed molecular mechanisms underlying the Ara-C resistance induced by FLT3-ITD, we performed a microarray gene expression analysis of the human leukemic cell line K562 transduced with FLT3-ITD (K562/FLT3-ITD) and identified RUNX3 as a downstream target of FLT3-ITD. The transcriptional induction of the RUNX3 expression by FLT3-ITD was noted on a Luciferase assay. The knockdown of the RUNX3 expression in the K562/FLT3-ITD cells increased the sensitivity to Ara-C, and the exogenous expression of RUNX3 per se resulted in the enhancement of Ara-C resistance in the K562 cells. A relationship between the FLT3-ITD-induced RUNX3 expression and Ara-C resistance was also observed in AML cells with an endogenous FLT3-ITD expression. Collectively, these findings demonstrate that RUNX3 is a prerequisite for Ara-C resistance via FLT3-ITD signaling.

  9. FLT3-ITDs Instruct a Myeloid Differentiation and Transformation Bias in Lymphomyeloid Multipotent Progenitors

    PubMed Central

    Mead, Adam J.; Kharazi, Shabnam; Atkinson, Deborah; Macaulay, Iain; Pecquet, Christian; Loughran, Stephen; Lutteropp, Michael; Woll, Petter; Chowdhury, Onima; Luc, Sidinh; Buza-Vidas, Natalija; Ferry, Helen; Clark, Sally-Ann; Goardon, Nicolas; Vyas, Paresh; Constantinescu, Stefan N.; Sitnicka, Ewa; Nerlov, Claus; Jacobsen, Sten Eirik W.

    2013-01-01

    Summary Whether signals mediated via growth factor receptors (GFRs) might influence lineage fate in multipotent progenitors (MPPs) is unclear. We explored this issue in a mouse knockin model of gain-of-function Flt3-ITD mutation because FLT3-ITDs are paradoxically restricted to acute myeloid leukemia even though Flt3 primarily promotes lymphoid development during normal hematopoiesis. When expressed in MPPs, Flt3-ITD collaborated with Runx1 mutation to induce high-penetrance aggressive leukemias that were exclusively of the myeloid phenotype. Flt3-ITDs preferentially expanded MPPs with reduced lymphoid and increased myeloid transcriptional priming while compromising early B and T lymphopoiesis. Flt3-ITD-induced myeloid lineage bias involved upregulation of the transcription factor Pu.1, which is a direct target gene of Stat3, an aberrantly activated target of Flt3-ITDs, further establishing how lineage bias can be inflicted on MPPs through aberrant GFR signaling. Collectively, these findings provide new insights into how oncogenic mutations might subvert the normal process of lineage commitment and dictate the phenotype of resulting malignancies. PMID:23727242

  10. Improved FLT3/ITD PCR assay predicts outcome following allogeneic transplant for AML

    PubMed Central

    Grunwald, Michael R.; Tseng, Li-Hui; Lin, Ming-Tseh; Pratz, Keith W.; Eshleman, James R.; Levis, Mark J.; Gocke, Christopher D.

    2014-01-01

    Acute myeloid leukemia (AML) patients harboring internal tandem duplication (ITD) mutations of the FMS-like tyrosine kinase 3 (FLT3) gene carry a poor prognosis. While allogeneic transplantation may improve outcomes, relapse occurs frequently. The FLT3/ITD mutation has been deemed an unsuitable minimal residual disease (MRD) marker because it is unstable and because the standard assay for the mutation is relatively insensitive. The FLT3 mutation is undetectable by polymerase chain reaction (PCR) at pre- or post-transplant time points in many FLT3/ITD AML patients who subsequently relapse following transplant. We report the application of a new technique, tandem duplication PCR (TD-PCR), for detecting MRD in FLT3/ITD AML patients. Between October 2004 and January 2012, 54 FLT3/ITD AML patients in remission underwent transplantation at our institution. Of 37 patients with available Day 60 marrow samples, 28 (76%) were evaluable for MRD detection. In seven (25%) of the 28 patients, the FLT3/ITD mutation was detectable by TD-PCR, but not by standard PCR, on day 60. Six out of the seven patients (86%) with MRD by TD-PCR have relapsed to date compared with only 2 of 21 (10%) patients who were negative for MRD (p = 0.0003). The ability to detect MRD by this sensitive technique may provide an opportunity for early clinical intervention. PMID:25240816

  11. Integration of Hedgehog and mutant FLT3 signaling in myeloid leukemia

    PubMed Central

    Lim, Yiting; Gondek, Lukasz; Li, Li; Wang, Qiuju; Ma, Haley; Chang, Emily; Huso, David L.; Foerster, Sarah; Marchionni, Luigi; McGovern, Karen; Watkins, D. Neil; Peacock, Craig D.; Levis, Mark; Smith, B. Douglas; Merchant, Akil A.; Small, Donald; Matsui, William

    2015-01-01

    FLT3 internal tandem duplication (ITD) mutations resulting in constitutive kinase activity are common in acute myeloid leukemia (AML) and carry a poor prognosis. Several agents targeting FLT3 have been developed, but their limited clinical activity suggests that the inhibition of other factors contributing to the malignant phenotype is required. We examined gene expression data sets as well as primary specimens and found that the expression of GLI2, a major effector of the Hedgehog (Hh) signaling pathway, was increased in FLT3-ITD compared to wild type FLT3 AML. To examine the functional role of the Hh pathway, we studied mice in which Flt3-ITD expression results in an indolent myeloproliferative state and found that constitutive Hh signaling accelerated the development of AML by enhancing STAT5 signaling and the proliferation of bone marrow myeloid progenitors. Furthermore, combined FLT3 and Hh pathway inhibition limited leukemic growth in vitro and in vivo, and this approach may serve as a therapeutic strategy for FLT3-ITD AML. PMID:26062848

  12. Inhibition of MEK5 by BIX02188 induces apoptosis in cells expressing the oncogenic mutant FLT3-ITD

    SciTech Connect

    Razumovskaya, Elena; Sun, Jianmin; Roennstrand, Lars

    2011-08-26

    Highlights: {yields} In this study we have demonstrated that FLT3 activation leads to activation of ERK5. {yields} We have demonstrated that ERK5 is involved in activation of AKT downstream of FLT3. {yields} (BIX02188) blocks activation of ERK5 and induces apoptosis in FLT3 Ba/F3 cells. {yields} (BIX02188) induce apoptosis in the two leukemic cell lines MV4-11 and MOLM-13. -- Abstract: Fms-like tyrosine kinase-3 (FLT3) is a growth factor receptor normally expressed on hematopoietic progenitor cells. Approximately one third of all patients with AML carry an activating mutation in FLT3 that drives proliferation and survival of the leukemic cells. The most common activating mutation is the so-called internal tandem duplication (ITD), which involves an in-frame duplication of a segment of varying length in the region of the FLT3 gene that encodes the juxtamembrane domain. The pathways downstream of FLT3-ITD are partially known but further knowledge regarding the downstream signal transduction molecules is important in order to develop alternative strategies for pharmacological intervention. In this paper we have studied the role of MEK/ERK5 in FLT3-ITD mediated transformation. We have found that both wild-type FLT3 and FLT3-ITD activate MEK5 leading to the activation of ERK5. By use of the selective inhibitor of MEK5, (BIX02188), we have shown that activation of AKT downstream of FLT3 is partially dependent on ERK5. Furthermore, inhibition of MEK5/ERK5 induces apoptosis of both FLT3-ITD transfected Ba/F3 cells as well as the FLT3-ITD carrying leukemic cell lines MV4-11 and MOLM-13. These results suggest that MEK5/ERK5 is important for FLT3-ITD induced hematopoietic transformation and may thus represent an alternative therapeutic target in the treatment of FLT3-ITD positive leukemia.

  13. A pharmacodynamic study of the FLT3 inhibitor KW-2449 yields insight into the basis for clinical response.

    PubMed

    Pratz, Keith W; Cortes, Jorge; Roboz, Gail J; Rao, Niranjan; Arowojolu, Omotayo; Stine, Adam; Shiotsu, Yukimasa; Shudo, Aiko; Akinaga, Shiro; Small, Donald; Karp, Judith E; Levis, Mark

    2009-04-23

    Internal tandem duplication mutations of FLT3 (FLT3/ITD mutations) are common in acute myeloid leukemia (AML) and confer a poor prognosis. This would suggest that FLT3 is an ideal therapeutic target, but FLT3 targeted therapy has produced only modest benefits in clinical trials. Due to technical obstacles, the assessment of target inhibition in patients treated with FLT3 inhibitors has been limited and generally only qualitative. KW-2449 is a novel multitargeted kinase inhibitor that induces cytotoxicity in Molm14 cells (which harbor an FLT3/ITD mutation). The cytotoxic effect occurs primarily at concentrations sufficient to inhibit FLT3 autophosphorylation to less than 20% of its baseline. We report here correlative data from a phase 1 trial of KW-2449, a trial in which typical transient reductions in the peripheral blast counts were observed. Using quantitative measurement of FLT3 inhibition over time in these patients, we confirmed that FLT3 was inhibited, but only transiently to less than 20% of baseline. Our results suggest that the failure to fully inhibit FLT3 in sustained fashion may be an underlying reason for the minimal success of FLT3 inhibitors to date, and stress the importance of confirming in vivo target inhibition when taking a targeted agent into the clinical setting.

  14. Targeting FLT3-ITD signaling mediates ceramide-dependent mitophagy and attenuates drug resistance in AML.

    PubMed

    Dany, Mohammed; Gencer, Salih; Nganga, Rose; Thomas, Raquela J; Oleinik, Natalia; Baron, Kyla D; Szulc, Zdzislaw M; Ruvolo, Peter; Kornblau, Steven; Andreeff, Michael; Ogretmen, Besim

    2016-10-13

    Signaling pathways regulated by mutant Fms-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD), which mediate resistance to acute myeloid leukemia (AML) cell death, are poorly understood. Here, we reveal that pro-cell death lipid ceramide generation is suppressed by FLT3-ITD signaling. Molecular or pharmacologic inhibition of FLT3-ITD reactivated ceramide synthesis, selectively inducing mitophagy and AML cell death. Mechanistically, FLT3-ITD targeting induced ceramide accumulation on the outer mitochondrial membrane, which then directly bound autophagy-inducing light chain 3 (LC3), involving its I35 and F52 residues, to recruit autophagosomes for execution of lethal mitophagy. Short hairpin RNA (shRNA)-mediated knockdown of LC3 prevented AML cell death in response to FLT3-ITD inhibition by crenolanib, which was restored by wild-type (WT)-LC3, but not mutants of LC3 with altered ceramide binding (I35A-LC3 or F52A-LC3). Mitochondrial ceramide accumulation and lethal mitophagy induction in response to FLT3-ITD targeting was mediated by dynamin-related protein 1 (Drp1) activation via inhibition of protein kinase A-regulated S637 phosphorylation, resulting in mitochondrial fission. Inhibition of Drp1 prevented ceramide-dependent lethal mitophagy, and reconstitution of WT-Drp1 or phospho-null S637A-Drp1 but not its inactive phospho-mimic mutant (S637D-Drp1), restored mitochondrial fission and mitophagy in response to crenolanib in FLT3-ITD(+) AML cells expressing stable shRNA against endogenous Drp1. Moreover, activating FLT3-ITD signaling in crenolanib-resistant AML cells suppressed ceramide-dependent mitophagy and prevented cell death. FLT3-ITD(+) AML drug resistance is attenuated by LCL-461, a mitochondria-targeted ceramide analog drug, in vivo, which also induced lethal mitophagy in human AML blasts with clinically relevant FLT3 mutations. Thus, these data reveal a novel mechanism which regulates AML cell death by ceramide-dependent mitophagy in response

  15. Profiling of somatic mutations in acute myeloid leukemia with FLT3-ITD at diagnosis and relapse

    PubMed Central

    Nagata, Yasunobu; Kanojia, Deepika; Mayakonda, Anand; Yoshida, Kenichi; Haridas Keloth, Sreya; Zang, Zhi Jiang; Okuno, Yusuke; Shiraishi, Yuichi; Chiba, Kenichi; Tanaka, Hiroko; Miyano, Satoru; Ding, Ling-Wen; Alpermann, Tamara; Sun, Qiao-Yang; Lin, De-Chen; Chien, Wenwen; Madan, Vikas; Liu, Li-Zhen; Tan, Kar-Tong; Sampath, Abhishek; Venkatesan, Subhashree; Inokuchi, Koiti; Wakita, Satoshi; Yamaguchi, Hiroki; Chng, Wee Joo; Kham, Shirley-Kow Yin; Yeoh, Allen Eng-Juh; Sanada, Masashi; Schiller, Joanna; Kreuzer, Karl-Anton; Kornblau, Steven M.; Kantarjian, Hagop M.; Haferlach, Torsten; Lill, Michael; Kuo, Ming-Chung; Shih, Lee-Yung; Blau, Igor-Wolfgang; Blau, Olga; Yang, Henry; Ogawa, Seishi; Koeffler, H. Phillip

    2015-01-01

    Acute myeloid leukemia (AML) with an FLT3 internal tandem duplication (FLT3-ITD) mutation is an aggressive hematologic malignancy with a grave prognosis. To identify the mutational spectrum associated with relapse, whole-exome sequencing was performed on 13 matched diagnosis, relapse, and remission trios followed by targeted sequencing of 299 genes in 67 FLT3-ITD patients. The FLT3-ITD genome has an average of 13 mutations per sample, similar to other AML subtypes, which is a low mutation rate compared with that in solid tumors. Recurrent mutations occur in genes related to DNA methylation, chromatin, histone methylation, myeloid transcription factors, signaling, adhesion, cohesin complex, and the spliceosome. Their pattern of mutual exclusivity and cooperation among mutated genes suggests that these genes have a strong biological relationship. In addition, we identified mutations in previously unappreciated genes such as MLL3, NSD1, FAT1, FAT4, and IDH3B. Mutations in 9 genes were observed in the relapse-specific phase. DNMT3A mutations are the most stable mutations, and this DNMT3A-transformed clone can be present even in morphologic complete remissions. Of note, all AML matched trio samples shared at least 1 genomic alteration at diagnosis and relapse, suggesting common ancestral clones. Two types of clonal evolution occur at relapse: either the founder clone recurs or a subclone of the founder clone escapes from induction chemotherapy and expands at relapse by acquiring new mutations. Relapse-specific mutations displayed an increase in transversions. Functional assays demonstrated that both MLL3 and FAT1 exert tumor-suppressor activity in the FLT3-ITD subtype. An inhibitor of XPO1 synergized with standard AML induction chemotherapy to inhibit FLT3-ITD growth. This study clearly shows that FLT3-ITD AML requires additional driver genetic alterations in addition to FLT3-ITD alone. PMID:26438511

  16. Oncogenic roles of PRL-3 in FLT3-ITD induced acute myeloid leukaemia

    PubMed Central

    Park, Jung Eun; Yuen, Hiu Fung; Zhou, Jian Biao; Al-aidaroos, Abdul Qader O; Guo, Ke; Valk, Peter J; Zhang, Shu Dong; Chng, Wee Joo; Hong, Cheng William; Mills, Ken; Zeng, Qi

    2013-01-01

    FLT3-ITD mutations are prevalent mutations in acute myeloid leukaemia (AML). PRL-3, a metastasis-associated phosphatase, is a downstream target of FLT3-ITD. This study investigates the regulation and function of PRL-3 in leukaemia cell lines and AML patients associated with FLT3-ITD mutations. PRL-3 expression is upregulated by the FLT3-STAT5 signalling pathway in leukaemia cells, leading an activation of AP-1 transcription factors via ERK and JNK pathways. PRL-3-depleted AML cells showed a significant decrease in cell growth. Clinically, high PRL-3 mRNA expression was associated with FLT3-ITD mutations in four independent AML datasets with 1158 patients. Multivariable Cox-regression analysis on our Cohort 1 with 221 patients identified PRL-3 as a novel prognostic marker independent of other clinical parameters. Kaplan–Meier analysis showed high PRL-3 mRNA expression was significantly associated with poorer survival among 491 patients with normal karyotype. Targeting PRL-3 reversed the oncogenic effects in FLT3-ITD AML models in vitro and in vivo. Herein, we suggest that PRL-3 could serve as a prognostic marker to predict poorer survival and as a promising novel therapeutic target for AML patients. PMID:23929599

  17. Regulation of the Flt3 Gene in Haematopoietic Stem and Early Progenitor Cells

    PubMed Central

    Volpe, Giacomo; Walton, David Scott; Vegiopoulos, Alexandros; Del Pozzo, Walter; O’Neill, Laura Patricia; Frampton, Jonathan; Dumon, Stéphanie

    2015-01-01

    The MYB transcription factor plays critical roles in normal and malignant haematopoiesis. We previously showed that MYB was a direct activator of FLT3 expression within the context of acute myeloid leukaemia. During normal haematopoiesis, increasing levels of FLT3 expression determine a strict hierarchy within the haematopoietic stem and early progenitor compartment, which associates with lymphoid and myeloid commitment potential. We use the conditional deletion of the Myb gene to investigate the influence of MYB in Flt3 transcriptional regulation within the haematopoietic stem cell (HSC) hierarchy. In accordance with previous report, in vivo deletion of Myb resulted in rapid biased differentiation of HSC with concomitant loss of proliferation capacity. We find that loss of MYB activity also coincided with decreased FLT3 expression. At the chromatin level, the Flt3 promoter is primed in immature HSC, but occupancy of further intronic elements determines expression. Binding to these locations, MYB and C/EBPα need functional cooperation to activate transcription of the locus. This cooperation is cell context dependent and indicates that MYB and C/EBPα activities are inter-dependent in controlling Flt3 expression to influence lineage commitment of multipotential progenitors. PMID:26382271

  18. An essential pathway links FLT3-ITD, HCK and CDK6 in acute myeloid leukemia

    PubMed Central

    Lopez, Sophie; Voisset, Edwige; Tisserand, Julie C.; Mosca, Cyndie; Prebet, Thomas; Santamaria, David; Dubreuil, Patrice; Sepulveda, Paulo De

    2016-01-01

    CDK4/CDK6 and RB proteins drive the progression through the G1 phase of the cell cycle. In acute myeloid leukemia (AML), the activity of the CDK/Cyclin D complex is increased. The mechanism involved is unknown, as are the respective roles played by CDK4 or CDK6 in this process. Here, we report that AML cells carrying FLT3-ITD mutations are dependent on CDK6 for cell proliferation while CDK4 is not essential. We showed that FLT3-ITD signaling is responsible for CDK6 overexpression, through a pathway involving the SRC-family kinase HCK. Accordingly, FLT3-ITD failed to transform primary hematopoietic progenitor cells from Cdk6−/− mice. Our results demonstrate that CDK6 is the primary target of CDK4/CDK6 inhibitors in FLT3-ITD positive AML. Furthermore, we delineate an essential protein kinase pathway -FLT3/HCK/CDK6- in the context of AML with FLT3-ITD mutations. PMID:27323399

  19. Oncogenic roles of PRL-3 in FLT3-ITD induced acute myeloid leukaemia.

    PubMed

    Park, Jung Eun; Yuen, Hiu Fung; Zhou, Jian Biao; Al-Aidaroos, Abdul Qader O; Guo, Ke; Valk, Peter J; Zhang, Shu Dong; Chng, Wee Joo; Hong, Cheng William; Mills, Ken; Zeng, Qi

    2013-09-01

    FLT3-ITD mutations are prevalent mutations in acute myeloid leukaemia (AML). PRL-3, a metastasis-associated phosphatase, is a downstream target of FLT3-ITD. This study investigates the regulation and function of PRL-3 in leukaemia cell lines and AML patients associated with FLT3-ITD mutations. PRL-3 expression is upregulated by the FLT3-STAT5 signalling pathway in leukaemia cells, leading an activation of AP-1 transcription factors via ERK and JNK pathways. PRL-3-depleted AML cells showed a significant decrease in cell growth. Clinically, high PRL-3 mRNA expression was associated with FLT3-ITD mutations in four independent AML datasets with 1158 patients. Multivariable Cox-regression analysis on our Cohort 1 with 221 patients identified PRL-3 as a novel prognostic marker independent of other clinical parameters. Kaplan-Meier analysis showed high PRL-3 mRNA expression was significantly associated with poorer survival among 491 patients with normal karyotype. Targeting PRL-3 reversed the oncogenic effects in FLT3-ITD AML models in vitro and in vivo. Herein, we suggest that PRL-3 could serve as a prognostic marker to predict poorer survival and as a promising novel therapeutic target for AML patients.

  20. SKLB-677, an FLT3 and Wnt/β-catenin signaling inhibitor, displays potent activity in models of FLT3-driven AML

    PubMed Central

    Ma, Shuang; Yang, Ling-Ling; Niu, Ting; Cheng, Chuan; Zhong, Lei; Zheng, Ming-Wu; Xiong, Yu; Li, Lin-Li; Xiang, Rong; Chen, Li-Juan; Zhou, Qiao; Wei, Yu-Quan; Yang, Sheng-Yong

    2015-01-01

    FLT3 has been identified as a valid target for the treatment of acute myeloid leukemia (AML), and some FLT3 inhibitors have shown very good efficacy in treating AML in clinical trials. Nevertheless, recent studies indicated that relapse and drug resistance are still difficult to avoid, and leukemia stem cells (LSCs) are considered one of the most important contributors. Here, we report the characterization of SKLB-677, a new FLT3 inhibitor developed by us recently. SKLB-677 exhibits low nanomolar potency in biochemical and cellular assays. It is efficacious in animal models at doses as low as 1mg/kg when administrated orally once daily. In particular, SKLB-677 but not first-generation and second-generation FLT3 inhibitors in clinical trials has the ability to inhibit Wnt/β-catenin signaling; Wnt/β-catenin signaling is required for the development of LSCs, but not necessary for the development of adult hematopoietic stem cells (HSCs). This compound indeed showed considerable suppression effects on leukemia stem-like cells in in vitro functional assays, but had no influence on normal HSCs. Collectively, SKLB-677 is an interesting lead compound for the treatment of AML, and deserves further investigations. PMID:26497577

  1. Deletion and deletion/insertion mutations in the juxtamembrane domain of the FLT3 gene in adult acute myeloid leukemia

    PubMed Central

    Deeb, Kristin K.; Smonskey, Matthew T.; DeFedericis, HanChun; Deeb, George; Sait, Sheila N.J.; Wetzler, Meir; Wang, Eunice S.; Starostik, Petr

    2014-01-01

    In contrast to FLT3 ITD mutations, in-frame deletions in the FLT3 gene have rarely been described in adult acute leukemia. We report two cases of AML with uncommon in-frame mutations in the juxtamembrane domain of the FLT3 gene: a 3-bp (c.1770_1774delCTACGinsGT; p.F590_V592delinsLF) deletion/insertion and a 12-bp (c.1780_1791delTTCAGAGAATAT; p.F594_Y597del) deletion. We verified by sequencing that the reading frame of the FLT3 gene was preserved and by cDNA analysis that the mRNA of the mutant allele was expressed in both cases. Given the recent development of FLT3 inhibitors, our findings may be of therapeutic value for AML patients harboring similar FLT3 mutations. PMID:25379410

  2. FLT3-ITD Mutations in Acute Myeloid Leukemia Patients in Northeast Thailand.

    PubMed

    Kumsaen, Piyawan; Fucharoen, Goonnapa; Sirijerachai, Chittima; Chainansamit, Su-On; Wisanuyothin, Nittaya; Kuwatjanakul, Pichayanan; Wiangnon, Surapon

    2016-01-01

    The FLT3-ITD mutation is one of the most frequent genetic abnormalities in acute myeloid leukemia (AML) where it is associated with a poor prognosis. The FLT3-ITD mutation could, therefore, be a potential molecular prognostic marker important for risk-stratified treatment options. We amplified the FLT3 gene at exon 14 and 15 in 52 AML patients (aged between 2 months and 74 years) from 4 referral centers (a university hospital and 3 regional hospitals in Northeast Thailand), using a simple PCR method. FLT3-ITD mutations were found in 10 patients (19.2%), being more common in adults than in children (21.1% vs. 14.3%) and more prevalent in patients with acute promyelocytic leukemia (AML-M3) than AML-non M3 (4 of 10 AML-M3 vs. 6 of 42 AML- non M3 patients). Duplication sequences varied in size-between 27 and 171 nucleotides (median=63.5) and in their location. FLT3-ITD mutations with common duplication sequences accounted for a significant percentage in AML patients in northeastern Thailand. This simple PCR method is feasible for routine laboratory practice and these data could help tailor use of the national protocol for AML.

  3. Flt3 is a target of coumestrol in protecting against UVB-induced skin photoaging.

    PubMed

    Park, Gaeun; Baek, Sohee; Kim, Jong-Eun; Lim, Tae-gyu; Lee, Charles C; Yang, Hee; Kang, Young-Gyu; Park, Jun Seong; Augustin, Martin; Mrosek, Michael; Lee, Chang Yong; Dong, Zigang; Huber, Robert; Lee, Ki Won

    2015-12-01

    While skin aging is a naturally occurring process by senescence, exposure to ultraviolet (UV) radiation accelerates wrinkle formation and sagging of skin. UV induces skin aging by degrading collagen via activating matrix metalloproteinases (MMPs). In this study, we show that coumestrol, a metabolite of the soybean isoflavone daidzein, has a preventive effect on skin photoaging in three-dimensional human skin equivalent model. Coumestrol inhibited UVB-induced MMP-1 expression and activity. Whole human kinase profiling assay identified FLT3 kinase as a novel target protein of coumestrol in UVB-induced signaling pathway in skin. Coumestrol suppresses FLT3 kinase activity, and subsequently, Ras/MEK/ERK and Akt/p70 ribosomal S6 kinase pathway. This suppresses AP-1 activity and in turn, diminishes MMP-1 gene transcription. Using X-ray crystallography, the binding of coumestrol to FLT3 was defined and implied ATP-competitive inhibition. Residues Lys644 and Phe830 showed local changes to accommodate coumestrol in the ATP-binding pocket. 4-APIA, a pharmacological inhibitor of FLT3, inhibited MMP-1 expression and induced signal transduction changes similar to coumestrol. Taken together, coumestrol inhibits UVB-induced MMP-1 expression by suppressing FLT3 kinase activity. These findings suggest that coumestrol is a novel dietary compound with potential application in preventing and improving UVB-associated skin aging.

  4. FLT3 is implicated in cytarabine transport by human equilibrative nucleoside transporter 1 in pediatric acute leukemia

    PubMed Central

    Català, Albert; Pastor-Anglada, Marçal; Caviedes-Cárdenas, Liska; Malatesta, Roberta; Rives, Susana; Vega-García, Nerea

    2016-01-01

    FLT3 abnormalities are negative prognostic markers in acute leukemia. Infant leukemias are a subgroup with frequent MLL (KMT2A) rearrangements, FLT3 overexpression and high sensitivity to cytarabine, but dismal prognosis. Cytarabine is transported into cells by Human Equilibrative Nucleoside Transporter-1 (hENT1, SLC29A1), but the mechanisms that regulate hENT1 in acute leukemia have been scarcely studied. We explored the expression and functional link between FLT3 and main cytarabine transporters in 50 pediatric patients diagnosed with acute lymphoblastic leukemia and MLL rearrangement (ALL-MLL+) and other subtypes of leukemia, and in leukemia cell lines. A significant positive correlation was found between FLT3 and hENT1 expression in patients. Cytarabine uptake into cells was mediated mainly by hENT1, hENT2 and hCNT1. hENT1-mediated uptake of cytarabine was transiently abolished by the FLT3 inhibitor PKC412, and this effect was associated with decreased hENT1 mRNA and protein levels. Noticeably, the cytotoxicity of cytarabine was lower when cells were first exposed to FLT3 inhibitors (PKC412 or AC220), probably due to decreased hENT1 activity, but we observed a higher cytotoxic effect if FLT3 inhibitors were administered after cytarabine. FLT3 regulates hENT1 activity and thereby affects cytarabine cytotoxicity. The sequence of administration of cytarabine and FLT3 inhibitors is important to maintain their efficacy. PMID:27391351

  5. Evolution of a FLT3-TKD mutated subclone at meningeal relapse in acute promyelocytic leukemia

    PubMed Central

    Bochtler, Tilmann; Fröhling, Stefan; Weichert, Wilko; Endris, Volker; Thiede, Christian; Hutter, Barbara; Hundemer, Michael; Ho, Anthony D.; Krämer, Alwin

    2016-01-01

    Here, we report the case of an acute promyelocytic leukemia (APL) patient who—although negative for FLT3 mutations at diagnosis—developed isolated FLT3 tyrosine kinase II domain (FLT3-TKD)-positive meningeal relapse, which, in retrospect, could be traced back to a minute bone marrow subclone present at first diagnosis. Initially, the 48-yr-old female diagnosed with high-risk APL had achieved complete molecular remission after standard treatment with all-trans retinoic acid (ATRA) and chemotherapy according to the AIDA (ATRA plus idarubicin) protocol. Thirteen months after the start of ATRA maintenance, the patient suffered clinically overt meningeal relapse along with minute molecular traces of PML/RARA (promyelocytic leukemia/retinoic acid receptor alpha) in the bone marrow. Following treatment with arsenic trioxide and ATRA in combination with intrathecal cytarabine and methotrexate, the patient achieved a complete molecular remission in both cerebrospinal fluid (CSF) and bone marrow, which currently lasts for 2 yr after completion of therapy. Whole-exome sequencing and subsequent ultradeep targeted resequencing revealed a heterozygous FLT3-TKD mutation in CSF leukemic cells (p.D835Y, c.2503G>T, 1000/1961 reads [51%]), which was undetectable in the concurrent bone marrow sample. Interestingly, the FLT3-TKD mutated meningeal clone originated from a small bone marrow subclone present in a variant allele frequency of 0.4% (6/1553 reads) at initial diagnosis. This case highlights the concept of clonal evolution with a subclone harboring an additional mutation being selected as the “fittest” and leading to meningeal relapse. It also further supports earlier suggestions that FLT3 mutations may play a role for migration and clonal expansion in the CSF sanctuary site. PMID:27626069

  6. The Flt3 Internal Tandem Duplication Alters Chemotherapy Response In Vitro and In Vivo in a p53-Dependent Manner

    PubMed Central

    Pardee, Timothy S.; Zuber, Johannes; Lowe, Scott W.

    2011-01-01

    Objective The FLT3 internal tandem duplication (Flt3-ITD) confers a worse prognosis for patients with acute myeloid leukemia (AML); however, the mechanisms involved are unknown. As AML is treated with cytarabine (Ara-C) and an anthracycline we sought to determine the effects of the Flt3-ITD on response to these agents. Methods A genetically defined mouse model of AML was used to examine the effects of the Flt3-ITD on response to cytarabine and doxorubicin in vitro and in vivo. Results In vitro, the Flt3-ITD conferred resistance to doxorubicin and doxorubicin plus Ara-C, but sensitivity to Ara-C alone. This resistance was reversible by the Flt3-ITD inhibitor sorafenib. The Flt3-ITD did not affect DNA damage levels following treatment but was associated with increased levels of p53. The p53 response was critical to the observed changes as the Flt3-ITD had no effect on chemotherapy response in the setting of p53 null AML. In vivo, the Flt3-ITD accelerated engraftment that was partially reversible by Ara-C but not doxorubicin. Additionally, Ara-C provided a significant reduction in disease burden and a survival advantage that was not increased by the addition of doxorubicin. Doxorubicin alone lead to only minimal disease reduction and no survival benefit. Conclusions These data demonstrate that the Flt3-ITD confers sensitivity to cytarabine, but resistance to doxorubicin in a manner that depends on p53. Thus, patients with Flt3-ITD positive AML may not benefit from treatment with an anthracycline. PMID:21288478

  7. Selective FLT3 inhibitor FI-700 neutralizes Mcl-1 and enhances p53-mediated apoptosis in AML cells with activating mutations of FLT3 through Mcl-1/Noxa axis.

    PubMed

    Kojima, K; Konopleva, M; Tsao, T; Andreeff, M; Ishida, H; Shiotsu, Y; Jin, L; Tabe, Y; Nakakuma, H

    2010-01-01

    Treatment using Fms-like tyrosine kinase-3 (FLT3) inhibitors is a promising approach to overcome the dismal prognosis of acute myeloid leukemia (AML) with activating FLT3 mutations. Current trials are combining FLT3 inhibitors with p53-activating conventional chemotherapy. The mechanisms of cytotoxicity of FLT3 inhibitors are poorly understood. We investigated the interaction of FLT3 and p53 pathways after their simultaneous blockade using the selective FLT3 inhibitor FI-700 and the MDM2 inhibitor Nutlin-3 in AML. We found that FI-700 immediately reduced antiapoptotic Mcl-1 levels and enhanced Nutlin-induced p53-mediated mitochondrial apoptosis in FLT3/internal tandem duplication cells through the Mcl-1/Noxa axis. FI-700 induced proteasome-mediated degradation of Mcl-1, resulting in the reduced ability of Mcl-1 to sequester proapoptotic Bim. Nutlin-3 induced Noxa, which displaced Bim from Mcl-1. The FI-700/Nutlin-3 combination profoundly activated Bax and induced apoptosis. Our findings suggest that FI-700 actively enhances p53 signaling toward mitochondrial apoptosis and that a combination strategy aimed at inhibiting FLT3 and activating p53 signaling could potentially be effective in AML.

  8. Plasma inhibitory activity (PIA): a pharmacodynamic assay reveals insights into the basis for cytotoxic response to FLT3 inhibitors

    PubMed Central

    Levis, Mark; Brown, Patrick; Smith, B. Douglas; Stine, Adam; Pham, Rosalyn; Stone, Richard; DeAngelo, Daniel; Galinsky, Ilene; Giles, Frank; Estey, Elihu; Kantarjian, Hagop; Cohen, Pamela; Wang, Yanfeng; Roesel, Johannes; Karp, Judith E.; Small, Donald

    2006-01-01

    We have developed a useful surrogate assay for monitoring the efficacy of FLT3 inhibition in patients treated with oral FLT3 inhibitors. The plasma inhibitory activity (PIA) for FLT3 correlates with clinical activity in patients treated with CEP-701 and PKC412. Using the PIA assay, along with in vitro phosphorylation and cytotoxicity assays in leukemia cells, we compared PKC412 and its metabolite, CGP52421, with CEP-701. While both drugs could effectively inhibit FLT3 in vitro, CEP-701 was more cytotoxic to primary samples at comparable levels of FLT3 inhibition. PKC412 appears to be more selective than CEP-701 and therefore less effective at inducing cytotoxicity in primary acute myeloid leukemia (AML) samples in vitro. However, the PKC412 metabolite CGP52421 is less selective than its parent compound, PKC412, and is more cytotoxic against primary blast samples at comparable levels of FLT3 inhibition. The plasma inhibitory activity assay represents a useful correlative tool in the development of small-molecule inhibitors. Our application of this assay has revealed that the metabolite CGP52421 may contribute a significant portion of the antileukemia activity observed in patients receiving oral PKC412. Additionally, our results suggest that nonselectivity may constitute an important component of the cytotoxic effect of FLT3 inhibitors in FLT3-mutant AML. PMID:16857987

  9. FLT3 and NPM-1 mutations in a cohort of acute promyelocytic leukemia patients from India

    PubMed Central

    Swaminathan, Suchitra; Garg, Swati; Madkaikar, Manisha; Gupta, Maya; Jijina, Farah; Ghosh, Kanjaksha

    2014-01-01

    Background: Acute promyelocytic leukemia (APL) with t (15;17) is a distinct category of acute myeloid leukemia (AML) and is reported to show better response to anthracyclin based chemotherapy. A favorable overall prognosis over other subtypes of AML has been reported for APL patients but still about 15% patients relapse. Methods: This study evaluated the presence of Famus like tyrosine kinase-3 (FLT3) and nucleophosmin-1 (NPM1) gene mutations in a cohort of 40 APL patients. Bone marrow/peripheral blood samples from patients at the time of diagnosis and follow-up were processed for immunophenotyping, cytogenetic markers and isolation of DNA and RNA. Samples were screened for the presence of mutations in FLT3 and NPM1 genes using polymerase chain reaction followed by sequencing. Results: Frequency of FLT3/internal tandem duplication and FLT3/tyrosine kinase domain was found to be 25% and 7% respectively. We observed a high frequency of NPM1 mutation (45%) in the present population of APL patients. PMID:25400345

  10. Facile identification of dual FLT3-Aurora A inhibitors: a computer-guided drug design approach.

    PubMed

    Chang Hsu, Yung; Ke, Yi-Yu; Shiao, Hui-Yi; Lee, Chieh-Chien; Lin, Wen-Hsing; Chen, Chun-Hwa; Yen, Kuei-Jung; Hsu, John T-A; Chang, Chungming; Hsieh, Hsing-Pang

    2014-05-01

    Computer-guided drug design is a powerful tool for drug discovery. Herein we disclose the use of this approach for the discovery of dual FMS-like receptor tyrosine kinase-3 (FLT3)-Aurora A inhibitors against cancer. An Aurora hit compound was selected as a starting point, from which 288 virtual molecules were screened. Subsequently, some of these were synthesized and evaluated for their capacity to inhibit FLT3 and Aurora kinase A. To further enhance FLT3 inhibition, structure-activity relationship studies of the lead compound were conducted through a simplification strategy and bioisosteric replacement, followed by the use of computer-guided drug design to prioritize molecules bearing a variety of different terminal groups in terms of favorable binding energy. Selected compounds were then synthesized, and their bioactivity was evaluated. Of these, one novel inhibitor was found to exhibit excellent inhibition of FLT3 and Aurora kinase A and exert a dramatic antiproliferative effect on MOLM-13 and MV4-11 cells, with an IC50 value of 7 nM. Accordingly, it is considered a highly promising candidate for further development.

  11. A novel bicistronic high-capacity gutless adenovirus vector that drives constitutive expression of herpes simplex virus type 1 thymidine kinase and tet-inducible expression of Flt3L for glioma therapeutics.

    PubMed

    Puntel, Mariana; Muhammad, A K M G; Candolfi, Marianela; Salem, Alireza; Yagiz, Kader; Farrokhi, Catherine; Kroeger, Kurt M; Xiong, Weidong; Curtin, James F; Liu, Chunyan; Bondale, Niyati S; Lerner, Jonathan; Pechnick, Robert N; Palmer, Donna; Ng, Philip; Lowenstein, Pedro R; Castro, Maria G

    2010-06-01

    Glioblastoma multiforme (GBM) is a deadly primary brain tumor. Conditional cytotoxic/immune-stimulatory gene therapy (Ad-TK and Ad-Flt3L) elicits tumor regression and immunological memory in rodent GBM models. Since the majority of patients enrolled in clinical trials would exhibit adenovirus immunity, which could curtail transgene expression and therapeutic efficacy, we used high-capacity adenovirus vectors (HC-Ads) as a gene delivery platform. Herein, we describe for the first time a novel bicistronic HC-Ad driving constitutive expression of herpes simplex virus type 1 thymidine kinase (HSV1-TK) and inducible Tet-mediated expression of Flt3L within a single-vector platform. We achieved anti-GBM therapeutic efficacy with no overt toxicities using this bicistronic HC-Ad even in the presence of systemic Ad immunity. The bicistronic HC-Ad-TK/TetOn-Flt3L was delivered into intracranial gliomas in rats. Survival, vector biodistribution, neuropathology, systemic toxicity, and neurobehavioral deficits were assessed for up to 1 year posttreatment. Therapeutic efficacy was also assessed in animals preimmunized against Ads. We demonstrate therapeutic efficacy, with vector genomes being restricted to the brain injection site and an absence of overt toxicities. Importantly, antiadenoviral immunity did not inhibit therapeutic efficacy. These data represent the first report of a bicistronic vector platform driving the expression of two therapeutic transgenes, i.e., constitutive HSV1-TK and inducible Flt3L genes. Further, our data demonstrate no promoter interference and optimum gene delivery and expression from within this single-vector platform. Analysis of the efficacy, safety, and toxicity of this bicistronic HC-Ad vector in an animal model of GBM strongly supports further preclinical testing and downstream process development of HC-Ad-TK/TetOn-Flt3L for a future phase I clinical trial for GBM.

  12. A Novel Tandem Duplication Assay to Detect Minimal Residual Disease in FLT3/ITD AML

    PubMed Central

    Lin, Ming-Tseh; Tseng, Li-Hui; Dudley, Jonathan C.; Riel, Stacey; Tsai, Harrison; Zheng, Gang; Pratz, Keith W.; Levis, Mark J.; Gocke, Christopher D.

    2015-01-01

    Background Internal tandem duplication (ITD) of the FLT3 gene is associated with poor prognosis in acute myeloid leukemia (AML) patients with a normal karyotype. The current standard PCR assay for FLT3/ITD detection is not sufficiently sensitive to monitor minimal residual disease (MRD). Clone-specific assays may have sufficient sensitivity but are not practical to implement, since each clone-specific primer/probe requires clinical validation. Objective To develop an assay for clinical molecular diagnostics laboratories to monitor MRD in FLT3/ITD AMLs. Methods We designed a simple novel assay, tandem duplication PCR (TD-PCR), and tested its sensitivity, specificity and clinical utility in FLT3/ITD AML patients. Results TD-PCR was capable of detecting a single ITD molecule and was applicable to 75% of ITD mutants tested. TD-PCR detected MRD in bone marrow prior to patient relapse. TD-PCR also identified low level ITD mutants not only in FLT3/ITD AMLs but also in initial diagnostic specimens reportedly negative by the standard assay in patients who progressed with the same ITDs detected by the TD-PCR assay. Conclusion Detection of MRD by TD-PCR may guide patient selection for early clinical intervention. In contrast to clone-specific approaches, TD-PCR assay can be more easily validated for MRD detection in clinical laboratories due to standardized primers and a universal positive control. In addition, our results on multi-clonality and low-level ITDs suggest that further studies are warranted to elucidate their clinical/biological significance. PMID:26446915

  13. ABT-869, a multitargeted receptor tyrosine kinase inhibitor: inhibition of FLT3 phosphorylation and signaling in acute myeloid leukemia.

    PubMed

    Shankar, Deepa B; Li, Junling; Tapang, Paul; Owen McCall, J; Pease, Lori J; Dai, Yujia; Wei, Ru-Qi; Albert, Daniel H; Bouska, Jennifer J; Osterling, Donald J; Guo, Jun; Marcotte, Patrick A; Johnson, Eric F; Soni, Niru; Hartandi, Kresna; Michaelides, Michael R; Davidsen, Steven K; Priceman, Saul J; Chang, Jenny C; Rhodes, Katrin; Shah, Neil; Moore, Theodore B; Sakamoto, Kathleen M; Glaser, Keith B

    2007-04-15

    In 15% to 30% of patients with acute myeloid leukemia (AML), aberrant proliferation is a consequence of a juxtamembrane mutation in the FLT3 gene (FMS-like tyrosine kinase 3-internal tandem duplication [FLT3-ITD]), causing constitutive kinase activity. ABT-869 (a multitargeted receptor tyrosine kinase inhibitor) inhibited the phosphorylation of FLT3, STAT5, and ERK, as well as Pim-1 expression in MV-4-11 and MOLM-13 cells (IC(50) approximately 1-10 nM) harboring the FLT3-ITD. ABT-869 inhibited the proliferation of these cells (IC(50) = 4 and 6 nM, respectively) through the induction of apoptosis (increased sub-G(0)/G(1) phase, caspase activation, and PARP cleavage), whereas cells harboring wild-type (wt)-FLT3 were less sensitive. In normal human blood spiked with AML cells, ABT-869 inhibited phosphorylation of FLT3 (IC(50) approximately 100 nM), STAT5, and ERK, and decreased Pim-1 expression. In methylcellulose-based colony-forming assays, ABT-869 had no significant effect up to 1000 nM on normal hematopoietic progenitor cells, whereas in AML patient samples harboring both FLT3-ITD and wt-FLT3, ABT-869 inhibited colony formation (IC(50) = 100 and 1000 nM, respectively). ABT-869 dose-dependently inhibited MV-4-11 and MOLM-13 flank tumor growth, prevented tumor formation, regressed established MV-4-11 xenografts, and increased survival by 20 weeks in an MV-4-11 engraftment model. In tumors, ABT-869 inhibited FLT3 phosphorylation, induced apoptosis (transferase-mediated dUTP nick-end labeling [TUNEL]) and decreased proliferation (Ki67). ABT-869 is under clinical development for AML.

  14. Potent Activity of Ponatinib (AP24534) in Models of FLT3-Driven Acute Myeloid Leukemia and Other Hematologic Malignancies

    PubMed Central

    Gozgit, Joseph M.; Wong, Matthew J.; Wardwell, Scott; Tyner, Jeffrey W.; Loriaux, Marc M.; Mohemmad, Qurish K.; Narasimhan, Narayana I.; Shakespeare, William C.; Wang, Frank; Druker, Brian J.; Clackson, Tim; Rivera, Victor M.

    2011-01-01

    Ponatinib (AP24534) is a novel multitargeted kinase inhibitor that potently inhibits native and mutant BCR-ABL at clinically achievable drug levels. Ponatinib also has in vitro inhibitory activity against a discrete set of kinases implicated in the pathogenesis of other hematologic malignancies, including FLT3, KIT, fibroblast growth factor receptor 1 (FGFR1), and platelet derived growth factor receptor α (PDGFRα). Here, using leukemic cell lines containing activated forms of each of these receptors, we show that ponatinib potently inhibits receptor phosphorylation and cellular proliferation with IC50 values comparable to those required for inhibition of BCR-ABL (0.3 to 20 nmol/L). The activity of ponatinib against the FLT3-ITD mutant, found in up to 30% of acute myeloid leukemia (AML) patients, was particularly notable. In MV4-11 (FLT3-ITD+/+) but not RS4;11 (FLT3-ITD−/−) AML cells, ponatinib inhibited FLT3 signaling and induced apoptosis at concentrations of less than 10 nmol/L. In an MV4-11 mouse xenograft model, once daily oral dosing of ponatinib led to a dose-dependent inhibition of signaling and tumor regression. Ponatinib inhibited viability of primary leukemic blasts from a FLT3-ITD positive AML patient (IC50 4 nmol/L) but not those isolated from 3 patients with AML expressing native FLT3. Overall, these results support the investigation of ponatinib in patients with FLT3-ITD–driven AML and other hematologic malignancies driven by KIT, FGFR1, or PDGFRα. PMID:21482694

  15. FLT3 mutational status is an independent risk factor for adverse outcomes after allogeneic transplantation in AML

    PubMed Central

    Li, Yumeng; Braun, Thomas; Chang, Lawrence; Bixby, Dale; Hanauer, David A.; Chughtai, Komal A.; Gatza, Erin; Couriel, Daniel; Goldstein, Steven; Pawarode, Attaphol; Reddy, Pavan; Riwes, Mary; Connelly, James; Harris, Andrew; Kitko, Carrie; Levine, John; Yanik, Greg

    2015-01-01

    Allogeneic HCT has been increasingly used in the setting of FLT3 mutated AML. However, its role in conferring durable relapse-free intervals remains in question. Herein, we sought to investigate FLT3 mutational status on transplant outcomes. We conducted a retrospective cohort study of 262 consecutive AML patients who underwent first-time allogeneic HCT (2008-2014), of whom 171 had undergone FLT3-ITD mutational testing. FLT3 mutated AML was associated with nearly twice the relapse risk (RR) compared with those without FLT3 mutation 3 years post-HCT (63% vs. 37%, P<0.001), and with a shorter median time to relapse (100 vs. 121 days). FLT3 mutational status remained significantly associated with this outcome after controlling for patient, disease, and transplant-related risk factors (P<0.05). Multivariate analysis showed a significant association of FLT3 mutation with increased 3-year RR (HR 3.63, 95% CI: 2.13, 6.19, P<0.001), and inferior disease-free survival (HR 2.05, 95% CI: 1.29, 3.27, P<0.01) and overall survival (HR 1.92, 95% CI: 1.14, 3.24, P<0.05). These data demonstrate high risk of early relapse after allogeneic HCT for FLT3 mutated AML that translates into adverse disease-free and overall survival outcomes. Additional targeted and coordinated interventions are needed to maintain durable remission after allogeneic HCT in this high-risk population. PMID:26191952

  16. Mll partial tandem duplication and Flt3 internal tandem duplication in a double knock-in mouse recapitulates features of counterpart human acute myeloid leukemias

    PubMed Central

    Zorko, Nicholas A.; Bernot, Kelsie M.; Whitman, Susan P.; Siebenaler, Ronald F.; Ahmed, Elshafa H.; Marcucci, Gabriele G.; Yanes, Daniel A.; McConnell, Kathleen K.; Mao, Charlene; Kalu, Chidimma; Zhang, Xiaoli; Jarjoura, David; Dorrance, Adrienne M.; Heerema, Nyla A.; Lee, Benjamin H.; Huang, Gang; Marcucci, Guido

    2012-01-01

    The MLL-partial tandem duplication (PTD) associates with high-risk cytogenetically normal acute myeloid leukemia (AML). Concurrent presence of FLT3-internal tandem duplication (ITD) is observed in 25% of patients with MLL-PTD AML. However, mice expressing either Mll-PTD or Flt3-ITD do not develop AML, suggesting that 2 mutations are necessary for the AML phenotype. Thus, we generated a mouse expressing both Mll-PTD and Flt3-ITD. MllPTD/WT:Flt3ITD/WT mice developed acute leukemia with 100% penetrance, at a median of 49 weeks. As in human MLL-PTD and/or the FLT3-ITD AML, mouse blasts exhibited normal cytogenetics, decreased Mll-WT-to-Mll-PTD ratio, loss of the Flt3-WT allele, and increased total Flt3. Highlighting the adverse impact of FLT3-ITD dosage on patient survival, mice with homozygous Flt3-ITD alleles, MllPTD/WT:Flt3ITD/ITD, demonstrated a nearly 30-week reduction in latency to overt AML. Here we demonstrate, for the first time, that Mll-PTD contributes to leukemogenesis as a gain-of-function mutation and describe a novel murine model closely recapitulating human AML. PMID:22674806

  17. PML/RARα and FLT3-ITD induce an APL-like disease in a mouse model

    PubMed Central

    Kelly, Louise M.; Kutok, Jeffrey L.; Williams, Ifor R.; Boulton, Christina L.; Amaral, Sonia M.; Curley, David P.; Ley, Timothy J.; Gilliland, D. Gary

    2002-01-01

    Acute promyelocytic leukemia (APL) cells invariably express aberrant fusion proteins involving the retinoic acid receptor α (RARα). The most common fusion partner is promyelocytic leukemia protein (PML), which is fused to RARα in the balanced reciprocal chromosomal translocation, t(15;17)(q22:q11). Expression of PML/RARα from the cathepsin G promoter in transgenic mice causes a nonfatal myeloproliferative syndrome in all mice; about 15% go on to develop APL after a long latent period, suggesting that additional mutations are required for the development of APL. A candidate target gene for a second mutation is FLT3, because it is mutated in approximately 40% of human APL cases. Activating mutations in FLT3, including internal tandem duplication (ITD) in the juxtamembrane domain, transform hematopoietic cell lines to factor independent growth. FLT3-ITDs also induce a myeloproliferative disease in a murine bone marrow transplant model, but are not sufficient to cause AML. Here, we test the hypothesis that PML/RARα can cooperate with FLT3-ITD to induce an APL-like disease in the mouse. Retroviral transduction of FLT3-ITD into bone marrow cells obtained from PML/RARα transgenic mice results in a short latency APL-like disease with complete penetrance. This disease resembles the APL-like disease that occurs with long latency in the PML/RARα transgenics, suggesting that activating mutations in FLT3 can functionally substitute for the additional mutations that occur during mouse APL progression. The leukemia is transplantable to secondary recipients and is ATRA responsive. These observations document cooperation between PML/RARα and FLT3-ITD in development of the murine APL phenotype. PMID:12060771

  18. A randomized assessment of adding the kinase inhibitor lestaurtinib to first-line chemotherapy for FLT3-mutated AML.

    PubMed

    Knapper, Steven; Russell, Nigel; Gilkes, Amanda; Hills, Robert K; Gale, Rosemary E; Cavenagh, James D; Jones, Gail; Kjeldsen, Lars; Grunwald, Michael R; Thomas, Ian; Konig, Heiko; Levis, Mark J; Burnett, Alan K

    2017-03-02

    The clinical benefit of adding FMS-like tyrosine kinase-3 (FLT3)-directed small molecule therapy to standard first-line treatment of acute myeloid leukemia (AML) has not yet been established. As part of the UK AML15 and AML17 trials, patients with previously untreated AML and confirmed FLT3-activating mutations, mostly younger than 60 years, were randomly assigned either to receive oral lestaurtinib (CEP701) or not after each of 4 cycles of induction and consolidation chemotherapy. Lestaurtinib was commenced 2 days after completing chemotherapy and administered in cycles of up to 28 days. The trials ran consecutively. Primary endpoints were overall survival in AML15 and relapse-free survival in AML17; outcome data were meta-analyzed. Five hundred patients were randomly assigned between lestaurtinib and control: 74% had FLT3-internal tandem duplication mutations, 23% FLT3-tyrosine kinase domain point mutations, and 2% both types. No significant differences were seen in either 5-year overall survival (lestaurtinib 46% vs control 45%; hazard ratio, 0.90; 95% CI 0.70-1.15; P = .3) or 5-year relapse-free survival (40% vs 36%; hazard ratio, 0.88; 95% CI 0.69-1.12; P = .3). Exploratory subgroup analysis suggested survival benefit with lestaurtinib in patients receiving concomitant azole antifungal prophylaxis and gemtuzumab ozogamicin with the first course of chemotherapy. Correlative studies included analysis of in vivo FLT3 inhibition by plasma inhibitory activity assay and indicated improved overall survival and significantly reduced rates of relapse in lestaurtinib-treated patients who achieved sustained greater than 85% FLT3 inhibition. In conclusion, combining lestaurtinib with intensive chemotherapy proved feasible in younger patients with newly diagnosed FLT3-mutated AML, but yielded no overall clinical benefit. The improved clinical outcomes seen in patients achieving sustained FLT3 inhibition encourage continued evaluation of FLT3-directed therapy alongside

  19. The AML1-ETO fusion gene and the FLT3 length mutation collaborate in inducing acute leukemia in mice

    PubMed Central

    Schessl, Christina; Rawat, Vijay P.S.; Cusan, Monica; Deshpande, Aniruddha; Kohl, Tobias M.; Rosten, Patricia M.; Spiekermann, Karsten; Humphries, R. Keith; Schnittger, Susanne; Kern, Wolfgang; Hiddemann, Wolfgang; Quintanilla-Martinez, Leticia; Bohlander, Stefan K.; Feuring-Buske, Michaela; Buske, Christian

    2005-01-01

    The molecular characterization of leukemia has demonstrated that genetic alterations in the leukemic clone frequently fall into 2 classes, those affecting transcription factors (e.g., AML1-ETO) and mutations affecting genes involved in signal transduction (e.g., activating mutations of FLT3 and KIT). This finding has favored a model of leukemogenesis in which the collaboration of these 2 classes of genetic alterations is necessary for the malignant transformation of hematopoietic progenitor cells. The model is supported by experimental data indicating that AML1-ETO and FLT3 length mutation (FLT3-LM), 2 of the most frequent genetic alterations in AML, are both insufficient on their own to cause leukemia in animal models. Here we report that AML1-ETO collaborates with FLT3-LM in inducing acute leukemia in a murine BM transplantation model. Moreover, in a series of 135 patients with AML1-ETO–positive AML, the most frequently identified class of additional mutations affected genes involved in signal transduction pathways including FLT3-LM or mutations of KIT and NRAS. These data support the concept of oncogenic cooperation between AML1-ETO and a class of activating mutations, recurrently found in patients with t(8;21), and provide a rationale for therapies targeting signal transduction pathways in AML1-ETO–positive leukemias. PMID:16025155

  20. UNC2025, a Potent and Orally Bioavailable MER/FLT3 Dual Inhibitor

    PubMed Central

    2015-01-01

    We previously reported a potent small molecule Mer tyrosine kinase inhibitor UNC1062. However, its poor PK properties prevented further assessment in vivo. We report here the sequential modification of UNC1062 to address DMPK properties and yield a new potent and highly orally bioavailable Mer inhibitor, 11, capable of inhibiting Mer phosphorylation in vivo, following oral dosing as demonstrated by pharmaco-dynamic (PD) studies examining phospho-Mer in leukemic blasts from mouse bone marrow. Kinome profiling versus more than 300 kinases in vitro and cellular selectivity assessments demonstrate that 11 has similar subnanomolar activity against Flt3, an additional important target in acute myelogenous leukemia (AML), with pharmacologically useful selectivity versus other kinases examined. PMID:25068800

  1. UNC2025, a potent and orally bioavailable MER/FLT3 dual inhibitor.

    PubMed

    Zhang, Weihe; DeRyckere, Deborah; Hunter, Debra; Liu, Jing; Stashko, Michael A; Minson, Katherine A; Cummings, Christopher T; Lee, Minjung; Glaros, Trevor G; Newton, Dianne L; Sather, Susan; Zhang, Dehui; Kireev, Dmitri; Janzen, William P; Earp, H Shelton; Graham, Douglas K; Frye, Stephen V; Wang, Xiaodong

    2014-08-28

    We previously reported a potent small molecule Mer tyrosine kinase inhibitor UNC1062. However, its poor PK properties prevented further assessment in vivo. We report here the sequential modification of UNC1062 to address DMPK properties and yield a new potent and highly orally bioavailable Mer inhibitor, 11, capable of inhibiting Mer phosphorylation in vivo, following oral dosing as demonstrated by pharmaco-dynamic (PD) studies examining phospho-Mer in leukemic blasts from mouse bone marrow. Kinome profiling versus more than 300 kinases in vitro and cellular selectivity assessments demonstrate that 11 has similar subnanomolar activity against Flt3, an additional important target in acute myelogenous leukemia (AML), with pharmacologically useful selectivity versus other kinases examined.

  2. The STAT5 Inhibitor Pimozide Displays Efficacy in Models of Acute Myelogenous Leukemia Driven by FLT3 Mutations

    PubMed Central

    Nelson, Erik A.; Walker, Sarah R.; Xiang, Michael; Weisberg, Ellen; Bar-Natan, Michal; Barrett, Rosemary; Liu, Suiyang; Kharbanda, Surender; Christie, Amanda L.; Nicolais, Maria; Griffin, James D.; Stone, Richard M.; Kung, Andrew L.

    2012-01-01

    Activation of the transcription factor STAT5 is essential for the pathogenesis of acute myelogenous leukemia (AML) containing the FLT3 internal tandem duplication (ITD) mutation. FLT3 ITD is a constitutively active tyrosine kinase that drives the activation of STAT5, leading to the growth and survival of AML cells. Although there has been some success in identifying tyrosine kinase inhibitors that block the function of FLT3 ITD, there remains a continued need for effective treatment of this disease. We have identified the psychotropic drug pimozide as an effective inhibitor of STAT5 function. Pimozide inhibits the tyrosine phosphorylation of STAT5, leading to the death of AML cells through the induction of apoptosis. Pimozide shows a combinatorial effect with the tyrosine kinase inhibitors midostaurin (PKC412) and sunitinib in the inhibition of STAT5 tyrosine phosphorylation and the induction of apoptosis. Significantly, pimozide reduces the tumor burden in a mouse model of FLT3-driven AML. Therefore, identifying STAT5 inhibitors may provide a new avenue for the treatment of AML, and these may be effective alone or in combination with tyrosine kinase inhibitors. PMID:23264850

  3. Phase I Trial of Maintenance Sorafenib after Allogeneic Hematopoietic Stem Cell Transplantation for FLT3-ITD AML

    PubMed Central

    Chen, Yi-Bin; Li, Shuli; Lane, Andrew A.; Connolly, Christine; Del Rio, Candice; Valles, Betsy; Curtis, Morgan; Ballen, Karen; Cutler, Corey; Dey, Bimalangshu R.; El-Jawahri, Areej; Fathi, Amir T.; Ho, Vincent T.; Joyce, Amy; McAfee, Steven; Rudek, Michelle; Rajkhowa, Trivikram; Verselis, Sigitas; Antin, Joseph H.; Spitzer, Thomas R.; Levis, Mark; Soiffer, Robert

    2014-01-01

    The FLT3-ITD mutation is associated with a high relapse rate for patients with AML even after allogeneic hematopoietic stem cell transplantation (HSCT). Sorafenib is a tyrosine kinase inhibitor which inhibits the FLT3 tyrosine kinase and has shown encouraging activity in FLT3-ITD AML. We conducted a phase I trial of maintenance sorafenib after HSCT in patients with FLT3-ITD AML (ClinicalTrials.gov NCT01398501). Patients received a variety of conditioning regimens and graft sources. A dose escalation 3+3 cohort design was used to define the maximum tolerated dose (MTD) with an additional 10 patients treated at the MTD. Sorafenib was initiated between days 45 and 120 after HSCT continued for twelve 28-day cycles. Twenty-two patients were enrolled (status at HSCT: CR1=16, CR2=3, refractory=3). The MTD was established at 400 mg BID with one DLT observed (pericardial effusion). Two patients died of transplant-related causes, both unrelated to sorafenib. Two patients stopped sorafenib after relapse and 5 stopped due to attributable toxicities after the DLT period. Median follow-up for surviving patients is 16.7 months after HSCT (range, 8.1–35.0). There was one case of grade II acute GVHD after starting sorafenib and the 12-month cumulative incidence of chronic GVHD was 38% (90% CI, 21%–56%). For all patients, one-year progression-free survival (PFS) is 85% (90% CI, 66%–94%) and one-year overall survival (OS) is 95% (90% CI, 79%–99%) after HSCT. For patients in CR1 / CR2 prior to HSCT (n=19), one-year PFS is 95% (90% CI, 76%–99%) and one-year OS is 100% with only one patient who has relapsed. Sorafenib is safe after HSCT for FLT3-ITD AML and merits further investigation for the prevention of relapse. PMID:25239228

  4. Prognostic value of FLT3 mutations in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and anthracycline monochemotherapy

    PubMed Central

    Barragán, Eva; Montesinos, Pau; Camos, Mireia; González, Marcos; Calasanz, Maria J.; Román-Gómez, José; Gómez-Casares, Maria T.; Ayala, Rosa; López, Javier; Fuster, Óscar; Colomer, Dolors; Chillón, Carmen; Larrayoz, María J.; Sánchez-Godoy, Pedro; González-Campos, José; Manso, Félix; Amador, Maria L.; Vellenga, Edo; Lowenberg, Bob; Sanz, Miguel A.

    2011-01-01

    Background Fms-like tyrosine kinase-3 (FLT3) gene mutations are frequent in acute promyelocytic leukemia but their prognostic value is not well established. Design and Methods We evaluated FLT3-internal tandem duplication and FLT3-D835 mutations in patients treated with all-trans retinoic acid and anthracycline-based chemotherapy enrolled in two subsequent trials of the Programa de Estudio y Tratamiento de las Hemopatías Malignas (PETHEMA) and Hemato-Oncologie voor Volwassenen Nederland (HOVON) groups between 1996 and 2005. Results FLT3-internal tandem duplication and FLT3-D835 mutation status was available for 306 (41%) and 213 (29%) patients, respectively. Sixty-eight (22%) and 20 (9%) patients had internal tandem duplication and D835 mutations, respectively. Internal tandem duplication was correlated with higher white blood cell and blast counts, lactate dehydrogenase, relapse-risk score, fever, hemorrhage, coagulopathy, BCR3 isoform, M3 variant subtype, and expression of CD2, CD34, human leukocyte antigen-DR, and CD11b surface antigens. The FLT3-D835 mutation was not significantly associated with any clinical or biological characteristic. Univariate analysis showed higher relapse and lower survival rates in patients with a FLT3-internal tandem duplication, while no impact was observed in relation to FLT3-D835. The prognostic value of the FLT3-internal tandem duplication was not retained in the multivariate analysis. Conclusions FLT3-internal tandem duplication mutations are associated with several hematologic features in acute promyelocytic leukemia, in particular with high white blood cell counts, but we were unable to demonstrate an independent prognostic value in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and anthracycline-based regimens. PMID:21685470

  5. Ihog and Boi elicit Hh signaling via Ptc but do not aid Ptc in sequestering the Hh ligand.

    PubMed

    Camp, Darius; Haitian He, Billy; Li, Sally; Althaus, Irene W; Holtz, Alexander M; Allen, Benjamin L; Charron, Frédéric; van Meyel, Donald J

    2014-10-01

    Hedgehog (Hh) proteins are secreted molecules essential for tissue development in vertebrates and invertebrates. Hh reception via the 12-pass transmembrane protein Patched (Ptc) elicits intracellular signaling through Smoothened (Smo). Hh binding to Ptc is also proposed to sequester the ligand, limiting its spatial range of activity. In Drosophila, Interference hedgehog (Ihog) and Brother of ihog (Boi) are two conserved and redundant transmembrane proteins that are essential for Hh pathway activation. How Ihog and Boi activate signaling in response to Hh remains unknown; each can bind both Hh and Ptc and so it has been proposed that they are essential for both Hh reception and sequestration. Using genetic epistasis we established here that Ihog and Boi, and their orthologs in mice, act upstream or at the level of Ptc to allow Hh signal transduction. In the Drosophila developing wing model we found that it is through Hh pathway activation that Ihog and Boi maintain the boundary between the anterior and posterior compartments. We dissociated the contributions of Ptc from those of Ihog/Boi and, surprisingly, found that cells expressing Ptc can retain and sequester the Hh ligand without Ihog and Boi, but that Ihog and Boi cannot do so without Ptc. Together, these results reinforce the central role for Ptc in Hh binding in vivo and demonstrate that, although Ihog and Boi are dispensable for Hh sequestration, they are essential for pathway activation because they allow Hh to inhibit Ptc and thereby relieve its repression of Smo.

  6. Detectable FLT3-ITD or RAS mutation at the time of transformation from MDS to AML predicts for very poor outcomes

    PubMed Central

    Badar, Talha; Patel, Keyur P; Thompson, Philip A; DiNardo, Courtney; Takahashi, Koichi; Cabrero, Monica; Borthakur, Gautam; Cortes, Jorge; Konopleva, Marina; Kadia, Tapan; Bohannan, Zach; Pierce, Sherry; Jabbour, Elias J; Ravandi, Farhad; Daver, Naval; Luthra, Raja; Kantarjian, Hagop; Garcia-Manero, Guillermo

    2016-01-01

    Background The molecular events that drive the transformation from myelodysplastic syndromes (MDS) to acute myeloid leukemia (AML) have yet to be fully characterized. We hypothesized that detection of these mutations at the time of transformation from MDS to AML may lead to poorer outcomes. Methods We analyzed 102 MDS patients who were admitted to our institution between 2004 and 2013, had wild-type (wt) FLT3-ITD and RAS at diagnosis, progressed to AML, and had serial mutation testing at both the MDS and AML stages. Results We detected FLT3-ITD and/or RAS mutations in twenty-seven (26%) patients at the time of transformation to AML. Twenty-two patients (81%) had RAS mutations and five (19%) had FLT3-ITD mutations. The median survival after leukemia transformation in patients who had detectable RAS and/or FLT3-ITD mutations was 2·4 months compared to 7·5 months in patients who retained wt RAS and FLT3-ITD (hazard ratio [HR]: 3·08, 95% confidence interval [CI]: 1·9–5·0, p < 0·0001). In multivariate analysis, FLT3-ITD and RAS mutations had independent prognostic significance for poor outcome. Conclusions We conclude that 26% of patients had detectable FLT3-ITD or RAS mutation at transformation to AML, and these mutations were associated with very poor outcome. PMID:26547258

  7. Expression of protein-tyrosine phosphatases in Acute Myeloid Leukemia cells: FLT3 ITD sustains high levels of DUSP6 expression

    PubMed Central

    2012-01-01

    Protein-tyrosine phosphatases (PTPs) are important regulators of cellular signaling and changes in PTP activity can contribute to cell transformation. Little is known about the role of PTPs in Acute Myeloid Leukemia (AML). The aim of this study was therefore to establish a PTP expression profile in AML cells and to explore the possible role of FLT3 ITD (Fms-like tyrosine kinase 3 with internal tandem duplication), an important oncoprotein in AML for PTP gene expression. PTP mRNA expression was analyzed in AML cells from patients and in cell lines using a RT-qPCR platform for detection of transcripts of 92 PTP genes. PTP mRNA expression was also analyzed based on a public microarray data set for AML patients. Highly expressed PTPs in AML belong to all PTP subfamilies. Very abundantly expressed PTP genes include PTPRC, PTPN2, PTPN6, PTPN22, DUSP1, DUSP6, DUSP10, PTP4A1, PTP4A2, PTEN, and ACP1. PTP expression was further correlated with the presence of FLT3 ITD, focusing on a set of highly expressed dual-specificity phosphatases (DUSPs). Elevated expression of DUSP6 in patients harboring FLT3 ITD was detected in this analysis. The mechanism and functional role of FLT3 ITD-mediated upregulation of DUSP6 was then explored using pharmacological inhibitors of FLT3 ITD signal transduction and si/shRNA technology in human and murine cell lines. High DUSP6 expression was causally associated with the presence of FLT3 ITD and dependent on FLT3 ITD kinase activity and ERK signaling. DUSP6 depletion moderately increased ERK1/2 activity but attenuated FLT3 ITD-dependent cell proliferation of 32D cells. In conclusion, DUSP6 may play a contributing role to FLT3 ITD-mediated cell transformation. PMID:22784513

  8. Tamoxifen-elicited uterotrophy: cross-species and cross-ligand analysis of the gene expression program

    PubMed Central

    Kwekel, Joshua C; Forgacs, Agnes L; Burgoon, Lyle D; Williams, Kurt J; Zacharewski, Timothy R

    2009-01-01

    Background Tamoxifen (TAM) is a well characterized breast cancer drug and selective estrogen receptor modulator (SERM) which also has been associated with a small increase in risk for uterine cancers. TAM's partial agonist activation of estrogen receptor has been characterized for specific gene promoters but not at the genomic level in vivo.Furthermore, reducing uncertainties associated with cross-species extrapolations of pharmaco- and toxicogenomic data remains a formidable challenge. Results A comparative ligand and species analysis approach was conducted to systematically assess the physiological, morphological and uterine gene expression alterations elicited across time by TAM and ethynylestradiol (EE) in immature ovariectomized Sprague-Dawley rats and C57BL/6 mice. Differential gene expression was evaluated using custom cDNA microarrays, and the data was compared to identify conserved and divergent responses. 902 genes were differentially regulated in all four studies, 398 of which exhibit identical temporal expression patterns. Conclusion Comparative analysis of EE and TAM differentially expressed gene lists suggest TAM regulates no unique uterine genes that are conserved in the rat and mouse. This demonstrates that the partial agonist activities of TAM extend to molecular targets in regulating only a subset of EE-responsive genes. Ligand-conserved, species-divergent expression of carbonic anhydrase 2 was observed in the microarray data and confirmed by real time PCR. The identification of comparable temporal phenotypic responses linked to related gene expression profiles demonstrates that systematic comparative genomic assessments can elucidate important conserved and divergent mechanisms in rodent estrogen signalling during uterine proliferation. PMID:19400957

  9. Silvestrol exhibits significant in vivo and in vitro antileukemic activities and inhibits FLT3 and miR-155 expressions in acute myeloid leukemia

    PubMed Central

    2013-01-01

    Background Activating mutations [internal tandem duplication (ITD)] or overexpression of the FMS-like tyrosine kinase receptor-3 (FLT3) gene are associated with poor outcome in acute myeloid leukemia (AML) patients, underscoring the need for novel therapeutic approaches. The natural product silvestrol has potent antitumor activity in several malignancies, but its therapeutic impact on distinct molecular high-risk AML subsets remains to be fully investigated. We examined here the preclinical activity of silvestrol in FLT3-ITD and FLT3 wild-type (wt) AML. Methods Silvestrol in vitro anti-leukemic activity was examined by colorimetric cell viability assay, colony-forming and flow cytometry assays assessing growth inhibition and apoptosis, respectively. Pharmacological activity of silvestrol on FLT3 mRNA translation, mRNA and protein expression was determined by RNA-immunoprecipitation, qRT-PCR and immunoblot analyses, respectively. Silvestrol in vivo efficacy was investigated using MV4-11 leukemia-engrafted mice. Results Silvestrol shows antileukemia activity at nanomolar concentrations both in FLT3-wt overexpressing (THP-1) and FLT3-ITD (MV4-11) expressing AML cell lines (IC50 = 3.8 and 2.7 nM, respectively) and patients’ primary blasts [IC50 = ~12 nM (FLT3-wt) and ~5 nM (FLT3-ITD)]. Silvestrol increased apoptosis (~4fold, P = 0.0001), and inhibited colony-formation (100%, P < 0.0001) in primary blasts. Silvestrol efficiently inhibited FLT3 translation reducing FLT3 protein expression by 80–90% and decreased miR-155 levels (~60%), a frequently co-regulated onco-miR in FLT3-ITD-positive AML. The median survival of silvestrol-treated vs vehicle-treated mice was 63 vs 29 days post-engraftment, respectively (P < 0.0001). Conclusions Silvestrol exhibits significant in vivo and in vitro antileukemic activities in AML through a novel mechanism resulting in inhibition of FLT3 and miR-155 expression. These encouraging results warrant a rapid

  10. Adaptation of the plasma inhibitory activity assay to detect Aurora, ABL and FLT3 kinase inhibition by AT9283 in pediatric leukemia.

    PubMed

    Podesta, Jennifer E; Sugar, Richard; Squires, Matt; Linardopoulos, Spiros; Pearson, Andrew D J; Moore, Andrew S

    2011-09-01

    Non-invasive assessment of biomarker modulation is important for evaluating targeted therapeutics, particularly in pediatrics. The plasma inhibitory activity (PIA) assay is used clinically to assess FLT3 inhibition ex vivo and guide dosing. AT9283 is a novel Aurora kinase inhibitor with secondary activity against FLT3 and ABL. We adapted the PIA assay to simultaneously detect inhibition of Aurora and FLT3 in AML, and Aurora and ABL in CML by AT9283. Furthermore, we optimized the assay for children, where limited blood volumes are available for pharmacodynamic studies. Simultaneously detecting multiple kinase inhibition may identify important mechanisms of action for novel anti-leukemic drugs.

  11. Long FLT3 internal tandem duplications and reduced PML-RARα expression at diagnosis characterize a high-risk subgroup of acute promyelocytic leukemia patients

    PubMed Central

    Chillón, María Carmen; Santamaría, Carlos; García-Sanz, Ramón; Balanzategui, Ana; María Eugenia, Sarasquete; Alcoceba, Miguel; Marín, Luis; Caballero, María Dolores; Vidriales, María Belén; Ramos, Fernando; Bernal, Teresa; Díaz-Mediavilla, Joaquín; de Coca, Alfonso García; Peñarrubia, María Jesús; Queizán, José Antonio; Giraldo, Pilar; San Miguel, Jesús F.; González, Marcos

    2010-01-01

    Background Internal tandem duplications of the FLT3 gene (FLT3-ITDs) are frequent in patients with acute promyelocytic leukemia (APL), however its clinical impact remains controversial. Design and Methods We analyzed the prognostic significance of FLT3-ITD mutant level and size, as well as FLT3-D835 point mutations, PML-RARα expression and other predictive factors in 129 APL patients at diagnosis enrolled on the Spanish LPA96 (n=43) or LPA99 (n=86) PETHEMA trials. Results FLT3-ITDs and D835 mutations were detected in 21% and 9% of patients, respectively. Patients with increased ITD mutant/wild-type ratio or longer ITD size displayed shorter 5-year relapse-free survival (RFS) (P=0.048 and P<0.0001, respectively). However, patients with D835 mutations did not show differences in RFS or overall survival (OS). Moreover, patients with initial normalized copy number (NCN) of PML-RARα transcripts less than the 25th percentile had adverse clinical features and shorter 5-year RFS (P<0.0001) and OS (P=0.004) compared to patients with higher NCN. Patients with low NCN showed increased incidence of ITDs (P=0.001), with higher ratios (P<0.0001) and/or longer sizes (P=0.007). Multivariate analysis showed that long FLT3-ITD (P=0.001), low PML-RARα levels (P=0.004) and elevated WBC counts (>10×109/L) (P=0.018) were independent predictors for shorter RFS. We identified a subgroup of patients with high WBC, long FLT3-ITD and low NCN of transcripts that showed an extremely bad prognosis (5-year RFS 23.4%, P<0.0001). Conclusions In conclusion, FLT3-ITD size and PML-RARα transcript levels at diagnosis could contribute to improve the risk stratification in APL. PMID:20133893

  12. Establishment of xenotransplantation model of human CN-AML with FLT3-ITD (mut) /NPM1 (-) in NOD/SCID mice.

    PubMed

    Shang, Zhen; Wang, Jue; Wang, Di; Xiao, Min; Li, Tong-juan; Wang, Na; Huang, Liang; Zhou, Jian-feng

    2013-06-01

    Patients with FLT3-ITD (mut) /NPM1 (-) cytogenetically normal acute myeloid leukemia (CN-AML), as high-risk molecular group in CN-AML, are associated with a worse prognosis than other CN-AML patients. It is beneficial to generate xenotransplantation model of FLT3-ITD (mut) /NPM1 (-) CN-AML to better understand the pathogenesis and therapeutic strategies of such AML subtype. The purpose of present study was to establish the xenotransplantation model in NOD/SCID mice with FLT3-ITD (mut) /NPM1 (-) CN-AML primary cells. The FLT3-ITD (mut) /NPM1 (-) CN-AML primary cells from 3 of 7 cases were successfully transplanted into NOD/SCID mice, and human CD45 positive cells were detected in the peripheral blood, spleen and bone marrow of mice by using flow cytometry. Infiltration of human leukemia cells in various organs of mice was observed by using immunohistochemistry. Gene analysis confirmed sustained FLT3/ITD mutation without NPM1 mutation in mice. By performing serial transplantation, it was found that characteristics of the leukemia cells in secondary and tertiary generation models remained unchanged. Moreover, in vivo cytarabine administration could extend survival of NOD/SCID mice, which was consistent with clinical observation. In conclusion, we successfully established xenotransplantation model of human FLT3-ITD (mut) /NPM1 (-) CN-AML in NOD/SCID mice. The model was able to present primary disease and suitable to evaluate the curative effects of new drugs or therapy strategies.

  13. Favorable outcome of patients with acute myeloid leukemia harboring a low-allelic burden FLT3-ITD mutation and concomitant NPM1 mutation: relevance to post-remission therapy.

    PubMed

    Pratcorona, Marta; Brunet, Salut; Nomdedéu, Josep; Ribera, Josep Maria; Tormo, Mar; Duarte, Rafael; Escoda, Lourdes; Guàrdia, Ramon; Queipo de Llano, M Paz; Salamero, Olga; Bargay, Joan; Pedro, Carmen; Martí, Josep Maria; Torrebadell, Montserrat; Díaz-Beyá, Marina; Camós, Mireia; Colomer, Dolors; Hoyos, Montserrat; Sierra, Jorge; Esteve, Jordi

    2013-04-04

    Risk associated to FLT3 internal tandem duplication (FLT3-ITD) in patients with acute myeloid leukemia (AML) may depend on mutational burden and its interaction with other mutations. We analyzed the effect of FLT3-ITD/FLT3 wild-type (FLT3wt) ratio depending on NPM1 mutation (NPM1mut) in 303 patients with intermediate-risk cytogenetics AML treated with intensive chemotherapy. Among NPM1mut patients, FLT3wt and low ratio (<0.5) subgroups showed similar overall survival, relapse risk, and leukemia-free survival, whereas high ratio (≥0.5) patients had a worse outcome. In NPM1wt AML, FLT3-ITD subgroups showed a comparable outcome, with higher risk of relapse and shortened overall survival than FLT3wt patients. Allogeneic stem cell transplantation in CR1 was associated with a reduced relapse risk in all molecular subgroups with the exception of NPM1mut AML with absent or low ratio FLT3-ITD. In conclusion, effect of FLT3 burden is modulated by NPM1 mutation, especially in patients with a low ratio.

  14. Flt3-L gene therapy enhances immunocytokine-mediated antitumor effects and induces long-term memory.

    PubMed

    Neal, Zane C; Sondel, Paul M; Bates, Mary Kay; Gillies, Stephen D; Herweijer, Hans

    2007-11-01

    Therapeutic treatment with hu14.18-IL-2 immunocytokine (IC) or Flt3-L (FL) protein is initially effective at resolving established intradermal NXS2 neuroblastoma tumors in mice. However, many treated animals develop recurrent disease. We previously found that tumors recurring following natural killer (NK) mediated IC treatment show augmented MHC class I expression, while the tumors that recurred following T cell dependent Flt3-L treatment exhibited decreased MHC class I expression. We hypothesized that this divergent MHC modulation on recurrent tumors was due to therapy-specific immunoediting. We further postulated that combining IC and Flt3-L treatments might decrease the likelihood of recurrent disease by preventing MHC modulation as a mechanism for immune escape. We now report that combinatorial treatment of FL plus hu14.18-IL-2 IC provides greater antitumor benefit than treatment with either alone, suppressing development of recurrent disease. We administered FL by gene therapy using a clinically relevant approach: hydrodynamic limb vein (HLV) delivery of DNA for transgene expression by myofibers. Delivery of FL DNA by HLV injection in mice resulted in systemic expression of >10 ng/ml of FL in blood at day 3, and promoted up to a fourfold and tenfold increase in splenic NK and dendritic cells (DCs), respectively. Furthermore, the combination of FL gene therapy plus suboptimal IC treatment induced a greater expansion in the absolute number of splenic NK and DCs than achieved by individual component treatments. Mice that received combined FL gene therapy plus IC exhibited complete and durable resolution of established NXS2 tumors, and demonstrated protection from subsequent rechallenge with NXS2 tumor.

  15. Characterization of children with FLT3-ITD acute myeloid leukemia: a report from the AIEOP AML-2002 study group.

    PubMed

    Manara, E; Basso, G; Zampini, M; Buldini, B; Tregnago, C; Rondelli, R; Masetti, R; Bisio, V; Frison, M; Polato, K; Cazzaniga, G; Menna, G; Fagioli, F; Merli, P; Biondi, A; Pession, A; Locatelli, F; Pigazzi, M

    2017-01-01

    Recurrent molecular markers have been routinely used in acute myeloid leukemia (AML) for risk assessment at diagnosis, whereas their post-induction monitoring still represents a debated issue. We evaluated the prognostic value and biological impact of minimal residual disease (MRD) and of the allelic ratio (AR) of FLT3-internal-tandem duplication (ITD) in childhood AML. We retrospectively screened 494 children with de novo AML for FLT3-ITD mutation, identifying 54 harboring the mutation; 51% of them presented high ITD-AR at diagnosis and had worse event-free survival (EFS, 19.2 versus 63.5% for low ITD-AR, <0.05). Forty-one percent of children with high levels of MRD after the 1st induction course, measured by a patient-specific real-time-PCR, had worse EFS (22.2 versus 59.4% in low-MRD patients, P<0.05). Next, we correlated these parameters with gene expression, showing that patients with high ITD-AR or persistent MRD had characteristic expression profiles with deregulated genes involved in methylation and acetylation. Moreover, patients with high CyclinA1 expression presented an unfavorable EFS (20.3 versus 51.2% in low CyclinA1 group, P<0.01). Our results suggest that ITD-AR levels and molecular MRD should be considered in planning clinical management of FLT3-ITD patients. Different transcriptional activation of epigenetic and oncogenic profiles may explain variability in outcome among these patients, for whom novel therapeutic approaches are desirable.

  16. FLT3 Mutations in Early T-Cell Precursor ALL Characterize a Stem Cell Like Leukemia and Imply the Clinical Use of Tyrosine Kinase Inhibitors

    PubMed Central

    Neumann, Martin; Coskun, Ebru; Fransecky, Lars; Mochmann, Liliana H.; Bartram, Isabelle; Farhadi Sartangi, Nasrin; Heesch, Sandra; Gökbuget, Nicola; Schwartz, Stefan; Brandts, Christian; Schlee, Cornelia; Haas, Rainer; Dührsen, Ulrich; Griesshammer, Martin; Döhner, Hartmut; Ehninger, Gerhard; Burmeister, Thomas; Blau, Olga; Thiel, Eckhard; Hoelzer, Dieter; Hofmann, Wolf-Karsten; Baldus, Claudia D.

    2013-01-01

    Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) has been identified as high-risk subgroup of acute T-lymphoblastic leukemia (T-ALL) with a high rate of FLT3-mutations in adults. To unravel the underlying pathomechanisms and the clinical course we assessed molecular alterations and clinical characteristics in a large cohort of ETP-ALL (n = 68) in comparison to non-ETP T-ALL adult patients. Interestingly, we found a high rate of FLT3-mutations in ETP-ALL samples (n = 24, 35%). Furthermore, FLT3 mutated ETP-ALL was characterized by a specific immunophenotype (CD2+/CD5-/CD13+/CD33-), a distinct gene expression pattern (aberrant expression of IGFBP7, WT1, GATA3) and mutational status (absence of NOTCH1 mutations and a low frequency, 21%, of clonal TCR rearrangements). The observed low GATA3 expression and high WT1 expression in combination with lack of NOTCH1 mutations and a low rate of TCR rearrangements point to a leukemic transformation at the pluripotent prothymocyte stage in FLT3 mutated ETP-ALL. The clinical outcome in ETP-ALL patients was poor, but encouraging in those patients with allogeneic stem cell transplantation (3-year OS: 74%). To further explore the efficacy of targeted therapies, we demonstrate that T-ALL cell lines transfected with FLT3 expression constructs were particularly sensitive to tyrosine kinase inhibitors. In conclusion, FLT3 mutated ETP-ALL defines a molecular distinct stem cell like leukemic subtype. These data warrant clinical studies with the implementation of FLT3 inhibitors in addition to early allogeneic stem cell transplantation for this high risk subgroup. PMID:23359050

  17. PRL-3, a metastasis associated tyrosine phosphatase, is involved in FLT3-ITD signaling and implicated in anti-AML therapy.

    PubMed

    Zhou, Jianbiao; Bi, Chonglei; Chng, Wee-Joo; Cheong, Lip-Lee; Liu, Shaw-Cheng; Mahara, Sylvia; Tay, Kian-Ghee; Zeng, Qi; Li, Jie; Guo, Ke; Tan, Cheng Peow Bobby; Yu, Hanry; Albert, Daniel H; Chen, Chien-Shing

    2011-05-12

    Combination with other small molecule drugs represents a promising strategy to improve therapeutic efficacy of FLT3 inhibitors in the clinic. We demonstrated that combining ABT-869, a FLT3 inhibitor, with SAHA, a HDAC inhibitor, led to synergistic killing of the AML cells with FLT3 mutations and suppression of colony formation. We identified a core gene signature that is uniquely induced by the combination treatment in 2 different leukemia cell lines. Among these, we showed that downregulation of PTP4A3 (PRL-3) played a role in this synergism. PRL-3 is downstream of FLT3 signaling and ectopic expression of PRL-3 conferred therapeutic resistance through upregulation of STAT (signal transducers and activators of transcription) pathway activity and anti-apoptotic Mcl-1 protein. PRL-3 interacts with HDAC4 and SAHA downregulates PRL-3 via a proteasome dependent pathway. In addition, PRL-3 protein was identified in 47% of AML cases, but was absent in myeloid cells in normal bone marrows. Our results suggest such combination therapies may significantly improve the therapeutic efficacy of FLT3 inhibitors. PRL-3 plays a potential pathological role in AML and it might be a useful therapeutic target in AML, and warrant clinical investigation.

  18. MiR-424 and miR-155 deregulated expression in cytogenetically normal acute myeloid leukaemia: correlation with NPM1 and FLT3 mutation status

    PubMed Central

    2012-01-01

    Background MicroRNA have a central role in normal haematopoiesis and are deregulated in acute myeloid leukaemia (AML). The purpose of the study was to investigate by qRT-PCR the expression of miRNAs involved in myeloid differentiation (miR-424, miR-155, miR-223, miR-17-5p) in 48 patients with cytogenetically normal AML well characterized for NPM1 and/or FLT3 mutations. Three types of normalization were used for the data validation. Findings We found that miR-424 was down-modulated in AMLs with NPM1mutA regardless of FLT3 status. On the contrary, miR-155 showed up-regulation in patients with FLT3 internal tandem duplications (ITD) with or without NPM1 mutations. No significant associations were found by analyzing miR-223 and miR-17-5p in relation to FLT3 and NPM1 status. Conclusions This study supports the view that major genetic subsets of CN-AML are associated with distinct miRNA signatures and suggests that miR-424 and miR-155 deregulation is involved in the pathogenesis of CN-AML with NPM1 and FLT3-ITD mutations, respectively. PMID:22681934

  19. Optimization of imidazo[4,5-b]pyridine-based kinase inhibitors: identification of a dual FLT3/Aurora kinase inhibitor as an orally bioavailable preclinical development candidate for the treatment of acute myeloid leukemia.

    PubMed

    Bavetsias, Vassilios; Crumpler, Simon; Sun, Chongbo; Avery, Sian; Atrash, Butrus; Faisal, Amir; Moore, Andrew S; Kosmopoulou, Magda; Brown, Nathan; Sheldrake, Peter W; Bush, Katherine; Henley, Alan; Box, Gary; Valenti, Melanie; de Haven Brandon, Alexis; Raynaud, Florence I; Workman, Paul; Eccles, Suzanne A; Bayliss, Richard; Linardopoulos, Spiros; Blagg, Julian

    2012-10-25

    Optimization of the imidazo[4,5-b]pyridine-based series of Aurora kinase inhibitors led to the identification of 6-chloro-7-(4-(4-chlorobenzyl)piperazin-1-yl)-2-(1,3-dimethyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine (27e), a potent inhibitor of Aurora kinases (Aurora-A K(d) = 7.5 nM, Aurora-B K(d) = 48 nM), FLT3 kinase (K(d) = 6.2 nM), and FLT3 mutants including FLT3-ITD (K(d) = 38 nM) and FLT3(D835Y) (K(d) = 14 nM). FLT3-ITD causes constitutive FLT3 kinase activation and is detected in 20-35% of adults and 15% of children with acute myeloid leukemia (AML), conferring a poor prognosis in both age groups. In an in vivo setting, 27e strongly inhibited the growth of a FLT3-ITD-positive AML human tumor xenograft (MV4-11) following oral administration, with in vivo biomarker modulation and plasma free drug exposures consistent with dual FLT3 and Aurora kinase inhibition. Compound 27e, an orally bioavailable dual FLT3 and Aurora kinase inhibitor, was selected as a preclinical development candidate for the treatment of human malignancies, in particular AML, in adults and children.

  20. Flt3L-mobilized dendritic cells bearing H2-Kbm1 apoptotic cells do not induce cross-tolerance to CD8+ T cells across a class I MHC mismatched barrier.

    PubMed

    del Rio, Maria-Luisa; Cote-Sierra, Javier; Rodriguez-Barbosa, Jose-Ignacio

    2011-05-01

    Tolerization of allogeneic CD8(+) T cells is still a pending issue in the field of transplantation research to achieve long-term survival. To test whether dendritic cells (DC) bearing allogeneic major histocompatibility complex (MHC) class I mismatched apoptotic cells could induce cross-tolerance to alloreactive CD8(+) T cells, the following experimental strategy was devised. Rag2/γ(c) KO B6 mice were treated with Fms-like tyrosine kinase 3 ligand (Flt3L)-transduced B16 melanoma cells to drive a rapid expansion and mobilization of DC in vivo. Of all DC populations expanded, splenic CD11c(+) CD103(+) CD8α(+) DC were selectively involved in the process of antigen clearance of X-ray irradiated apoptotic thymocytes in vivo. Considering that CD11c(+) CD103(+) CD8α(+) DC selectively take up apoptotic cells and that they are highly specialized in cross-presenting antigen to CD8(+) T cells, we investigated whether B6 mice adoptively transferred with Flt3L-derived DC loaded with donor-derived apoptotic thymocytes could induce tolerance to bm1 skin allografts. Our findings on host anti-donor alloresponse, as revealed by skin allograft survival and cytotoxic T lymphocyte assays, indicated that the administration of syngeneic DC presenting K(bm1) donor-derived allopeptides through the indirect pathway of antigen presentation was not sufficient to induce cross-tolerance to alloreactive CD8(+) T cells responding to bm1 alloantigens in a murine model of skin allograft transplantation across an MHC class I mismatched barrier.

  1. FLT3L and Plerixafor Combination Increases Hematopoietic Stem Cell Mobilization and Leads to Improved Transplantation Outcome

    PubMed Central

    He, Shun; Chu, Jianhong; Vasu, Sumithira; Deng, Youcai; Yuan, Shunzong; Zhang, Jianying; Fan, Zhijin; Hofmeister, Craig C.; He, Xiaoming; Marsh, Henry C.; Devine, Steven M.; Yu, Jianhua

    2014-01-01

    Hematopoietic stem cell (HSC) transplantation has curative potential for patients with hematological malignancies. Clinically, HSCs derived from mobilized peripheral blood are used more frequently than bone marrow. However, current standard mobilizing agents yield grafts that may not contain sufficient HSCs. Here, using murine models, we discovered that FLT3L synergized with Plerixafor to mobilize phenotypically defined HSCs, and their combination (FP) was superior to G-CSF alone or in combination with Plerixafor (GP). Additionally, FP mobilized more Treg cells, NK cells, and plasmacytoid dendritic cells compared with G-CSF alone or GP. Both syngeneic and allogeneic grafts mobilized by FP led to long-term survival in transplanted mice. Collectively, FP represents a promising novel and potent mobilization regimen with potential clinical application in both the autologous and allogeneic transplantation settings. PMID:24365795

  2. The value of molecular stratification for CEBPA(DM) and NPM1(MUT) FLT3(WT) genotypes in older patients with acute myeloid leukaemia.

    PubMed

    Dickson, Glenda J; Bustraan, Sophia; Hills, Robert K; Ali, Akbar; Goldstone, Anthony H; Burnett, Alan K; Linch, David C; Gale, Rosemary E

    2016-02-01

    Older adult patients (≥60 years) with acute myeloid leukaemia (AML) are generally considered to be poor-risk and there is limited information available regarding risk stratification based on molecular characterization in this age group, particularly for the double-mutant CEBPA (CEBPA(DM) ) genotype. To investigate whether a molecular favourable-risk genotype can be identified, we investigated CEBPA, NPM1 and FLT3 status and prognostic impact in a cohort of 301 patients aged 60 years or more with intermediate-risk cytogenetics, all treated intensively. Overall survival (OS) at 1 year was highest in the 12 patients (4%) that were CEBPA(DM) compared to the 76 (28%) with a mutant NPM1 and wild-type FLT3 (NPM1(MUT) FLT3(WT) ) genotype or all other patients (75%, 54%, 33% respectively), with median survival 15·2, 13·6 and 6·6 months, although the benefit was short-term (OS at 3 years 17%, 29%, 12% respectively). Combination of the CEBPA(DM) and NPM1(MUT) FLT3(WT) genotype patients defined a molecular group with favourable prognosis (P < 0·0001 in multivariate analysis), with 57% of patients alive at 1 year compared to 33% for all other patients. Knowledge of genotype in older cytogenetically intermediate-risk patients might influence therapy decisions.

  3. 3,3'-Diindolylmethane Inhibits Flt3L/GM-CSF-induced-bone Marrow-derived CD103+ Dendritic Cell Differentiation Regulating Phosphorylation of STAT3 and STAT5

    PubMed Central

    Choi, Ah-Jeong; Kim, Soo-Ji; Jeong, So-Yeon

    2015-01-01

    The intestinal immune system maintains oral tolerance to harmless antigens or nutrients. One mechanism of oral tolerance is mediated by regulatory T cell (Treg)s, of which differentiation is regulated by a subset of dendritic cell (DC)s, primarily CD103+ DCs. The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, plays an important role in regulating immunity. The intestines are exposed to various AhR ligands, including endogenous metabolites and phytochemicals. It was previously reported that AhR activation induced tolerogenic DCs in mice or in cultures of bone marrow-derived DCs. However, given the variety of tolerogenic DCs, which type of tolerogenic DCs is regulated by AhR remains unknown. In this study, we found that AhR ligand 3,3'-diindolylmethane (DIM) inhibited the development of CD103+ DCs from mouse bone marrow cells stimulated with Flt3L and GM-CSF. DIM interfered with phosphorylation of STAT3 and STAT5 inhibiting the expression of genes, including Id2, E2-2, IDO-1, and Aldh1a2, which are associated with DC differentiation and functions. Finally, DIM suppressed the ability of CD103+ DCs to induce Foxp3+ Tregs. PMID:26770182

  4. Tandem duplication PCR: an ultra-sensitive assay for the detection of internal tandem duplications of the FLT3 gene

    PubMed Central

    Lin, Ming-Tseh; Tseng, Li-Hui; Beierl, Katie; Hsieh, Antony; Thiess, Michele; Chase, Nadine; Stafford, Amanda; Levis, Mark J.; Eshleman, James R.; Gocke, Christopher D.

    2013-01-01

    Internal tandem duplication (ITD) mutations of the FLT3 gene have been associated with a poor prognosis in acute myeloid leukemia (AML). Detection of ITD-positive minor clones at the initial diagnosis and during the minimal residual disease (MRD) stage may be essential. We previously designed a delta-PCR strategy to improve the sensitivity to 0.1% ITD-positive leukemia cells and showed that minor mutants with an allele burden of less than 1% can be clinically significant. In this study, we report on tandem duplication PCR (TD-PCR), a modified inverse PCR assay, and demonstrate a limit of detection of a few molecules of ITD mutants. The TD-PCR was initially designed to confirm ITD mutation of an amplicon which was undetectable by capillary electrophoresis and was incidentally isolated by a molecular fraction collecting tool. Subsequently, TD-PCR detected ITD mutation in 2 of 77 patients previously reported as negative for ITD mutation by a standard PCR assay. TD-PCR can also potentially be applied to monitor MRD with high analytic sensitivity in a portion of ITD-positive AML patients. Further studies using TD-PCR to detect ITD mutants at diagnosis may clarify the clinical significance of those ITD mutants with extremely low allele burden. PMID:23846441

  5. Retinal Photoreceptor Expresses Toll-Like Receptors (TLRs) and Elicits Innate Responses Following TLR Ligand and Bacterial Challenge

    PubMed Central

    Singh, Pawan Kumar; Kumar, Ashok

    2015-01-01

    Toll-like receptors (TLRs) play an important role in host defense against microbial pathogens. Our previous studies have shown that TLRs are expressed on various retinal cells (Microglia and Müller glia) and orchestrate retinal innate responses in bacterial endophthalmitis. In this study, we used a well-characterized mouse cone photoreceptor cell line (661W); and demonstrated that these cells express all known TLRs. Although the stimulation of 661W cells with TLR ligands (Pam3Cys, PolyI:C, LPS, Flagellin, Poly DT, and ODN) did not alter TLR expression, downstream TLR-signaling pathways (NF-κB, p38, and ERK) are activated. Moreover, TLR-activated 661W cells secreted significant amounts of inflammatory mediators (IL-6, IL-1β, MIP-2, and KC) in their culture supernatant, as assessed by ELISA. A similar trend was observed in 661W cells challenged with live bacteria (Staphylococcus aureus). Interestingly, the neutralization of TLR2, a major receptor for S. aureus recognition, did not significantly attenuate bacterial-induced inflammatory mediators, suggesting the existence of TLR2-independent mechanisms in photoreceptor cells. Together, these results indicate that photoreceptors constitutively express functional TLRs and possess the ability to initiate innate responses following pathogen challenge, implicating their role in retinal innate immunity. PMID:25767877

  6. Characterizing and Overriding the Structural Mechanism of the Quizartinib-resistant FLT3 “Gatekeeper” F691L Mutation with PLX3397

    PubMed Central

    Smith, Catherine C.; Zhang, Chao; Lin, Kimberly; Lasater, Elisabeth A.; Zhang, Ying; Massi, Evan; Damon, Lauren E.; Pendleton, Matthew; Bashir, Ali; Sebra, Robert; Perl, Alexander; Kasarskis, Andrew; Shellooe, Rafe; Tsang, Garson; Carias, Heidi; Powell, Ben; Burton, Elizabeth A.; Matusow, Bernice; Zhang, Jiazhong; Spevak, Wayne; Ibrahim, Prabha N.; Le, Mai H.; Hsu, Henry H.; Habets, Gaston; West, Brian L.; Bollag, Gideon; Shah, Neil P.

    2015-01-01

    Tyrosine kinase domain mutations are a common cause of acquired clinical resistance to tyrosine kinase inhibitors (TKIs) used to treat cancer, including the FLT3 inhibitor quizartinib. Mutation of kinase “gatekeeper” residues, which control access to an allosteric pocket adjacent to the ATP-binding site, have been frequently implicated in TKI resistance. The molecular underpinnings of gatekeeper mutation-mediated resistance are incompletely understood. We report the first co-crystal structure of FLT3 with the TKI quizartinib, which demonstrates that quizartinib binding relies on essential edge-to-face aromatic interactions with the gatekeeper F691 residue, and F830 within the highly conserved DFG motif in the activation loop. This reliance makes quizartinib critically vulnerable to gatekeeper and activation loop substitutions while minimizing the impact of mutations elsewhere. Moreover, we identify PLX3397, a novel FLT3 inhibitor that retains activity against the F691L mutant due to a binding mode that depends less vitally on specific interactions with the gatekeeper position. PMID:25847190

  7. High rate of hematological responses to sorafenib in FLT3-ITD acute myeloid leukemia relapsed after allogeneic hematopoietic stem cell transplantation.

    PubMed

    De Freitas, Tiago; Marktel, Sarah; Piemontese, Simona; Carrabba, Matteo G; Tresoldi, Cristina; Messina, Carlo; Lupo Stanghellini, Maria Teresa; Assanelli, Andrea; Corti, Consuelo; Bernardi, Massimo; Peccatori, Jacopo; Vago, Luca; Ciceri, Fabio

    2016-06-01

    Relapse represents the most significant cause of failure of allogeneic hematopoietic stem cell transplantation (HSCT) for FLT3-ITD-positive acute myeloid leukemia (AML), and available therapies are largely unsatisfactory. In this study, we retrospectively collected data on the off-label use of the tyrosine kinase inhibitor sorafenib, either alone or in association with hypomethylating agents and adoptive immunotherapy, in 13 patients with post-transplantation FLT3-ITD-positive AML relapses. Hematological response was documented in 12 of 13 patients (92%), and five of 13 (38%) achieved complete bone marrow remission. Treatment was overall manageable in the outpatient setting, although all patients experienced significant adverse events, especially severe cytopenias (requiring a donor stem cell boost in five patients) and typical hand-foot syndrome. None of the patients developed graft-vs.-host disease following sorafenib alone, whereas this was frequently observed when this was given in association with donor T-cell infusions. Six patients are alive and in remission at the last follow-up, and four could be bridged to a second allogeneic HSCT, configuring a 65 ± 14% overall survival at 100 d from relapse. Taken together, our data suggest that sorafenib might represent a valid treatment option for patients with FLT3-ITD-positive post-transplantation relapses, manageable also in combination with other therapeutic strategies.

  8. Novel anticancer compound [trifluoromethyl-substituted pyrazole N-nucleoside] inhibits FLT3 activity to induce differentiation in acute myeloid leukemia cells.

    PubMed

    Saleh, Ayman M; Taha, Mutasem O; Aziz, Mohammad A; Al-Qudah, Mahmoud A; AbuTayeh, Reem F; Rizvi, Syed A

    2016-06-01

    Anticancer properties of chemically synthesized compounds have continuously been optimized for better efficacy and selectivity. Derivatives of heterocyclic compounds are well known to have selective antiproliferative effect against many types of cancer. In this study, we investigated the ability of an indigenously synthesized anticancer molecule, G-11 [1-(2",3",4",6"-Tetra-O-acetyl-β-D-glucopyranosyl)-4-(3'-trifluoromethylphenylhydrazono)-3-trifluoromethyl-1,4-dihydropyrazol-5-one], to cause selective cytotoxicity and induce differentiation in the acute myeloid leukemia HL-60 cells. G-11 was able to exert cytotoxic effect on hematological (Jurkat, U937, K562, HL-60, CCRF-SB) and solid tumor (MCF-7, HepG2, HeLa, Caco-2) cell lines, with IC50 values significantly lower than noncancerous cells (HEK-293, BJ and Vero) and normal peripheral blood mononuclear cells. G-11 induced differentiation of HL-60 cells to granulocytes and monocytes/macrophages by inhibiting the activation of FLT3 (CD135 tyrosine kinase). ITD-FLT3 mutation found in many acute myeloid leukemia patients could also be targeted by G-11 as exhibited by its inhibitory effect on MOLM-13 and MV4-11 cell lines. Molecular docking studies suggest the involvement of Leu616, Asp698, Cys694 and Cys828 residues in binding of G-11 to FLT3. The ability of G-11 to cause selective cytotoxicity and induce differentiation in cancer cells could be clinically relevant for therapeutic gains.

  9. PCB153-elicited hepatic responses in the immature, ovariectomized C57BL/6 mice: Comparative toxicogenomic effects of dioxin and non-dioxin-like ligands

    SciTech Connect

    Kopec, Anna K.; Burgoon, Lyle D.; Ibrahim-Aibo, Daher; Mets, Bryan D.; Tashiro, Colleen; Potter, Dave; Sharratt, Bonnie; Harkema, Jack R.; Zacharewski, Timothy R.

    2010-03-15

    Polychlorinated biphenyls (PCBs) are ubiquitous contaminants found as complex mixtures of coplanar and non-coplanar congeners. The hepatic temporal and dose-dependent effects of the most abundant non-dioxin-like congener, 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153), were examined in immature, ovariectomized C57BL/6 mice, and compared to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the prototypical aryl hydrocarbon receptor (AhR) ligand. Animals were gavaged once with 300 mg/kg PCB153 or sesame oil vehicle and sacrificed 4, 12, 24, 72 or 168 h post dose. In the dose-response study, mice were gavaged with 1, 3, 10, 30, 100 or 300 mg/kg PCB153 or sesame oil for 24 h. Significant increases in relative liver weights were induced with 300 mg/kg PCB153 between 24 and 168 h, accompanied by slight vacuolization and hepatocellular hypertrophy. The hepatic differential expression of 186 and 177 genes was detected using Agilent 4 x 44 K microarrays in the time course (|fold change| >= 1.5, P1(t) >= 0.999) and dose-response (|fold change| >= 1.5, P1(t) >= 0.985) studies, respectively. Comparative analysis with TCDD suggests that the differential gene expression elicited by PCB153 was not mediated by the AhR. Furthermore, constitutive androstane and pregnane X receptor (CAR/PXR) regulated genes including Cyp2b10, Cyp3a11, Ces2, Insig2 and Abcc3 were dose-dependently induced by PCB153. Collectively, these results suggest that the hepatocellular effects elicited by PCB153 are qualitatively and quantitatively different from TCDD and suggestive of CAR/PXR regulation.

  10. Common and biased signaling pathways of the chemokine receptor CCR7 elicited by its ligands CCL19 and CCL21 in leukocytes.

    PubMed

    Hauser, Mark A; Legler, Daniel F

    2016-06-01

    Chemokines are pivotal regulators of cell migration during continuous immune surveillance, inflammation, homeostasis, and development. Chemokine binding to their 7-transmembrane domain, G-protein-coupled receptors causes conformational changes that elicit intracellular signaling pathways to acquire and maintain an asymmetric architectural organization and a polarized distribution of signaling molecules necessary for directional cell migration. Leukocytes rely on the interplay of chemokine-triggered migration modules to promote amoeboid-like locomotion. One of the most important chemokine receptors for adaptive immune cell migration is the CC-chemokine receptor CCR7. CCR7 and its ligands CCL19 and CCL21 control homing of T cells and dendritic cells to areas of the lymph nodes where T cell priming and the initiation of the adaptive immune response occur. Moreover, CCR7 signaling also contributes to T cell development in the thymus and to lymphorganogenesis. Although the CCR7-CCL19/CCL21 axis evolved to benefit the host, inappropriate regulation or use of these proteins can contribute or cause pathobiology of chronic inflammation, tumorigenesis, and metastasis, as well as autoimmune diseases. Therefore, it appears as the CCR7-CCL19/CCL21 axis is tightly regulated at numerous intersections. Here, we discuss the multiple regulatory mechanism of CCR7 signaling and its influence on CCR7 function. In particular, we focus on the functional diversity of the 2 CCR7 ligands, CCL19 and CCL21, as well as on their impact on biased signaling. The understanding of the molecular determinants of biased signaling and the multiple layers of CCR7 regulation holds the promise for potential future therapeutic intervention.

  11. Structure-based design of oxygen-linked macrocyclic kinase inhibitors: discovery of SB1518 and SB1578, potent inhibitors of Janus kinase 2 (JAK2) and Fms-like tyrosine kinase-3 (FLT3)

    NASA Astrophysics Data System (ADS)

    Poulsen, Anders; William, Anthony; Blanchard, Stéphanie; Lee, Angeline; Nagaraj, Harish; Wang, Haishan; Teo, Eeling; Tan, Evelyn; Goh, Kee Chuan; Dymock, Brian

    2012-04-01

    Macrocycles from our Aurora project were screened in a kinase panel and were found to be active on other kinase targets, mainly JAKs, FLT3 and CDKs. Subsequently these compounds became leads in our JAK2 project. Macrocycles with a basic nitrogen in the linker form a salt bridge with Asp86 in CDK2 and Asp698 in FLT3. This residue is conserved in most CDKs resulting in potent pan CDK inhibition. One of the main project objectives was to achieve JAK2 potency with 100-fold selectivity against CDKs. Macrocycles with an ether linker have potent JAK2 activity with the ether oxygen forming a hydrogen bond to Ser936. A hydrogen bond to the equivalent residues of JAK3 and most CDKs cannot be formed resulting in good selectivity for JAK2 over JAK3 and CDKs. Further optimization of the macrocyclic linker and side chain increased JAK2 and FLT3 activity as well as improving DMPK properties. The selective JAK2/FLT3 inhibitor 11 (Pacritinib, SB1518) has successfully finished phase 2 clinical trials for myelofibrosis and lymphoma. Another selective JAK2/FLT3 inhibitor, 33 (SB1578), has entered phase 1 clinical development for the non-oncology indication rheumatoid arthritis.

  12. Rapid Exercise-Induced Mobilization of Dendritic Cells Is Potentially Mediated by a Flt3L- and MMP-9-Dependent Process in Multiple Sclerosis.

    PubMed

    Deckx, Nathalie; Wens, Inez; Nuyts, Amber H; Lee, Wai-Ping; Hens, Niel; Koppen, Gudrun; Goossens, Herman; Van Damme, Pierre; Berneman, Zwi N; Eijnde, Bert O; Cools, Nathalie

    2015-01-01

    In healthy individuals, one exercise bout induces a substantial increase in the number of circulating leukocytes, while their function is transiently suppressed. The effect of one exercise bout in multiple sclerosis (MS) is less studied. Since recent evidence suggests a role of dendritic cells (DC) in the pathogenesis of MS, we investigated the effect of one combined endurance/resistance exercise bout on the number and function of DC in MS patients and healthy controls. Our results show a rapid increase in the number of DC in response to physical exercise in both MS patients and controls. Further investigation revealed that in particular DC expressing the migratory molecules CCR5 and CD62L were increased upon acute physical activity. This may be mediated by Flt3L- and MMP-9-dependent mobilization of DC, as demonstrated by increased circulating levels of Flt3L and MMP-9 following one exercise bout. Circulating DC display reduced TLR responsiveness after acute exercise, as evidenced by a less pronounced upregulation of activation markers, HLA-DR and CD86, on plasmacytoid DC and conventional DC, respectively. Our results indicate mobilization of DC, which may be less prone to drive inflammatory processes, following exercise. This may present a negative feedback mechanism for exercise-induced tissue damage and inflammation.

  13. Outcome of allogeneic hematopoietic stem cell transplantation for cytogenetically normal AML and identification of high-risk subgroup using WT1 expression in association with NPM1 and FLT3-ITD mutations.

    PubMed

    Yoon, Jae-Ho; Kim, Hee-Je; Jeon, Young-Woo; Lee, Sung-Eun; Cho, Byung-Sik; Eom, Ki-Seong; Kim, Yoo-Jin; Lee, Seok; Min, Chang-Ki; Cho, Seok-Goo; Kim, Dong-Wook; Lee, Jong-Wook; Min, Woo-Sung

    2015-06-06

    According to recent guidelines, cytogenetically normal acute myeloid leukemia (CN AML) is divided into four molecular subgroups based on nucleophosmin-1 (NPM1) and FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutations. All subgroups except for isolated NPM1mut are associated with poor prognosis. We retrospectively analyzed 223 patients with CN AML, 156 of whom were treated with standard chemotherapy. For postremission therapy, patients with available donors underwent allogeneic (allo) hematopoietic stem cell transplantation (HSCT) and the rest were treated with autologous HSCT or chemotherapy alone. We first compared the 4 conventional molecular subgroups, and then created another 4 subgroups based on WT1 expression: isolated NPM1mut, NPM1wt/FLT3-ITD-neg with low WT1 or high WT1, and FLT3-ITD-pos CN AML. We finally evaluated 89 patients who were treated with allo HSCT and achieved complete remission after standard chemotherapy. FLT3-ITD CN AML showed the worst outcome irrespective of NPM1mut, and isolated NPM1mut CN AML showed no significant differences compared with NPM1wt/FLT3-ITD-neg CN AML. In contrast, two newly stratified low-risk subgroups (NPM1wt/FLT3-ITD-neg with low WT1 and isolated NPM1mut CN AML) showed higher remission rates with superior overall survival (OS) compared with the other two high-risk subgroups, which showed a higher relapse rate even after allo HSCT. Further analysis showed that higher pre-HSCT expression of WT1 resulted in a higher relapse rate and poorer OS after allo HSCT. For CN AML, a risk-adapted approach using allo HSCT with novel agents should be evaluated with stratification specified by WT1. © 2015 Wiley Periodicals, Inc.

  14. Single-cell mass cytometry reveals intracellular survival/proliferative signaling in FLT3-ITD-mutated AML stem/progenitor cells.

    PubMed

    Han, Lina; Qiu, Peng; Zeng, Zhihong; Jorgensen, Jeffrey L; Mak, Duncan H; Burks, Jared K; Schober, Wendy; McQueen, Teresa J; Cortes, Jorge; Tanner, Scott D; Roboz, Gail J; Kantarjian, Hagop M; Kornblau, Steven M; Guzman, Monica L; Andreeff, Michael; Konopleva, Marina

    2015-04-01

    Understanding the unique phenotypes and complex signaling pathways of leukemia stem cells (LSCs) will provide insights and druggable targets that can be used to eradicate acute myeloid leukemia (AML). Current work on AML LSCs is limited by the number of parameters that conventional flow cytometry (FCM) can analyze because of cell autofluorescence and fluorescent dye spectral overlap. Single-cell mass cytometry (CyTOF) substitutes rare earth elements for fluorophores to label antibodies, which allows measurements of up to 120 parameters in single cells without correction for spectral overlap. The aim of this study was the evaluation of intracellular signaling in antigen-defined stem/progenitor cell subsets in primary AML. CyTOF and conventional FCM yielded comparable results on LSC phenotypes defined by CD45, CD34, CD38, CD123, and CD99. Intracellular phosphoprotein responses to ex vivo cell signaling inhibitors and cytokine stimulation were assessed in myeloid leukemia cell lines and one primary AML sample. CyTOF and conventional FCM results were confirmed by western blotting. In the primary AML sample, we investigated the cell responses to ex vivo stimulation with stem cell factor and BEZ235-induced inhibition of PI3K and identified activation patterns in multiple PI3K downstream signaling pathways including p-4EBP1, p-AKT, and p-S6, particularly in CD34(+) subsets. We evaluated multiple signaling pathways in antigen-defined subpopulations in primary AML cells with FLT3-ITD mutations. The data demonstrated the heterogeneity of cell phenotype distribution and distinct patterns of signaling activation across AML samples and between AML and normal samples. The mTOR targets p-4EBP1 and p-S6 were exclusively found in FLT3-ITD stem/progenitor cells, but not in their normal counterparts, suggesting both as novel targets in FLT3 mutated AML. Our data suggest that CyTOF can identify functional signaling pathways in antigen-defined subpopulations in primary AML, which may

  15. Support for 5-HT2C receptor functional selectivity in vivo utilizing structurally diverse, selective 5-HT2C receptor ligands and the 2,5-dimethoxy-4-iodoamphetamine elicited head-twitch response model.

    PubMed

    Canal, Clinton E; Booth, Raymond G; Morgan, Drake

    2013-07-01

    There are seemingly conflicting data in the literature regarding the role of serotonin (5-HT) 5-HT2C receptors in the mouse head-twitch response (HTR) elicited by the hallucinogenic 5-HT2A/2B/2C receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI). Namely, both 5-HT2C receptor agonists and antagonists, regarding 5-HT2C receptor-mediated Gq-phospholipase C (PLC) signaling, reportedly attenuate the HTR response. The present experiments tested the hypothesis that both classes of 5-HT2C receptor compounds could attenuate the DOI-elicited-HTR in a single strain of mice, C57Bl/6J. The expected results were considered in accordance with ligand functional selectivity. Commercially-available 5-HT2C agonists (CP 809101, Ro 60-0175, WAY 161503, mCPP, and 1-methylpsilocin), novel 4-phenyl-2-N,N-dimethyl-aminotetralin (PAT)-type 5-HT2C agonists (with 5-HT2A/2B antagonist activity), and antagonists selective for 5-HT2A (M100907), 5-HT2C (SB-242084), and 5-HT2B/2C (SB-206553) receptors attenuated the DOI-elicited-HTR. In contrast, there were differential effects on locomotion across classes of compounds. The 5-HT2C agonists and M100907 decreased locomotion, SB-242084 increased locomotion, SB-206553 resulted in dose-dependent biphasic effects on locomotion, and the PATs did not alter locomotion. In vitro molecular pharmacology studies showed that 5-HT2C agonists potent for attenuating the DOI-elicited-HTR also reduced the efficacy of DOI to activate mouse 5-HT2C receptor-mediated PLC signaling in HEK cells. Although there were differences in affinities of a few compounds at mouse compared to human 5-HT2A or 5-HT2C receptors, all compounds tested retained their selectivity for either receptor, regardless of receptor species. Results indicate that 5-HT2C receptor agonists and antagonists attenuate the DOI-elicited-HTR in C57Bl/6J mice, and suggest that structurally diverse 5-HT2C ligands result in different 5-HT2C receptor signaling outcomes compared to DOI.

  16. Exogenous fms-like tyrosine kinase 3 ligand overrides brain immune privilege and facilitates recognition of a neo-antigen without causing autoimmune neuropathology

    PubMed Central

    Larocque, Daniel; Sanderson, Nicholas S. R.; Bergeron, Josée; Curtin, James F.; Girton, Joe; Wibowo, Mia; Bondale, Niyati; Kroeger, Kurt M.; Yang, Jieping; Lacayo, Liliana M.; Reyes, Kevin C.; Farrokhi, Catherine; Pechnick, Robert N.; Castro, Maria G.; Lowenstein, Pedro R.

    2010-01-01

    Soluble antigens diffuse out of the brain and can thus stimulate a systemic immune response, whereas particulate antigens (from infectious agents or tumor cells) remain within brain tissue, thus failing to stimulate a systemic immune response. Immune privilege describes how the immune system responds to particulate antigens localized selectively within the brain parenchyma. We believe this immune privilege is caused by the absence of antigen presenting dendritic cells from the brain. We tested the prediction that expression of fms-like tyrosine kinase ligand 3 (Flt3L) in the brain will recruit dendritic cells and induce a systemic immune response against exogenous influenza hemagglutinin in BALB/c mice. Coexpression of Flt3L with HA in the brain parenchyma induced a robust systemic anti-HA immune response, and a small response against myelin basic protein and proteolipid protein epitopes. Depletion of CD4+CD25+ regulatory T cells (Tregs) enhanced both responses. To investigate the autoimmune impact of these immune responses, we characterized the neuropathological and behavioral consequences of intraparenchymal injections of Flt3L and HA in BALB/c and C57BL/6 mice. T cell infiltration in the forebrain was time and strain dependent, and increased in animals treated with Flt3L and depleted of Tregs; however, we failed to detect widespread defects in myelination throughout the forebrain or spinal cord. Results of behavioral tests were all normal. These results demonstrate that Flt3L overcomes the brain's immune privilege, and supports the clinical development of Flt3L as an adjuvant to stimulate clinically effective immune responses against brain neo-antigens, for example, those associated with brain tumors. PMID:20660723

  17. Detection of minor clones with internal tandem duplication mutations of FLT3 gene in acute myeloid leukemia using delta-PCR.

    PubMed

    Beierl, Katie; Tseng, Li-Hui; Beierl, Russell; Haley, Lisa; Gocke, Christopher D; Eshleman, James R; Lin, Ming-Tseh

    2013-03-01

    Internal tandem duplication (ITD) mutations of the FLT3 gene have been associated with inferior prognosis of acute myeloid leukemia. Detection of minor clones or minimal residual clones with ITD mutations is desirable, but is challenging when the mutant signal determined by polymerase chain reaction (PCR) and capillary electrophoresis is weak. In this study, we applied delta-PCR, which is a triple-primer strategy, to ensure PCR specificity and improve the sensitivity to 0.1% leukemic cells with ITD mutation. We also applied a reference peak to calculate ITD allelic burdens of <2% threshold of technical limitation for evaluating the relative ratio of 2 signals by capillary electrophoresis. Delta-PCR was able to detect single or multiple ITD mutations with an allelic burden (peak height ratio of mutant allele and wild-type allele) ranging from 0.4% to >100% among all 31 cases with previous documented ITD mutations. In one of the 3 cases with previously reported negative ITD mutation in the initial diagnostic specimen and ITD-positive results in the follow-up specimens, an ITD of 0.04% allele burden was retrospectively detected in the initial diagnosis specimen using delta-PCR. We also demonstrated that minor ITD mutant clones with an allelic burden of <1% present at diagnosis may become a dominant clone at the later refractory status, suggesting that detection of leukemic clones with allelic burdens of <1% may be clinically significant. Delta-PCR can detect ITD mutations with improved sensitivity and specificity and may be useful for the detection of minimal residual leukemia.

  18. FLT3-ITD and MLL-PTD influence the expression of MDR-1, MRP-1, and BCRP mRNA but not LRP mRNA assessed with RQ-PCR method in adult acute myeloid leukemia.

    PubMed

    Nasilowska-Adamska, Barbara; Solarska, Iwona; Paluszewska, Monika; Malinowska, Iwona; Jedrzejczak, Wieslaw W; Warzocha, Krzysztof

    2014-04-01

    Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) and mixed-lineage leukemia gene-partial tandem duplication (MLL-PTD) are aberrations associated with leukemia which indicate unsatisfactory prognosis. Downstream regulatory targets of FLT3-ITD and MLL-PTD are not well defined. We have analyzed the expression of MDR-1, multidrug resistant protein-1 (MRP-1), breast cancer resistance protein (BCRP), and lung resistance protein (LRP) messenger RNA (mRNA) in relation to the mutational status of FLT3-ITD and MLL-PTD in 185 acute myeloid leukemia (AML) adult patients. The real-time quantitative polymerase chain reaction method was performed to assess the expression of the MDR-1, MRP-1, BCRP, and LRP mRNA, and the results were presented as coefficients calculated using an intermediate method according to Pfaffl's rule. Significantly higher expressions of MDR-1 mRNA were found in patients who did not harbor FLT3-ITD (0.20 vs. 0.05; p = 0.0001) and MRP-1 mRNA in patients with this mutation (0.96 vs. 0.70; p = 0.002) and of BCRP mRNA in patients with MLL-PTD (0.61 vs. 0.38; p = 0.03). In univariate analysis, the high expression of MDR-1 mRNA (≥0.1317) negatively influenced the outcome of induction therapy (p = 0.05), whereas the high expression of BCRP mRNA (≥1.1487) was associated with a high relapse rate (RR) (p = 0.013). We found that the high expression of MDR-1 (≥0.1317), MRP-1 (≥0.8409), and BCRP mRNA (≥1.1487) significantly influenced disease-free survival (DFS; p = 0.059, 0.032, and 0.009, respectively) and overall survival (0.048, 0.014, and 0.059, respectively). Moreover, a high expression of BCRP mRNA (≥1.1487) proved to be an independent prognostic factor for RR (p = 0.01) and DFS (p = 0.002) in multivariate analysis. The significant correlation between the expression of MDR-1, MRP-1, and BCRP mRNA and FLT3-ITD or MLL-PTD in AML patients requires further investigation.

  19. STK-1, the human homolog of Flk-2/Flt-3, is selectively expressed in CD34+ human bone marrow cells and is involved in the proliferation of early progenitor/stem cells.

    PubMed Central

    Small, D; Levenstein, M; Kim, E; Carow, C; Amin, S; Rockwell, P; Witte, L; Burrow, C; Ratajczak, M Z; Gewirtz, A M

    1994-01-01

    We cloned the cDNA for stem cell tyrosine kinase 1 (STK-1), the human homolog of murine Flk-2/Flt-3, from a CD34+ hematopoietic stem cell-enriched library and investigated its expression in subsets of normal human bone marrow. The cDNA encodes a protein of 993 aa with 85% identity and 92% similarity to Flk-2/Flt-3. STK-1 is a member of the type III receptor tyrosine kinase family that includes KIT (steel factor receptor), FMS (colony-stimulating factor 1R), and platelet-derived growth factor receptor. STK-1 expression in human blood and marrow is restricted to CD34+ cells, a population greatly enriched for stem/progenitor cells. Anti-STK-1 antiserum recognizes polypeptides of 160 and 130 kDa in several STK-1-expressing cell lines and in 3T3 cells transfected with a STK-1 expression vector. Antisense oligonucleotides directed against STK-1 sequences inhibited hematopoietic colony formation, most strongly in long-term bone marrow cultures. These data suggest that STK-1 may function as a growth factor receptor on hematopoietic stem and/or progenitor cells. Images Fig. 2 Fig. 3 Fig. 4 PMID:7507245

  20. Fms-like tyrosine kinase 3 ligand is required for thymic dendritic cell generation from bone marrow-derived CD117⁺ hematopoietic progenitor cells.

    PubMed

    Xu, Yunyun; Jiang, Dong; Hu, Yizhou; Li, Yiping; Zhang, Xueguang; Wang, Jian; Wang, Yong

    2015-11-01

    Thymic dendritic cells (TDCs) are a type of dendritic cell (DC) in the thymus, which can enhance the proliferation of thymic T lymphocytes, regulate negative selection and induce central tolerance through autoantigen presentation. However, further investigations using TDCs has been restricted due to insufficient numbers. Therefore, an effective expansion method for TDCs in vitro is urgently required to further examine their biological characteristics. In the present study, a novel system was established using fetal thymus organ culture (FTOC) and a hanging drop culture system in the presence of fms‑like tyrosine kinase 3 ligand (Flt3L), termed the Flt3L/FTOC system. TDCs were successfully generated and expanded from CD117+ bone marrow hematopoietic progenitor cells. Conventional DCs (cDCs; CD11c+B220‑ DCs) and plasmacytoid DCs (pDCs; CD11c+B220+ DCs) were found in the TDCs generated using the Flt3L/FTOC system. These cells exhibited the specific morphological features of DCs, which were confirmed using Giemsa staining. Furthermore, the cytokine and surface marker profiles were also analyzed. Higher expression levels of interferon‑α and interleukin‑12 were observed in the pDCs, compared with the cDCs, and higher expression levels of toll‑like receptor (TLR)7 and TLR9 were found in the pDCs than in the cDCs. In addition, the Flt3L/FTOC‑derived TDCs also exhibited the ability to stimulate the allogenic T cell response. In conclusion, a novel in vitro culture system of thymic cDCs and pDCs using Flt3L was established, and this may provide a methodological basis for understanding the properties of TDCs.

  1. Ludic Elicitation: Using Games for Knowledge Elicitation

    ERIC Educational Resources Information Center

    Cao, Yan

    2014-01-01

    Knowledge elicitation from human beings is important for many fields, such as decision support systems, risk communication, and customer preference studying. Traditional approaches include observations, questionnaires, structured and semi-structured interviews, and group discussions. Many publications have been studying different techniques for a…

  2. Retracted: FLT3: beyond good and evil.

    PubMed

    Lagunas-Rangel, Francisco Alejandro; Cortes-Penagos, Carlos; Viveros-Sandoval, Martha Eva

    2016-07-29

    The above article, published online on 29 July 2016 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the Journal Editor in Chief, Journal Production Manager, and John Wiley & Sons, Ltd. The retraction has been agreed due to 48% similar significant between this article and an article published in Nature Reviews Cancer journal.

  3. Issues in Requirements Elicitation

    DTIC Science & Technology

    1992-09-01

    systems approach: characterized by Checkland’s Soft Systems Methodology (SSM), which is discussed briefly in Appendix A.6; emphasizes the subjectivity of...methodology for requirements elicitation. A.6 Notes on SSM Both the definition of methodology and the philosophy behind soft systems methodology (SSM...1986. [Checkland 89a] Checkland, Peter. Soft Systems Methodology . Rational Analysis for a Problematic World. New York: John Wiley & Sons, 71-100

  4. Notch-ligand expression by NALT dendritic cells regulates mucosal Th1- and Th2-type responses

    SciTech Connect

    Fukuyama, Yoshiko; Tokuhara, Daisuke; Sekine, Shinichi; Kataoka, Kosuke; Markham, Jonathan D.; Irwin, Allyson R.; Moon, Grace H.; Tokuhara, Yuka; Fujihashi, Keiko; Davydova, Julia; Yamamoto, Masato; Gilbert, Rebekah S.; Fujihashi, Kohtaro

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer Nasal Ad-FL effectively up-regulates APC function by CD11c{sup +} DCs in mucosal tissues. Black-Right-Pointing-Pointer Nasal Ad-FL induces Notch ligand (L)-expressing CD11c{sup +} DCs. Black-Right-Pointing-Pointer Notch L-expressing DCs support the induction of Th1- and Th2-type cytokine responses. -- Abstract: Our previous studies showed that an adenovirus (Ad) serotype 5 vector expressing Flt3 ligand (Ad-FL) as nasal adjuvant activates CD11c{sup +} dendritic cells (DCs) for the enhancement of antigen (Ag)-specific IgA antibody (Ab) responses. In this study, we examined the molecular mechanism for activation of CD11c{sup +} DCs and their roles in induction of Ag-specific Th1- and Th2-cell responses. Ad-FL activated CD11c{sup +} DCs expressed increased levels of the Notch ligand (L)-expression and specific mRNA. When CD11c{sup +} DCs from various mucosal and systemic lymphoid tissues of mice given nasal OVA plus Ad-FL were cultured with CD4{sup +} T cells isolated from non-immunized OVA TCR-transgenic (OT II) mice, significantly increased levels of T cell proliferative responses were noted. Furthermore, Ad-FL activated DCs induced IFN-{gamma}, IL-2 and IL-4 producing CD4{sup +} T cells. Of importance, these APC functions by Ad-FL activated DCs were down-regulated by blocking Notch-Notch-L pathway. These results show that Ad-FL induces CD11c{sup +} DCs to the express Notch-ligands and these activated DCs regulate the induction of Ag-specific Th1- and Th2-type cytokine responses.

  5. A Knowledge Elicitation Study of Military Planning

    DTIC Science & Technology

    1990-02-01

    66027-0347 Ba. NAME OF FUNDING/SPONSORING 8b. OFFICE SYMBOL 9. PROCUREMENT INSTRUMENT IDENTIFICATION NUMBER ORPANIZATION U.S. Army Research (If...Method (CDM) has been developed as a knowledge elicitation tool for probing proficient decision making. This report describes the use of CDM during...knowledge elicitation can be used in Army Command and Control (Ce) exercises as a means of understanding decision-making dynamics. Such a tool could

  6. Stereoscopy Amplifies Emotions Elicited by Facial Expressions.

    PubMed

    Hakala, Jussi; Kätsyri, Jari; Häkkinen, Jukka

    2015-12-01

    Mediated facial expressions do not elicit emotions as strongly as real-life facial expressions, possibly due to the low fidelity of pictorial presentations in typical mediation technologies. In the present study, we investigated the extent to which stereoscopy amplifies emotions elicited by images of neutral, angry, and happy facial expressions. The emotional self-reports of positive and negative valence (which were evaluated separately) and arousal of 40 participants were recorded. The magnitude of perceived depth in the stereoscopic images was manipulated by varying the camera base at 15, 40, 65, 90, and 115 mm. The analyses controlled for participants' gender, gender match, emotional empathy, and trait alexithymia. The results indicated that stereoscopy significantly amplified the negative valence and arousal elicited by angry expressions at the most natural (65 mm) camera base, whereas stereoscopy amplified the positive valence elicited by happy expressions in both the narrowed and most natural (15-65 mm) base conditions. Overall, the results indicate that stereoscopy amplifies the emotions elicited by mediated emotional facial expressions when the depth geometry is close to natural. The findings highlight the sensitivity of the visual system to depth and its effect on emotions.

  7. Stereoscopy Amplifies Emotions Elicited by Facial Expressions

    PubMed Central

    Kätsyri, Jari; Häkkinen, Jukka

    2015-01-01

    Mediated facial expressions do not elicit emotions as strongly as real-life facial expressions, possibly due to the low fidelity of pictorial presentations in typical mediation technologies. In the present study, we investigated the extent to which stereoscopy amplifies emotions elicited by images of neutral, angry, and happy facial expressions. The emotional self-reports of positive and negative valence (which were evaluated separately) and arousal of 40 participants were recorded. The magnitude of perceived depth in the stereoscopic images was manipulated by varying the camera base at 15, 40, 65, 90, and 115 mm. The analyses controlled for participants’ gender, gender match, emotional empathy, and trait alexithymia. The results indicated that stereoscopy significantly amplified the negative valence and arousal elicited by angry expressions at the most natural (65 mm) camera base, whereas stereoscopy amplified the positive valence elicited by happy expressions in both the narrowed and most natural (15–65 mm) base conditions. Overall, the results indicate that stereoscopy amplifies the emotions elicited by mediated emotional facial expressions when the depth geometry is close to natural. The findings highlight the sensitivity of the visual system to depth and its effect on emotions. PMID:27551358

  8. Rational elicitation of cold-sensitive phenotypes

    PubMed Central

    Baliga, Chetana; Majhi, Sandipan; Mondal, Kajari; Bhattacharjee, Antara; Varadarajan, Raghavan

    2016-01-01

    Cold-sensitive phenotypes have helped us understand macromolecular assembly and biological phenomena, yet few attempts have been made to understand the basis of cold sensitivity or to elicit it by design. We report a method for rational design of cold-sensitive phenotypes. The method involves generation of partial loss-of-function mutants, at either buried or functional sites, coupled with selective overexpression strategies. The only essential input is amino acid sequence, although available structural information can be used as well. The method has been used to elicit cold-sensitive mutants of a variety of proteins, both monomeric and dimeric, and in multiple organisms, namely Escherichia coli, Saccharomyces cerevisiae, and Drosophila melanogaster. This simple, yet effective technique of inducing cold sensitivity eliminates the need for complex mutations and provides a plausible molecular mechanism for eliciting cold-sensitive phenotypes. PMID:27091994

  9. Safety profile, efficacy, and biodistribution of a bicistronic high-capacity adenovirus vector encoding a combined immunostimulation and cytotoxic gene therapy as a prelude to a phase I clinical trial for glioblastoma

    SciTech Connect

    Puntel, Mariana; Ghulam, Muhammad A.K.M.; Farrokhi, Catherine; VanderVeen, Nathan; Paran, Christopher; Appelhans, Ashley; Kroeger, Kurt M.; Salem, Alireza; Lacayo, Liliana; Pechnick, Robert N.; Kelson, Kyle R.; Kaur, Sukhpreet; Kennedy, Sean; Palmer, Donna; Ng, Philip; and others

    2013-05-01

    Adenoviral vectors (Ads) are promising gene delivery vehicles due to their high transduction efficiency; however, their clinical usefulness has been hampered by their immunogenicity and the presence of anti-Ad immunity in humans. We reported the efficacy of a gene therapy approach for glioma consisting of intratumoral injection of Ads encoding conditionally cytotoxic herpes simplex type 1 thymidine kinase (Ad-TK) and the immunostimulatory cytokine fms-like tyrosine kinase ligand 3 (Ad-Flt3L). Herein, we report the biodistribution, efficacy, and neurological and systemic effects of a bicistronic high-capacity Ad, i.e., HC-Ad-TK/TetOn-Flt3L. HC-Ads elicit sustained transgene expression, even in the presence of anti-Ad immunity, and can encode large therapeutic cassettes, including regulatory elements to enable turning gene expression “on” or “off” according to clinical need. The inclusion of two therapeutic transgenes within a single vector enables a reduction of the total vector load without adversely impacting efficacy. Because clinically the vectors will be delivered into the surgical cavity, normal regions of the brain parenchyma are likely to be transduced. Thus, we assessed any potential toxicities elicited by escalating doses of HC-Ad-TK/TetOn-Flt3L (1 × 10{sup 8}, 1 × 10{sup 9}, or 1 × 10{sup 10} viral particles [vp]) delivered into the rat brain parenchyma. We assessed neuropathology, biodistribution, transgene expression, systemic toxicity, and behavioral impact at acute and chronic time points. The results indicate that doses up to 1 × 10{sup 9} vp of HC-Ad-TK/TetOn-Flt3L can be safely delivered into the normal rat brain and underpin further developments for its implementation in a phase I clinical trial for glioma. - Highlights: ► High capacity Ad vectors elicit sustained therapeutic gene expression in the brain. ► HC-Ad-TK/TetOn-Flt3L encodes two therapeutic genes and a transcriptional switch. ► We performed a dose escalation study at

  10. CD40 ligand immunotherapy in cancer: an efficient approach.

    PubMed

    Kuwashima, N; Kageyama, S; Eto, Y; Urashima, M

    2001-01-01

    Cancer cells do not elicit a clinically sufficient anti-tumor immune response that results in tumor rejection. Recently, many investigators have been trying to enhance anti-tumor immunity and encouraging results have been reported. This review will discuss current anti-cancer immunotherapy; interleukin-2 therapy, tumor vaccine secreting Granulocyte macrophage-colony stimulating factor, dendritic cells fused with tumor cells, and CD40 ligand immunotherapy. Moreover, we introduce our two kinds of CD40 ligand immuno-genetherapy; (1) oral CD40 ligand gene therapy against lymphoma using attenuated Salmonella typhimurium (published in BLOOD 2000), (2) cancer vaccine transfected with CD40 ligand ex vivo for neuroblastoma (unpublished). Both approaches resulted in a high degree of protection against the tumor progression and they are simple and safe in the murine system.

  11. Methods of Eliciting Information from Experts

    DTIC Science & Technology

    1987-10-01

    Itzhak Perlman) or a concertmeister in an orchestra, or simply one of its violinists . Differences in amount of expertise may supply different...would lead to becoming a world class violinist . Underlying Assumptions In attempting to elicit information from experts, one makes a number of

  12. Realistic Real World Contexts: Model Eliciting Activities

    ERIC Educational Resources Information Center

    Doruk, Bekir Kürsat

    2016-01-01

    Researchers have proposed a variety of methods to make a connection between real life and mathematics so that it can be learned in a practical way and enable people to utilise mathematics in their daily lives. Model-eliciting activities (MEAs) were developed to fulfil this need and are very capable of serving this purpose. The reason MEAs are so…

  13. Eliciting User Requirements Using Appreciative Inquiry

    ERIC Educational Resources Information Center

    Gonzales, Carol Kernitzki

    2010-01-01

    Many software development projects fail because they do not meet the needs of users, are over-budget, and abandoned. To address this problem, the user requirements elicitation process was modified based on principles of Appreciative Inquiry. Appreciative Inquiry, commonly used in organizational development, aims to build organizations, processes,…

  14. Video Elicitation of the Semiotic Self.

    ERIC Educational Resources Information Center

    Stockall, Nancy

    2001-01-01

    Illustrates the power of video recording to elicit the thinking of teachers as they reflect on their teaching practices. Draws on the work of Wiley (1994), which identified the internal conversation as a critical feature of the semiotic self. (Author/VWL)

  15. Learning from Expert Elicitation in Climate Change

    NASA Astrophysics Data System (ADS)

    Morgan, M. G.

    2009-12-01

    Since the early 1990's the author has been involved in the design and execution of six detailed expert elicitations that, among other things, have obtained subjective judgments from experts that reflect their best judgment in the form of subjective probability density functions, about the value of key climate variables, climate impacts and a technology for mitigation (Morgan and Keith, 1995; Morgan Pitelka and Shevliakova, 2001; Morgan, Adams and Keith, 2006; Zickfeld et al, 2007; Curtright, Morgan and Keith, 2008; Zickfeld, Morgan Keith and Frame, in review). This paper builds on that experience to draw insights about the design and use of expert elicitation in the assessment and analysis of climate change and its impacts. Several trends in responses will be noted. Methodological pitfalls will be discussed. Comparisons will be drawn with the consensus-based methods employed by IPCC, which appear to have produced tighter uncertainty bounds than individual elicitation. The paper will close with thoughts on the possible use of expert elicitation in future IPCC assessments. Support for this work is from the Climate Decision Making Center through a cooperative agreement between the National Science Foundation (SES-0345798) and Carnegie Mellon University. References: M. Granger Morgan and David Keith, "Subjective Judgments by Climate Experts," Environmental Science & Technology, 29(10), 468A-476A, October 1995. M. Granger Morgan, Louis F. Pitelka and Elena Shevliakova, "Elicitation of Expert Judgments of Climate Change Impacts on Forest Ecosystems," Climatic Change, 49, 279-307, 2001. M. Granger Morgan, Peter Adams, and David W. Keith, "Elicitation of Expert Judgments of Aerosol Forcing," Climatic Change, 75, 195-214, 2006. Kirsten Zickfeld, Anders Levermann, Till Kuhlbrodt. Stefan Rahmstorf, M. Granger Morgan and David Keith, "Expert Judgements on the Response on the Atlantic Meridional Overturning Circulation to Climate Change," Climatic Change, 82, 235-265, 2007

  16. [Endorphines--the endogenous ligands of opiate receptors (author's transl)].

    PubMed

    Teschemacher, H

    1978-01-01

    The demonstration of opiate receptors in the nervous tissue of vertebrates in 1973 was the starting point of an intensive search for the endogenous ligands of these receptors. During the following years, several of such "edogenous opiates", called "endorphines", were isolated from various tissues of the mammalian organism. These are peptides which are able to elicit the same effects as do opiates. Possibly, they play a role in the reaction of the organism to stress.

  17. Rod-shaped nanocrystals elicit neuronal activity in vivo.

    PubMed

    Malvindi, Maria Ada; Carbone, Luigi; Quarta, Alessandra; Tino, Angela; Manna, Liberato; Pellegrino, Teresa; Tortiglione, Claudia

    2008-10-01

    The development of novel nanomaterials has raised great interest in efforts to evaluate their effect on biological systems, ranging from single cells to whole animals. In particular, there exists an open question regarding whether nanoparticles per se can elicit biological responses, which could interfere with the phenomena they are intended to measure. Here it is reported that challenging the small cnidaria Hydra vulgaris in vivo with rod-shaped semiconductor nanoparticles, also known as quantum rods (QRs), results in an unexpected tentacle-writhing behavior, which is Ca(2+) dependent and relies on the presence of tentacle neurons. Due to the absence of surface functionalization of the QRs with specific ligands, and considering that spherical nanoparticles with same composition as the QRs fail to induce any in vivo behavior on the same experimental model, it is suggested that unique shape-tunable electrical properties of the QRs may account for the neuronal stimulation. This model system may represent a widely applicable tool for screening neuronal response to nanoparticles in vivo.

  18. Elicitation of secondary metabolism in actinomycetes.

    PubMed

    Abdelmohsen, Usama Ramadan; Grkovic, Tanja; Balasubramanian, Srikkanth; Kamel, Mohamed Salah; Quinn, Ronald J; Hentschel, Ute

    2015-11-01

    Genomic sequence data have revealed the presence of a large fraction of putatively silent biosynthetic gene clusters in the genomes of actinomycetes that encode for secondary metabolites, which are not detected under standard fermentation conditions. This review focuses on the effects of biological (co-cultivation), chemical, as well as molecular elicitation on secondary metabolism in actinomycetes. Our review covers the literature until June 2014 and exemplifies the diversity of natural products that have been recovered by such approaches from the phylum Actinobacteria.

  19. Essays on probability elicitation scoring rules

    NASA Astrophysics Data System (ADS)

    Firmino, Paulo Renato A.; dos Santos Neto, Ademir B.

    2012-10-01

    In probability elicitation exercises it has been usual to considerer scoring rules (SRs) to measure the performance of experts when inferring about a given unknown, Θ, for which the true value, θ*, is (or will shortly be) known to the experimenter. Mathematically, SRs quantify the discrepancy between f(θ) (the distribution reflecting the expert's uncertainty about Θ) and d(θ), a zero-one indicator function of the observation θ*. Thus, a remarkable characteristic of SRs is to contrast expert's beliefs with the observation θ*. The present work aims at extending SRs concepts and formulas for the cases where Θ is aleatory, highlighting advantages of goodness-of-fit and entropy-like measures. Conceptually, it is argued that besides of evaluating the personal performance of the expert, SRs may also play a role when comparing the elicitation processes adopted to obtain f(θ). Mathematically, it is proposed to replace d(θ) by g(θ), the distribution that model the randomness of Θ, and do also considerer goodness-of-fit and entropylike metrics, leading to SRs that measure the adherence of f(θ) to g(θ). The implications of this alternative perspective are discussed and illustrated by means of case studies based on the simulation of controlled experiments. The usefulness of the proposed approach for evaluating the performance of experts and elicitation processes is investigated.

  20. Ligand modeling and design

    SciTech Connect

    Hay, B.P.

    1997-10-01

    The purpose of this work is to develop and implement a molecular design basis for selecting organic ligands that would be used in the cost-effective removal of specific radionuclides from nuclear waste streams. Organic ligands with metal ion specificity are critical components in the development of solvent extraction and ion exchange processes that are highly selective for targeted radionuclides. The traditional approach to the development of such ligands involves lengthy programs of organic synthesis and testing, which in the absence of reliable methods for screening compounds before synthesis, results in wasted research effort. The author`s approach breaks down and simplifies this costly process with the aid of computer-based molecular modeling techniques. Commercial software for organic molecular modeling is being configured to examine the interactions between organic ligands and metal ions, yielding an inexpensive, commercially or readily available computational tool that can be used to predict the structures and energies of ligand-metal complexes. Users will be able to correlate the large body of existing experimental data on structure, solution binding affinity, and metal ion selectivity to develop structural design criteria. These criteria will provide a basis for selecting ligands that can be implemented in separations technologies through collaboration with other DOE national laboratories and private industry. The initial focus will be to select ether-based ligands that can be applied to the recovery and concentration of the alkali and alkaline earth metal ions including cesium, strontium, and radium.

  1. Programming of donor T cells using allogeneic δ-like ligand 4-positive dendritic cells to reduce GVHD in mice.

    PubMed

    Mochizuki, Kazuhiro; Meng, Lijun; Mochizuki, Izumi; Tong, Qing; He, Shan; Liu, Yongnian; Purushe, Janaki; Fung, Henry; Zaidi, M Raza; Zhang, Yanyun; Reshef, Ran; Blazar, Bruce R; Yagita, Hideo; Mineishi, Shin; Zhang, Yi

    2016-06-23

    Alloreactive T cells play a critical role in eliminating hematopoietic malignant cells but are also the mediators of graft-versus-host disease (GVHD), a major complication that subverts the success of allogeneic hematopoietic stem cell transplantation (HSCT). However, induction of alloreactive T cells does not necessarily lead to GVHD. Here we report the development of a cellular programming approach to render alloreactive T cells incapable of causing severe GVHD in both major histocompatibility complex (MHC)-mismatched and MHC-identical but minor histocompatibility antigen-mismatched mouse models. We established a novel platform that produced δ-like ligand 4-positive dendritic cells (Dll4(hi)DCs) from murine bone marrow using Flt3 ligand and Toll-like receptor agonists. Upon allogeneic Dll4(hi)DC stimulation, CD4(+) naïve T cells underwent effector differentiation and produced high levels of interferon γ (IFN-γ) and interleukin-17 in vitro, depending on Dll4 activation of Notch signaling. Following transfer, allogeneic Dll4(hi)DC-induced T cells were unable to mediate severe GVHD but preserved antileukemic activity, significantly improving the survival of leukemic mice undergoing allogeneic HSCT. This effect of Dll4(hi)DC-induced T cells was associated with their impaired expansion in GVHD target tissues. IFN-γ was important for Dll4(hi)DC programming to reduce GVHD toxicities of alloreactive T cells. Absence of T-cell IFN-γ led to improved survival and expansion of Dll4(hi)DC-induced CD4(+) T cells in transplant recipients and caused lethal GVHD. Our findings demonstrate that Dll4(hi)DC programming can overcome GVHD toxicity of donor T cells and produce leukemia-reactive T cells for effective immunotherapy.

  2. Ligand modeling and design

    SciTech Connect

    Hay, B.

    1996-10-01

    The purpose of this work is to develop and implement a molecular design basis for selecting organic ligands that would be used tin applications for the cost-effective removal of specific radionuclides from nuclear waste streams.

  3. Antagonist-Elicited Cannabis Withdrawal in Humans

    PubMed Central

    Gorelick, David A.; Goodwin, Robert S.; Schwilke, Eugene; Schwope, David M.; Darwin, William D.; Kelly, Deanna L.; McMahon, Robert P.; Liu, Fang; Ortemann-Renon, Catherine; Bonnet, Denis; Huestis, Marilyn A.

    2013-01-01

    Cannabinoid CB1 receptor antagonists have potential therapeutic benefits, but antagonist-elicited cannabis withdrawal has not been reported in humans. Ten male daily cannabis smokers received 8 days of increasingly frequent 20-mg oral Δ9-tetrahydrocannabinol (THC) dosages (40–120 mg/d) around-the-clock to standardize cannabis dependence while residing on a closed research unit. On the ninth day, double-blind placebo or 20- (suggested therapeutic dose) or 40-mg oral rimonabant, a CB1-cannabinoid receptor antagonist, was administered. Cannabis withdrawal signs and symptoms were assessed before and for 23.5 hours after rimonabant. Rimonabant, THC, and 11-hydroxy-THC plasma concentrations were quantified by mass spectrometry. The first 6 subjects received 20-mg rimonabant (1 placebo); the remaining 4 subjects received 40-mg rimonabant (1 placebo). Fourteen subjects enrolled; 10 completed before premature termination because of withdrawal of rimonabant from clinical development. Three of 5 subjects in the 20-mg group, 1 of 3 in the 40-mg group, and none of 2 in the placebo group met the prespecified withdrawal criterion of 150% increase or higher in at least 3 visual analog scales for cannabis withdrawal symptoms within 3 hours of rimonabant dosing. There were no significant associations between visual analog scale, heart rate, or blood pressure changes and peak rimonabant plasma concentration, area-under-the-rimonabant-concentration-by-time curve (0–8 hours), or peak rimonabant/THC or rimonabant/(THC + 11-hydroxy-THC) plasma concentration ratios. In summary, prespecified criteria for antagonist-elicited cannabis withdrawal were not observed at the 20- or 40-mg rimonabant doses. These data do not preclude antagonist-elicited withdrawal at higher rimonabant doses. PMID:21869692

  4. Unsaturated Zone Flow Model Expert Elicitation Project

    SciTech Connect

    Coppersmith, K. J.

    1997-05-30

    This report presents results of the Unsaturated Zone Flow Model Expert Elicitation (UZFMEE) project at Yucca Mountain, Nevada. This project was sponsored by the US Department of Energy (DOE) and managed by Geomatrix Consultants, Inc. (Geomatrix), for TRW Environmental Safety Systems, Inc. The objective of this project was to identify and assess the uncertainties associated with certain key components of the unsaturated zone flow system at Yucca Mountain. This assessment reviewed the data inputs, modeling approaches, and results of the unsaturated zone flow model (termed the ''UZ site-scale model'') being developed by Lawrence Berkeley National Laboratory (LBNL) and the US Geological Survey (USGS). In addition to data input and modeling issues, the assessment focused on percolation flux (volumetric flow rate per unit cross-sectional area) at the potential repository horizon. An understanding of unsaturated zone processes is critical to evaluating the performance of the potential high-level nuclear waste repository at Yucca Mountain. A major goal of the project was to capture the uncertainties involved in assessing the unsaturated flow processes, including uncertainty in both the models used to represent physical controls on unsaturated zone flow and the parameter values used in the models. To ensure that the analysis included a wide range of perspectives, multiple individual judgments were elicited from members of an expert panel. The panel members, who were experts from within and outside the Yucca Mountain project, represented a range of experience and expertise. A deliberate process was followed in facilitating interactions among the experts, in training them to express their uncertainties, and in eliciting their interpretations. The resulting assessments and probability distributions, therefore, provide a reasonable aggregate representation of the knowledge and uncertainties about key issues regarding the unsaturated zone at the Yucca Mountain site.

  5. Antagonist-elicited cannabis withdrawal in humans.

    PubMed

    Gorelick, David A; Goodwin, Robert S; Schwilke, Eugene; Schwope, David M; Darwin, William D; Kelly, Deanna L; McMahon, Robert P; Liu, Fang; Ortemann-Renon, Catherine; Bonnet, Denis; Huestis, Marilyn A

    2011-10-01

    Cannabinoid CB1 receptor antagonists have potential therapeutic benefits, but antagonist-elicited cannabis withdrawal has not been reported in humans. Ten male daily cannabis smokers received 8 days of increasingly frequent 20-mg oral Δ⁹-tetrahydrocannabinol (THC) dosages (40-120 mg/d) around-the-clock to standardize cannabis dependence while residing on a closed research unit. On the ninth day, double-blind placebo or 20- (suggested therapeutic dose) or 40-mg oral rimonabant, a CB1-cannabinoid receptor antagonist, was administered. Cannabis withdrawal signs and symptoms were assessed before and for 23.5 hours after rimonabant. Rimonabant, THC, and 11-hydroxy-THC plasma concentrations were quantified by mass spectrometry. The first 6 subjects received 20-mg rimonabant (1 placebo); the remaining 4 subjects received 40-mg rimonabant (1 placebo). Fourteen subjects enrolled; 10 completed before premature termination because of withdrawal of rimonabant from clinical development. Three of 5 subjects in the 20-mg group, 1 of 3 in the 40-mg group, and none of 2 in the placebo group met the prespecified withdrawal criterion of 150% increase or higher in at least 3 visual analog scales for cannabis withdrawal symptoms within 3 hours of rimonabant dosing. There were no significant associations between visual analog scale, heart rate, or blood pressure changes and peak rimonabant plasma concentration, area-under-the-rimonabant-concentration-by-time curve (0-8 hours), or peak rimonabant/THC or rimonabant/(THC + 11-hydroxy-THC) plasma concentration ratios. In summary, prespecified criteria for antagonist-elicited cannabis withdrawal were not observed at the 20- or 40-mg rimonabant doses. These data do not preclude antagonist-elicited withdrawal at higher rimonabant doses.

  6. GPCR biased ligands as novel heart failure therapeutics.

    PubMed

    Violin, Jonathan D; Soergel, David G; Boerrigter, Guido; Burnett, John C; Lark, Michael W

    2013-10-01

    G protein-coupled receptors have been successfully targeted by numerous therapeutics including drugs that have transformed the management of cardiovascular disease. However, many GPCRs, when activated or blocked by drugs, elicit both beneficial and adverse pharmacology. Recent work has demonstrated that in some cases, the salutary and deleterious signals linked to a specific GPCR can be selectively targeted by "biased ligands" that entrain subsets of a receptor's normal pharmacology. This review briefly summarizes the advances and current state of the biased ligand field, focusing on an example: biased ligands targeting the angiotensin II type 1 receptor. These compounds exhibit unique pharmacology, distinct from classic agonists or antagonists, and one such molecule is now in clinical development for the treatment of acute heart failure.

  7. Eliciting promises from children reduces cheating.

    PubMed

    Heyman, Gail D; Fu, Genyue; Lin, Jianyan; Qian, Miao K; Lee, Kang

    2015-11-01

    Widespread cheating can undermine rules that are necessary for maintaining social order. Preventing cheating can be a challenge, especially with regard to children, who as a result of their limited executive function skills may have particular difficulty with resisting temptation to cheat. We examined one approach designed to help children resist this temptation: eliciting a verbal commitment to not cheat. We tested 4- to 7-year-olds (total N = 330) and found that starting at 5 years of age, a verbal commitment to not cheat led to a substantial reduction in cheating. The results suggest that verbal commitments can be used to help children overcome temptations and comply with rules.

  8. Cell mechanosensory recognizes ligand compliance at biomaterial interface.

    PubMed

    Cosenza, Chiara; Lettera, Vincenzo; Causa, Filippo; Scognamiglio, Pasqualina Liana; Battista, Edmondo; Netti, Paolo Antonio

    2016-01-01

    Cells activate signalling through ligand-receptor bonds by sensing the mechanical properties of the surrounding extracellular matrix (ECM). Ligands, indeed, have to withstand the pulling force elicited by cell receptors through focal adhesions (FAs). On this basis, we developed functional ligands to be simply adsorbed on surfaces and constituted by a two-domain peptide: one derived from ECM proteins and available to receptors to offer biochemical cues, and another adsorbed on material to withstand the tension upon receptor engagement. Tuneable compliance of the anchoring domain of the peptide ligand was verified by single peptide analysis through molecular dynamics and adsorption measurements. We showed that the highest adsorbed peptides combined with integrin cell-binding motifs allow for the cell recognition and polarization with larger mature FA areas. On the contrary, the lowest adsorbed sequences did not provide mechanical resistance to the integrin pulling action, leading to more rounded cells with smaller FA areas. This evidence demonstrates that cell mechanosensory can discriminate ligands on surfaces and should be considered as a criterion in ligand design for material bioactivation.

  9. Ligand binding analysis and screening by chemical denaturation shift.

    PubMed

    Schön, Arne; Brown, Richard K; Hutchins, Burleigh M; Freire, Ernesto

    2013-12-01

    The identification of small molecule ligands is an important first step in drug development, especially drugs that target proteins with no intrinsic activity. Toward this goal, it is important to have access to technologies that are able to measure binding affinities for a large number of potential ligands in a fast and accurate way. Because ligand binding stabilizes the protein structure in a manner dependent on concentration and binding affinity, the magnitude of the protein stabilization effect elicited by binding can be used to identify and characterize ligands. For example, the shift in protein denaturation temperature (Tm shift) has become a popular approach to identify potential ligands. However, Tm shifts cannot be readily transformed into binding affinities, and the ligand rank order obtained at denaturation temperatures (≥60°C) does not necessarily coincide with the rank order at physiological temperature. An alternative approach is the use of chemical denaturation, which can be implemented at any temperature. Chemical denaturation shifts allow accurate determination of binding affinities with a surprisingly wide dynamic range (high micromolar to sub nanomolar) and in situations where binding changes the cooperativity of the unfolding transition. In this article, we develop the basic analytical equations and provide several experimental examples.

  10. Gene therapy-mediated reprogramming tumor infiltrating T cells using IL-2 and inhibiting NF-κB signaling improves the efficacy of immunotherapy in a brain cancer model.

    PubMed

    Mineharu, Yohei; Muhammad, A K M Ghulam; Yagiz, Kader; Candolfi, Marianela; Kroeger, Kurt M; Xiong, Weidong; Puntel, Mariana; Liu, Chunyan; Levy, Eva; Lugo, Claudia; Kocharian, Adrina; Allison, James P; Curran, Michael A; Lowenstein, Pedro R; Castro, Maria G

    2012-10-01

    Immune-mediated gene therapy using adenovirus expressing Flt3 ligand and thymidine kinase followed by ganciclovir administration (Flt3/TK) effectively elicits tumor regression in preclinical glioma models. Herein, we assessed new strategies to optimize Flt3L/TK therapeutic efficacy in a refractory RG2 orthotopic glioblastoma model. Specifically, we aimed to optimize the therapeutic efficacy of Flt3L/TK treatment in the RG2 model by overexpressing the following genes within the brain tumor microenvironment: 1) a TK mutant with enhanced cytotoxicity (SR39 mutant TK), 2) Flt3L-IgG fusion protein that has a longer half-life, 3) CD40L to stimulate DC maturation, 4) T helper cell type 1 polarizing dendritic cell cytokines interleukin-12 or C-X-C motif ligand 10 chemokine (CXCL)-10, 5) C-C motif ligand 2 chemokine (CCL2) or C-C motif ligand 3 chemokine (CCL3) to enhance dendritic cell recruitment into the tumor microenvironment, 6) T helper cell type 1 cytokines interferon-γ or interleukin-2 to enhance effector T-cell functions, and 7) IκBα or p65RHD (nuclear factor kappa-B [NF-κB] inhibitors) to suppress the function of Foxp3+ Tregs and enhanced effector T-cell functions. Anti-tumor immunity and tumor specific effector T-cell functions were assessed by cytotoxic T lymphocyte assay and intracellular IFN-γ staining. Our data showed that overexpression of interferon-γ or interleukin-2, or inhibition of the nuclear factor kappa-B within the tumor microenvironment, enhanced cytotoxic T lymphocyte-mediated immune responses and successfully extended the median survival of rats bearing intracranial RG2 when combined with Flt3L/TK. These findings indicate that enhancement of T-cell functions constitutes a critical therapeutic target to overcome immune evasion and enhance therapeutic efficacy for brain cancer. In addition, our study provides novel targets to be used in combination with immune-therapeutic strategies for glioblastoma, which are currently being tested in the

  11. Multiple faces elicit augmented neural activity

    PubMed Central

    Puce, Aina; McNeely, Marie E.; Berrebi, Michael E.; Thompson, James C.; Hardee, Jillian; Brefczynski-Lewis, Julie

    2013-01-01

    How do our brains respond when we are being watched by a group of people?Despite the large volume of literature devoted to face processing, this question has received very little attention. Here we measured the effects on the face-sensitive N170 and other ERPs to viewing displays of one, two and three faces in two experiments. In Experiment 1, overall image brightness and contrast were adjusted to be constant, whereas in Experiment 2 local contrast and brightness of individual faces were not manipulated. A robust positive-negative-positive (P100-N170-P250) ERP complex and an additional late positive ERP, the P400, were elicited to all stimulus types. As the number of faces in the display increased, N170 amplitude increased for both stimulus sets, and latency increased in Experiment 2. P100 latency and P250 amplitude were affected by changes in overall brightness and contrast, but not by the number of faces in the display per se. In Experiment 1 when overall brightness and contrast were adjusted to be constant, later ERP (P250 and P400) latencies showed differences as a function of hemisphere. Hence, our data indicate that N170 increases its magnitude when multiple faces are seen, apparently impervious to basic low-level stimulus features including stimulus size. Outstanding questions remain regarding category-sensitive neural activity that is elicited to viewing multiple items of stimulus categories other than faces. PMID:23785327

  12. Isolated sleep paralysis elicited by sleep interruption.

    PubMed

    Takeuchi, T; Miyasita, A; Sasaki, Y; Inugami, M; Fukuda, K

    1992-06-01

    We elicited isolated sleep paralysis (ISP) from normal subjects by a nocturnal sleep interruption schedule. On four experimental nights, 16 subjects had their sleep interrupted for 60 minutes by forced awakening at the time when 40 minutes of nonrapid eye movement (NREM) sleep had elapsed from the termination of rapid eye movement (REM) sleep in the first or third sleep cycle. This schedule produced a sleep onset REM period (SOREMP) after the interruption at a high rate of 71.9%. We succeeded in eliciting six episodes of ISP in the sleep interruptions performed (9.4%). All episodes of ISP except one occurred from SOREMP, indicating a close correlation between ISP and SOREMP. We recorded verbal reports about ISP experiences and recorded the polysomnogram (PSG) during ISP. All of the subjects with ISP experienced inability to move and were simultaneously aware of lying in the laboratory. All but one reported auditory/visual hallucinations and unpleasant emotions. PSG recordings during ISP were characterized by a REM/W stage dissociated state, i.e. abundant alpha electroencephalographs and persistence of muscle atonia shown by the tonic electromyogram. Judging from the PSG recordings, ISP differs from other dissociated states such as lucid dreaming, nocturnal panic attacks and REM sleep behavior disorders. We compare some of the sleep variables between ISP and non-ISP nights. We also discuss the similarities and differences between ISP and sleep paralysis in narcolepsy.

  13. TGF-α ligands can substitute for the neuregulin Vein in Drosophila development.

    PubMed

    Austin, Christina L; Manivannan, Sathiya N; Simcox, Amanda

    2014-11-01

    ErbB receptors, including the epidermal growth factor receptor (Egfr), are activated by EGF ligands to govern cell proliferation, survival, migration and differentiation. The different EGF-induced cell responses in development are regulated by deployment of multiple ligands. These inputs, however, engage only a limited number of intracellular pathways and are thought to elicit specific responses by regulating the amplitude or duration of the intracellular signal. The single Drosophila Egfr has four ligands: three of the TGF-α-type and a single neuregulin-like called vein (vn). Here, we used mutant combinations and gene replacement to determine the constraints of ligand specificity in development. Mutant analysis revealed extensive ligand redundancy in embryogenesis and wing development. Surprisingly, we found that the essential role of vn in development could be largely replaced by expression of any TGF-α ligand, including spitz (spi), in the endogenous vn pattern. vn mutants die as white undifferentiated pupae, but the rescued individuals showed global differentiation of adult body parts. Spi is more potent than Vn, and the best morphological rescue occurred when Spi expression was reduced to achieve an intracellular signaling level comparable to that produced by Vn. Our results show that the developmental repertoire of a strong ligand like Spi is flexible and at the appropriate level can emulate the activity of a weak ligand like Vn. These findings align with a model whereby cells respond similarly to an equivalent quantitative level of an intracellular signal generated by two distinct ligands regardless of ligand identity.

  14. Fluorescent Approaches for Understanding Interactions of Ligands with G Protein Coupled Receptors

    PubMed Central

    Sridharan, Rajashri; Zuber, Jeffrey; Connelly, Sara M.; Mathew, Elizabeth; Dumont, Mark E.

    2014-01-01

    G Protein Coupled Receptors (GPCRs) are responsible for a wide variety of signaling responses in diverse cell types. Despite major advances in the determination of structures of this class of receptors, the underlying mechanisms by which binding of different types of ligands specifically elicits particular signaling responses remains unclear. The use of fluorescence spectroscopy can provide important information about the process of ligand binding and ligand dependent conformational changes in receptors, especially kinetic aspects of these processes, that can be difficult to extract from x-ray structures. We present an overview of the extensive array of fluorescent ligands that have been used in studies of GPCRs and describe spectroscopic approaches for assaying binding and probing the environment of receptor-bound ligands with particular attention to examples involving yeast pheromone receptors. In addition, we discuss the use of fluorescence spectroscopy for detecting and characterizing conformational changes in receptors induced by the binding of ligands. Such studies have provided strong evidence for diversity of receptor conformations elicited by different ligands, consistent with the idea that GPCRs are not simple on and off switches. This diversity of states constitutes an underlying mechanistic basis for biased agonism, the observation that different stimuli can produce different responses from a single receptor. It is likely that continued technical advances will allow fluorescence spectroscopy to play an important role in continued probing of structural transitions in GPCRs. PMID:24055822

  15. Assessing pragmatic skills in elicited production.

    PubMed

    de Villiers, Peter

    2004-02-01

    In developing a test of pragmatic skills for children ages 4 to 9 years, we focused on a number of functional language skills that are important for children's success in early schooling and for the development of fluent reading and writing. They included (1) wh-question asking, (2) communicative role taking, (3) linking events in a cohesive narrative, and (4) articulating the mental states of the characters in a story. All of the proposed items provide specific referential support and pragmatic motivation for the forms and content to be produced by the child. The pictured materials and elicitation prompts constrain the range of appropriate utterances, so the children's productions are more easily scored than an open-ended spontaneous speech sample. All tasks described show a clear developmental trend, a clear separation between the performance of typically developing and language-impaired children, and no performance differences between African American English- and Mainstream American English-speaking children.

  16. Ligand fitting with CCP4

    PubMed Central

    2017-01-01

    Crystal structures of protein–ligand complexes are often used to infer biology and inform structure-based drug discovery. Hence, it is important to build accurate, reliable models of ligands that give confidence in the interpretation of the respective protein–ligand complex. This paper discusses key stages in the ligand-fitting process, including ligand binding-site identification, ligand description and conformer generation, ligand fitting, refinement and subsequent validation. The CCP4 suite contains a number of software tools that facilitate this task: AceDRG for the creation of ligand descriptions and conformers, Lidia and JLigand for two-dimensional and three-dimensional ligand editing and visual analysis, Coot for density interpretation, ligand fitting, analysis and validation, and REFMAC5 for macromolecular refinement. In addition to recent advancements in automatic carbohydrate building in Coot (LO/Carb) and ligand-validation tools (FLEV), the release of the CCP4i2 GUI provides an integrated solution that streamlines the ligand-fitting workflow, seamlessly passing results from one program to the next. The ligand-fitting process is illustrated using instructive practical examples, including problematic cases such as post-translational modifications, highlighting the need for careful analysis and rigorous validation. PMID:28177312

  17. Expert elicitation of population-level effects of disturbance

    USGS Publications Warehouse

    Fleishman, Erica; Burgman, Mark; Runge, Michael C.; Schick, Robert S; Krauss, Scott; Popper, Arthur N.; Hawkins, Anthony

    2016-01-01

    Expert elicitation is a rigorous method for synthesizing expert knowledge to inform decision making and is reliable and practical when field data are limited. We evaluated the feasibility of applying expert elicitation to estimate population-level effects of disturbance on marine mammals. Diverse experts estimated parameters related to mortality and sublethal injury of North Atlantic right whales (Eubalaena glacialis). We are now eliciting expert knowledge on the movement of right whales among geographic regions to parameterize a spatial model of health. Expert elicitation complements methods such as simulation models or extrapolations from other species, sometimes with greater accuracy and less uncertainty.

  18. Expert Elicitation of Population-Level Effects of Disturbance.

    PubMed

    Fleishman, Erica; Burgman, Mark; Runge, Michael C; Schick, Robert S; Kraus, Scott

    2016-01-01

    Expert elicitation is a rigorous method for synthesizing expert knowledge to inform decision making and is reliable and practical when field data are limited. We evaluated the feasibility of applying expert elicitation to estimate population-level effects of disturbance on marine mammals. Diverse experts estimated parameters related to mortality and sublethal injury of North Atlantic right whales (Eubalaena glacialis). We are now eliciting expert knowledge on the movement of right whales among geographic regions to parameterize a spatial model of health. Expert elicitation complements methods such as simulation models or extrapolations from other species, sometimes with greater accuracy and less uncertainty.

  19. LigandRNA: computational predictor of RNA-ligand interactions.

    PubMed

    Philips, Anna; Milanowska, Kaja; Lach, Grzegorz; Bujnicki, Janusz M

    2013-12-01

    RNA molecules have recently become attractive as potential drug targets due to the increased awareness of their importance in key biological processes. The increase of the number of experimentally determined RNA 3D structures enabled structure-based searches for small molecules that can specifically bind to defined sites in RNA molecules, thereby blocking or otherwise modulating their function. However, as of yet, computational methods for structure-based docking of small molecule ligands to RNA molecules are not as well established as analogous methods for protein-ligand docking. This motivated us to create LigandRNA, a scoring function for the prediction of RNA-small molecule interactions. Our method employs a grid-based algorithm and a knowledge-based potential derived from ligand-binding sites in the experimentally solved RNA-ligand complexes. As an input, LigandRNA takes an RNA receptor file and a file with ligand poses. As an output, it returns a ranking of the poses according to their score. The predictive power of LigandRNA favorably compares to five other publicly available methods. We found that the combination of LigandRNA and Dock6 into a "meta-predictor" leads to further improvement in the identification of near-native ligand poses. The LigandRNA program is available free of charge as a web server at http://ligandrna.genesilico.pl.

  20. Eliciting and using expert knowledge in metrology

    NASA Astrophysics Data System (ADS)

    O'Hagan, Anthony

    2014-08-01

    The expression of uncertainty has hitherto been seen as an add-on—first an estimate is obtained and then uncertainty in that estimate is evaluated. We argue that quantification of uncertainty should be an intrinsic part of measurement and that the measurement result should be a probability distribution for the measurand. Full quantification of uncertainties in measurement, recognizing and quantifying all sources of uncertainty, is rarely simple. Many potential sources of uncertainty can effectively only be quantified by the application of expert judgement. Scepticism about the validity or reliability of expert judgement has meant that these sources of uncertainty have often been overlooked, ignored or treated in a qualitative, narrative way. But the consequence of this is that reported expressions of uncertainty regularly understate the true degree of uncertainty in measurements. This article first discusses the concept of quantifying uncertainty in measurement, and then considers some of the areas where expert judgement is needed in order to quantify fully the uncertainties in measurement. The remainder of the article is devoted to describing methodology for eliciting expert knowledge.

  1. Basic emotions elicited by odors and pictures.

    PubMed

    Croy, Ilona; Olgun, Selda; Joraschky, Peter

    2011-12-01

    The sense of olfaction is often reported to have a special relationship with emotional processing. Memories triggered by olfactory cues often have a very emotional load. On the other hand, basic negative or positive emotional states should be sufficient to cover the most significant functions of the olfactory system including ingestion, hazard avoidance, and social communication. Thus, we investigated whether different basic emotions can be evoked in healthy people through the sense of olfaction. We asked 119 participants which odor evokes one of the six basic emotions (happiness, disgust, anger, anxiety, sadness, and surprise); another 97 participants were asked about pictures evoking those emotions. The results showed that almost every participant could name an olfactory elicitor for happiness or disgust. Olfactory elicitors of anxiety were reported less frequently, but they were still reported by three-quarters of the participants. However, for sadness and anger only about half of the participants reported an olfactory elicitor, whereas significantly more named a visual cue. Olfactory emotion elicitors were mainly related to the classes of culture, plants, and food, and visual emotion elicitors were largely related to humans. This data supports the hypothesis that in the vast majority of people, few differentiated emotions can be elicited through the olfactory channel. These emotions are happiness, disgust, and anxiety.

  2. Acting green elicits a literal warm glow

    NASA Astrophysics Data System (ADS)

    Taufik, Danny; Bolderdijk, Jan Willem; Steg, Linda

    2015-01-01

    Environmental policies are often based on the assumption that people only act environmentally friendly if some extrinsic reward is implicated, usually money. We argue that people might also be motivated by intrinsic rewards: doing the right thing (such as acting environmentally friendly) elicits psychological rewards in the form of positive feelings, a phenomenon known as warm glow. Given the fact that people's psychological state may affect their thermal state, we expected that this warm glow could express itself quite literally: people who act environmentally friendly may perceive the temperature to be higher. In two studies, we found that people who learned they acted environmentally friendly perceived a higher temperature than people who learned they acted environmentally unfriendly. The underlying psychological mechanism pertains to the self-concept: learning you acted environmentally friendly signals to yourself that you are a good person. Together, our studies show that acting environmentally friendly can be psychologically rewarding, suggesting that appealing to intrinsic rewards can be an alternative way to encourage pro-environmental actions.

  3. Octave illusion elicited by overlapping narrowband noises.

    PubMed

    Jonas Brännström, K; Nilsson, Patrik

    2011-05-01

    The octave or Deutsch illusion occurs when two tones, separated by about one octave, are presented simultaneously but alternating between ears, such that when the low tone is presented to the left ear the high tone is presented to the right ear and vice versa. Most subjects hear a single tone that alternates both between ears and in pitch; i.e., they hear a low pitched tone in one ear alternating with a high pitched tone in the other ear. The present study examined whether the illusion can be elicited by aperiodic signals consisting of low-frequency band-pass filtered noises with overlapping spectra. The amount of spectral overlap was held constant, but the high- and low-frequency content of the signals was systematically varied. The majority of subjects perceived an auditory illusion in terms of a dominant ear for pitch and lateralization by frequency, as proposed by Deutsch [(1975a) Sci. Am. 233, 92-104]. Furthermore, the salience of the illusion increased as the high frequency of the content in the signal increased. Since no harmonics were present in the stimuli, it is highly unlikely that this illusion is perceived on the basis of binaural diplacusis or harmonic binaural fusion.

  4. Analysis of macromolecules, ligands and macromolecule-ligand complexes

    DOEpatents

    Von Dreele, Robert B.

    2008-12-23

    A method for determining atomic level structures of macromolecule-ligand complexes through high-resolution powder diffraction analysis and a method for providing suitable microcrystalline powder for diffraction analysis are provided. In one embodiment, powder diffraction data is collected from samples of polycrystalline macromolecule and macromolecule-ligand complex and the refined structure of the macromolecule is used as an approximate model for a combined Rietveld and stereochemical restraint refinement of the macromolecule-ligand complex. A difference Fourier map is calculated and the ligand position and points of interaction between the atoms of the macromolecule and the atoms of the ligand can be deduced and visualized. A suitable polycrystalline sample of macromolecule-ligand complex can be produced by physically agitating a mixture of lyophilized macromolecule, ligand and a solvent.

  5. EGF receptor ligands: recent advances

    PubMed Central

    Singh, Bhuminder; Carpenter, Graham; Coffey, Robert J.

    2016-01-01

    Seven ligands bind to and activate the mammalian epidermal growth factor (EGF) receptor (EGFR/ERBB1/HER1): EGF, transforming growth factor-alpha (TGFA), heparin-binding EGF-like growth factor (HBEGF), betacellulin (BTC), amphiregulin (AREG), epiregulin (EREG), and epigen (EPGN). Of these, EGF, TGFA, HBEGF, and BTC are thought to be high-affinity ligands, whereas AREG, EREG, and EPGN constitute low-affinity ligands. This focused review is meant to highlight recent studies related to actions of the individual EGFR ligands, the interesting biology that has been uncovered, and relevant advances related to ligand interactions with the EGFR. PMID:27635238

  6. DEVELOPMENT OF AN INSTRUMENT FOR ELICITING AND EVALUATING VOCATIONAL IMAGERY.

    ERIC Educational Resources Information Center

    MATHEWSON, ROBERT H.; ORTON, JOHN W.

    THIS STUDY WAS CONDUCTED TO (1) PRODUCE AN INSTRUMENT FOR ELICITING VOCATIONAL IMAGERY FOR USE IN THE EDUCATIONAL-VOCATIONAL ORIENTATION AND COUNSELING OF HIGH SCHOOL YOUTH, AND (2) DEVELOP A SCALE FOR EVALUATING THE MATURITY OF THE VOCATIONAL IMAGERY ELICITED BY THE INSTRUMENT. A PREVIOUSLY DESIGNED INSTRUMENT "WHAT I THINK OF MYSELF"…

  7. Freeze or Flee? Negative Stimuli Elicit Selective Responding

    ERIC Educational Resources Information Center

    Estes, Zachary; Verges, Michelle

    2008-01-01

    Humans preferentially attend to negative stimuli. A consequence of this automatic vigilance for negative valence is that negative words elicit slower responses than neutral or positive words on a host of cognitive tasks. Some researchers have speculated that negative stimuli elicit a general suppression of motor activity, akin to the freezing…

  8. Eliciting Spontaneous Speech in Bilingual Students: Methods & Techniques.

    ERIC Educational Resources Information Center

    Cornejo, Ricardo J.; And Others

    Intended to provide practical information pertaining to methods and techniques for speech elicitation and production, the monograph offers specific methods and techniques to elicit spontaneous speech in bilingual students. Chapter 1, "Traditional Methodologies for Language Production and Recording," presents an overview of studies using…

  9. Introducing Forum Theatre to Elicit and Advocate Children's Views

    ERIC Educational Resources Information Center

    Hammond, Nick

    2013-01-01

    Eliciting and advocating the voice of the child remains at the heart of international political agenda and also remains a central role for educational psychologists (EPs). Previous research indicates that EPs tend to use language-based methods for eliciting and advocating views of children. However, these approaches are often limited. Taking a…

  10. Pedagogical use of ELICIT for Leadership Training: Survey and Recommendations

    DTIC Science & Technology

    2011-06-01

    classroom training as part of leadership development programs. ELICIT is particularly helpful in teaching the strengths and weakness of various...organizational structures (edge organization vs. traditional command and control hierarchy.) This paper reviews some of the pedagogical uses of the classroom ...training to date, and provides recommendations for the use of ELICIT in various classroom settings. 15. SUBJECT TERMS 16. SECURITY CLASSIFICATION OF

  11. Elicited Emotions and Cognitive Functioning in Preschool Children

    ERIC Educational Resources Information Center

    Blau, Rivka; Klein, Pnina S.

    2010-01-01

    In this study, the effects of eliciting positive and negative emotions on various cognitive functions of four- to five-year-old preschool children were examined. Emotions were elicited through presentations of "happy" and "sad" video clips, before the children performed the cognitive tasks. Behavioural (facial expressions) and…

  12. Somatic and axonal LIGHT signaling elicit degenerative and regenerative responses in motoneurons, respectively

    PubMed Central

    Otsmane, Belkacem; Moumen, Anice; Aebischer, Julianne; Coque, Emmanuelle; Sar, Chamroeun; Sunyach, Claire; Salsac, Céline; Valmier, Jean; Salinas, Sara; Bowerman, Melissa; Raoul, Cédric

    2014-01-01

    A receptor–ligand interaction can evoke a broad range of biological activities in different cell types depending on receptor identity and cell type-specific post-receptor signaling intermediates. Here, we show that the TNF family member LIGHT, known to act as a death-triggering factor in motoneurons through LT-βR, can also promote axon outgrowth and branching in motoneurons through the same receptor. LIGHT-induced axonal elongation and branching require ERK and caspase-9 pathways. This distinct response involves a compartment-specific activation of LIGHT signals, with somatic activation-inducing death, while axonal stimulation promotes axon elongation and branching in motoneurons. Following peripheral nerve damage, LIGHT increases at the lesion site through expression by invading B lymphocytes, and genetic deletion of Light significantly delays functional recovery. We propose that a central and peripheral activation of the LIGHT pathway elicits different functional responses in motoneurons. PMID:24668263

  13. Estrogen Receptor Ligands: A Review (2013–2015)

    PubMed Central

    Farzaneh, Shabnam; Zarghi, Afshin

    2016-01-01

    Estrogen receptors (ERs) are a group of compounds named for their importance in both menstrual and estrous reproductive cycles. They are involved in the regulation of various processes ranging from tissue growth maintenance to reproduction. Their action is mediated through ER nuclear receptors. Two subtypes of the estrogen receptor, ERα and ERβ, exist and exhibit distinct cellular and tissue distribution patterns. In humans, both receptor subtypes are expressed in many cells and tissues, and they control key physiological functions in various organ systems. Estrogens attract great attention due to their wide applications in female reproductive functions and treatment of some estrogen-dependent cancers and osteoporosis. This paper provides a general review of ER ligands published in international journals patented between 2013 and 2015. The broad physiological profile of estrogens has attracted the attention of many researchers to develop new estrogen ligands as therapeutic molecules for various clinical purposes. After the discovery of the ERβ receptor, subtype-selective ligands could be used to elicit beneficial estrogen-like activities and reduce adverse side effects, based on the different distributions and relative levels of the two ER subtypes in different estrogen target tissues. Therefore, recent literature has focused on selective estrogen ligands as highly promising agents for the treatment of some types of cancer, as well as for cardiovascular, inflammatory, and neurodegenerative diseases. Estrogen receptors are nuclear transcription factors that are involved in the regulation of many complex physiological functions in humans. Selective estrogen ligands are highly promising targets for treatment of some types of cancer, as well as for cardiovascular, inflammatory and neurodegenerative diseases. Extensive structure-activity relationship studies of ER ligands based on small molecules indicate that many different structural scaffolds may provide high

  14. Conformational Nature of the Borrelia burgdorferi Decorin Binding Protein A Epitopes That Elicit Protective Antibodies

    PubMed Central

    Ulbrandt, Nancy D.; Cassatt, David R.; Patel, Nita K.; Roberts, William C.; Bachy, Christine M.; Fazenbaker, Christine A.; Hanson, Mark S.

    2001-01-01

    Decorin binding protein A (DbpA) has been shown by several laboratories to be a protective antigen for the prevention of experimental Borrelia burgdorferi infection in the mouse model of Lyme borreliosis. However, different recombinant forms of the antigen having either lipidated amino termini, approximating the natural secretion and posttranslational processing, or nonprocessed cytosolic forms have elicited disparate levels of protection in the mouse model. We have now used the unique functional properties of this molecule to investigate the structural requirements needed to elicit a protective immune response. Genetic and physicochemical alterations to DbpA showed that the ability to bind to the ligand decorin is indicative of a potent immunogen but is not conclusive. By mutating the two carboxy-terminal nonconserved cysteines of DbpA from B. burgdorferi strain N40, we have determined that the stability afforded by the putative disulfide bond is essential for the generation of protective antibodies. This mutated protein was more sensitive to thermal denaturation and proteolysis, suggesting that it is in a less ordered state. Immunization with DbpA that was thermally denatured and functionally inactivated stimulated an immune response that was not protective and lacked bactericidal antibodies. Antibodies against conformationally altered forms of DbpA also failed to kill heterologous B. garinii and B. afzelii strains. Additionally, nonsecreted recombinant forms of DbpAN40 were found to be inferior to secreted lipoprotein DbpAN40 in terms of functional activity and antigenic potency. These data suggest that elicitation of a bactericidal and protective immune response to DbpA requires a properly folded conformation for the production of functional antibodies. PMID:11447153

  15. Differentiating a Ligand's Chemical Requirements for Allosteric Interactions from Those for Protein Binding. Phenylalanine Inhibition of Pyruvate Kinase

    SciTech Connect

    Williams,R.; Holyoak, T.; McDonald, G.; Gui, C.; Fenton, A.

    2006-01-01

    The isoform of pyruvate kinase from brain and muscle of mammals (M1-PYK) is allosterically inhibited by phenylalanine. Initial observations in this model allosteric system indicate that Ala binds competitively with Phe, but elicits a minimal allosteric response. Thus, the allosteric ligand of this system must have requirements for eliciting an allosteric response in addition to the requirements for binding. Phe analogues have been used to dissect what chemical properties of Phe are responsible for eliciting the allosteric response. We first demonstrate that the L-2-aminopropanaldehyde substructure of the amino acid ligand is primarily responsible for binding to M1-PYK. Since the allosteric response to Ala is minimal and linear addition of methyl groups beyond the -carbon increase the magnitude of the allosteric response, we conclude that moieties beyond the -carbon are primarily responsible for allostery. Instead of an all-or-none mechanism of allostery, these findings support the idea that the bulk of the hydrophobic side chain, but not the aromatic nature, is the primary determinant of the magnitude of the observed allosteric inhibition. The use of these results to direct structural studies has resulted in a 1.65 Angstroms structure of M1-PYK with Ala bound. The coordination of Ala in the allosteric amino acid binding site confirms the binding role of the L-2-aminopropanaldehyde substructure of the ligand. Collectively, this study confirms that a ligand can have chemical regions specific for eliciting the allosteric signal in addition to the chemical regions necessary for binding.

  16. Picturing Masculinities: Using Photo-elicitation in Men's Health Research.

    PubMed

    Creighton, Genevieve M; Brussoni, Mariana; Oliffe, John L; Han, Christina

    2015-10-18

    This article explores the use of photo-elicitation methods in two men's health studies. Discussed are the ways that photo-elicitation can facilitate conversation about health issues that might be otherwise challenging to access. In the first study, researchers explored 35 young men's experiences of grief following the accidental death of a male peer. In the second study, researchers describe 64 fathers' perceptions about their roles and identity with respect to child safety and risk. Photographs and accompanying narratives were analyzed and results were theorized using a masculinities framework. Discussed are the benefits of photo-elicitation, which include facilitating conversation about emotions, garnering insight into the structures and identities of masculinity in the context of men's health. Considered also are some methodological challenges amid recommendations for ensuring reflexive practices. Based on the findings it is concluded that photo-elicitation can innovatively advance qualitative research in men's health.

  17. CCSI Risk Estimation: An Application of Expert Elicitation

    SciTech Connect

    Engel, David W.; Dalton, Angela C.

    2012-10-01

    The Carbon Capture Simulation Initiative (CCSI) is a multi-laboratory simulation-driven effort to develop carbon capture technologies with the goal of accelerating commercialization and adoption in the near future. One of the key CCSI technical challenges is representing and quantifying the inherent uncertainty and risks associated with developing, testing, and deploying the technology in simulated and real operational settings. To address this challenge, the CCSI Element 7 team developed a holistic risk analysis and decision-making framework. The purpose of this report is to document the CCSI Element 7 structured systematic expert elicitation to identify additional risk factors. We review the significance of and established approaches to expert elicitation, describe the CCSI risk elicitation plan and implementation strategies, and conclude by discussing the next steps and highlighting the contribution of risk elicitation toward the achievement of the overarching CCSI objectives.

  18. Weaker Ligands Can Dominate an Odor Blend due to Syntopic Interactions

    PubMed Central

    2013-01-01

    Most odors in natural environments are mixtures of several compounds. Perceptually, these can blend into a new “perfume,” or some components may dominate as elements of the mixture. In order to understand such mixture interactions, it is necessary to study the events at the olfactory periphery, down to the level of single-odorant receptor cells. Does a strong ligand present at a low concentration outweigh the effect of weak ligands present at high concentrations? We used the fruit fly receptor dOr22a and a banana-like odor mixture as a model system. We show that an intermediate ligand at an intermediate concentration alone elicits the neuron’s blend response, despite the presence of both weaker ligands at higher concentration, and of better ligands at lower concentration in the mixture. Because all of these components, when given alone, elicited significant responses, this reveals specific mixture processing already at the periphery. By measuring complete dose–response curves we show that these mixture effects can be fully explained by a model of syntopic interaction at a single-receptor binding site. Our data have important implications for how odor mixtures are processed in general, and what preprocessing occurs before the information reaches the brain. PMID:23315042

  19. Subjective Probability Distribution Elicitation in Cost Risk Analysis: A Review

    DTIC Science & Technology

    2007-01-01

    where reasonable, to counteract known biases in elicitation). 1 For the triangle distribution, the probability is set to zero outside the endpoints...probability is set to zero outside the endpoints, while between the endpoints the density rises linearly from the lower value to the most-likely values...Wheeler, T. A., S. C. Hora , W. R. Cramond, and S. D. Unwin, Analysis of Core Damage Frequency from Internal Events: Expert Judgment Elicitation

  20. Knowledge Elicitation: Phase 1 Final Report. Volume 1

    DTIC Science & Technology

    1989-06-01

    artificial intelligence has been focused on methods for eliciting and representing expert knowledge (e.g., Newell and Simon, 1972; Schank and Abelson...methods. These issues have not yet been dealt with in a satisfactory way, either in the artificial intelligence community or by cognitive psychologists...ELICITATION 2.1 The Myth of "Mining for Nugets" of Knowledge The central question in the field of artificial intelligence, whether computers can be

  1. Melatonin: functions and ligands.

    PubMed

    Singh, Mahaveer; Jadhav, Hemant R

    2014-09-01

    Melatonin is a chronobiotic substance that acts as synchronizer by stabilizing bodily rhythms. Its synthesis occurs in various locations throughout the body, including the pineal gland, skin, lymphocytes and gastrointestinal tract (GIT). Its synthesis and secretion is controlled by light and dark conditions, whereby light decreases and darkness increases its production. Thus, melatonin is also known as the 'hormone of darkness'. Melatonin and analogs that bind to the melatonin receptors are important because of their role in the management of depression, insomnia, epilepsy, Alzheimer's disease (AD), diabetes, obesity, alopecia, migraine, cancer, and immune and cardiac disorders. In this review, we discuss the mechanism of action of melatonin in these disorders, which could aid in the design of novel melatonin receptor ligands.

  2. Bifunctional DTPA-type ligand

    SciTech Connect

    Gansow, O.A.; Brechbiel, M.W.

    1990-03-26

    The subject matter of the invention relates to bifunctional cyclohexyl DTPA ligands and methods of using these compounds. Specifically, such ligands are useful for radiolabeling proteins with radioactive metals, and can consequently be utilized with respect to radioimmunoimaging and/or radioimmunotherapy.

  3. Murine germinal center B cells require functional Fms-like tyrosine kinase 3 signaling for IgG1 class-switch recombination.

    PubMed

    Svensson, Mattias N D; Andersson, Karin M E; Wasén, Caroline; Erlandsson, Malin C; Nurkkala-Karlsson, Merja; Jonsson, Ing-Marie; Brisslert, Mikael; Bemark, Mats; Bokarewa, Maria I

    2015-12-01

    Switched antibody classes are important for efficient immune responses. Aberrant antibody production to otherwise harmless antigens may result in autoimmunity. The protein kinase fms-like tyrosine kinase 3 receptor (Flt3) has an important role during early B-cell development, but the role of Flt3 in peripheral B cells has not been assessed before. Herein we describe a previously unappreciated role for Flt3 in IgG1 class-switch recombination (CSR) and production. We show that Flt3 is reexpressed on B-cell lymphoma 6(+) germinal center B cells in vivo and following LPS activation of peripheral B cells in vitro. Absence of Flt3 signaling in Flt3 ligand-deficient mice results in impaired IgG1 CSR and accumulation of IgM-secreting plasma cells. On activated B cells, Flt3 is coexpressed and functions in synergy with the common-gamma chain receptor family. B cells from Flt3 ligand-deficient mice have impaired IL-4R signaling, with reduced phosphorylation of signal transducer and activator of transcription (Stat) 6, and demonstrate a failure to initiate CSR to IgG1 with low expression of γ1 germ-line transcripts, resulting in impaired IgG1 production. Thus, functional synergy between Flt3 and IL-4R signaling is critical for Stat-mediated regulation of sterile γ1 germ-line transcripts and CSR to IgG1.

  4. Pseudo-Mannosylated DC-SIGN Ligands as Immunomodulants.

    PubMed

    Berzi, Angela; Ordanini, Stefania; Joosten, Ben; Trabattoni, Daria; Cambi, Alessandra; Bernardi, Anna; Clerici, Mario

    2016-10-13

    DC-SIGN, a C-type lectin mainly expressed by DCs, mediates antigen uptake and can induce specific immune responses, depending on the ligand involved. Owing to these properties, DC-SIGN is an attracting target for approaches aimed at tailoring the immune response towards specific immunologic outcomes. A multivalent DC-SIGN ligand (Polyman26), containing at its core a fluorescent "rod-like" spacer and able to inhibit DC-SIGN mediated HIV infection in nanomolar concentration, has been recently developed by our group. We investigated the internalization pattern and the ability of Polyman26 to elicit innate immune responses. Results obtained by confocal microscopy indicate that Polyman26 is internalized by DCs via receptor- mediated endocytosis and is then routed to endolysosomal compartments, thus being presented together with MHC class II molecules, with important implications for the development of vaccines. Moreover, Polyman26 up-regulated the production of β-chemokines and pro-inflammatory cytokines (including IL-1β, IL-6, IL-12, and TNFα) as well as the expression of TLR9 and CD40L. These results indicate that glycomimetic DC-SIGN ligands should be further investigated and suggest that these compounds could be used to differentially stimulate immune responses.

  5. Beta-arrestin-biased ligands at seven-transmembrane receptors.

    PubMed

    Violin, Jonathan D; Lefkowitz, Robert J

    2007-08-01

    Seven-transmembrane receptors (7TMRs), the most common molecular targets of modern drug therapy, are critically regulated by beta-arrestins, which both inhibit classic G-protein signaling and initiate distinct beta-arrestin signaling. The interplay of G-protein and beta-arrestin signals largely determines the cellular consequences of 7TMR-targeted drugs. Until recently, a drug's efficacy for beta-arrestin recruitment was believed to be proportional to its efficacy for G-protein activities. This paradigm restricts 7TMR drug effects to a linear spectrum of responses, ranging from inhibition of all responses to stimulation of all responses. However, it is now clear that 'biased ligands' can selectively activate G-protein or beta-arrestin functions and thus elicit novel biological effects from even well-studied 7TMRs. Here, we discuss the current state of beta-arrestin-biased ligand research and the prospects for beta-arrestin bias as a therapeutic target. Consideration of ligand bias might have profound influences on the way scientists approach 7TMR-targeted drug discovery.

  6. Pseudo-Mannosylated DC-SIGN Ligands as Immunomodulants

    PubMed Central

    Berzi, Angela; Ordanini, Stefania; Joosten, Ben; Trabattoni, Daria; Cambi, Alessandra; Bernardi, Anna; Clerici, Mario

    2016-01-01

    DC-SIGN, a C-type lectin mainly expressed by DCs, mediates antigen uptake and can induce specific immune responses, depending on the ligand involved. Owing to these properties, DC-SIGN is an attracting target for approaches aimed at tailoring the immune response towards specific immunologic outcomes. A multivalent DC-SIGN ligand (Polyman26), containing at its core a fluorescent “rod-like” spacer and able to inhibit DC-SIGN mediated HIV infection in nanomolar concentration, has been recently developed by our group. We investigated the internalization pattern and the ability of Polyman26 to elicit innate immune responses. Results obtained by confocal microscopy indicate that Polyman26 is internalized by DCs via receptor- mediated endocytosis and is then routed to endolysosomal compartments, thus being presented together with MHC class II molecules, with important implications for the development of vaccines. Moreover, Polyman26 up-regulated the production of β-chemokines and pro-inflammatory cytokines (including IL-1β, IL-6, IL-12, and TNFα) as well as the expression of TLR9 and CD40L. These results indicate that glycomimetic DC-SIGN ligands should be further investigated and suggest that these compounds could be used to differentially stimulate immune responses. PMID:27734954

  7. Elicitability of muscle cramps in different leg and foot muscles.

    PubMed

    Minetto, Marco Alessandro; Botter, Alberto

    2009-10-01

    To explore the efficacy of muscle motor point stimulation in eliciting muscle cramps, 11 subjects underwent eight sessions of electrical stimulation of the following muscles bilaterally: abductor hallucis flexor hallucis brevis, and both heads of the gastrocnemius muscles. Bursts of 150 square wave stimuli (duration: 152 micros; current intensity: 30% supramaximal) were applied. The stimulation frequency was increased from 4 pulses per second (pps) at increments of 2 pps until a cramp was induced. The number of cramps that could be elicited was smaller in flexor hallucis brevis than in abductor hallucis (16 vs. 22 out of 22 trials each; P < 0.05) and in the lateral gastrocnemius than in the medial gastrocnemius (5 vs. 20 out of 22 trials each; P < 0.0001). We show that leg and foot muscles have different cramp susceptibility, and the intermuscle variability in the elicitability profile for electrically induced cramps supports the use of the proposed method for cramp research.

  8. Validity of ligand efficiency metrics.

    PubMed

    Murray, Christopher W; Erlanson, Daniel A; Hopkins, Andrew L; Keserü, György M; Leeson, Paul D; Rees, David C; Reynolds, Charles H; Richmond, Nicola J

    2014-06-12

    A recent viewpoint article (Improving the plausibility of success with inefficient metrics. ACS Med. Chem. Lett. 2014, 5, 2-5) argued that the standard definition of ligand efficiency (LE) is mathematically invalid. In this viewpoint, we address this criticism and show categorically that the definition of LE is mathematically valid. LE and other metrics such as lipophilic ligand efficiency (LLE) can be useful during the multiparameter optimization challenge faced by medicinal chemists.

  9. Biologically inspired robots elicit a robust fear response in zebrafish

    NASA Astrophysics Data System (ADS)

    Ladu, Fabrizio; Bartolini, Tiziana; Panitz, Sarah G.; Butail, Sachit; Macrı, Simone; Porfiri, Maurizio

    2015-03-01

    We investigate the behavioral response of zebrafish to three fear-evoking stimuli. In a binary choice test, zebrafish are exposed to a live allopatric predator, a biologically-inspired robot, and a computer-animated image of the live predator. A target tracking algorithm is developed to score zebrafish behavior. Unlike computer-animated images, the robotic and live predator elicit a robust avoidance response. Importantly, the robotic stimulus elicits more consistent inter-individual responses than the live predator. Results from this effort are expected to aid in hypothesis-driven studies on zebrafish fear response, by offering a valuable approach to maximize data-throughput and minimize animal subjects.

  10. Ligand Identification Scoring Algorithm (LISA)

    PubMed Central

    Zheng, Zheng; Merz, Kenneth M.

    2011-01-01

    A central problem in de novo drug design is determining the binding affinity of a ligand with a receptor. A new scoring algorithm is presented that estimates the binding affinity of a protein-ligand complex given a three-dimensional structure. The method, LISA (Ligand Identification Scoring Algorithm), uses an empirical scoring function to describe the binding free energy. Interaction terms have been designed to account for van der Waals (VDW) contacts, hydrogen bonding, desolvation effects and metal chelation to model the dissociation equilibrium constants using a linear model. Atom types have been introduced to differentiate the parameters for VDW, H-bonding interactions and metal chelation between different atom pairs. A training set of 492 protein-ligand complexes was selected for the fitting process. Different test sets have been examined to evaluate its ability to predict experimentally measured binding affinities. By comparing with other well known scoring functions, the results show that LISA has advantages over many existing scoring functions in simulating protein-ligand binding affinity, especially metalloprotein-ligand binding affinity. Artificial Neural Network (ANN) was also used in order to demonstrate that the energy terms in LISA are well designed and do not require extra cross terms. PMID:21561101

  11. Ligand Identification Scoring Algorithm (LISA).

    PubMed

    Zheng, Zheng; Merz, Kenneth M

    2011-06-27

    A central problem in de novo drug design is determining the binding affinity of a ligand with a receptor. A new scoring algorithm is presented that estimates the binding affinity of a protein-ligand complex given a three-dimensional structure. The method, LISA (Ligand Identification Scoring Algorithm), uses an empirical scoring function to describe the binding free energy. Interaction terms have been designed to account for van der Waals (VDW) contacts, hydrogen bonding, desolvation effects, and metal chelation to model the dissociation equilibrium constants using a linear model. Atom types have been introduced to differentiate the parameters for VDW, H-bonding interactions, and metal chelation between different atom pairs. A training set of 492 protein-ligand complexes was selected for the fitting process. Different test sets have been examined to evaluate its ability to predict experimentally measured binding affinities. By comparing with other well-known scoring functions, the results show that LISA has advantages over many existing scoring functions in simulating protein-ligand binding affinity, especially metalloprotein-ligand binding affinity. Artificial Neural Network (ANN) was also used in order to demonstrate that the energy terms in LISA are well designed and do not require extra cross terms.

  12. Elicitation of distinct populations of monoclonal antibodies specific for the variable domains of monoclonal anti-fluorescein antibody 4-4-20.

    PubMed

    Weidner, K M; Denzin, L K; Kim, M L; Mallender, W D; Miklasz, S D; Voss, E W

    1993-08-01

    Armenian hamsters were immunized with non-liganded, partially liganded or affinity-labeled anti-fluorescein Mab 4-4-20. Seventeen hybridoma producing monoclonal anti-4-4-20 antibodies were characterized from chemically-mediated fusions of immune hamster lymphocytes with murine Sp2/O-Ag14 myeloma cells. Distinct populations of anti-4-4-20 monoclonal antibodies were isolated from hamsters receiving immunizations with partially liganded Mab 4-4-20 relative to those receiving affinity-labeled 4-4-20. Two of the three monoclonal antibodies produced in response to partially liganded 4-4-20 were inhibited in their interaction with 4-4-20 by fluorescyl ligand. These two clones, 1F4 and 1B7, recognized unique epitopes on the 4-4-20 molecules, as demonstrated by non-reactivity with members of the 4-4-20 idiotype family. Additionally, 1F4 and 1B7 demonstrated the ability to delay the association of fluorescein with Mab 4-4-20. The 14 characterized non-ligand-inhibitable Mabs elicited to affinity-labeled 4-4-20 were classified into four separate groups based on various binding properties with members of the 4-4-20 idiotype family and binding to resolved H- and L-chains in a western blot. Members of three of the four groups showed strong reactivity with both 04-01 Ig and 04-01 SCA, which utilizes the same L-chain as Mab 4-4-20. Six non-ligand-inhibitable Mabs, 4A6, P1E11, 3A5-1, 2C3, 2C4, and 1A4, delayed the dissociation rate of ligand from Mab 4-4-20 and mutant 4-4-20 SCA L32phe.

  13. Modification of commercially available simulators to elicit decision making behavior.

    PubMed

    Salud, Jonathan; Ononye, Chiagozie; Salud, Lawrence; Pugh, Carla

    2012-01-01

    In recent years, simulation training has emerged as an innovative method for decreasing error and teaching complex procedures. Simulation has also been a valuable tool for evaluating investigatory and analytic thinking. By adding a specific, clinically oriented modification to a commercially available simulator, we were able to elicit first-year emergency medicine resident perceptions, actions, and decisions.

  14. Elicited Production of Relative Clauses in Children with Williams Syndrome

    ERIC Educational Resources Information Center

    Zukowski, Andrea

    2009-01-01

    Relative clauses have been implicated alternately as a strength and a weakness in the language of people with Williams Syndrome (WS). To clarify the facts, an elicited production test was administered to 10 people with WS (age 10-16 years), 10 typically developing children (age 4-7 years), and 12 typically developing adults. Nearly every WS…

  15. Intermediary's Information Seeking, Inquiring Minds, and Elicitation Styles.

    ERIC Educational Resources Information Center

    Wu, Mei-Mei; Liu, Ying-Hsang

    2003-01-01

    Explores how intermediaries seek information from patrons by analyzing intermediaries' elicitation utterances through three dimensions: linguistic forms, utterance purposes, and communicative functions. Reports on results of questionnaires given to patrons and intermediaries in academic and research libraries in Taiwan and investigates…

  16. A Study of the Affective Responses Elicited by Occupational Stimuli

    ERIC Educational Resources Information Center

    Schoon, Craig G.

    1976-01-01

    The semantic differential was used to assess the properties of affect elicited by occupational stimuli. Vocationally committed men studying medicine, business, and engineering responded to a semantic differential containing occupational concepts. Results show a semantic space for all three groups composed of three orthogonal dimensions of affect…

  17. Eliciting Student Views Using an Interview-About-Instances Technique.

    ERIC Educational Resources Information Center

    Gilbert, John K.; And Others

    An exposition is made of a method to elicit students' comprehension of the meaning of words commonly used in science, which may be influential in determining what they learn. The method's aim is to elucidate features in students' understanding of childrens' science (that found before formal teaching of science), student science (that found after…

  18. Transfer of Aversive Respondent Elicitation in Accordance with Equivalence Relations

    ERIC Educational Resources Information Center

    Valverde, Miguel Rodriguez; Luciano, Carmen; Barnes-Holmes, Dermot

    2009-01-01

    The present study investigates the transfer of aversively conditioned respondent elicitation through equivalence classes, using skin conductance as the measure of conditioning. The first experiment is an attempt to replicate Experiment 1 in Dougher, Augustson, Markham, Greenway, and Wulfert (1994), with different temporal parameters in the…

  19. The Role of Elicited Verbal Imitation in Toddlers' Word Learning

    ERIC Educational Resources Information Center

    Hodges, Rosemary; Munro, Natalie; Baker, Elise; McGregor, Karla; Docking, Kimberley; Arciuli, Joanne

    2016-01-01

    This study is about the role of elicited verbal imitation in toddler word learning. Forty-eight toddlers were taught eight nonwords linked to referents. During training, they were asked to imitate the nonwords. Naming of the referents was tested at three intervals (one minute later [uncued], five minutes, and 1-7 days later [cued]) and recognition…

  20. Photo-Elicitation: Reflexivity on Method, Analysis, and Graphic Portraits

    ERIC Educational Resources Information Center

    Richard, Veronica M.; Lahman, Maria K. E.

    2015-01-01

    In this methodological discussion, the authors detail and reflect on the processes of using photo-elicitation interviewing as a way to align with positive qualitative methodologies, to gain access to participant beliefs and values, and to highlight participant voices through their choices of words and visuals. A review of the literature and an…

  1. Engaging Young Children in Research through Photo Elicitation

    ERIC Educational Resources Information Center

    Pyle, Angela

    2013-01-01

    Embracing the new sociology of childhood, this paper describes a participatory research method built on a belief in the competency of young children. The paper begins with a critical review of the photo elicitation literature exploring the varied levels of children's participation. Drawing on the strengths of the previous research, a multi-step…

  2. Using Automatic Speech Recognition Technology with Elicited Oral Response Testing

    ERIC Educational Resources Information Center

    Cox, Troy L.; Davies, Randall S.

    2012-01-01

    This study examined the use of automatic speech recognition (ASR) scored elicited oral response (EOR) tests to assess the speaking ability of English language learners. It also examined the relationship between ASR-scored EOR and other language proficiency measures and the ability of the ASR to rate speakers without bias to gender or native…

  3. Web-Based Elicitation Tasks in SLA Research.

    ERIC Educational Resources Information Center

    Ayoun, Dalila

    2000-01-01

    Studied second language acquisition (SLA) designed with Web-based elicitation tasks to obtain greater internal and external validity. Tested the acquisition of the properties subsumed under the verb movement parameter and the null subject parameter by English native speakers enrolled in college French. (Author/VWL)

  4. Eliciting Students' Beliefs about Who Is Good at Mathematics

    ERIC Educational Resources Information Center

    Morge, Shelby P.

    2007-01-01

    This article highlights a series of activities designed to elicit students' mathematics-related beliefs, particularly those related to gender. As a result of the activities, females in upper-level classes rated themselves as having less confidence than males, and viewing a movie clip was sufficient for some students to modify their descriptions of…

  5. Elicitation Support Requirements of Multi-Expertise Teams

    ERIC Educational Resources Information Center

    Bitter-Rijpkema, Marlies; Martens, Rob; Jochems, Wim

    2005-01-01

    Tools to support knowledge elicitation are used more and more in situations where employees or students collaborate using the computer. Studies indicate that differences exist between experts and novices regarding their methods of work and reasoning. However, the commonly preferred approach tends to deal with team members as a single system with…

  6. Preparing Beginning Teachers to Elicit and Interpret Students' Mathematical Thinking

    ERIC Educational Resources Information Center

    Sleep, Laurie; Boerst, Timothy A.

    2012-01-01

    This study investigated how teacher education assignments can be designed to support beginning teachers in learning to do the work of teaching. We examined beginners' formative assessment practices--in particular, their eliciting and interpreting of students' mathematical thinking--in the context of an elementary mathematics methods assignment,…

  7. Creating a Framework: Art Therapy Elicits the Narrative

    ERIC Educational Resources Information Center

    Harber, Karen

    2011-01-01

    A case study illustrates how art therapy was used to elicit the narrative of an adolescent male student in transition from incarceration to a transfer school setting. Childhood trauma was addressed in individual sessions and within a literacy group co-led by a reading specialist. The art therapist responded to the client's needs by broadening the…

  8. Eliciting Design Patterns for E-Learning Systems

    ERIC Educational Resources Information Center

    Retalis, Symeon; Georgiakakis, Petros; Dimitriadis, Yannis

    2006-01-01

    Design pattern creation, especially in the e-learning domain, is a highly complex process that has not been sufficiently studied and formalized. In this paper, we propose a systematic pattern development cycle, whose most important aspects focus on reverse engineering of existing systems in order to elicit features that are cross-validated through…

  9. Clinicians as Communication Partners: Developing a Mediated Discourse Elicitation Protocol

    ERIC Educational Resources Information Center

    Hengst, Julie A.; Duff, Melissa C.

    2007-01-01

    This article presents the development and piloting of a mediated discourse elicitation protocol. Grounded in situated theories of communication and informed by mediated discourse analysis, this protocol selectively samples familiar discourse types in a manner designed to preserve interactional aspects of communication. Critically, the mediated…

  10. Eliciting Production of L2 Target Structures through Priming Activities

    ERIC Educational Resources Information Center

    McDonough, Kim; Trofimovich, Pavel; Neumann, Heike

    2015-01-01

    This study focuses on the pedagogical applications of structural priming research in an English for academic purposes (EAP) context, investigating whether priming activities are an effective tool for eliciting production of target grammatical structures. University students across four EAP classes carried out a total of 6 information-exchange…

  11. Visual emotional context modulates brain potentials elicited by unattended tones.

    PubMed

    Sugimoto, Sayaka; Nittono, Hiroshi; Hori, Tadao

    2007-10-01

    To examine whether brain electrical responses to environmental stimuli were influenced by emotional contexts, event-related potentials (ERPs) elicited by nonstartle probe tones were recorded from 13 student volunteers while they were viewing emotionally positive, neutral, and negative slides of the International Affective Picture System. The auditory stimuli consisted of high-deviant (2000 Hz, p=.08), low-deviant (1050 Hz, p=.08), and standard (1000 Hz, p=.84) tones with a mean onset-to-onset interval of 600 ms. Participants were told to ignore the tones. High-deviant tones elicited a larger N1 (peaking around 100 ms) when participants were viewing negative slides than when viewing positive slides. The amplitude of the P2 elicited by standard tones (peaking around 170 ms) was smaller when participants were viewing positive slides than when viewing negative and neutral slides. The amplitude of the mismatch negativity (150-200 ms) tended to reduce during positive slide presentation, but this difference appeared to be due to reduction of the P2 elicited by standard tones. These findings suggest that visually induced emotional states have a sequential effect on auditory information processing, in that the influence of negative emotion appears at an earlier stage than that of positive emotion.

  12. Photo-Elicitation Method Gives Voice and Reactions of Subjects.

    ERIC Educational Resources Information Center

    Smith, C. Zoe; Woodward, Anne-Marie

    1999-01-01

    Describes a research assignment (called "photo-elicitation") in a graduate course on the role of photography in society in which students interview people similar to the subjects in the photographs to discover how the photographs affect them. Includes material from one research project interviewing three recovering drug addicts responding to Larry…

  13. Computational Support for Early Elicitation and Classification of Tone

    ERIC Educational Resources Information Center

    Bird, Steven; Lee, Haejoong

    2014-01-01

    Investigating a tone language involves careful transcription of tone on words and phrases. This is challenging when the phonological categories--the tones or melodies--have not been identified. Effects such as coarticulation, sandhi, and phrase-level prosody appear as obstacles to early elicitation and classification of tone. This article presents…

  14. A Task that Elicits Reasoning: A Dual Analysis

    ERIC Educational Resources Information Center

    Yankelewitz, Dina; Mueller, Mary; Maher, Carolyn A.

    2010-01-01

    This paper reports on the forms of reasoning elicited as fourth grade students in a suburban district and sixth grade students in an urban district worked on similar tasks involving reasoning with the use of Cuisenaire rods. Analysis of the two data sets shows similarities in the reasoning used by both groups of students on specific tasks, and the…

  15. Catalytic and mechanistic studies into the epoxidation of styrenes using manganese complexes of structurally similar polyamine ligands.

    PubMed

    Ilyashenko, Gennadiy; De Faveri, Giorgio; Follier, Thomas; Al-Safadi, Rawan; Motevalli, Majid; Watkinson, Michael

    2014-02-21

    Two structurally similar polyamine ligands (7 and 8) have been prepared, which differ only by the presence of either a secondary or tertiary nitrogen donor within their N5 donor set. The ligands, in combination with iron and manganese salts, have been screened for their efficacy as catalysts for the epoxidation of styrene, using both hydrogen peroxide and peracetic acid as oxidants. Clear differences in activity between the two systems were observed, with 7 proving most effective in the presence of MnSO4 with H2O2, whereas ligand 8 proved to be effective with Mn(OTf)2, MnCl2 and Mn(ClO4)2 using peracetic acid as the oxidant. A Hammett analysis of the initial rate kinetics of the optimal systems, combined with analysis by UV-vis spectroscopy, indicates that the small structural differences in the ligands elicit profound changes in the nature of the active species formed.

  16. Single channel currents at six microsecond resolution elicited by acetylcholine in mouse myoballs

    PubMed Central

    Parzefall, Franz; Wilhelm, Robert; Heckmann, Manfred; Dudel, Josef

    1998-01-01

    A patch-clamp set-up was optimized for low noise and high time resolution. An Axoclamp 200B amplifier was modified to incorporate a Teflon connector to the electrode. An electrode puller was equipped with a hydrogen-oxygen burner to produce quartz-glass pipettes with optimally 0.2μm openings and 20 MΩ resistance.The r.m.s. (root mean square) noise of sealed pipettes in the bath ranged from 3.6 fA with 100 Hz filter cut-off to 1.5 pA with 61 kHz filter cut-off. At these extremes currents of 17 fA and more than 3 ms, or 9 pA and more than 6μs could be resolved with a negligible error rate.The system was tested on mouse myoballs, recording 9–10 pA single channel currents on-cell at −200 mV polarization which were elicited by 0.1–5000μm acetylcholine (ACh).Distributions of open and closed times and of correlations of open times to the preceding closed time defined several open states: single openings with mean durations of 1.2 and 25μs, from single-liganded receptors, and bursts of 10 ms mean duration containing on average 800μs openings and 16μs closings, from double liganded receptors. Above 0.1 mm ACh these openings are interrupted increasingly by on average 18μs and 72μs channel blocks by ACh. PMID:9729627

  17. Novel photoaffinity ligands for the GA-receptor

    SciTech Connect

    Suttle, J.C.; Hultstrand, J.F.; Tanaka, F.S. )

    1990-05-01

    Previous studies from this laboratory have shown that certain N-substituted phthalimides (NSPs) exhibit GA-like activity in a range of specific bioassays and that bioactive NSPs compete with ({sup 3}H)-GA{sub 4} for soluble binding sites in cucumber homogenates. As such, these compounds may prove useful in the purification and characterization of GA receptor proteins. To this end, five azido-NSPs have been synthesized and are currently being screened for biological activity and photochemical stability. Three azido-NSPs elicit {alpha}-amylase production in barley half-seeds and stimulate tissue elongation in d{sub 5} maize, lettuce, sunflower, and soybean. Further evaluations are in progress and these data as well as the utility of these compounds as photo-affinity ligands will be discussed.

  18. Why mercury prefers soft ligands

    SciTech Connect

    Riccardi, Demian M; Guo, Hao-Bo; Gu, Baohua; Parks, Jerry M; Summers, Anne; Miller, S; Liang, Liyuan; Smith, Jeremy C

    2013-01-01

    Mercury (Hg) is a major global pollutant arising from both natural and anthropogenic sources. Defining the factors that determine the relative affinities of different ligands for the mercuric ion, Hg2+, is critical to understanding its speciation, transformation, and bioaccumulation in the environment. Here, we use quantum chemistry to dissect the relative binding free energies for a series of inorganic anion complexes of Hg2+. Comparison of Hg2+ ligand interactions in the gaseous and aqueous phases shows that differences in interactions with a few, local water molecules led to a clear periodic trend within the chalcogenide and halide groups and resulted in the well-known experimentally observed preference of Hg2+ for soft ligands such as thiols. Our approach establishes a basis for understanding Hg speciation in the biosphere.

  19. What are Nuclear Receptor Ligands?

    PubMed Central

    Sladek, Frances M.

    2010-01-01

    Nuclear receptors (NRs) are a family of highly conserved transcription factors that regulate transcription in response to small lipophilic compounds. They play a role in every aspect of development, physiology and disease in humans. They are also ubiquitous in and unique to the animal kingdom suggesting that they may have played an important role in their evolution. In contrast to the classical endocrine receptors that originally defined the family, recent studies suggest that the first NRs might have been sensors of their environment, binding ligands that were external to the host organism. The purpose of this review is to provide a broad perspective on NR ligands and address the issue of exactly what constitutes a NR ligand from historical, biological and evolutionary perspectives. This discussion will lay the foundation for subsequent reviews in this issue as well as pose new questions for future investigation. PMID:20615454

  20. Molecular Recognition and Ligand Association

    NASA Astrophysics Data System (ADS)

    Baron, Riccardo; McCammon, J. Andrew

    2013-04-01

    We review recent developments in our understanding of molecular recognition and ligand association, focusing on two major viewpoints: (a) studies that highlight new physical insight into the molecular recognition process and the driving forces determining thermodynamic signatures of binding and (b) recent methodological advances in applications to protein-ligand binding. In particular, we highlight the challenges posed by compensating enthalpic and entropic terms, competing solute and solvent contributions, and the relevance of complex configurational ensembles comprising multiple protein, ligand, and solvent intermediate states. As more complete physics is taken into account, computational approaches increase their ability to complement experimental measurements, by providing a microscopic, dynamic view of ensemble-averaged experimental observables. Physics-based approaches are increasingly expanding their power in pharmacology applications.

  1. Nonlinear Analyses of Elicited Modal, Raised, and Pressed Rabbit Phonation

    PubMed Central

    Awan, Shaheen N.; Novaleski, Carolyn K.; Rousseau, Bernard

    2014-01-01

    Objectives/Hypothesis The purpose of this study was to use nonlinear dynamic analysis methods such as phase space portraits and correlation dimension (D2) as well as descriptive spectrographic analyses to characterize acoustic signals produced during evoked rabbit phonation. Methods Seventeen New Zealand white breeder rabbits were used to perform the study. A Grass S-88 stimulator (SA Instrumentation, Encinitas, CA) and constant current isolation unit (Grass Telefactor, model PSIU6; West Warwick, RI) were used to provide electrical stimulation to laryngeal musculature, and transglottal airflow rate and stimulation current (mA) were manipulated to elicit modal, raised intensity, and pressed phonations. Central 1 second portions of the most stable portion of the acoustic waveform for modal, raised intensity, and pressed phonations were edited, and then analyzed via phase space portraits, Poincaré sections, and the estimation of the correlation dimension (D2). In an attempt to limit the effects of the highly variable and nonstationary characteristics of some of the signals being analyzed, D2 analysis was also performed on the most stable central 200 ms portion of the acoustic waveform. Descriptive analysis of each phonation was also conducted using sound spectrograms. Results Results showed that the complexity of phonation and the subsequent acoustic waveform is increased as transglottal airflow rate and degree of glottal adduction is manipulated in the evoked rabbit phonation model. In particular, phonatory complexity, as quantified via correlation dimension analyses and demonstrated via spectrographic characteristics, increases from “modal” (i.e., phonation elicited at just above the phonation threshold pressure) to raised intensity (phonation elicited by increasing transglottal airflow rate) to pressed (phonation elicited by increasing the stimulation current delivered to the larynx). Variations in a single dynamic dimension (airflow rate or adductory force

  2. Ligand-induced Epitope Masking

    PubMed Central

    Mould, A. Paul; Askari, Janet A.; Byron, Adam; Takada, Yoshikazu; Jowitt, Thomas A.; Humphries, Martin J.

    2016-01-01

    We previously demonstrated that Arg-Gly-Asp (RGD)-containing ligand-mimetic inhibitors of integrins are unable to dissociate pre-formed integrin-fibronectin complexes (IFCs). These observations suggested that amino acid residues involved in integrin-fibronectin binding become obscured in the ligand-occupied state. Because the epitopes of some function-blocking anti-integrin monoclonal antibodies (mAbs) lie near the ligand-binding pocket, it follows that the epitopes of these mAbs may become shielded in the ligand-occupied state. Here, we tested whether function-blocking mAbs directed against α5β1 can interact with the integrin after it forms a complex with an RGD-containing fragment of fibronectin. We showed that the anti-α5 subunit mAbs JBS5, SNAKA52, 16, and P1D6 failed to disrupt IFCs and hence appeared unable to bind to the ligand-occupied state. In contrast, the allosteric anti-β1 subunit mAbs 13, 4B4, and AIIB2 could dissociate IFCs and therefore were able to interact with the ligand-bound state. However, another class of function-blocking anti-β1 mAbs, exemplified by Lia1/2, could not disrupt IFCs. This second class of mAbs was also distinguished from 13, 4B4, and AIIB2 by their ability to induce homotypic cell aggregation. Although the epitope of Lia1/2 was closely overlapping with those of 13, 4B4, and AIIB2, it appeared to lie closer to the ligand-binding pocket. A new model of the α5β1-fibronectin complex supports our hypothesis that the epitopes of mAbs that fail to bind to the ligand-occupied state lie within, or very close to, the integrin-fibronectin interface. Importantly, our findings imply that the efficacy of some therapeutic anti-integrin mAbs could be limited by epitope masking. PMID:27484800

  3. Multifunctional Ligands in Transition Metal Catalysis

    SciTech Connect

    Crabtree, Robert H

    2011-01-01

    Sophisticated ligands are now being designed that do far more than just fulfil their traditional spectator roles by binding to the metal and providing a sterically-defined binding pocket for the substrate in homogeneous transition metal catalysis. This Focus review emphasizes selected cases in which ligands carry additional functional groups that change the properties of the ligand as a result of an external stimulus or undergo catalytically-relevant ligand-based reactivity. These include proton responsive ligands capable of gaining or losing one or more protons, ligands having a hydrogen bonding function, electroresponsive ligands capable of gaining or losing one or more electrons, and photoresponsive ligands capable of undergoing a useful change of properties upon irradiation. Molecular recognition ligands and proton coupled electron transfer (PCET) are briefly discussed.

  4. Elicitation of specific syntactic structures in primary progressive aphasia.

    PubMed

    Deleon, Jessica; Gesierich, Benno; Besbris, Max; Ogar, Jennifer; Henry, Maya L; Miller, Bruce L; Gorno-Tempini, Maria Luisa; Wilson, Stephen M

    2012-12-01

    Many patients with primary progressive aphasia (PPA) are impaired in syntactic production. Because most previous studies of expressive syntax in PPA have relied on quantitative analysis of connected speech samples, which is a relatively unconstrained task, it is not well understood which specific syntactic structures are most challenging for these patients. We used an elicited syntactic production task to identify which syntactic structures pose difficulties for 31 patients with three variants of PPA: non-fluent/agrammatic, semantic and logopenic. Neurodegenerative and healthy age-matched participants were included as controls. As expected, non-fluent/agrammatic patients made the most syntactic errors. The structures that resulted in the most errors were constructions involving third person singular present agreement, and constructions involving embedded clauses. Deficits on this elicited production task were associated with atrophy of the left posterior inferior frontal gyrus.

  5. Expert elicitation for a national-level volcano hazard model

    NASA Astrophysics Data System (ADS)

    Bebbington, Mark; Stirling, Mark; Cronin, Shane; Wang, Ting; Jolly, Gill

    2016-04-01

    The quantification of volcanic hazard at national level is a vital pre-requisite to placing volcanic risk on a platform that permits meaningful comparison with other hazards such as earthquakes. New Zealand has up to a dozen dangerous volcanoes, with the usual mixed degrees of knowledge concerning their temporal and spatial eruptive history. Information on the 'size' of the eruptions, be it in terms of VEI, volume or duration, is sketchy at best. These limitations and the need for a uniform approach lend themselves to a subjective hazard analysis via expert elicitation. Approximately 20 New Zealand volcanologists provided estimates for the size of the next eruption from each volcano and, conditional on this, its location, timing and duration. Opinions were likewise elicited from a control group of statisticians, seismologists and (geo)chemists, all of whom had at least heard the term 'volcano'. The opinions were combined via the Cooke classical method. We will report on the preliminary results from the exercise.

  6. Conditioned craving cues elicit an automatic approach tendency.

    PubMed

    Van Gucht, Dinska; Vansteenwegen, Debora; Van den Bergh, Omer; Beckers, Tom

    2008-10-01

    In two experiments, we used a Pavlovian differential conditioning procedure to induce craving for chocolate. As a result of repeated pairing with chocolate intake, initially neutral cues came to elicit an automatic approach tendency in a speeded stimulus-response compatibility reaction time task. This automatic approach tendency, moreover, seemed to be sensitive to manipulations of extinction and renewal in the Pavlovian conditioning procedure. These findings corroborate and extend previous reports of automatic approach tendencies elicited by substance-relevant cues in addiction, while controlling for alternative accounts for such observations. Moreover, our data lend support to and extend learning models of cue-induced craving and addiction. Finally, we argue that the procedure we present here provides an ecologically valid behavioural tool that allows studying processes involved in cue-induced craving, addiction and relapse without relying on verbal report.

  7. Simplified Expert Elicitation Procedure for Risk Assessment of Operating Events

    SciTech Connect

    Ronald L. Boring; David Gertman; Jeffrey Joe; Julie Marble; William Galyean; Larry Blackwood; Harold Blackman

    2005-06-01

    This report describes a simplified, tractable, and usable procedure within the US Nuclear Regulator Commission (NRC) for seeking expert opinion and judgment. The NRC has increased efforts to document the reliability and risk of nuclear power plants (NPPs) through Probabilistic Risk Assessment (PRA) and Human Reliability Analysis (HRA) models. The Significance Determination Process (SDP) and Accident Sequence Precursor (ASP) programs at the NRC utilize expert judgment on the probability of failure, human error, and the operability of equipment in cases where otherwise insufficient operational data exist to make meaningful estimates. In the past, the SDP and ASP programs informally sought the opinion of experts inside and outside the NRC. This document represents a formal, documented procedure to take the place of informal expert elicitation. The procedures outlined in this report follow existing formal expert elicitation methodologies, but are streamlined as appropriate to the degree of accuracy required and the schedule for producing SDP and ASP analyses.

  8. Elicitation of Diacetylenic Compounds in Suspension Cultured Cells of Eggplant

    PubMed Central

    Imoto, Setsuko; Ohta, Yoshimoto

    1988-01-01

    Induction of stress metabolites in the suspension cultured cells of eggplant (Solanum melongena L.) was examined. When autoclaved RNase A or nigeran, both of which are nonspecific phytoalexin elicitors in bean cells, were added to the cell culture of eggplant, greatly enhanced levels of three compounds were observed. One of them was cis-pentadeca-6-ene-1,3-diyne-5,15-diol, a novel diacetylenic compound. This compound has considerable fungitoxic activity. Also identified was falcarindiol, another fungitoxic diacetylenic compound previously reported as one of the phytoalexins in infected tomato fruits and leaves. Elicited compounds preferentially accumulated in the culture medium rather than in the cells and decreased to original levels during prolonged culturing. The elicitation of these compounds was closely correlated with cellular damage in terms of the decrease of growth rate and was inhibited by 10 micromolar cycloheximide. PMID:16665862

  9. Modified repetition in poems elicited from young children.

    PubMed

    Dowker, A D

    1991-10-01

    Attempts were made to elicit poems from 133 children between the ages of 2;3 and 6;11. Seventy-eight of the children produced 606 poems between them. Forty-five per cent of the poems contained the syntactic device of modified repetition: a substitution exercise where a grammatical frame is repeated and the substitution occurs in part of the grammatical frame. This was so despite the fact that modified repetition was not present in the examples that were used to elicit poems from the children. The frequency and types of modified repetition used by the children did not vary much with age. The question of whether children's use of modified repetition in their rhythmical poems has the function of helping them to practise grammatical forms, or whether it is simply one reflection of a general human tendency towards the use of pattern in language, is discussed.

  10. Multicoordinate ligands for actinide/lanthanide separations.

    PubMed

    Dam, Henk H; Reinhoudt, David N; Verboom, Willem

    2007-02-01

    In nuclear waste treatment processes there is a need for improved ligands for the separation of actinides (An(III)) and lanthanides (Ln(III)). Several research groups are involved in the design and synthesis of new An(III) ligands and in the confinement of these and existing An(III) ligands onto molecular platforms giving multicoordinate ligands. The preorganization of ligands considerably improves the An(III) extraction properties, which are largely dependent on the solubility and rigidity of the platform. This tutorial review summarizes the most important An(III) ligands with emphasis on the preorganization strategy using (macrocyclic) platforms.

  11. Kinetics of ligand binding to nucleic acids.

    PubMed

    Arakelyan, V B; Babayan, S Y; Tairyan, V I; Arakelyan, A V; Parsadanyan, M A; Vardevanyan, P O

    2006-02-01

    Ligand binding to nucleic acids (NA) is considered as a stationary Markov process. It is shown that the probabilistic description of ligand-NA binding allows one to describe not only the kinetics of the change of number of bound ligands at arbitrary fillings but also to calculate stationary values of the number of bound ligands and its dispersion. The general analysis of absorption isotherms and kinetics of ligand binding to NA make it possible to determine of rate constants of ligand-NA complex formation and dissociation.

  12. Saturated Zone Flow and Transport Expert Elicitation Project

    SciTech Connect

    Coppersmith, Kevin J.; Perman, Roseanne C.

    1998-01-01

    This report presents results of the Saturated Zone Flow and Transport Expert Elicitation (SZEE) project for Yucca Mountain, Nevada. This project was sponsored by the US Department of Energy (DOE) and managed by Geomatrix Consultants, Inc. (Geomatrix), for TRW Environmental Safety Systems, Inc. The DOE's Yucca Mountain Site Characterization Project (referred to as the YMP) is intended to evaluate the suitability of the site for construction of a mined geologic repository for the permanent disposal of spent nuclear fuel and high-level radioactive waste. The SZEE project is one of several that involve the elicitation of experts to characterize the knowledge and uncertainties regarding key inputs to the Yucca Mountain Total System Performance Assessment (TSPA). The objective of the current project was to characterize the uncertainties associated with certain key issues related to the saturated zone system in the Yucca Mountain area and downgradient region. An understanding of saturated zone processes is critical to evaluating the performance of the potential high-level nuclear waste repository at Yucca Mountain. A major goal of the project was to capture the uncertainties involved in assessing the saturated flow processes, including uncertainty in both the models used to represent the physical processes controlling saturated zone flow and transport, and the parameter values used in the models. So that the analysis included a wide range of perspectives, multiple individual judgments were elicited from members of an expert panel. The panel members, who were experts from within and outside the Yucca Mountain project, represented a range of experience and expertise. A deliberate process was followed in facilitating interactions among the experts, in training them to express their uncertainties, and in eliciting their interpretations. The resulting assessments and probability distributions, therefore, provide a reasonable aggregate representation of the knowledge and

  13. A Step-Wise Approach to Elicit Triangular Distributions

    NASA Technical Reports Server (NTRS)

    Greenberg, Marc W.

    2013-01-01

    Adapt/combine known methods to demonstrate an expert judgment elicitation process that: 1.Models expert's inputs as a triangular distribution, 2.Incorporates techniques to account for expert bias and 3.Is structured in a way to help justify expert's inputs. This paper will show one way of "extracting" expert opinion for estimating purposes. Nevertheless, as with most subjective methods, there are many ways to do this.

  14. Nicotine Elicits Convulsive Seizures by Activating Amygdalar Neurons

    PubMed Central

    Iha, Higor A.; Kunisawa, Naofumi; Shimizu, Saki; Tokudome, Kentaro; Mukai, Takahiro; Kinboshi, Masato; Ikeda, Akio; Ito, Hidefumi; Serikawa, Tadao; Ohno, Yukihiro

    2017-01-01

    Nicotinic acetylcholine (nACh) receptors are implicated in the pathogenesis of epileptic disorders; however, the mechanisms of nACh receptors in seizure generation remain unknown. Here, we performed behavioral and immunohistochemical studies in mice and rats to clarify the mechanisms underlying nicotine-induced seizures. Treatment of animals with nicotine (1–4 mg/kg, i.p.) produced motor excitement in a dose-dependent manner and elicited convulsive seizures at 3 and 4 mg/kg. The nicotine-induced seizures were abolished by a subtype non-selective nACh antagonist, mecamylamine (MEC). An α7 nACh antagonist, methyllycaconitine, also significantly inhibited nicotine-induced seizures whereas an α4β2 nACh antagonist, dihydro-β-erythroidine, affected only weakly. Topographical analysis of Fos protein expression, a biological marker of neural excitation, revealed that a convulsive dose (4 mg/kg) of nicotine region-specifically activated neurons in the piriform cortex, amygdala, medial habenula, paratenial thalamus, anterior hypothalamus and solitary nucleus among 48 brain regions examined, and this was also suppressed by MEC. In addition, electric lesioning of the amygdala, but not the piriform cortex, medial habenula and thalamus, specifically inhibited nicotine-induced seizures. Furthermore, microinjection of nicotine (100 and 300 μg/side) into the amygdala elicited convulsive seizures in a dose-related manner. The present results suggest that nicotine elicits convulsive seizures by activating amygdalar neurons mainly via α7 nACh receptors. PMID:28232801

  15. Conditioned cues for smoking elicit preparatory responses in healthy smokers

    PubMed Central

    Winkler, Markus H.; Weyers, Peter; Mucha, Ronald F.; Stippekohl, Bastian; Stark, Rudolf

    2010-01-01

    Rationale Smoking cues are theorized to be conditioned stimuli (CSs) formed by repeated pairing with drug. Smoking paraphernalia can elicit subjective and physiological responses in smokers, indicative of positive affect and motivation to consume. Although these responses are probably the result of conditioning, direct evidence from human conditioning studies with physiological measures of motivational valence is rare. Objective The present study investigated the motivational properties of experimentally conditioned cues for smoking. Methods Thirty-nine smokers completed a differential conditioning protocol. Abstract pictures were used as CSs and single puffs on a cigarette as unconditioned stimulus (US). Skin conductance responses and facial electromyography of the zygomatic, corrugator, and orbicularis oris muscles were measured during conditioning. Results The conditioned cue for smoking (CS+) elicited stronger skin conductance responses and more activity of the zygomatic and orbicularis oris muscles than the CS−. Conclusions These results support the notion that through pairing with smoking, neutral stimuli acquire the ability to elicit preparatory physiological responses, which are assumed to play an important role in the maintenance of addiction and relapse in the natural environment. PMID:20953588

  16. Elicited versus emitted behavior: Time to abandon the distinction.

    PubMed

    Domjan, Michael

    2016-03-01

    The concept of emitted behavior was formulated as a part of the original argument for the validity of a new kind of learning called operant conditioning. The rationale for operant conditioning contrasted it with Pavlovian or classical conditioning, which was (and remains) fundamentally based on responses to conditioned and unconditioned stimuli. Classical conditioned responses were said to be elicited. In contrast, operant behavior was viewed as emitted and controlled primarily by response consequences rather than antecedents. I argue that the distinction between emitted and elicited behavior is no longer warranted for three major reasons. First, the distinction was based on a view of Pavlovian conditioning that is no longer viable. Second, the distinction is incompatible with both empirical data and contemporary conceptualizations of operant behavior. Third, the only way to overcome these problems is to define emitted and elicited in terms of the type of conditioning (operant and classical) that produces these behaviors, but that approach makes the definitions circular and does not avoid implications of the terms that are misleading and counterproductive in light of contemporary research and thinking.

  17. Elicitation of macrophages from the peritoneal cavity of channel catfish

    USGS Publications Warehouse

    Jenkins, J.A.; Klesius, P.H.

    1998-01-01

    Four chemicals were evaluated for elicitation of macrophages in peritoneal cavities of 250-300g healthy channel catfish Ictalurus punctatus. Cellular exudates were collected at 3, 5, 7, 10, 14, and 20 d following intraperitoneal injections with squalene, Freund's incomplete adjuvant (FIA), goat serum, thioglycollate, or as a control, phosphate-buffered saline. Injection with either squalene or FIA induced significantly greater (P ??? 0.0001) macrophage recruitment than the other chemicals. The effectiveness of squalene and FIA was compared further by macrophage collection daily for 7 d. Squalene and FIA elicited similarly high macrophage responses (P ??? 0.0450), the highest being 3.43 x 106 macrophages/mL (SE, 2.4 x l06) at 99% purity at day 2 and 2.1 X 106 macrophages/mL (SE, 0.7 x 106) at day 14 at 80% purity, respectively. In both experiments, the time after injection was not statistically significant, nor was there an interaction between time and chemicals. The occurrence of cells other than macrophages decreased with time to yield macrophage recoveries of 47-99% for squalene and 30-80% for FIA. Two subsets of macrophages were observed by means of flow cytometry. As demonstrated by chemiluminescence, the squalene-elicited cells produced high-energy oxygen compounds important to the phagocytic process.

  18. Physiological responses induced by emotion-eliciting films.

    PubMed

    Fernández, Cristina; Pascual, Juan C; Soler, Joaquim; Elices, Matilde; Portella, Maria J; Fernández-Abascal, Enrique

    2012-06-01

    Emotion-eliciting films are commonly used to evoke subjective emotional responses in experimental settings. The main aim of the present study was to investigate whether a set of film clips with discrete emotions were capable to elicit measurable objective physiological responses. The convergence between subjective and objective measures was evaluated. Finally, the effect of gender on emotional responses was investigated. A sample of 123 subjects participated in the study. Individuals were asked to view a set of emotional film clips capable to induce seven emotions: anger, fear, sadness, disgust, amusement, tenderness and neutral state. Skin conductance level (SCL), heart rate (HR) and subjective emotional responses were measured for each film clip. In comparison with neutral films, SCL was significantly increased after viewing fear films, and HR was also significantly incremented for anger and fear films. Physiological variations were associated with arousal measures indicating a convergence between subjective and objective reactions. Women appeared to display significantly greater SCL and HR responses for films inducing sadness. The findings suggest that physiological activation would be more easily induced by emotion-eliciting films that tap into emotions with higher subjective arousal such as anger and fear.

  19. Elicitation of andrographolide in the suspension cultures of Andrographis paniculata.

    PubMed

    Gandi, Suryakala; Rao, Kiranmayee; Chodisetti, Bhuvaneswari; Giri, Archana

    2012-12-01

    Andrographis paniculata belonging to the family Acanthaceae produces a group of diterpene lactones, one of which is the pharmaceutically important-andrographolide. It is known to possess various important biological properties like anticancer, anti-HIV, anti-inflammatory, etc. This is the first report on the production of andrographolide in the cell suspension cultures of Andrographis paniculata by 'elicitation'. Elicitation was attempted to enhance the andrographolide content in the suspension cultures of Andrographis paniculata and also to ascertain its stimulation under stress conditions or in response to pathogen attack. The maximum andrographolide production was found to be 1.53 mg/g dry cell weight (DCW) at the end of stationary phase during the growth curve. The biotic elicitors (yeast, Escherichia coli, Bacillus subtilis, Agrobacterium rhizogenes 532 and Agrobacterium tumefaciens C 58) were more effective in eliciting the response when compared to the abiotic elicitors (CdCl(2), AgNO(3), CuCl(2) and HgCl(2)). Yeast has shown to stimulate maximum accumulation of 13.5 mg/g DCW andrographolide, which was found to be 8.82-fold higher than the untreated cultures.

  20. Local and global ligand-induced changes in the structure of the GABA(A) receptor.

    PubMed

    Muroi, Yukiko; Czajkowski, Cynthia; Jackson, Meyer B

    2006-06-13

    Ligand-gated channels mediate synaptic transmission through conformational transitions triggered by the binding of neurotransmitters. These transitions are well-defined in terms of ion conductance, but their structural basis is poorly understood. To probe these changes in structure, GABA(A) receptors were expressed in Xenopus oocytes and labeled at selected sites with environment-sensitive fluorophores. With labels at two different residues in the alpha1 subunit in loop E of the GABA-binding pocket, GABA elicited fluorescence changes opposite in sign. This pattern of fluorescence changes is consistent with a closure of the GABA-binding cavity at the subunit interface. The competitive antagonist SR-95531 inverted this pattern of fluorescence change, but the noncompetitive antagonist picrotoxin failed to elicit optical signals. In response to GABA (but not SR-95531), labels at the homologous residues in the beta2 subunit showed the same pattern of fluorescence change as the alpha1-subunit labels, indicating a global transition with comparable movements in homologous regions of different subunits. Incorporation of the gamma2 subunit altered the fluorescence changes of alpha1-subunit labels and eliminated them in beta2-subunit labels. Thus, the ligand-induced structural changes in the GABA(A) receptor can extend over considerable distances or remain highly localized, depending upon subunit composition and ligand.

  1. Glyconanomaterials: synthesis, characterization, and ligand presentation.

    PubMed

    Wang, Xin; Ramström, Olof; Yan, Mingdi

    2010-05-04

    Glyconanomaterials, nanomaterials carrying surface-tethered carbohydrate ligands, have emerged and demonstrated increasing potential in biomedical imaging, therapeutics, and diagnostics. These materials combine the unique properties of nanometer-scale objects with the ability to present multiple copies of carbohydrate ligands, greatly enhancing the weak affinity of individual ligands to their binding partners. Critical to the performance of glyconanomaterials is the proper display of carbohydrate ligands, taking into consideration of the coupling chemistry, the type and length of the spacer linkage, and the ligand density. This article provides an overview of the coupling chemistry for attaching carbohydrate ligands to nanomaterials, and discusses the need for thorough characterization of glyconanomaterials, especially quantitative analyses of the ligand density and binding affinities. Using glyconanoparticles synthesized by a versatile photocoupling chemistry, methods for determining the ligand density by colorimetry and the binding affinity with lectins by a fluorescence competition assay are determined. The results show that the multivalent presentation of carbohydrate ligands significantly enhances the binding affinity by several orders of magnitude in comparison to the free ligands in solution. The effect is sizeable even at low surface ligand density. The type and length of the spacer linkage also affect the binding affinity, with the longer linkage promoting the association of bound ligands with the corresponding lectins.

  2. Glyconanomaterials: Synthesis, Characterization, and Ligand Presentation

    PubMed Central

    Wang, Xin

    2010-01-01

    Glyconanomaterials, nanomaterials carrying surface-tethered carbohydrate ligands, have emerged and demonstrated increasing potential in biomedical imaging, therapeutics, and diagnostics. These materials combine the unique properties of nanometer-scale objects with the ability to present multiple copies of carbohydrate ligands, greatly enhancing the weak affinity of individual ligands to their binding partners. Critical to the performance of glyconanomaterials is the proper display of carbohydrate ligands, taking into consideration of the coupling chemistry, the type and length of the spacer linkage, and the ligand density. This article provides an overview of the coupling chemistry for attaching carbohydrate ligands to nanomaterials, and discusses the need for thorough characterization of glyconanomaterials, especially quantitative analyses of the ligand density and binding affinities. Using glyconanoparticles synthesized by a versatile photocoupling chemistry, methods for determining the ligand density by colorimetry and the binding affinity with lectins by a fluorescence competition assay are determined. The results show that the multivalent presentation of carbohydrate ligands significantly enhances the binding affinity by several orders of magnitude in comparison to the free ligands in solution. The effect is sizeable even at low surface ligand density. The type and length of the spacer linkage also affect the binding affinity, with the longer linkage promoting the association of bound ligands with the corresponding lectins. PMID:20301131

  3. Developing Socio-Cultural Scaffolding Model to Elicit Learners's Speech Production

    ERIC Educational Resources Information Center

    Englishtina, Inti

    2015-01-01

    This study is concerned with developing scaffolding model to elicit bilingual kindergarten children's English speech production. It is aimed at describing what the teachers need in eliciting their students' speech production; how a scaffolding model should be developed to elicit the children's speech production; and how effective is the…

  4. Research Dilemmas Associated with Photo Elicitation in Comparative Early Childhood Education Research

    ERIC Educational Resources Information Center

    Birkeland, Asta

    2013-01-01

    Photo elicitation has become an important method to produce data in qualitative research. There is quite an extensive literature indicating the benefits of photo elicitation in order to facilitate collaboration in meaning making between researcher and the interviewee. This article addresses dilemmas associated with using photo elicitation in a…

  5. Sample Size for Measuring Grammaticality in Preschool Children from Picture-Elicited Language Samples

    ERIC Educational Resources Information Center

    Eisenberg, Sarita L.; Guo, Ling-Yu

    2015-01-01

    Purpose: The purpose of this study was to investigate whether a shorter language sample elicited with fewer pictures (i.e., 7) would yield a percent grammatical utterances (PGU) score similar to that computed from a longer language sample elicited with 15 pictures for 3-year-old children. Method: Language samples were elicited by asking forty…

  6. Sustained Hypoxia Elicits Competing Spinal Mechanisms of Phrenic Motor Facilitation

    PubMed Central

    Devinney, Michael J.; Nichols, Nicole L.

    2016-01-01

    Acute intermittent hypoxia (AIH) induces phrenic long-term facilitation (pLTF), a form of spinal motor plasticity. Competing mechanisms give rise to phrenic motor facilitation (pMF; a general term including pLTF) depending on the severity of hypoxia within episodes. In contrast, moderate acute sustained hypoxia (mASH) does not elicit pMF. By varying the severity of ASH and targeting competing mechanisms of pMF, we sought to illustrate why moderate AIH (mAIH) elicits pMF but mASH does not. Although mAIH elicits serotonin-dependent pLTF, mASH does not; thus, mAIH-induced pLTF is pattern sensitive. In contrast, severe AIH (sAIH) elicits pLTF through adenosine-dependent mechanisms, likely from greater extracellular adenosine accumulation. Because serotonin- and adenosine-dependent pMF interact via cross talk inhibition, we hypothesized that pMF is obscured because the competing mechanisms of pMF are balanced and offsetting during mASH. Here, we demonstrate the following: (1) blocking spinal A2A receptors with MSX-3 reveals mASH-induced pMF; and (2) sASH elicits A2A-dependent pMF. In anesthetized rats pretreated with intrathecal A2A receptor antagonist injections before mASH (PaO2 = 40–54 mmHg) or sASH (PaO2 = 25–36 mmHg), (1) mASH induced a serotonin-dependent pMF and (2) sASH induced an adenosine-dependent pMF, which was enhanced by spinal serotonin receptor inhibition. Thus, competing adenosine- and serotonin-dependent mechanisms contribute differentially to pMF depending on the pattern/severity of hypoxia. Understanding interactions between these mechanisms has clinical relevance as we develop therapies to treat severe neuromuscular disorders that compromise somatic motor behaviors, including breathing. Moreover, these results demonstrate how competing mechanisms of plasticity can give rise to pattern sensitivity in pLTF. SIGNIFICANCE STATEMENT Intermittent hypoxia elicits pattern-sensitive spinal plasticity and improves motor function after spinal injury or

  7. Antibody networks and imaging: elicitation of anti-fluorescein antibodies in response to the metatypic state of fluorescein-specific monoclonal antibodies.

    PubMed

    Cedergren, A M; Miklasz, S D; Voss, E W

    1996-01-01

    Studies are described regarding generation of anti-hapten antibodies starting with a monoclonal Ig immunogen in the ligand-induced conformation or metatypic state. Liganded monoclonal Ab1 antibodies represent the unique feature of the study since previous reports investigating internal imaging in the original Idiotype Network Hypothesis [Jerne, 1974 (Ann. Immun. 125C, 373-389)] were based on the non-liganded or idiotypic state [as reviewed in: Rodkey, 1980 (Microbiol. Rev. 44, 631-659); Kohler et al., 1979 (In: Methods in Enzymology: Antibodies, Antigens and Molecular Mimicry, pp. 3-35); Greenspan and Bona, 1993 (FASEB J. 7,437-444)]. Affinity-labeled liganded murine monoclonal anti-fluorescein antibodies served as immunogens administered both in the syngenic and xenogenic modes to determine if the metatypic state elicited anti-hapten antibodies through imaging-like mechanisms. Polyclonal and monoclonal anti-Ab1 reagents in various hosts were assayed for anti-fluorescein and/or anti-metatype specificity. Significant anti-fluorescein responses were measured indicating that the metatypic state directly or indirectly stimulates an anti-hapten antibody population.

  8. Ligand chain length conveys thermochromism.

    PubMed

    Ganguly, Mainak; Panigrahi, Sudipa; Chandrakumar, K R S; Sasmal, Anup Kumar; Pal, Anjali; Pal, Tarasankar

    2014-08-14

    Thermochromic properties of a series of non-ionic copper compounds have been reported. Herein, we demonstrate that Cu(II) ion with straight-chain primary amine (A) and alpha-linolenic (fatty acid, AL) co-jointly exhibit thermochromic properties. In the current case, we determined that thermochromism becomes ligand chain length-dependent and at least one of the ligands (A or AL) must be long chain. Thermochromism is attributed to a balanced competition between the fatty acids and amines for the copper(II) centre. The structure-property relationship of the non-ionic copper compounds Cu(AL)2(A)2 has been substantiated by various physical measurements along with detailed theoretical studies based on time-dependent density functional theory. It is presumed from our results that the compound would be a useful material for temperature-sensor applications.

  9. Nicotine elicits prolonged calcium signaling along ventral hippocampal axons.

    PubMed

    Zhong, Chongbo; Talmage, David A; Role, Lorna W

    2013-01-01

    Presynaptic nicotinic acetylcholine receptors (nAChRs) have long been implicated in the modulation of CNS circuits. We previously reported that brief exposure to low concentrations of nicotine induced sustained potentiation of glutamatergic transmission at ventral hippocampal (vHipp)-striatal synapses. Here, we exploited nAChR subtype-selective antagonists and agonists and α7*nAChR knockout mutant mice (α7-/-) to elucidate the signaling mechanisms underlying nAChR-mediated modulation of synaptic transmission. Using a combination of micro-slices culture from WT and α7-/-mice, calcium imaging, and immuno-histochemical techniques, we found that nicotine elicits localized and oscillatory increases in intracellular Ca(2+) along vHipp axons that persists for up to 30 minutes. The sustained phase of the nicotine-induced Ca(2+) response was blocked by α-BgTx but not by DHβE and was mimicked by α7*nAChR agonists but not by non-α7*nAChR agonists. In vHipp slices from α7-/- mice, nicotine elicited only transient increases of axonal Ca(2+) signals and did not activate CaMKII. The sustained phase of the nicotine-induced Ca(2+) response required localized activation of CaMKII, phospholipase C, and IP3 receptor mediated Ca(2+)-induced Ca(2+) release (CICR). In conclusion, activation of presynaptic nAChRs by nicotine elicits Ca(2+) influx into the presynaptic axons, the sustained phase of the nicotine-induced Ca(2+) response requires that axonal α7*nAChR activate a downstream signaling network in the vHipp axons.

  10. Estimating unknown parameters in haemophilia using expert judgement elicitation.

    PubMed

    Fischer, K; Lewandowski, D; Janssen, M P

    2013-09-01

    The increasing attention to healthcare costs and treatment efficiency has led to an increasing demand for quantitative data concerning patient and treatment characteristics in haemophilia. However, most of these data are difficult to obtain. The aim of this study was to use expert judgement elicitation (EJE) to estimate currently unavailable key parameters for treatment models in severe haemophilia A. Using a formal expert elicitation procedure, 19 international experts provided information on (i) natural bleeding frequency according to age and onset of bleeding, (ii) treatment of bleeds, (iii) time needed to control bleeding after starting secondary prophylaxis, (iv) dose requirements for secondary prophylaxis according to onset of bleeding, and (v) life-expectancy. For each parameter experts provided their quantitative estimates (median, P10, P90), which were combined using a graphical method. In addition, information was obtained concerning key decision parameters of haemophilia treatment. There was most agreement between experts regarding bleeding frequencies for patients treated on demand with an average onset of joint bleeding (1.7 years): median 12 joint bleeds per year (95% confidence interval 0.9-36) for patients ≤ 18, and 11 (0.8-61) for adult patients. Less agreement was observed concerning estimated effective dose for secondary prophylaxis in adults: median 2000 IU every other day The majority (63%) of experts expected that a single minor joint bleed could cause irreversible damage, and would accept up to three minor joint bleeds or one trauma related joint bleed annually on prophylaxis. Expert judgement elicitation allowed structured capturing of quantitative expert estimates. It generated novel data to be used in computer modelling, clinical care, and trial design.

  11. Presentation of Ligands on Hydroxylapatite

    NASA Technical Reports Server (NTRS)

    Chu, Barbara C. F.; Orgel, Leslie E.

    1997-01-01

    Conjugates of biotin with the decamer of glutamic acid (glu(sub 10)) and the trimer of D,L-2-amino-5-phosphonovaleric acid (I) have been synthesized, and it has been shown that they mediate the binding of avidin to hydroxylapatite. In a similar way a conjugate of methotrexate with glu(sub 10) mediates the binding of dihydrofolate reductase to the mineral. The presentation of ligands on the hydroxylapatite component of bone may find applications in clinical medicine.

  12. Electronic health records: eliciting behavioral health providers' beliefs.

    PubMed

    Shank, Nancy; Willborn, Elizabeth; Pytlikzillig, Lisa; Noel, Harmonijoie

    2012-04-01

    Interviews with 32 community behavioral health providers elicited perceived benefits and barriers of using electronic health records. Themes identified were (a) quality of care, (b) privacy and security, and (c) delivery of services. Benefits to quality of care were mentioned by 100% of the providers, and barriers by 59% of providers. Barriers involving privacy and security concerns were mentioned by 100% of providers, and benefits by 22%. Barriers to delivery of services were mentioned by 97% of providers, and benefits by 66%. Most providers (81%) expressed overall positive support for electronic behavioral health records.

  13. Understanding, eliciting and negotiating clients' multicultural health beliefs.

    PubMed

    Jackson, L E

    1993-04-01

    People of many cultures explain and treat illness in ways that are different from and that may conflict with the biomedical beliefs and practices on which the American health care system is based. Eliciting clients' health beliefs and negotiating treatment plans with them can help avoid problems caused by discrepancies in belief systems. This article presents three major categories of belief systems commonly found in the United States as well as other countries. Questions designed to discover clients' health beliefs are included, along with guidelines for arriving at plans of care that accommodate those beliefs. Case studies are provided that illustrate this process of negotiation.

  14. Simplifying Probability Elicitation and Uncertainty Modeling in Bayesian Networks

    SciTech Connect

    Paulson, Patrick R; Carroll, Thomas E; Sivaraman, Chitra; Neorr, Peter A; Unwin, Stephen D; Hossain, Shamina S

    2011-04-16

    In this paper we contribute two methods that simplify the demands of knowledge elicitation for particular types of Bayesian networks. The first method simplify the task of providing probabilities when the states that a random variable takes can be described by a new, fully ordered state set in which a state implies all the preceding states. The second method leverages Dempster-Shafer theory of evidence to provide a way for the expert to express the degree of ignorance that they feel about the estimates being provided.

  15. Tumor Targeting via Integrin Ligands

    PubMed Central

    Marelli, Udaya Kiran; Rechenmacher, Florian; Sobahi, Tariq Rashad Ali; Mas-Moruno, Carlos; Kessler, Horst

    2013-01-01

    Selective and targeted delivery of drugs to tumors is a major challenge for an effective cancer therapy and also to overcome the side-effects associated with current treatments. Overexpression of various receptors on tumor cells is a characteristic structural and biochemical aspect of tumors and distinguishes them from physiologically normal cells. This abnormal feature is therefore suitable for selectively directing anticancer molecules to tumors by using ligands that can preferentially recognize such receptors. Several subtypes of integrin receptors that are crucial for cell adhesion, cell signaling, cell viability, and motility have been shown to have an upregulated expression on cancer cells. Thus, ligands that recognize specific integrin subtypes represent excellent candidates to be conjugated to drugs or drug carrier systems and be targeted to tumors. In this regard, integrins recognizing the RGD cell adhesive sequence have been extensively targeted for tumor-specific drug delivery. Here we review key recent examples on the presentation of RGD-based integrin ligands by means of distinct drug-delivery systems, and discuss the prospects of such therapies to specifically target tumor cells. PMID:24010121

  16. Tools for ligand validation in Coot

    PubMed Central

    Emsley, Paul

    2017-01-01

    Coot is a molecular-graphics program primarily aimed at model building using X-ray data. Recently, tools for the manipulation and representation of ligands have been introduced. Here, these new tools for ligand validation and comparison are described. Ligands in the wwPDB have been scored by density-fit, distortion and atom-clash metrics. The distributions of these scores can be used to assess the relative merits of the particular ligand in the protein–ligand complex of interest by means of ‘sliders’ akin to those now available for each accession code on the wwPDB websites. PMID:28291755

  17. Using 3 TLR ligands as a combination adjuvant induces qualitative changes in T cell responses needed for antiviral protection in mice

    PubMed Central

    Zhu, Qing; Egelston, Colt; Gagnon, Susan; Sui, Yongjun; Belyakov, Igor M.; Klinman, Dennis M.; Berzofsky, Jay A.

    2010-01-01

    TLR ligands are promising candidates for the development of novel vaccine adjuvants that can elicit protective immunity against emerging infectious diseases. Adjuvants have been used most frequently to increase the quantity of an immune response. However, the quality of a T cell response can be more important than its quantity. Stimulating certain pairs of TLRs induces a synergistic response in terms of activating dendritic cells and eliciting/enhancing T cell responses through clonal expansion, which increases the number of responding T cells. Here, we have found that utilizing ligands for 3 TLRs (TLR2/6, TLR3, and TLR9) greatly increased the protective efficacy of vaccination with an HIV envelope peptide in mice when compared with using ligands for only any 2 of these TLRs; surprisingly, increased protection was induced without a marked increase in the number of peptide-specific T cells. Rather, the combination of these 3 TLR ligands augmented the quality of the T cell responses primarily by amplifying their functional avidity for the antigen, which was necessary for clearance of virus. The triple combination increased production of DC IL-15 along with its receptor, IL-15Rα, which contributed to high avidity, and decreased expression of programmed death–ligand 1 and induction of Tregs. Therefore, selective TLR ligand combinations can increase protective efficacy by increasing the quality rather than the quantity of T cell responses. PMID:20101095

  18. Ligand identification using electron-density mapcorrelations

    SciTech Connect

    Terwilliger, Thomas C.; Adams, Paul D.; Moriarty, Nigel W.; Cohn,Judith D.

    2006-12-01

    A procedure for the identification of ligands bound incrystal structuresof macromolecules is described. Two characteristics ofthe density corresponding to a ligand are used in the identificationprocedure. One is the correlation of the ligand density with each of aset of test ligands after optimization of the fit of that ligand to thedensity. The other is the correlation of a fingerprint of the densitywith the fingerprint of model density for each possible ligand. Thefingerprints consist of an ordered list of correlations of each the testligands with the density. The two characteristics are scored using aZ-score approach in which the correlations are normalized to the mean andstandard deviation of correlations found for a variety of mismatchedligand-density pairs, so that the Z scores are related to the probabilityof observing a particular value of the correlation by chance. Theprocedure was tested with a set of 200 of the most commonly found ligandsin the Protein Data Bank, collectively representing 57 percent of allligands in the Protein Data Bank. Using a combination of these twocharacteristics of ligand density, ranked lists of ligand identificationswere made for representative (F-o-F-c) exp(i phi(c)) difference densityfrom entries in the Protein Data Bank. In 48 percent of the 200 cases,the correct ligand was at the top of the ranked list of ligands. Thisapproach may be useful in identification of unknown ligands in newmacromolecular structures as well as in the identification of whichligands in a mixture have bound to a macromolecule.

  19. NKG2D ligands as therapeutic targets

    PubMed Central

    Spear, Paul; Wu, Ming-Ru; Sentman, Marie-Louise; Sentman, Charles L.

    2013-01-01

    The Natural Killer Group 2D (NKG2D) receptor plays an important role in protecting the host from infections and cancer. By recognizing ligands induced on infected or tumor cells, NKG2D modulates lymphocyte activation and promotes immunity to eliminate ligand-expressing cells. Because these ligands are not widely expressed on healthy adult tissue, NKG2D ligands may present a useful target for immunotherapeutic approaches in cancer. Novel therapies targeting NKG2D ligands for the treatment of cancer have shown preclinical success and are poised to enter into clinical trials. In this review, the NKG2D receptor and its ligands are discussed in the context of cancer, infection, and autoimmunity. In addition, therapies targeting NKG2D ligands in cancer are also reviewed. PMID:23833565

  20. Interviewing strategically to elicit admissions from guilty suspects.

    PubMed

    Tekin, Serra; Granhag, Pär Anders; Strömwall, Leif; Giolla, Erik Mac; Vrij, Aldert; Hartwig, Maria

    2015-06-01

    In this article we introduce a novel interviewing tactic to elicit admissions from guilty suspects. By influencing the suspects' perception of the amount of evidence the interviewer holds against them, we aimed to shift the suspects' counterinterrogation strategies from less to more forthcoming. The proposed tactic (SUE-Confrontation) is a development of the Strategic Use of Evidence (SUE) framework and aims to affect the suspects' perception by confronting them with statement-evidence inconsistencies. Participants (N = 90) were asked to perform several mock criminal tasks before being interviewed using 1 of 3 interview techniques: (a) SUE-Confrontation, (b) Early Disclosure of Evidence, or (c) No Disclosure of Evidence. As predicted, the SUE-Confrontation interview generated more statement-evidence inconsistencies from suspects than the Early Disclosure interview. Importantly, suspects in the SUE-Confrontation condition (vs. Early and No disclosure conditions) admitted more self-incriminating information and also perceived the interviewer to have had more information about the critical phase of the crime (the phase where the interviewer lacked evidence). The findings show the adaptability of the SUE-technique and how it may be used as a tool for eliciting admissions.

  1. Experimental elicitation with hydroxyisohexyl-3-cyclohexene carboxaldehyde-containing deodorants.

    PubMed

    Jørgensen, Pia Haslund; Jensen, Charlotte Devantier; Rastogi, Suresh; Andersen, Klaus Ejner; Johansen, Jeanne Duus

    2007-03-01

    Hydroxyisohexyl-3-cyclohexene carboxaldehyde (HICC) known as Lyral is a frequent allergen. It is used in more than 50% of marketed deodorants. The aim of the present study was to determine elicitation thresholds for HICC under simulated conditions of deodorant use. 15 patients with previously diagnosed contact allergy to HICC were patch tested with 5 solutions of HICC-scented and HICC-unscented deodorants. Patients and 10 healthy controls performed a use test in the axillae using deodorants scented with HICC in increasing concentrations and unscented deodorants as control. The concentration of HICC was increased every second week (200, 600, and 1800 p.p.m.) until either a reaction developed or for 6 weeks. 14 patients completed the study, and all developed unilateral eczema from the HICC-containing deodorant, while controls were all negative (P= 0.004). In 9/14 patients, a positive use test developed during the first 2 weeks to the deodorant containing 200 p.p.m. HICC. Positive correlations were found between the day of positive use and patch test threshold concentration of the HICC solutions (r= 0.71, P= 0.01) as well as the patch test thresholds of the HICC-scented deodorants (r= 0.74, P= 0.007). In conclusion, HICC elicits allergic contact dermatitis in a high proportion of sensitized individuals at common usage concentrations in deodorants.

  2. Expert elicitation survey on future wind energy costs

    NASA Astrophysics Data System (ADS)

    Wiser, Ryan; Jenni, Karen; Seel, Joachim; Baker, Erin; Hand, Maureen; Lantz, Eric; Smith, Aaron

    2016-10-01

    Wind energy supply has grown rapidly over the last decade. However, the long-term contribution of wind to future energy supply, and the degree to which policy support is necessary to motivate higher levels of deployment, depends—in part—on the future costs of both onshore and offshore wind. Here, we summarize the results of an expert elicitation survey of 163 of the world’s foremost wind experts, aimed at better understanding future costs and technology advancement possibilities. Results suggest significant opportunities for cost reductions, but also underlying uncertainties. Under the median scenario, experts anticipate 24-30% reductions by 2030 and 35-41% reductions by 2050 across the three wind applications studied. Costs could be even lower: experts predict a 10% chance that reductions will be more than 40% by 2030 and more than 50% by 2050. Insights gained through expert elicitation complement other tools for evaluating cost-reduction potential, and help inform policy and planning, R&D and industry strategy.

  3. Vibrio elicits targeted transcriptional responses from copepod hosts.

    PubMed

    Almada, Amalia A; Tarrant, Ann M

    2016-06-01

    Copepods are abundant crustaceans that harbor diverse bacterial communities, yet the nature of their interactions with microbiota are poorly understood. Here, we report that Vibrio elicits targeted transcriptional responses in the estuarine copepod Eurytemora affinis We pre-treated E. affinis with an antibiotic cocktail and exposed them to either a zooplankton specialist (Vibrio sp. F10 9ZB36) or a free-living species (Vibrio ordalii 12B09) for 24 h. We then identified via RNA-Seq a total of 78 genes that were differentially expressed following Vibrio exposure, including homologs of C-type lectins, chitin-binding proteins and saposins. The response differed between the two Vibrio treatments, with the greatest changes elicited upon inoculation with V. sp. F10 We suggest that these differentially regulated genes play important roles in cuticle integrity, the innate immune response, and general stress response, and that their expression may enable E. affinis to recognize and regulate symbiotic vibrios. We further report that V. sp. F10 culturability is specifically altered upon colonization of E. affinis These findings suggest that rather than acting as passive environmental vectors, copepods discriminately interact with vibrios, which may ultimately impact the abundance and activity of copepod-associated bacteria.

  4. Cognitive constraints on constituent order: Evidence from elicited pantomime

    PubMed Central

    Hall, Matthew L.; Mayberry, Rachel I.; Ferreira, Victor S.

    2014-01-01

    To what extent does human cognition influence the structure of human language? Recent experiments using elicited pantomime suggest that the prevalence of Subject-Object-Verb (SOV) order across the world's languages may arise in part because SOV order is most compatible with how we conceptually represent transitive events (Goldin-Meadow, So, Özyürek, & Mylander, 2008). However, this raises the question as to why non-SOV orders exist. Two recent studies (Meir, Lifshitz, Ilkbasaran, & Padden, 2010; Gibson et al., 2013) suggest that SOV might be suboptimal for describing events in which both the agent and patient are plausible agents (e.g. a woman pushing a boy); we call these “reversible” events. We replicate these findings using elicited pantomime and offer a new interpretation. Meir et al.'s (2010) account is framed largely in terms of constraints on comprehension, while Gibson et al.'s (2013) account involves minimizing the risk of information loss or memory degradation. We offer an alternative hypothesis that is grounded in constraints on production. We consider the implications of these findings for the distribution of constituent order in the world's spoken languages and for the structure of emerging sign languages. PMID:23792806

  5. Expert elicitation survey on future wind energy costs

    SciTech Connect

    Wiser, Ryan; Jenni, Karen; Seel, Joachim; Baker, Erin; Hand, Maureen; Lantz, Eric; Smith, Aaron

    2016-09-12

    Wind energy supply has grown rapidly over the last decade. However, the long-term contribution of wind to future energy supply, and the degree to which policy support is necessary to motivate higher levels of deployment, depends -- in part -- on the future costs of both onshore and offshore wind. Here, we summarize the results of an expert elicitation survey of 163 of the world's foremost wind experts, aimed at better understanding future costs and technology advancement possibilities. Results suggest significant opportunities for cost reductions, but also underlying uncertainties. Under the median scenario, experts anticipate 24-30% reductions by 2030 and 35-41% reductions by 2050 across the three wind applications studied. Costs could be even lower: experts predict a 10% chance that reductions will be more than 40% by 2030 and more than 50% by 2050. Insights gained through expert elicitation complement other tools for evaluating cost-reduction potential, and help inform policy and planning, R&D and industry strategy.

  6. Bee threat elicits alarm call in African elephants.

    PubMed

    King, Lucy E; Soltis, Joseph; Douglas-Hamilton, Iain; Savage, Anne; Vollrath, Fritz

    2010-04-26

    Unlike the smaller and more vulnerable mammals, African elephants have relatively few predators that threaten their survival. The sound of disturbed African honeybees Apis meliffera scutellata causes African elephants Loxodonta africana to retreat and produce warning vocalizations that lead other elephants to join the flight. In our first experiment, audio playbacks of bee sounds induced elephants to retreat and elicited more head-shaking and dusting, reactive behaviors that may prevent bee stings, compared to white noise control playbacks. Most importantly, elephants produced distinctive "rumble" vocalizations in response to bee sounds. These rumbles exhibited an upward shift in the second formant location, which implies active vocal tract modulation, compared to rumbles made in response to white noise playbacks. In a second experiment, audio playbacks of these rumbles produced in response to bees elicited increased headshaking, and further and faster retreat behavior in other elephants, compared to control rumble playbacks with lower second formant frequencies. These responses to the bee rumble stimuli occurred in the absence of any bees or bee sounds. This suggests that these elephant rumbles may function as referential signals, in which a formant frequency shift alerts nearby elephants about an external threat, in this case, the threat of bees.

  7. Emotion Elicitation: A Comparison of Pictures and Films

    PubMed Central

    Uhrig, Meike K.; Trautmann, Nadine; Baumgärtner, Ulf; Treede, Rolf-Detlef; Henrich, Florian; Hiller, Wolfgang; Marschall, Susanne

    2016-01-01

    Pictures and film clips are widely used and accepted stimuli to elicit emotions. Based on theoretical arguments it is often assumed that the emotional effects of films exceed those of pictures, but to date this assumption has not been investigated directly. The aim of the present study was to compare pictures and films in terms of their capacity to induce emotions verified by means of explicit measures. Stimuli were (a) single pictures presented for 6 s, (b) a set of three consecutive pictures with emotionally congruent contents presented for 2 s each, (c) short film clips with a duration of 6 s. A total of 144 participants rated their emotion and arousal states following stimulus presentation. Repeated-measures ANOVAs revealed that the film clips and 3-picture version were as effective as the classical 1-picture method to elicit positive emotions, however, modulation toward positive valence was little. Modulation toward negative valence was more effective in general. Film clips were less effective than pictorial stimuli in producing the corresponding emotion states (all p < 0.001) and were less arousing (all p ≤ 0.02). Possible reasons for these unexpected results are discussed. PMID:26925007

  8. Autoreactivity and Exceptional CDR Plasticity (but Not Unusual Polyspecificity) Hinder Elicitation of the Anti-HIV Antibody 4E10

    PubMed Central

    Finton, Kathryn A. K.; Larimore, Kevin; Larman, H. Benjamin; Friend, Della; Correnti, Colin; Rupert, Peter B.; Elledge, Stephen J.; Greenberg, Philip D.; Strong, Roland K.

    2013-01-01

    The broadly-neutralizing anti-HIV antibody 4E10 recognizes an epitope in the membrane-proximal external region of the HIV envelope protein gp41. Previous attempts to elicit 4E10 by vaccination with envelope-derived or reverse-engineered immunogens have failed. It was presumed that the ontogeny of 4E10-equivalent responses was blocked by inherent autoreactivity and exceptional polyreactivity. We generated 4E10 heavy-chain knock-in mice, which displayed significant B cell dysregulation, consistent with recognition of autoantigen/s by 4E10 and the presumption that tolerance mechanisms may hinder the elicitation of 4E10 or 4E10-equivalent responses. Previously proposed candidate 4E10 autoantigens include the mitochondrial lipid cardiolipin and a nuclear splicing factor, 3B3. However, using carefully-controlled assays, 4E10 bound only weakly to cardiolipin-containing liposomes, but also bound negatively-charged, non-cardiolipin-containing liposomes comparably poorly. 4E10/liposome binding was predominantly mediated by electrostatic interactions rather than presumed hydrophobic interactions. The crystal structure of 4E10 free of bound ligands showed a dramatic restructuring of the combining site, occluding the HIV epitope binding site and revealing profound flexibility, but creating an electropositive pocket consistent with non-specific binding of phospholipid headgroups. These results strongly suggested that antigens other than cardiolipin mediate 4E10 autoreactivity. Using a synthetic peptide library spanning the human proteome, we determined that 4E10 displays limited and focused, but unexceptional, polyspecificity. We also identified a novel autoepitope shared by three ER-resident inositol trisphosphate receptors, validated through binding studies and immunohistochemistry. Tissue staining with 4E10 demonstrated reactivity consistent with the type 1 inositol trisphosphate receptor as the most likely candidate autoantigen, but is inconsistent with splicing factor 3B3

  9. Novel Ligands for a Purine Riboswitch Discovered by RNA-Ligand Docking

    PubMed Central

    Daldrop, Peter; Reyes, Francis E.; Robinson, David A.; Hammond, Colin M.; Lilley, David M.; Batey, Robert T.; Brenk, Ruth

    2011-01-01

    Summary The increasing number of RNA crystal structures enables a structure-based approach to the discovery of new RNA-binding ligands. To develop the poorly explored area of RNA-ligand docking, we have conducted a virtual screening exercise for a purine riboswitch to probe the strengths and weaknesses of RNA-ligand docking. Using a standard protein-ligand docking program with only minor modifications, four new ligands with binding affinities in the micromolar range were identified, including two compounds based on molecular scaffolds not resembling known ligands. RNA-ligand docking performed comparably to protein-ligand docking indicating that this approach is a promising option to explore the wealth of RNA structures for structure-based ligand design. PMID:21439477

  10. Ligand placement based on prior structures: the guided ligand-replacement method

    SciTech Connect

    Klei, Herbert E.; Moriarty, Nigel W. Echols, Nathaniel; Terwilliger, Thomas C.; Baldwin, Eric T.; Pokross, Matt; Posy, Shana; Adams, Paul D.

    2014-01-01

    A new module, Guided Ligand Replacement (GLR), has been developed in Phenix to increase the ease and success rate of ligand placement when prior protein-ligand complexes are available. The process of iterative structure-based drug design involves the X-ray crystal structure determination of upwards of 100 ligands with the same general scaffold (i.e. chemotype) complexed with very similar, if not identical, protein targets. In conjunction with insights from computational models and assays, this collection of crystal structures is analyzed to improve potency, to achieve better selectivity and to reduce liabilities such as absorption, distribution, metabolism, excretion and toxicology. Current methods for modeling ligands into electron-density maps typically do not utilize information on how similar ligands bound in related structures. Even if the electron density is of sufficient quality and resolution to allow de novo placement, the process can take considerable time as the size, complexity and torsional degrees of freedom of the ligands increase. A new module, Guided Ligand Replacement (GLR), was developed in Phenix to increase the ease and success rate of ligand placement when prior protein–ligand complexes are available. At the heart of GLR is an algorithm based on graph theory that associates atoms in the target ligand with analogous atoms in the reference ligand. Based on this correspondence, a set of coordinates is generated for the target ligand. GLR is especially useful in two situations: (i) modeling a series of large, flexible, complicated or macrocyclic ligands in successive structures and (ii) modeling ligands as part of a refinement pipeline that can automatically select a reference structure. Even in those cases for which no reference structure is available, if there are multiple copies of the bound ligand per asymmetric unit GLR offers an efficient way to complete the model after the first ligand has been placed. In all of these applications, GLR

  11. Voice Pitch Elicited Frequency Following Response in Chinese Elderlies

    PubMed Central

    Wang, Shuo; Hu, Jiong; Dong, Ruijuan; Liu, Dongxin; Chen, Jing; Musacchia, Gabriella; Liu, Bo

    2016-01-01

    Background: Perceptual and electrophysiological studies have found reduced speech discrimination in quiet and noisy environment, delayed neural timing, decreased neural synchrony, and decreased temporal processing ability in elderlies, even those with normal hearing. However, recent studies have also demonstrated that language experience and auditory training enhance the temporal dynamics of sound encoding in the auditory brainstem response (ABR). The purpose of this study was to explore the pitch processing ability at the brainstem level in an aging population that has a tonal language background. Method: Mandarin speaking younger (n = 12) and older (n = 12) adults were recruited for this study. All participants had normal audiometric test results and normal suprathreshold click-evoked ABR. To record frequency following responses (FFRs) elicited by Mandarin lexical tones, two Mandarin Chinese syllables with different fundamental frequency pitch contours (Flat Tone and Falling Tone) were presented at 70 dB SPL. Fundamental frequencies (f0) of both the stimulus and the responses were extracted and compared to individual brainstem responses. Two indices were used to examine different aspects of pitch processing ability at the brainstem level: Pitch Strength and Pitch Correlation. Results: Lexical tone elicited FFR were overall weaker in the older adult group compared to their younger adult counterpart. Measured by Pitch Strength and Pitch Correlation, statistically significant group differences were only found when the tone with a falling f0 (Falling Tone) were used as the stimulus. Conclusion: Results of this study demonstrated that in a tonal language speaking population, pitch processing ability at the brainstem level of older adults are not as strong and robust as their younger counterparts. Findings of this study are consistent with previous reports on brainstem responses of older adults whose native language is English. On the other hand, lexical tone elicited

  12. Ligands for Ionotropic Glutamate Receptors

    PubMed Central

    Swanson, Geoffrey T.; Sakai, Ryuichi

    2010-01-01

    Marine-derived small molecules and peptides have played a central role in elaborating pharmacological specificities and neuronal functions of mammalian ionotropic glutamate receptors (iGluRs), the primary mediators of excitatory synaptic transmission in the central nervous system (CNS). As well, the pathological sequelae elicited by one class of compounds (the kainoids) constitute a widely-used animal model for human mesial temporal lobe epilepsy (mTLE). New and existing molecules could prove useful as lead compounds for the development of therapeutics for neuropathologies that have aberrant glutamatergic signaling as a central component. In this chapter we discuss natural source origins and pharmacological activities of those marine compounds that target ionotropic glutamate receptors. PMID:19184587

  13. A structure-function approach to optimizing TLR4 ligands for human vaccines

    PubMed Central

    Carter, Darrick; Fox, Christopher B; Day, Tracey A; Guderian, Jeffrey A; Liang, Hong; Rolf, Tom; Vergara, Julie; Sagawa, Zachary K; Ireton, Greg; Orr, Mark T; Desbien, Anthony; Duthie, Malcolm S; Coler, Rhea N; Reed, Steven G

    2016-01-01

    Adjuvants are combined with vaccine antigens to enhance and modify immune responses, and have historically been primarily crude, undefined entities. Introducing toll-like receptor (TLR) ligands has led to a new generation of adjuvants, with TLR4 ligands being the most extensively used in human vaccines. The TLR4 crystal structures demonstrate extensive contact with their ligands and provide clues as to how they discriminate a broad array of molecules and activate or attenuate innate, as well as adaptive, responses resulting from these interactions. Leveraging this discerning ability, we made subtle chemical alterations to the structure of a synthetic monophosphoryl lipid-A molecule to produce SLA, a designer TLR4 ligand that had a number of desirable adjuvant effects. The SLA molecule stimulated human TLR4 and induced Th1 biasing cytokines and chemokines. On human cells, the activity of SLA plateaued at lower concentrations than the lipid A comparator, and induced cytokine profiles distinct from other known TLR4 agonists, indicating the potential for superior adjuvant performance. SLA was formulated in an oil-in-water emulsion, producing an adjuvant that elicited potent Th1-biased adaptive responses. This was verified using a recombinant Leishmania vaccine antigen, first in mice, then in a clinical study in which the antigen-specific Th1-biased responses observed in mice were recapitulated in humans. These results demonstrated that using structure-based approaches one can predictably design and produce modern adjuvant formulations for safe and effective human vaccines. PMID:27990284

  14. Ligand-Based Peptide Design and Combinatorial Peptide Libraries to Target G Protein-Coupled Receptors

    PubMed Central

    Gruber, Christian W.; Muttenthaler, Markus; Freissmuth, Michael

    2016-01-01

    G protein-coupled receptors (GPCRs) are considered to represent the most promising drug targets; it has been repeatedly said that a large fraction of the currently marketed drugs elicit their actions by binding to GPCRs (with cited numbers varying from 30–50%). Closer scrutiny, however, shows that only a modest fraction of (~60) GPCRs are, in fact, exploited as drug targets, only ~20 of which are peptide-binding receptors. The vast majority of receptors in the humane genome have not yet been explored as sites of action for drugs. Given the drugability of this receptor class, it appears that opportunities for drug discovery abound. In addition, GPCRs provide for binding sites other than the ligand binding sites (referred to as the “orthosteric site”). These additional sites include (i) binding sites for ligands (referred to as “allosteric ligands”) that modulate the affinity and efficacy of orthosteric ligands, (ii) the interaction surface that recruits G proteins and arrestins, (iii) the interaction sites of additional proteins (GIPs, GPCR interacting proteins that regulate G protein signaling or give rise to G protein-independent signals). These sites can also be targeted by peptides. Combinatorial and natural peptide libraries are therefore likely to play a major role in identifying new GPCR ligands at each of these sites. In particular the diverse natural peptide libraries such as the venom peptides from marine cone-snails and plant cyclotides have been established as a rich source of drug leads. High-throughput screening and combinatorial chemistry approaches allow for progressing from these starting points to potential drug candidates. This will be illustrated by focusing on the ligand-based drug design of oxytocin (OT) and vasopressin (AVP) receptor ligands using natural peptide leads as starting points. PMID:20687879

  15. Reliance on luck: identifying which achievement goals elicit superstitious behavior.

    PubMed

    Hamerman, Eric J; Morewedge, Carey K

    2015-03-01

    People often resort to superstitious behavior to facilitate goal achievement. We examined whether the specific type of achievement goal pursued influences the propensity to engage in superstitious behavior. Across six studies, we found that performance goals were more likely than learning goals to elicit superstitious behavior. Participants were more likely to engage in superstitious behavior at high than at low levels of chronic performance orientation, but superstitious behavior was not influenced by chronic learning orientation (Studies 1 and 2). Similarly, participants exhibited stronger preferences for lucky items when primed to pursue performance goals rather than learning goals (Studies 3 and 4). As uncertainty of goal achievement increased, superstitious behavior increased when participants pursued performance goals but not learning goals (Study 5). Finally, assignment to use a lucky (vs. unlucky) item resulted in greater confidence of achieving performance goals but not learning goals (Study 6).

  16. Inflectional morphology in primary progressive aphasia: an elicited production study.

    PubMed

    Wilson, Stephen M; Brandt, Temre H; Henry, Maya L; Babiak, Miranda; Ogar, Jennifer M; Salli, Chelsey; Wilson, Lisa; Peralta, Karen; Miller, Bruce L; Gorno-Tempini, Maria Luisa

    2014-09-01

    Inflectional morphology lies at the intersection of phonology, syntax and the lexicon, three language domains that are differentially impacted in the three main variants of primary progressive aphasia (PPA). To characterize spared and impaired aspects of inflectional morphology in PPA, we elicited inflectional morphemes in 48 individuals with PPA and 13 healthy age-matched controls. We varied the factors of regularity, frequency, word class, and lexicality, and used voxel-based morphometry to identify brain regions where atrophy was predictive of deficits on particular conditions. All three PPA variants showed deficits in inflectional morphology, with the specific nature of the deficits dependent on the anatomical and linguistic features of each variant. Deficits in inflecting low-frequency irregular words were associated with semantic PPA, with lexical/semantic deficits, and with left temporal atrophy. Deficits in inflecting pseudowords were associated with non-fluent/agrammatic and logopenic variants, with phonological deficits, and with left frontal and parietal atrophy.

  17. Inflectional morphology in primary progressive aphasia: An elicited production study

    PubMed Central

    Wilson, Stephen M.; Brandt, Temre H.; Henry, Maya L.; Babiak, Miranda; Ogar, Jennifer M.; Salli, Chelsey; Wilson, Lisa; Peralta, Karen; Miller, Bruce L.; Gorno-Tempini, Maria Luisa

    2014-01-01

    Inflectional morphology lies at the intersection of phonology, syntax and the lexicon, three language domains that are differentially impacted in the three main variants of primary progressive aphasia (PPA). To characterize spared and impaired aspects of inflectional morphology in PPA, we elicited inflectional morphemes in 48 individuals with PPA and 13 healthy age-matched controls. We varied the factors of regularity, frequency, word class, and lexicality, and used voxel-based morphometry to identify brain regions where atrophy was predictive of deficits on particular conditions. All three PPA variants showed deficits in inflectional morphology, with the specific nature of the deficits dependent on the anatomical and linguistic features of each variant. Deficits in inflecting low-frequency irregular words were associated with semantic PPA, with lexical/semantic deficits, and with left temporal atrophy. Deficits in inflecting pseudowords were associated with non-fluent/agrammatic and logopenic variants, with phonological deficits, and with left frontal and parietal atrophy. PMID:25129631

  18. Eliciting the Level of Health Inequality Aversion in England.

    PubMed

    Robson, Matthew; Asaria, Miqdad; Cookson, Richard; Tsuchiya, Aki; Ali, Shehzad

    2016-09-20

    Health inequality aversion parameters can be used to represent alternative value judgements about policy concern for reducing health inequality versus improving total health. In this study, we use data from an online survey of the general public in England (n = 244) to elicit health inequality aversion parameters for both Atkinson and Kolm social welfare functions. We find median inequality aversion parameters of 10.95 for Atkinson and 0.15 for Kolm. These values suggest substantial concern for health inequality among the English general public which, at current levels of quality adjusted life expectancy, implies weighting health gains to the poorest fifth of people in society six to seven times as highly as health gains to the richest fifth. Copyright © 2016 John Wiley & Sons, Ltd.

  19. Elicitation: a tool for enriching the bioactive composition of foods.

    PubMed

    Baenas, Nieves; García-Viguera, Cristina; Moreno, Diego A

    2014-09-01

    Elicitation is a good strategy to induce physiological changes and stimulate defense or stress-induced responses in plants. The elicitor treatments trigger the synthesis of phytochemical compounds in fruits, vegetables and herbs. These metabolites have been widely investigated as bioactive compounds responsible of plant cell adaptation to the environment, specific organoleptic properties of foods, and protective effects in human cells against oxidative processes in the development of neurodegenerative and cardiovascular diseases and certain types of cancer. Biotic (biological origin), abiotic (chemical or physical origin) elicitors and phytohormones have been applied alone or in combinations, in hydroponic solutions or sprays, and in different selected time points of the plant growth or during post-harvest. Understanding how plant tissues and their specific secondary metabolic pathways respond to specific treatments with elicitors would be the basis for designing protocols to enhance the production of secondary metabolites, in order to produce quality and healthy fresh foods.

  20. Some properties of probability inversion algorithms to elicit expert opinion.

    NASA Astrophysics Data System (ADS)

    Lark, Murray

    2015-04-01

    Probability inversion methods have been developed to infer underlying expert utility functions from rankings that experts offer of subsets of scenarios. The method assumes that the expert ranking reflects an underlying utility, which can be modelled as a function of predictive covariates. This is potentially useful as a method for the extraction of expert opinions for prediction in new scenarios. Two particular algorithms are considered here, the IPF algorithm and the PURE algorithm. The former always converges for consistent sets of rankings and finds a solution which minimizes the mutual information of the estimated utilities and an initial random sample of proposed utilities drawn in the algorithm. In this poster I report some empirical studies on the probability inversion procedure, investigating the effects of the size of the expert panel, the consistency and quality of the expert panel and the validity of the predictive covariates. These results have practical implications for the design of elicitation by probability inversion methods.

  1. Biomimetic Antigenic Nanoparticles Elicit Controlled Protective Immune Response to Influenza

    PubMed Central

    Patterson, Dustin P.; Rynda-Apple, Agnieszka; Harmsen, Ann L.; Harmsen, Allen G.; Douglas, Trevor

    2013-01-01

    Here we present a biomimetic strategy towards nanoparticle design for controlled immune response through encapsulation of conserved internal influenza proteins on the interior of virus like particles (VLPs) to direct CD8+ cytotoxic T cell protection. Programmed encapsulation and sequestration of the conserved nucleoprotein (NP) from influenza on the interior of a VLP, derived from the bacteriophage P22, results in a vaccine that provides multi-strain protection against 100 times lethal doses of influenza in an NP specific CD8+ T cell-dependent manner. VLP assembly and encapsulation of the immunogenic NP cargo protein is the result of a genetically programmed self-assembly making this strategy amendable to the quick production of vaccines to rapidly emerging pathogens. Addition of adjuvants or targeting molecules were not required for eliciting the protective response. PMID:23540530

  2. Molecular mimicry and horror autotoxicus: do chlamydial infections elicit autoimmunity?

    PubMed

    Swanborg, Robert H; Boros, Dov L; Whittum-Hudson, Judith A; Hudson, Alan P

    2006-11-30

    All species of the order Chlamydiales are obligate intracellular eubacterial pathogens of their various hosts. Two chlamydial species, Chlamydia trachomatis and Chlamydia pneumoniae, are primarily human pathogens, and each is known to cause important diseases. Some strains of C. trachomatis are sexually transmitted and frequently cause severe reproductive problems, primarily in women. Other strains of the organism serve as the aetiological agents for blinding trachoma, still the leading cause of preventable blindness in underdeveloped nations. C. pneumoniae is a respiratory pathogen known to cause community-acquired pneumonia. Importantly, both organisms engender an immunopathogenic response in the human host, and both have been associated with widely diverse, relatively common and currently idiopathic chronic diseases, most of which include an important autoimmune component. In this article, we explore the available experimental data regarding the possible elicitation of autoimmunity in various contexts by chlamydial infection, and we suggest several avenues for research to explore this potentially important issue further.

  3. A universal rule for organic ligand exchange.

    PubMed

    You, Hongjun; Wang, Wenjin; Yang, Shengchun

    2014-11-12

    Most synthetic routes to high-quality nanocrystals with tunable morphologies predominantly employ long hydro-carbon molecules as ligands, which are detrimental for electronic and catalytic applications. Here, a rule is found that the adsorption energy of an organic ligand is related to its carbon-chain length. Using the density functional theory method, the adsorption energies of some commonly used ligand molecules with different carbon-chain lengths are calculated, including carboxylate, hydroxyl, and amine molecules adsorbed on metal or metal oxide crystal surface. The results indicate that the adsorption energy of the ligand molecule with a long carbon chain is weaker than that of a smaller molecule with same functional group. This rule provides a theoretical support for a new kind of ligand exchange method in which large organic ligand molecules can be exchanged by small molecules with same functional group to improve the catalytic properties.

  4. Cripto-1 vaccination elicits protective immunity against metastatic melanoma.

    PubMed

    Ligtenberg, M A; Witt, K; Galvez-Cancino, F; Sette, A; Lundqvist, A; Lladser, A; Kiessling, R

    2016-05-01

    Metastatic melanoma is a fatal disease that responds poorly to classical treatments but can be targeted by T cell-based immunotherapy. Cancer vaccines have the potential to generate long-lasting cytotoxic CD8(+) T cell responses able to eradicate established and disseminated tumors. Vaccination against antigens expressed by tumor cells with enhanced metastatic potential represents a highly attractive strategy to efficiently target deadly metastatic disease. Cripto-1 is frequently over-expressed in human carcinomas and melanomas, but is expressed only at low levels on normal differentiated tissues. Cripto-1 is particularly upregulated in cancer-initiating cells and is involved in cellular processes such as cell migration, invasion and epithelial-mesenchymal transition, which are hallmarks of aggressive cancer cells able to initiate metastatic disease. Here, we explored the potential of Cripto-1 vaccination to target metastatic melanoma in a preclinical model. Cripto-1 was overexpressed in highly metastatic B16F10 cells as compared to poorly metastatic B16F1 cells. Moreover, B16F10 cells grown in sphere conditions to enrich for cancer stem cells (CSC) progressively upregulated cripto1 expression. Vaccination of C57Bl/6 mice with a DNA vaccine encoding mouse Cripto-1 elicited a readily detectable/strong cytotoxic CD8(+) T cell response specific for a H-2 Kb-restricted epitope identified based on its ability to bind H-2(b) molecules. Remarkably, Cripto-1 vaccination elicited a protective response against lung metastasis and subcutaneous challenges with highly metastatic B16F10 melanoma cells. Our data indicate that vaccination against Cripto-1 represents a novel strategy to be tested in the clinic.

  5. Cripto-1 vaccination elicits protective immunity against metastatic melanoma

    PubMed Central

    Ligtenberg, M. A.; Witt, K.; Galvez-Cancino, F.; Sette, A.; Lundqvist, A.; Lladser, A.; Kiessling, R.

    2016-01-01

    ABSTRACT Metastatic melanoma is a fatal disease that responds poorly to classical treatments but can be targeted by T cell-based immunotherapy. Cancer vaccines have the potential to generate long-lasting cytotoxic CD8+ T cell responses able to eradicate established and disseminated tumors. Vaccination against antigens expressed by tumor cells with enhanced metastatic potential represents a highly attractive strategy to efficiently target deadly metastatic disease. Cripto-1 is frequently over-expressed in human carcinomas and melanomas, but is expressed only at low levels on normal differentiated tissues. Cripto-1 is particularly upregulated in cancer-initiating cells and is involved in cellular processes such as cell migration, invasion and epithelial–mesenchymal transition, which are hallmarks of aggressive cancer cells able to initiate metastatic disease. Here, we explored the potential of Cripto-1 vaccination to target metastatic melanoma in a preclinical model. Cripto-1 was overexpressed in highly metastatic B16F10 cells as compared to poorly metastatic B16F1 cells. Moreover, B16F10 cells grown in sphere conditions to enrich for cancer stem cells (CSC) progressively upregulated cripto1 expression. Vaccination of C57Bl/6 mice with a DNA vaccine encoding mouse Cripto-1 elicited a readily detectable/strong cytotoxic CD8+ T cell response specific for a H-2 Kb-restricted epitope identified based on its ability to bind H-2b molecules. Remarkably, Cripto-1 vaccination elicited a protective response against lung metastasis and subcutaneous challenges with highly metastatic B16F10 melanoma cells. Our data indicate that vaccination against Cripto-1 represents a novel strategy to be tested in the clinic. PMID:27467944

  6. A novel mimetic antigen eliciting protective antibody to Neisseria meningitidis.

    PubMed

    Granoff, D M; Moe, G R; Giuliani, M M; Adu-Bobie, J; Santini, L; Brunelli, B; Piccinetti, F; Zuno-Mitchell, P; Lee, S S; Neri, P; Bracci, L; Lozzi, L; Rappuoli, R

    2001-12-01

    Molecular mimetic Ags are of considerable interest as vaccine candidates. Yet there are few examples of mimetic Ags that elicit protective Ab against a pathogen, and the functional activity of anti-mimetic Abs has not been studied in detail. As part of the Neisseria meningitidis serogroup B genome sequencing project, a large number of novel proteins were identified. Herein, we provide evidence that genome-derived Ag 33 (GNA33), a lipoprotein with homology to Escherichia coli murein transglycosylase, elicits protective Ab to meningococci as a result of mimicking an epitope on loop 4 of porin A (PorA) in strains with serosubtype P1.2. Epitope mapping of a bactericidal anti-GNA33 mAb using overlapping peptides shows that the mAb recognizes peptides from GNA33 and PorA that share a QTP sequence that is necessary but not sufficient for binding. By flow cytometry, mouse antisera prepared against rGNA33 and the anti-GNA33 mAb bind as well as an anti-PorA P1.2 mAb to the surface of eight of nine N. meningitidis serogroup B strains tested with the P1.2 serosubtype. Anti-GNA33 Abs also are bactericidal for most P1.2 strains and, for susceptible strains, the activity of an anti-GNA33 mAb is similar to that of an anticapsular mAb but less active than an anti-P1.2 mAb. Anti-GNA Abs also confer passive protection against bacteremia in infant rats challenged with P1.2 strains. Thus, GNA33 represents one of the most effective immunogenic mimetics yet described. These results demonstrate that molecular mimetics have potential as meningococcal vaccine candidates.

  7. Tactile stimulation of the oropharynx elicits sympathoexcitation in conscious humans

    PubMed Central

    Muller, Matthew D.; Mast, Jessica L.; Cui, Jian; Heffernan, Matthew J.; McQuillan, Patrick M.

    2013-01-01

    Tactile stimulation of the oropharynx (TSO) elicits the gag reflex and increases heart rate (HR) and mean arterial pressure (MAP) in anesthetized patients. However, the interaction between upper-airway defense reflexes and the sympathetic nervous system has not been investigated in conscious humans. In Experiment 1, beat-by-beat measurements of HR, MAP, muscle sympathetic nerve activity (MSNA), and renal vascular resistance (RVR) were measured during TSO and tactile stimulation of the hard palate (Sham) in the supine posture. In Experiment 2, TSO was performed before (pre) and after (post) inhalation of 4% lidocaine via nebulizer. Rate pressure product (RPP) was determined. Compared with Sham, TSO elicited the gag reflex and increased RPP [absolute change (Δ)36 ± 6 vs. 17 ± 5%], MSNA (Δ122 ± 39 vs. 19 ± 19%), and RVR (Δ55 ± 11 vs. 4 ± 4%). This effect occurred within one to two cardiac cycles of TSO. The ΔMAP (12 ± 3 vs. 6 ± 1 mmHg) and the ΔHR (10 ± 3 vs. 3 ± 3 beats/min) were also greater following TSO compared with Sham. Lidocaine inhalation blocked the gag reflex and attenuated increases in MAP (Δpre: 16 ± 2; Δpost: 5 ± 2 mmHg) and HR (Δpre: 12 ± 3; Δpost: 2 ± 2 beats/min) in response to TSO. When mechanically stimulated, afferents in the oropharynx not only serve to protect the airway but also cause reflex increases in MSNA, RVR, MAP, and HR. An augmented sympathoexcitatory response during intubation and laryngoscopy may contribute to perioperative cardiovascular morbidity and mortality. PMID:23599399

  8. Nest sanitation elicits egg discrimination in cuckoo hosts.

    PubMed

    Yang, Canchao; Chen, Min; Wang, Longwu; Liang, Wei; Møller, Anders Pape

    2015-11-01

    Nest sanitation is a nearly universal behavior in birds, while egg discrimination is a more specific adaptation that has evolved to counter brood parasitism. These two behaviors are closely related with nest sanitation being the ancestral behavior, and it has been hypothesized to constitute a preadaptation for egg discrimination. However, previous studies found little evidence to support this hypothesis. Here, we conducted an empirical test of the association between nest sanitation and egg discrimination in the barn swallow (Hirundo rustica) by inserting a single non-mimetic model egg or a non-mimetic model egg plus half a peanut shell into host nests. Compared to the rejection rate of single model eggs, barn swallows significantly increased egg rejection frequency if a half peanut shell was simultaneously introduced. Our result for the first time shows the impact of nest sanitation on egg discrimination and demonstrates that nest sanitation can elicit egg discrimination in hosts of brood parasites. This study provided evidence for nest sanitation being a preadaptation to egg discrimination by facilitating egg rejection, thereby significantly advancing our understanding of avian cognition of foreign objects. Furthermore, we suggest that egg discrimination behavior in many accepters and intermediate rejecters may be lost or diluted. Such egg discrimination can be elicited and restored after nest sanitation, implying a sensitive and rapid phenotypic response to increased risk of parasitism. Our study offers a novel perspective for investigating the role of so-called intermediate rejecter individuals or species in the long-term coevolutionary cycle between brood parasites and their hosts.

  9. Outer membrane vesicles displaying engineered glycotopes elicit protective antibodies

    PubMed Central

    Valentine, Jenny L.; Huang, Chung-Jr; Endicott, Christine E.; Moeller, Tyler D.; Rasmussen, Jed A.; Fletcher, Joshua R.; Boll, Joseph M.; Rosenthal, Joseph A.; Dobruchowska, Justyna; Wang, Zhirui; Heiss, Christian; Azadi, Parastoo; Putnam, David; Trent, M. Stephen; Jones, Bradley D.; DeLisa, Matthew P.

    2016-01-01

    The O-antigen polysaccharide (O-PS) component of lipopolysaccharides on the surface of gram-negative bacteria is both a virulence factor and a B-cell antigen. Antibodies elicited by O-PS often confer protection against infection; therefore, O-PS glycoconjugate vaccines have proven useful against a number of different pathogenic bacteria. However, conventional methods for natural extraction or chemical synthesis of O-PS are technically demanding, inefficient, and expensive. Here, we describe an alternative methodology for producing glycoconjugate vaccines whereby recombinant O-PS biosynthesis is coordinated with vesiculation in laboratory strains of Escherichia coli to yield glycosylated outer membrane vesicles (glycOMVs) decorated with pathogen-mimetic glycotopes. Using this approach, glycOMVs corresponding to eight different pathogenic bacteria were generated. For example, expression of a 17-kb O-PS gene cluster from the highly virulent Francisella tularensis subsp. tularensis (type A) strain Schu S4 in hypervesiculating E. coli cells yielded glycOMVs that displayed F. tularensis O-PS. Immunization of BALB/c mice with glycOMVs elicited significant titers of O-PS–specific serum IgG antibodies as well as vaginal and bronchoalveolar IgA antibodies. Importantly, glycOMVs significantly prolonged survival upon subsequent challenge with F. tularensis Schu S4 and provided complete protection against challenge with two different F. tularensis subsp. holarctica (type B) live vaccine strains, thereby demonstrating the vaccine potential of glycOMVs. Given the ease with which recombinant glycotopes can be expressed on OMVs, the strategy described here could be readily adapted for developing vaccines against many other bacterial pathogens. PMID:27274048

  10. Knowledge elicitation techniques and application to nuclear plant maintenance

    NASA Astrophysics Data System (ADS)

    Doyle, E. Kevin

    The new millennium has brought with it the opportunity of global trade which in turn requires the utmost in efficiency from each individual industry. This includes the nuclear power industry, a point which was emphasized when the electrical generation industry began to be de regulated across North America the late 1990s and re-emphasized when the northeast power grid of North America collapsed in the summer of 2003. This dissertation deals with reducing the cost of the maintenance function of Candu nuclear power plants and initiating a strong link between universities and the Canadian nuclear industry. Various forms of RCM (reliability-centred maintenance) have been the tools of choice in industry for improving the maintenance function during the last 20 years. In this project, pilot studies, conducted at Bruce Power between 1999 and 2005, and reported on in this dissertation, lay out a path to implement statistical improvements as the next step after RCM in reducing the cost of the maintenance. Elicitation protocols, designed for the age group being elicited, address the much-documented issue of a lack of data. Clear, graphical, inferential statistical interfaces are accentuated and developed to aid in building the teams required to implement the various methodologies and to help in achieving funding targets. Graphical analysis and Crow/AMSAA (army materials systems analysis activity) plots are developed and demonstrated from the point of view of justifying the expenditures of cost reduction efforts. This dissertation ultimately speaks to the great opportunity being presented by this approach at this time: of capturing the baby-boom generation's huge pool of knowledge before those people retire. It is expected that the protocols and procedures referenced here will have applicability across the many disciplines where collecting expert information from a similar age group is required.

  11. Evaluating Residents' Readiness to Elicit Advance Care Plans

    PubMed Central

    Levy, Deborah; Strand, Jacob; McMahon, Graham T.

    2015-01-01

    Background Trainees are responsible for conducting advance care discussions but are often stressed by this role. Objective We developed an instrument to determine whether residents could identify a clinical scenario that necessitated an examination of a patient's goals and preferences as they pertain to clinical care, and subsequently measured their readiness to engage in such discussions. Methods Participants responded verbally to open-ended case presentations and completed survey items. We scored responses according to proximity to idealized answers. Results The sample consisted of 44 internal medicine residents, 12 students, 5 hospitalists, and 3 palliative care attendings, all of whom volunteered for the study and participated in standard interviews. Residents had widely varying scores (range 0–12, maximum score of 15) on the scored open response items. For eliciting values, mean score increased with training, and students, trainees, and attending physicians had mean scores of 3.7, 5.7, and 8.7, respectively (P = .01). For recommending care, mean scores were 3.0, 6.5, and 9.3, respectively (P < .001). Scores were correlated closely with increasing clinical experience and inversely with self-reported stress when conducting a goals-of-care discussion. The Kuder-Richardson Formula 20 reliability for the instrument was 0.52. Interrater reliability for sections about eliciting and recommending care were 0.64 (P < .001) and 0.50 (P < .001), respectively. The 1-week test-retest reliability was 0.91 for open response items and 0.76 for Likert responses. Conclusions A verbally administered instrument can readily and rapidly characterize a trainee's readiness to participate in advance care planning with patients. PMID:26457140

  12. Promoting environmental sustainability via an expert elicitation process

    SciTech Connect

    Swor, Tom; Canter, Larry

    2011-09-15

    Environmental sustainability (ES) planning was applied to the 981-mile, commercially navigable Ohio River. Navigation improvement needs were identified within the broad study along with actions to restore aquatic and riparian ecological resources to a higher state of sustainability. The actions were identified via an Expert Elicitation Process (EEP) involving aquatic and riparian/terrestrial experts knowledgeable of Ohio River resources. The received information was synthesized into goals for the selected resources (Valued Ecosystem Components - or VECs), actions or measures to attain the goals, and monitoring to evaluate conditions. Finally, 26 types of ES actions were identified and classified into three ES alternatives. These alternatives were then evaluated relative to key decision criteria, and such evaluations, based on pertinent decision criteria, were also conducted for four navigation improvement alternatives. Finally, the best combination of ES and navigation alternatives was identified. The key lessons derived from this use of EEP were that: (1) EEP can support the preliminary identification of ES measures; however, more detailed study of specific designs and cost evaluations will be necessary; (2) the method promotes collaboration between key scientists and policymakers from governmental agencies and private sectors, and such collaboration will ultimately provide the foundation for implementation of sustainability actions; and (3) an effective EEP does not occur by accident, it requires careful planning, implementation, and documentation. - Research Highlights: > Use of an Expert Elicitation Process (EEP) is demonstrated in this study. > EEP was used to identify Environmental Sustainability (ES) needs for the Ohio River. > EEP helped develop consensus among resource experts on ES needs. > EEP promotes collaboration to identify and contribute to common resource goals. > EEP may be used in assessing cumulative effects and formulating restoration plans.

  13. Insights into an original pocket-ligand pair classification: a promising tool for ligand profile prediction.

    PubMed

    Pérot, Stéphanie; Regad, Leslie; Reynès, Christelle; Spérandio, Olivier; Miteva, Maria A; Villoutreix, Bruno O; Camproux, Anne-Claude

    2013-01-01

    Pockets are today at the cornerstones of modern drug discovery projects and at the crossroad of several research fields, from structural biology to mathematical modeling. Being able to predict if a small molecule could bind to one or more protein targets or if a protein could bind to some given ligands is very useful for drug discovery endeavors, anticipation of binding to off- and anti-targets. To date, several studies explore such questions from chemogenomic approach to reverse docking methods. Most of these studies have been performed either from the viewpoint of ligands or targets. However it seems valuable to use information from both ligands and target binding pockets. Hence, we present a multivariate approach relating ligand properties with protein pocket properties from the analysis of known ligand-protein interactions. We explored and optimized the pocket-ligand pair space by combining pocket and ligand descriptors using Principal Component Analysis and developed a classification engine on this paired space, revealing five main clusters of pocket-ligand pairs sharing specific and similar structural or physico-chemical properties. These pocket-ligand pair clusters highlight correspondences between pocket and ligand topological and physico-chemical properties and capture relevant information with respect to protein-ligand interactions. Based on these pocket-ligand correspondences, a protocol of prediction of clusters sharing similarity in terms of recognition characteristics is developed for a given pocket-ligand complex and gives high performances. It is then extended to cluster prediction for a given pocket in order to acquire knowledge about its expected ligand profile or to cluster prediction for a given ligand in order to acquire knowledge about its expected pocket profile. This prediction approach shows promising results and could contribute to predict some ligand properties critical for binding to a given pocket, and conversely, some key pocket

  14. CB receptor ligands from plants.

    PubMed

    Woelkart, Karin; Salo-Ahen, Outi M H; Bauer, Rudolf

    2008-01-01

    Advances in understanding the physiology and pharmacology of the endogenous cannabinoid system have potentiated the interest of cannabinoid receptors as potential therapeutic targets. Cannabinoids have been shown to modulate a variety of immune cell functions and have therapeutic implications on central nervous system (CNS) inflammation, chronic inflammatory conditions such as arthritis, and may be therapeutically useful in treating autoimmune conditions such as multiple sclerosis. Many of these drug effects occur through cannabinoid receptor signalling mechanisms and the modulation of cytokines and other gene products. Further, endocannabinoids have been found to have many physiological and patho-physiological functions, including mood alteration and analgesia, control of energy balance, gut motility, motor and co-ordination activities, as well as alleviation of neurological, psychiatric and eating disorders. Plants offer a wide range of chemical diversity and have been a growing domain in the search for effective cannabinoid ligands. Cannabis sativa L. with the known plant cannabinoid, Delta(9-)tetrahydrocannabinol (THC) and Echinacea species with the cannabinoid (CB) receptor-binding lipophilic alkamides are the best known herbal cannabimimetics. This review focuses on the state of the art in CB ligands from plants, as well their possible therapeutic and immunomodulatory effects.

  15. Measurement of protein-ligand complex formation.

    PubMed

    Lowe, Peter N; Vaughan, Cara K; Daviter, Tina

    2013-01-01

    Experimental approaches to detect, measure, and quantify protein-ligand binding, along with their theoretical bases, are described. A range of methods for detection of protein-ligand interactions is summarized. Specific protocols are provided for a nonequilibrium procedure pull-down assay, for an equilibrium direct binding method and its modification into a competition-based measurement and for steady-state measurements based on the effects of ligands on enzyme catalysis.

  16. Asymmetric catalysis with chiral ferrocene ligands.

    PubMed

    Dai, Li-Xin; Tu, Tao; You, Shu-Li; Deng, Wei-Ping; Hou, Xue-Long

    2003-09-01

    Chiral ferrocene ligands have been widely used in asymmetric catalysis. The advantages of using ferrocene as a scaffold for chiral ligands are described, particularly those regarding planar chirality, rigid bulkiness, and ease of derivatization. The role of planar chirality in 1,2- and 1,1'-disubstituted ferrocene systems is discussed. By using a bulky ferrocene fragment, novel ferrocene ligands were designed, and high enantioselectivity and regioselectivity were achieved in the allylic substitution reaction of monosubstituted allyl substrates. Using the tunable electronic properties of a diphosphine-oxazoline ferrocenyl ligand, the regioselectivity of the intermolecular asymmetric Heck reaction was also examined.

  17. Integrated ligand based pharmacophore model derived from diverse FAAH covalent ligand classes.

    PubMed

    Shen, Lingling; Huang, Hongwei; Makriyannis, Alexandros; Fisher, Luke S

    2012-12-01

    3D pharmacophore modeling is an important computational methodology for ligand-enzyme binding interactions in drug discovery. More specifically, a consensus pharmacophore model derived from diverse ligands is a key determinant upon which the prediction power of computational models is based for designing novel ligands. In this work, by merging the important pharmacophore features based on four classes of covalent FAAH ligands, and then integrating the exclusion volume spheres derived from the crystal structure, we created for the first time an integrated FAAH pharmacophore model to describe the ligand-enzyme binding interactions. This new integrated FAAH pharmacophore model can correctly predict the covalent ligand binding mode, which correlates with the SAR data. The study is expected to provide insights into novel covalent ligand-FAAH binding interactions, and facilitate the design of covalent ligands against FAAH.

  18. Targeting Ligand-Dependent and Ligand-Independent Androgen Receptor Signaling in Prostate Cancer

    DTIC Science & Technology

    2013-10-01

    sub 10nM range efficacy. Our primary objective was to establish a series of compounds blocking the AR ligand-dependent and ligand-independent gene ...of AR driven genes to be more comprehensive and more in line with what is currently known about AR-driven signaling in prostate cancer. We have...developed a robust panel of genes for AR signaling that is reflective of the clinical findings in both ligand dependent and ligand-independent androgen

  19. Cis-interactions between Notch and its ligands block ligand-independent Notch activity

    PubMed Central

    Palmer, William Hunt; Jia, Dongyu; Deng, Wu-Min

    2014-01-01

    The Notch pathway is integrated into numerous developmental processes and therefore is fine-tuned on many levels, including receptor production, endocytosis, and degradation. Notch is further characterized by a twofold relationship with its Delta-Serrate (DSL) ligands, as ligands from opposing cells (trans-ligands) activate Notch, whereas ligands expressed in the same cell (cis-ligands) inhibit signaling. We show that cells without both cis- and trans-ligands can mediate Notch-dependent developmental events during Drosophila oogenesis, indicating ligand-independent Notch activity occurs when the receptor is free of cis- and trans-ligands. Furthermore, cis-ligands can reduce Notch activity in endogenous and genetically induced situations of elevated trans-ligand-independent Notch signaling. We conclude that cis-expressed ligands exert their repressive effect on Notch signaling in cases of trans-ligand-independent activation, and propose a new function of cis-inhibition which buffers cells against accidental Notch activity. DOI: http://dx.doi.org/10.7554/eLife.04415.001 PMID:25486593

  20. Proneurotrophin-3 is a neuronal apoptotic ligand: evidence for retrograde-directed cell killing

    PubMed Central

    Yano, Hiroko; Torkin, Risa; Martin, Laura Andrés; Chao, Moses V.; Teng, Kenneth K.

    2010-01-01

    While mature neurotrophins are well-described trophic factors that elicit retrograde survival signaling, the precursor forms of neurotrophins (i.e., proneurotrophins) can function as high affinity apoptotic ligands for selected neural populations. An outstanding question is whether target-derived proneurotrophins might affect neuronal survival/death decisions through a retrograde transport mechanism. Since neurotrophin-3 (NT-3) is highly expressed in non-neural tissues that receive peripheral innervation, we investigated the localized actions of its precursor (proNT-3) on sympathetic neurons in the present study. Pharmacological inhibition of intracellular furin proteinase activity in 293T cells resulted in proNT-3 release instead of mature NT-3 while membrane depolarization in cerebellar granule neurons stimulated endogenous proNT-3 secretion, suggesting that proNT-3 is an inducible bona fide ligand in the nervous system. Our data also indicate that recombinant proNT-3 induced sympathetic neuron death that is p75NTR- and sortilin-dependent, with hallmark features of apoptosis including JNK activation and nuclear fragmentation. Using compartmentalized culture systems that segregate neuronal cell bodies from axons, proNT-3, acting within the distal axon compartment, elicited sympathetic neuron death and overrode the survival promoting actions of NGF. Together these results raise the intriguing possibility that dysregulation of proneurotrophin processing/release by innervated targets can be deleterious to the neurons projecting to these sites. PMID:19940174

  1. Application and Evaluation of an Expert Judgment Elicitation Procedure for Correlations

    PubMed Central

    Zondervan-Zwijnenburg, Mariëlle; van de Schoot-Hubeek, Wenneke; Lek, Kimberley; Hoijtink, Herbert; van de Schoot, Rens

    2017-01-01

    The purpose of the current study was to apply and evaluate a procedure to elicit expert judgments about correlations, and to update this information with empirical data. The result is a face-to-face group elicitation procedure with as its central element a trial roulette question that elicits experts' judgments expressed as distributions. During the elicitation procedure, a concordance probability question was used to provide feedback to the experts on their judgments. We evaluated the elicitation procedure in terms of validity and reliability by means of an application with a small sample of experts. Validity means that the elicited distributions accurately represent the experts' judgments. Reliability concerns the consistency of the elicited judgments over time. Four behavioral scientists provided their judgments with respect to the correlation between cognitive potential and academic performance for two separate populations enrolled at a specific school in the Netherlands that provides special education to youth with severe behavioral problems: youth with autism spectrum disorder (ASD), and youth with diagnoses other than ASD. Measures of face-validity, feasibility, convergent validity, coherence, and intra-rater reliability showed promising results. Furthermore, the current study illustrates the use of the elicitation procedure and elicited distributions in a social science application. The elicited distributions were used as a prior for the correlation, and updated with data for both populations collected at the school of interest. The current study shows that the newly developed elicitation procedure combining the trial roulette method with the elicitation of correlations is a promising tool, and that the results of the procedure are useful as prior information in a Bayesian analysis. PMID:28197115

  2. Ligand binding by PDZ domains.

    PubMed

    Chi, Celestine N; Bach, Anders; Strømgaard, Kristian; Gianni, Stefano; Jemth, Per

    2012-01-01

    The postsynaptic density protein-95/disks large/zonula occludens-1 (PDZ) protein domain family is one of the most common protein-protein interaction modules in mammalian cells, with paralogs present in several hundred human proteins. PDZ domains are found in most cell types, but neuronal proteins, for example, are particularly rich in these domains. The general function of PDZ domains is to bring proteins together within the appropriate cellular compartment, thereby facilitating scaffolding, signaling, and trafficking events. The many functions of PDZ domains under normal physiological as well as pathological conditions have been reviewed recently. In this review, we focus on the molecular details of how PDZ domains bind their protein ligands and their potential as drug targets in this context.

  3. Cytofluorometric Quantification of Cell Death Elicited by NLR Proteins.

    PubMed

    Sica, Valentina; Manic, Gwenola; Kroemer, Guido; Vitale, Ilio; Galluzzi, Lorenzo

    2016-01-01

    Nucleotide-binding domain and leucine-rich repeat containing (NLR) proteins, also known as NOD-like receptors, are critical components of the molecular machinery that senses intracellular danger signals to initiate an innate immune response against invading pathogens or endogenous sources of hazard. The best characterized effect of NLR signaling is the secretion of various cytokines with immunostimulatory effects, including interleukin (IL)-1β and IL-18. Moreover, at least under specific circumstances, NLRs can promote regulated variants of cell death. Here, we detail two protocols for the cytofluorometric quantification of cell death-associated parameters that can be conveniently employed to assess the lethal activity of specific NLRs or their ligands.

  4. Ligand placement based on prior structures: the guided ligand-replacement method

    PubMed Central

    Klei, Herbert E.; Moriarty, Nigel W.; Echols, Nathaniel; Terwilliger, Thomas C.; Baldwin, Eric T.; Pokross, Matt; Posy, Shana; Adams, Paul D.

    2014-01-01

    The process of iterative structure-based drug design involves the X-ray crystal structure determination of upwards of 100 ligands with the same general scaffold (i.e. chemotype) complexed with very similar, if not identical, protein targets. In conjunction with insights from computational models and assays, this collection of crystal structures is analyzed to improve potency, to achieve better selectivity and to reduce liabilities such as absorption, distribution, metabolism, excretion and toxicology. Current methods for modeling ligands into electron-density maps typically do not utilize information on how similar ligands bound in related structures. Even if the electron density is of sufficient quality and resolution to allow de novo placement, the process can take considerable time as the size, complexity and torsional degrees of freedom of the ligands increase. A new module, Guided Ligand Replacement (GLR), was developed in Phenix to increase the ease and success rate of ligand placement when prior protein–ligand complexes are available. At the heart of GLR is an algorithm based on graph theory that associates atoms in the target ligand with analogous atoms in the reference ligand. Based on this correspondence, a set of coordinates is generated for the target ligand. GLR is especially useful in two situations: (i) modeling a series of large, flexible, complicated or macrocyclic ligands in successive structures and (ii) modeling ligands as part of a refinement pipeline that can automatically select a reference structure. Even in those cases for which no reference structure is available, if there are multiple copies of the bound ligand per asymmetric unit GLR offers an efficient way to complete the model after the first ligand has been placed. In all of these applications, GLR leverages prior knowledge from earlier structures to facilitate ligand placement in the current structure. PMID:24419386

  5. Flexible ligand docking using a genetic algorithm

    NASA Astrophysics Data System (ADS)

    Oshiro, C. M.; Kuntz, I. D.; Dixon, J. Scott

    1995-04-01

    Two computational techniques have been developed to explore the orientational and conformational space of a flexible ligand within an enzyme. Both methods use the Genetic Algorithm (GA) to generate conformationally flexible ligands in conjunction with algorithms from the DOCK suite of programs to characterize the receptor site. The methods are applied to three enzyme-ligand complexes: dihydrofolate reductase-methotrexate, thymidylate synthase-phenolpthalein and HIV protease-thioketal haloperidol. Conformations and orientations close to the crystallographically determined structures are obtained, as well as alternative structures with low energy. The potential for the GA method to screen a database of compounds is also examined. A collection of ligands is evaluated simultaneously, rather than docking the ligands individually into the enzyme.

  6. Biomimetic affinity ligands for protein purification.

    PubMed

    Sousa, Isabel T; Taipa, M Angela

    2014-01-01

    The development of sophisticated molecular modeling software and new bioinformatic tools, as well as the emergence of data banks containing detailed information about a huge number of proteins, enabled the de novo intelligent design of synthetic affinity ligands. Such synthetic compounds can be tailored to mimic natural biological recognition motifs or to interact with key surface-exposed residues on target proteins and are designated as "biomimetic ligands." A well-established methodology for generating biomimetic or synthetic affinity ligands integrates rational design with combinatorial solid-phase synthesis and screening, using the triazine scaffold and analogues of amino acids side chains to create molecular diversity.Triazine-based synthetic ligands are nontoxic, low-cost, highly stable compounds that can replace advantageously natural biological ligands in the purification of proteins by affinity-based methodologies.

  7. Micafungin Elicits an Immunomodulatory Effect in Galleria mellonella and Mice.

    PubMed

    Fuchs, Beth Burgwyn; Li, Yan; Li, Dedong; Johnston, Tatiana; Hendricks, Gabriel; Li, Gang; Rajamuthiah, Rajmohan; Mylonakis, Eleftherios

    2016-02-01

    The echinocandin family of drugs is well characterized for antifungal function that inhibits β-D-glucan synthesis. The aim of this work was to study whether micafungin, a member of the echinocandin family, elicits additional activities that prime the host's immune response. We found that in a Galleria mellonella model, prophylactic treatment with micafungin extended the life of Staphylococcus aureus-infected larvae (a pathogen to which the drug demonstrates no direct antimicrobial activity) compared to insects that did not receive micafungin (P < 0.05). The inhibition of pathogens in the G. mellonella infection model was characterized by a 2.43-fold increase in hemocyte density, compared to larvae inoculated with PBS. In a murine model where animals were provided micafungin prophylaxis 3 days prior to macrophage collection, macrophages were found associated with an average 0.9 more fungal cells per macrophage as compared to saline-treated animals. Interestingly, micafungin-stimulated macrophages killed 11.6 ± 6.2 % of fungal cells compared to 3.8 ± 2.4 % of macrophages from saline-treated animals. The prophylactic provision of micafungin prior to Candida albicans infection was characterized by an increase in the proinflammatory cytokines CXCL13 and SPP1 by 11- and 6.9-fold, respectively. In conclusion, micafungin demonstrated the ability to stimulate phagocytic cells and promote an immune response that can inhibit microbial infections.

  8. Dreamed movement elicits activation in the sensorimotor cortex.

    PubMed

    Dresler, Martin; Koch, Stefan P; Wehrle, Renate; Spoormaker, Victor I; Holsboer, Florian; Steiger, Axel; Sämann, Philipp G; Obrig, Hellmuth; Czisch, Michael

    2011-11-08

    Since the discovery of the close association between rapid eye movement (REM) sleep and dreaming, much effort has been devoted to link physiological signatures of REM sleep to the contents of associated dreams [1-4]. Due to the impossibility of experimentally controlling spontaneous dream activity, however, a direct demonstration of dream contents by neuroimaging methods is lacking. By combining brain imaging with polysomnography and exploiting the state of "lucid dreaming," we show here that a predefined motor task performed during dreaming elicits neuronal activation in the sensorimotor cortex. In lucid dreams, the subject is aware of the dreaming state and capable of performing predefined actions while all standard polysomnographic criteria of REM sleep are fulfilled [5, 6]. Using eye signals as temporal markers, neural activity measured by functional magnetic resonance imaging (fMRI) and near-infrared spectroscopy (NIRS) was related to dreamed hand movements during lucid REM sleep. Though preliminary, we provide first evidence that specific contents of REM-associated dreaming can be visualized by neuroimaging.

  9. Multiple Membrane Interactions and Versatile Vesicle Deformations Elicited by Melittin

    PubMed Central

    Takahashi, Tomoyoshi; Nomura, Fumimasa; Yokoyama, Yasunori; Tanaka-Takiguchi, Yohko; Homma, Michio; Takiguchi, Kingo

    2013-01-01

    Melittin induces various reactions in membranes and has been widely studied as a model for membrane-interacting peptide; however, the mechanism whereby melittin elicits its effects remains unclear. Here, we observed melittin-induced changes in individual giant liposomes using direct real-time imaging by dark-field optical microscopy, and the mechanisms involved were correlated with results obtained using circular dichroism, cosedimentation, fluorescence quenching of tryptophan residues, and electron microscopy. Depending on the concentration of negatively charged phospholipids in the membrane and the molecular ratio between lipid and melittin, melittin induced the “increasing membrane area”, “phased shrinkage”, or “solubilization” of liposomes. In phased shrinkage, liposomes formed small particles on their surface and rapidly decreased in size. Under conditions in which the increasing membrane area, phased shrinkage, or solubilization were mainly observed, the secondary structure of melittin was primarily estimated as an α-helix, β-like, or disordered structure, respectively. When the increasing membrane area or phased shrinkage occurred, almost all melittin was bound to the membranes and reached more hydrophobic regions of the membranes than when solubilization occurred. These results indicate that the various effects of melittin result from its ability to adopt various structures and membrane-binding states depending on the conditions. PMID:23594437

  10. Reading the lesson: eliciting requirements for a mammography training application

    NASA Astrophysics Data System (ADS)

    Hartswood, M.; Blot, L.; Taylor, P.; Anderson, S.; Procter, R.; Wilkinson, L.; Smart, L.

    2009-02-01

    Demonstrations of a prototype training tool were used to elicit requirements for an intelligent training system for screening mammography. The prototype allowed senior radiologists (mentors) to select cases from a distributed database of images to meet the specific training requirements of junior colleagues (trainees) and then provided automated feedback in response to trainees' attempts at interpretation. The tool was demonstrated to radiologists and radiographers working in the breast screening service at four evaluation sessions. Participants highlighted ease of selecting cases that can deliver specific learning objectives as important for delivering effective training. To usefully structure a large data set of training images we undertook a classification exercise of mentor authored free text 'learning points' attached to training case obtained from two screening centres (n=333, n=129 respectively). We were able to adduce a hierarchy of abstract categories representing classes of lesson that groups of cases were intended to convey (e.g. Temporal change, Misleading juxtapositions, Position of lesion, Typical/Atypical presentation, and so on). In this paper we present the method used to devise this classification, the classification scheme itself, initial user-feedback, and our plans to incorporated it into a software tool to aid case selection.

  11. Genuine eye contact elicits self-referential processing.

    PubMed

    Hietanen, Jonne O; Hietanen, Jari K

    2017-03-16

    The effect of eye contact on self-awareness was investigated with implicit measures based on the use of first-person singular pronouns in sentences. The measures were proposed to tap into self-referential processing, that is, information processing associated with self-awareness. In addition, participants filled in a questionnaire measuring explicit self-awareness. In Experiment 1, the stimulus was a video clip showing another person and, in Experiment 2, the stimulus was a live person. In both experiments, participants were divided into two groups and presented with the stimulus person either making eye contact or gazing downward, depending on the group assignment. During the task, the gaze stimulus was presented before each trial of the pronoun-selection task. Eye contact was found to increase the use of first-person pronouns, but only when participants were facing a real person, not when they were looking at a video of a person. No difference in self-reported self-awareness was found between the two gaze direction groups in either experiment. The results indicate that eye contact elicits self-referential processing, but the effect may be stronger, or possibly limited to, live interaction.

  12. PEGylated graphene oxide elicits strong immunological responses despite surface passivation

    PubMed Central

    Luo, Nana; Weber, Jeffrey K.; Wang, Shuang; Luan, Binquan; Yue, Hua; Xi, Xiaobo; Du, Jing; Yang, Zaixing; Wei, Wei; Zhou, Ruhong; Ma, Guanghui

    2017-01-01

    Engineered nanomaterials promise to transform medicine at the bio–nano interface. However, it is important to elucidate how synthetic nanomaterials interact with critical biological systems before such products can be safely utilized in humans. Past evidence suggests that polyethylene glycol-functionalized (PEGylated) nanomaterials are largely biocompatible and elicit less dramatic immune responses than their pristine counterparts. We here report results that contradict these findings. We find that PEGylated graphene oxide nanosheets (nGO-PEGs) stimulate potent cytokine responses in peritoneal macrophages, despite not being internalized. Atomistic molecular dynamics simulations support a mechanism by which nGO-PEGs preferentially adsorb onto and/or partially insert into cell membranes, thereby amplifying interactions with stimulatory surface receptors. Further experiments demonstrate that nGO-PEG indeed provokes cytokine secretion by enhancing integrin β8-related signalling pathways. The present results inform that surface passivation does not always prevent immunological reactions to 2D nanomaterials but also suggest applications for PEGylated nanomaterials wherein immune stimulation is desired. PMID:28233871

  13. Naturalistic Enactment to Elicit and Recognize Caregiver State Anxiety.

    PubMed

    Miranda, Darien; Favela, Jesus; Ibarra, Catalina; Cruz, Netzahualcoyotl

    2016-09-01

    Caring for people with dementia imposes significant stress on family members and caregivers. Often, these informal caregivers have no coping strategy to deal with these behaviors. Anxiety and stress episodes are often triggered by problematic behaviors exhibited by the person who suffers from dementia. Detecting these behaviors could help them in dealing with them and reduce caregiver burden. However, work on anxiety detection using physiological signals has mostly been done under controlled conditions. In this paper we describe an experiment aimed at inducing anxiety among caregivers of people with dementia under naturalistic conditions. We report an experiment, using the naturalistic enactment technique, in which 10 subjects were asked to care for an older adult who acts as if she experiences dementia. We record physiological signals from the participants (GSR, HR, EEG) during the sessions that lasted for approximately 30 min. We explain how we obtained ground truth from self-report and observation data. We conducted two different tests using the Support Vector Machine technique. We obtained an average precision of 77.8 % and 38.1 % recall when classifying two different possible states: "Anxious" and "Not anxious". Analysis of the data provides evidence that the experiment elicits state anxiety and that it can be detected using wearable sensors. Furthermore, if episodes of problematic behaviors can also be detected, the recognition of anxiety in the caregiver can be improved, leading to the enactment of appropriate interventions to help caregivers cope with anxiety episodes.

  14. Comparison of physiological responses to affect eliciting pictures and music.

    PubMed

    Kim, Jongwan; Wedell, Douglas H

    2016-03-01

    Recent investigations of the neural correlates of affect elicited from different modalities have found both modality-general and modality-specific representations (Chikazoe et al., 2014). The implications for how physiological responses to affect differ across stimulus modalities have not been fully investigated. This study examined similarities and differences between physiological signatures of affect derived from two different modes of presentation: visual pictures and auditory music sampled from an affective space defined by valence and arousal. Electromyography recordings for the zygomaticus major (EMGZ) and corrugator supercilii (EMGC) were measured along with heart rate and skin conductance level (SCL). Multidimensional scaling was used to visualize relationships from physiological and behavioral responses, and the observed relationships were statistically evaluated using multivariate and univariate analyses. Results for physiological measures demonstrated that valence was represented in the same general way across modalities, primarily reflected in EMGC responses. Arousal, however, was represented in a modality-specific manner, with SCL and EMGZ sensitive to music-based arousal but not picture-based arousal. Stimulus modality itself was predicted from EMGC. Thus, physiological responses to valence were similar across modalities but physiological responses to arousal differed across modalities. These results support the utility of testing for affective markers across modalities within the same experimental setting to reveal how physiological responses are linked to either affect, stimulus modality or both.

  15. PEGylated graphene oxide elicits strong immunological responses despite surface passivation

    NASA Astrophysics Data System (ADS)

    Luo, Nana; Weber, Jeffrey K.; Wang, Shuang; Luan, Binquan; Yue, Hua; Xi, Xiaobo; Du, Jing; Yang, Zaixing; Wei, Wei; Zhou, Ruhong; Ma, Guanghui

    2017-02-01

    Engineered nanomaterials promise to transform medicine at the bio-nano interface. However, it is important to elucidate how synthetic nanomaterials interact with critical biological systems before such products can be safely utilized in humans. Past evidence suggests that polyethylene glycol-functionalized (PEGylated) nanomaterials are largely biocompatible and elicit less dramatic immune responses than their pristine counterparts. We here report results that contradict these findings. We find that PEGylated graphene oxide nanosheets (nGO-PEGs) stimulate potent cytokine responses in peritoneal macrophages, despite not being internalized. Atomistic molecular dynamics simulations support a mechanism by which nGO-PEGs preferentially adsorb onto and/or partially insert into cell membranes, thereby amplifying interactions with stimulatory surface receptors. Further experiments demonstrate that nGO-PEG indeed provokes cytokine secretion by enhancing integrin β8-related signalling pathways. The present results inform that surface passivation does not always prevent immunological reactions to 2D nanomaterials but also suggest applications for PEGylated nanomaterials wherein immune stimulation is desired.

  16. Qualification of a free ligand assay in the presence of anti-ligand antibody Fab fragments.

    PubMed

    Hansen, Ryan J; Brown, Robin M; Lu, Jirong; Wroblewski, Victor J

    2013-01-01

    The aim of this work was to develop and characterize an ELISA to measure free ligand concentrations in rat serum in the presence of a Fab to the same ligand. A variety of experiments were conducted to understand optimal assay conditions and to verify that only free ligand was detected. The parameters explored included sample incubation time on plate, the initial concentrations of Fab and ligand, and the pre-incubation time required for the Fab-ligand complex concentrations to reach equilibrium. We found the optimal experimental conditions to include a 10-minute on-plate incubation of ligand-containing samples, with a 24-hour pre-incubation time for test samples of Fab and ligand to reach equilibrium. An alternative approach, involving removal of Fab-ligand complexes from the solution prior to measuring concentrations of the ligand, was also used to verify that the assay only measured free ligand. Rats were dosed subcutaneously with Fab and the assay was used to demonstrate dose-dependent suppression of endogenous free ligand levels in vivo.

  17. Naturalistic Observations of Elicited Expressive Communication of Children with Autism: An Analysis of Teacher Instructions

    ERIC Educational Resources Information Center

    Chiang, Hsu-Min

    2009-01-01

    This study observed expressive communication of 17 Australian and 15 Taiwanese children with autism who were mute or had limited spoken language during 2 hour regular school routines and analyzed teacher instructions associated with elicited expressive communication. Results indicated: (a) the frequency of occurrence of elicited expressive…

  18. Stone Soup: Photo-Elicitation as a Learning Tool in the Food Geography Classroom

    ERIC Educational Resources Information Center

    Kurtz, Hilda E.; Wood, Jason

    2014-01-01

    This paper showcases self-reflective and inclusive pedagogy using photo-elicitation in a food geography course assignment. The Stone Soup project positions students as both researchers and participant-subjects in a participant-driven photo-elicitation (PDPE) study of students' foodways. Student papers for this assignment demonstrate rich…

  19. Model-Eliciting Activities (MEAs) as a Bridge between Engineering Education Research and Mathematics Education Research

    ERIC Educational Resources Information Center

    Hamilton, Eric; Lesh, Richard; Lester, Frank; Brilleslyper, Michael

    2008-01-01

    This article introduces Model-Eliciting Activities (MEAs) as a form of case study team problem-solving. MEA design focuses on eliciting from students conceptual models that they iteratively revise in problem-solving. Though developed by mathematics education researchers to study the evolution of mathematical problem-solving expertise in middle…

  20. Translation Elicitation Techniques and Mother-Tongue Interference: Any Significant Connection?

    ERIC Educational Resources Information Center

    Mattar, Hameed

    1999-01-01

    Examined the validity of Dulay, Burt, and Krashen's hypothesis that the use of translation as an elicitation technique in foreign or second language (L2) research artificially increases L2 learners' reliance on the mother tongue, and accordingly, the proportion of interference errors. Examined interference errors on two elicitation tasks: an…

  1. Uncovering Pre-Service Teacher Beliefs about Young Children: A Photographic Elicitation Methodology

    ERIC Educational Resources Information Center

    Stockall, Nancy; Davis, Sara

    2011-01-01

    This illustrative paper provides an introduction to using mixed qualitative methods of photo-elicitation, face to face interviews and semiotic analysis to uncover pre-service students' beliefs about young children. The researchers share their experience on conducting a study using photo-elicitation and engaging pre-service teachers in a discussion…

  2. Effects of Race and Dialect of Examiner on Language Samples Elicited from Southern African American Preschoolers.

    ERIC Educational Resources Information Center

    Agerton, Emily P.; Moran, Michael J.

    1995-01-01

    Language samples were elicited from 17 African American preschoolers by 3 examiners; a white female using standard English and 2 African American females using either standard English or black English. Samples elicited by the African American examiners contained more different Black English features, with examiner usage of Black English eliciting…

  3. Eliciting Covert Mental Operations, Concepts and Oral Language Skills in Young Bilingual Children.

    ERIC Educational Resources Information Center

    Hollomon, John W.; And Others

    The purpose of this investigation was to design and test an information-eliciting question instrument in order to determine whether the structures in the verbal responses of young Mexican-American, bilingual children entering school would reveal the covert mental operations, concepts and oral language skills elicited. The basic objective was to…

  4. Identifying and Responding to Personal Stressors: Utilizing Photo Elicitation in Health Education Courses

    ERIC Educational Resources Information Center

    Hunter, Theresa

    2014-01-01

    The "Photo Elicitation Project" teaching idea applies the techniques of photo elicitation to stress prevention and management. This activity is designed to help students identify their personal stressors and to determine which stress prevention strategies are most useful for them. Objectives: students will be able to (a) identify current…

  5. Numerical Algorithms for the Analysis of Expert Opinions Elicited in Text Format

    DTIC Science & Technology

    2013-04-01

    applied to subject matter expert text data elicited through carefully constructed decision support workshops. In the main these workshops address...various stake-holder groups. These workshops are carefully designed to address specific defence questions and to elicit, record and analyse expert...each of these topics. • Differential Analysis: Differential analysis concerns identifying and quantifying the differences between subsets of text, where

  6. Photo-Elicitation and Visual Semiotics: A Unique Methodology for Studying Inclusion for Children with Disabilities

    ERIC Educational Resources Information Center

    Stockall, Nancy

    2013-01-01

    The methodology in this paper discusses the use of photographs as an elicitation strategy that can reveal the thinking processes of participants in a qualitatively rich manner. Photo-elicitation techniques combined with a Piercian semiotic perspective offer a unique method for creating a frame of action for later participant analysis. Illustrative…

  7. Concurrent Data Elicitation Procedures, Processes, and the Early Stages of L2 Learning: A Critical Overview

    ERIC Educational Resources Information Center

    Leow, Ronald P.; Grey, Sarah; Marijuan, Silvia; Moorman, Colleen

    2014-01-01

    Given the current methodological interest in eliciting direct data on the cognitive processes L2 learners employ as they interact with L2 data during the early stages of the learning process, this article takes a critical and comparative look at three concurrent data elicitation procedures currently employed in the SLA literature: Think aloud (TA)…

  8. Chemistry of Marine Ligands and Siderophores

    NASA Astrophysics Data System (ADS)

    Vraspir, Julia M.; Butler, Alison

    2009-01-01

    Marine microorganisms are presented with unique challenges to obtain essential metal ions required to survive and thrive in the ocean. The production of organic ligands to complex transition metal ions is one strategy to both facilitate uptake of specific metals, such as iron, and to mitigate the potential toxic effects of other metal ions, such as copper. A number of important trace metal ions are complexed by organic ligands in seawater, including iron, cobalt, nickel, copper, zinc, and cadmium, thus defining the speciation of these metal ions in the ocean. In the case of iron, siderophores have been identified and structurally characterized. Siderophores are low molecular weight iron-binding ligands produced by marine bacteria. Although progress has been made toward the identity of in situ iron-binding ligands, few compounds have been identified that coordinate the other trace metals. Deciphering the chemical structures and production stimuli of naturally produced organic ligands and the organisms they come from is fundamental to understanding metal speciation and bioavailability. The current evidence for marine ligands, with an emphasis on siderophores, and discussion of the importance and implications of metal-binding ligands in controlling metal speciation and cycling within the world's oceans are presented.

  9. Characteristics of Late Negative ERG Responses Elicited By Sawtooth Flicker

    PubMed Central

    Gowrisankaran, Sowjanya; Genead, Mohamed A.; Anastasakis, Anastasios; Alexander, Kenneth R.

    2012-01-01

    Background This study aimed to determine whether the properties of the late negative responses (LNRs) of the electroretinogram (ERG) elicited by sawtooth flicker are consistent with the characteristics of the photopic negative response generated by a light pulse (PhNRpulse). Methods ERG recordings were obtained from 10 visually normal individuals and from 6 patients with optic atrophy (OA) in response to 8-Hz rapid-on and rapid-off sawtooth flicker and to brief (4 ms) light pulses. All stimuli were either long-wavelength (R), middle-wavelength (G), or a combination of equal luminances of long and middle-wavelengths (Y) presented on a short-wavelength, rod-saturating adapting field. Amplitudes of LNRs were obtained in response to rapid-on (LNRon) and rapid-off (LNRoff) sawtooth flicker, and were also derived from the sum of the ERG waveforms to the two sawtooth phases (LNRadd). Results For the control subjects, PhNRpulse amplitude varied with stimulus wavelength, being largest in response to a long-wavelength pulse, as expected. However, the amplitudes of LNRon, LNRoff, and LNRadd were not significantly different for R, Y, and G sawtooth flicker. Despite the absence of a chromatic effect, LNRoff and LNRadd amplitudes were significantly smaller in the OA patients than in the controls, similar to the results for the PhNRpulse, implying an inner retinal origin for the LNRoff and LNRadd. However, LNRon amplitudes did not differ significantly between the OA patients and controls, although there was a significant correlation between the LNRon and PhNRpulse for R stimuli. Conclusion We conclude that LNRoff and LNRadd but not LNRon can be useful measures to assess the integrity of the inner retina that can complement the PhNRpulse. PMID:23054159

  10. Role of phosphodiesterase-4 on ethanol elicited locomotion and narcosis.

    PubMed

    Baliño, Pablo; Ledesma, Juan Carlos; Aragon, Carlos M G

    2016-02-01

    The cAMP signaling pathway has emerged as an important modulator of the pharmacological effects of ethanol. In this respect, the cAMP-dependent protein kinase has been shown to play an important role in the modulation of several ethanol-induced behavioral actions. Cellular levels of cAMP are maintained by the activity of adenylyl cyclases and phosphodiesterases. In the present work we have focused on ascertaining the role of PDE4 in mediating the neurobehavioral effects of ethanol. For this purpose, we have used the selective PDE4 inhibitor Ro 20-1724. This compound has been proven to enhance cellular cAMP response by PDE4 blockade and can be administered systemically. Swiss mice were injected intraperitoneally (i.p.) with Ro 20-1724 (0-5 mg/kg; i.p.) at different time intervals before ethanol (0-4 g/kg; i.p.) administration. Immediately after the ethanol injection, locomotor activity, loss of righting reflex, PKA footprint and enzymatic activity were assessed. Pretreatment with Ro 20-1724 increased ethanol-induced locomotor stimulation in a dose-dependent manner. Doses that increased locomotor stimulation did not modify basal locomotion or the suppression of motor activity produced by high doses of this alcohol. Ro 20-1724 did not alter the locomotor activation produced by amphetamine or cocaine. The time of loss of righting reflex evoked by ethanol was increased after pretreatment with Ro 20-1724. This effect was selective for the narcotic effects of ethanol since Ro 20-1724 did not affect pentobarbital-induced narcotic effects. Moreover, Ro 20-1724 administration increased the PKA footprint and enzymatic activity response elicited by ethanol. These data provide further evidence of the key role of the cAMP signaling pathway in the central effects of ethanol.

  11. Feeding-elicited cataplexy in orexin knockout mice.

    PubMed

    Clark, E L; Baumann, C R; Cano, G; Scammell, T E; Mochizuki, T

    2009-07-21

    Mice lacking orexin/hypocretin signaling have sudden episodes of atonia and paralysis during active wakefulness. These events strongly resemble cataplexy, episodes of sudden muscle weakness triggered by strong positive emotions in people with narcolepsy, but it remains unknown whether murine cataplexy is triggered by positive emotions. To determine whether positive emotions elicit murine cataplexy, we placed orexin knockout (KO) mice on a scheduled feeding protocol with regular or highly palatable food. Baseline sleep/wake behavior was recorded with ad libitum regular chow. Mice were then placed on a scheduled feeding protocol in which they received 60% of their normal amount of chow 3 h after dark onset for the next 10 days. Wild-type and KO mice rapidly entrained to scheduled feeding with regular chow, with more wake and locomotor activity prior to the feeding time. On day 10 of scheduled feeding, orexin KO mice had slightly more cataplexy during the food-anticipation period and more cataplexy in the second half of the dark period, when they may have been foraging for residual food. To test whether more palatable food increases cataplexy, mice were then switched to scheduled feeding with an isocaloric amount of Froot Loops, a food often used as a reward in behavioral studies. With this highly palatable food, orexin KO mice had much more cataplexy during the food-anticipation period and throughout the dark period. The increase in cataplexy with scheduled feeding, especially with highly palatable food, suggests that positive emotions may trigger cataplexy in mice, just as in people with narcolepsy. Establishing this connection helps validate orexin KO mice as an excellent model of human narcolepsy and provides an opportunity to better understand the mechanisms that trigger cataplexy.

  12. Functional Changes in the Snail Statocyst System Elicited by Microgravity

    PubMed Central

    Balaban, Pavel M.; Malyshev, Aleksey Y.; Ierusalimsky, Victor N.; Aseyev, Nikolay; Korshunova, Tania A.; Bravarenko, Natasha I.; Lemak, M. S.; Roshchin, Matvey; Zakharov, Igor S.; Popova, Yekaterina; Boyle, Richard

    2011-01-01

    Background The mollusk statocyst is a mechanosensing organ detecting the animal's orientation with respect to gravity. This system has clear similarities to its vertebrate counterparts: a weight-lending mass, an epithelial layer containing small supporting cells and the large sensory hair cells, and an output eliciting compensatory body reflexes to perturbations. Methodology/Principal Findings In terrestrial gastropod snail we studied the impact of 16- (Foton M-2) and 12-day (Foton M-3) exposure to microgravity in unmanned orbital missions on: (i) the whole animal behavior (Helix lucorum L.), (ii) the statoreceptor responses to tilt in an isolated neural preparation (Helix lucorum L.), and (iii) the differential expression of the Helix pedal peptide (HPep) and the tetrapeptide FMRFamide genes in neural structures (Helix aspersa L.). Experiments were performed 13–42 hours after return to Earth. Latency of body re-orientation to sudden 90° head-down pitch was significantly reduced in postflight snails indicating an enhanced negative gravitaxis response. Statoreceptor responses to tilt in postflight snails were independent of motion direction, in contrast to a directional preference observed in control animals. Positive relation between tilt velocity and firing rate was observed in both control and postflight snails, but the response magnitude was significantly larger in postflight snails indicating an enhanced sensitivity to acceleration. A significant increase in mRNA expression of the gene encoding HPep, a peptide linked to ciliary beating, in statoreceptors was observed in postflight snails; no differential expression of the gene encoding FMRFamide, a possible neurotransmission modulator, was observed. Conclusions/Significance Upregulation of statocyst function in snails following microgravity exposure parallels that observed in vertebrates suggesting fundamental principles underlie gravi-sensing and the organism's ability to adapt to gravity changes. This

  13. Metaphors of Distress: Photo-Elicitation Enhances a Discourse Analysis of Parents' Accounts.

    PubMed

    Kantrowitz-Gordon, Ira; Vandermause, Roxanne

    2016-07-01

    In research on sensitive topics, photo-elicitation can be a profound aid to data collection and interpretation processes. Photo-elicitation methods were used in this manner in a discourse analysis of parents' distress at least 6 months after preterm birth. After an initial interview, participants were asked to take digital photographs representing their distress and to return for a second interview to discuss the photographs. The elicited photo representations supported participants' engagement with their current or past distress and generated new meanings from the reappraisal of old photographs. Photo-elicitation demonstrated the embodiment of parents' distress in the child and the placement of distress in specific locations. Photographs of documents showed the power of the written word in generating and maintaining distress. Participants used existing photographs from their child's photo history to generate rich metaphors for their distress as parents. These findings have implications for enhancing interpretive health research by incorporating photo-elicitation methods.

  14. Comparison of elicitation methods for moral and affective beliefs in the theory of planned behaviour.

    PubMed

    Dean, M; Arvola, A; Vassallo, M; Lähteenmäki, L; Raats, M M; Saba, A; Shepherd, R

    2006-09-01

    Although the theory of planned behaviour (TPB) has been applied successfully in the area of food choice, it has been criticized for its pure utilitarian approach to the factors determining behaviour. Despite the increase in predictive power of the model with added components such as affective attitude and moral and ethical concerns, in most studies the elicitation process still only addresses people's utilitarian beliefs about the behaviour with little attention paid to other aspects. This study compares the traditional method of elicitation of advantages and disadvantages with two other methods (word association and open-ended) in the elicitations of beliefs, attitudes and moral concerns in relation to the consumption of organic foods. Results show the traditional method to be best for eliciting cognitive beliefs, open-ended emotion task for eliciting emotional beliefs and open-ended beliefs task best for moral concerns. The advantages and disadvantages of each method are discussed.

  15. Notch Ligand DLL4 Alleviates Allergic Airway Inflammation via Induction of a Homeostatic Regulatory Pathway

    PubMed Central

    Huang, Miao-Tzu; Chen, Yi-Lien; Lien, Chia-I; Liu, Wei-Liang; Hsu, Li-Chung; Yagita, Hideo; Chiang, Bor-Luen

    2017-01-01

    Notch is a pleiotropic signaling family that has been implicated in pathogenesis of allergic airway diseases; however, the distinct function of individual Notch ligands remains elusive. We investigated whether Notch ligands, Jagged1 and DLL4, exert differential effects in OVA-induced allergic asthma. We found that whilst Jagged1 inhibition mitigated Th2-dominated airway inflammation, blockage of DLL4 aggravated the Th2-mediated asthma phenotypes. Additionally, Jagged1 signaling blockage enhanced IL-17 production and neutrophilic airway infiltration. In vitro, exogenous Jagged1 induced Th2-skewed responses, whereas augmented DLL4 signaling displayed a dual role by promoting expansion of both Tregs and Th17. In vivo, DLL4 blockage impaired Treg differentiation which plausibly resulted in exaggerated asthma phenotypes. On the contrary, administration of DLL4-expressing antigen-presenting cells promoted endogenous Treg expansion and ameliorated the allergic responses. Therefore, whilst Jagged1 induces Th2-skewed inflammation, DLL4 elicits an essential self-regulatory mechanism via Treg-mediated pathway that counterbalances Jagged1-induced Th2 responses and facilitates resolution of the airway inflammation to restore homeostasis. These findings uncover a disparate function of Jagged1 and DLL4 in allergic airway diseases, hinting feasibility of Notch ligand-specific targeting in therapy of allergic airway diseases. PMID:28262821

  16. ProPose: steered virtual screening by simultaneous protein-ligand docking and ligand-ligand alignment.

    PubMed

    Seifert, Markus H J

    2005-01-01

    The 'model-free' screening engine ProPose implements a general method for performing simultaneous protein-ligand docking, ligand-ligand alignment, pharmacophore queries-and combinations thereof-in order to incorporate a priori information into screening protocols. In this manuscript we describe a case study on herpes simplex virus thymidine kinase, an important antiviral drug target, where we evaluate different approaches for handling a specific type of a priori information, i.e., multiple target structures. We demonstrate that a simultaneous alignment on two target structures--in conjunction with logic operations on interactions and docking constraints derived from protein structure--is an effective means of (i) improving the enrichment of chemical substructures that are compatible with the a priori known ligands, (ii) ensuring the steric fit into the target protein, and (iii) handling target flexibility. The combination of ligand- and receptor-based methods steers the virtual screening by ranking molecules according to the similarity of their interaction pattern with known ligands, thereby--to some extent--outweighing the deficiencies of simple scoring functions often used in initial virtual screening.

  17. Prostate cancer stem cells are targets of both innate and adaptive immunity and elicit tumor-specific immune responses

    PubMed Central

    Jachetti, Elena; Mazzoleni, Stefania; Grioni, Matteo; Ricupito, Alessia; Brambillasca, Chiara; Generoso, Luca; Calcinotto, Arianna; Freschi, Massimo; Mondino, Anna; Galli, Rossella; Bellone, Matteo

    2013-01-01

    According to the cancer stem cell (CSC) theory, therapies that do not target the CSC compartment have limited, if any, chances to eradicate established tumors. While cytotoxic T lymphocytes (CTLs) have the potential to recognize and kill single neoplastic cells within a tissue, whether CSCs can be targeted by the immune system during spontaneous or vaccination-elicited responses is poorly defined. Here, we provide experimental evidence showing that CSC lines established from the prostate of transgenic adenocarcinoma of the mouse prostate (TRAMP) mice expressed prostate cancer-associated antigens, MHC Class I and II molecules as well as ligands for natural killer (NK) cell receptors. Indeed, CSC were targets for both NK cell- and CTL-mediated cytotoxicity, both in vitro and in vivo. The administration of dendritic cells pulsed with irradiated CSCs induced a tumor-specific immune response that was more robust than that induced by dendritic cells pulsed with differentiated tumor cells, delayed tumor growth in mice challenged with prostate CSCs and caused tumor regression in TRAMP mice. Thus, CSC are targeted by both innate and adaptive immune responses and might be exploited for the design of novel immunotherapeutic approaches against cancer. PMID:23762811

  18. Preclinical evidence that PD-1 blockade cooperates with cancer vaccine TEGVAX to elicit regression of established tumors

    PubMed Central

    Fu, Juan; Malm, Ian-James; Kadayakkara, Deepak K.; Levitsky, Hy; Pardoll, Drew; Kim, Young J.

    2015-01-01

    Biomarker studies have shown that expression of the T cell co-regulatory ligand PD-L1 on tumor cells correlates with clinical responsiveness to the PD-1 antibody nivolumab. Here we report the findings of a preclinical cancer vaccine study demonstrating a similiar correlate where PD-L1 is upregulated in the tumor microenvironment. We formulated an IFNγ-inducing cancer vaccine called TEGVAX that combined GM-CSF and multiple toll-like receptor agonists to increase the number of activated dendritic cells. Treatment of established tumors with TEGVAX retarded tumor growth in a manner associated with enhanced systemic anti-tumor immunity. Unexpectedly, TEGVAX also upregulated PD-L1 expression in the tumor microenvironment, possibly explaining why tumors were not eliminated completely. In support of this likelihood, PD-L1 upregulation in this setting relied upon IFNγ-expressing tumor-infiltrating CD4+ and CD8+ T cells and administration of a PD-1 blocking antibody with TEGVAX elicited complete regression of established tumors. Taken together, our findings provide a mechanistic rationale to combine IFNγ inducing cancer vaccines with immune checkpoint blockade. PMID:24812273

  19. Preclinical evidence that PD1 blockade cooperates with cancer vaccine TEGVAX to elicit regression of established tumors.

    PubMed

    Fu, Juan; Malm, Ian-James; Kadayakkara, Deepak K; Levitsky, Hy; Pardoll, Drew; Kim, Young J

    2014-08-01

    Biomarker studies have shown that expression of the T-cell coregulatory ligand PDL1 on tumor cells correlates with clinical responsiveness to the PD1 antibody nivolumab. Here, we report the findings of a preclinical cancer vaccine study demonstrating vaccine-dependent PDL1 upregulation in the tumor microenvironment. We formulated an IFNγ-inducing cancer vaccine called TEGVAX that combined GM-CSF and multiple Toll-like receptor agonists to increase the number of activated dendritic cells. Treatment of established tumors with TEGVAX retarded tumor growth in a manner associated with enhanced systemic antitumor immunity. Unexpectedly, TEGVAX also upregulated PDL1 expression in the tumor microenvironment, possibly explaining why tumors were not eliminated completely. In support of this likelihood, PDL1 upregulation in this setting relied upon IFNγ-expressing tumor-infiltrating CD4(+) and CD8(+) T cells and administration of a PD1-blocking antibody with TEGVAX elicited complete regression of established tumors. Taken together, our findings provide a mechanistic rationale to combine IFNγ-inducing cancer vaccines with immune checkpoint blockade.

  20. Minocycline inhibits D-amphetamine-elicited action potential bursts in a central snail neuron.

    PubMed

    Chen, Y-H; Lin, P-L; Wong, R-W; Wu, Y-T; Hsu, H-Y; Tsai, M-C; Lin, M-J; Hsu, Y-C; Lin, C-H

    2012-10-25

    Minocycline is a second-generation tetracycline that has been reported to have powerful neuroprotective properties. In our previous studies, we found that d-amphetamine (AMPH) elicited action potential bursts in an identifiable RP4 neuron of the African snail, Achatina fulica Ferussac. This study sought to determine the effects of minocycline on the AMPH-elicited action potential pattern changes in the central snail neuron, using the two-electrode voltage clamping method. Extracellular application of AMPH at 300 μM elicited action potential bursts in the RP4 neuron. Minocycline dose-dependently (300-900 μM) inhibited the action potential bursts elicited by AMPH. The inhibitory effects of minocycline on AMPH-elicited action potential bursts were restored by forskolin (50 μM), an adenylate cyclase activator, and by dibutyryl cAMP (N(6),2'-O-Dibutyryladenosine 3',5'-cyclic monophosphate; 1mM), a membrane-permeable cAMP analog. Co-administration of forskolin (50 μM) plus tetraethylammonium chloride (TEA; 5mM) or co-administration of TEA (5mM) plus dibutyryl cAMP (1mM) also elicited action potential bursts, which were prevented and inhibited by minocycline. In addition, minocycline prevented and inhibited forskolin (100 μM)-elicited action potential bursts. Notably, TEA (50mM)-elicited action potential bursts in the RP4 neuron were not affected by minocycline. Minocycline did not affect steady-state outward currents of the RP4 neuron. However, minocycline did decrease the AMPH-elicited steady-state current changes. Similarly, minocycline decreased the effects of forskolin-elicited steady-state current changes. Pretreatment with H89 (N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; 10 μM), a protein kinase A inhibitor, inhibited AMPH-elicited action potential bursts and decreased AMPH-elicited steady-state current changes. These results suggest that the cAMP-protein kinase A signaling pathway and the steady-state current are involved in

  1. Ligand engineering of nanoparticle solar cells

    NASA Astrophysics Data System (ADS)

    Voros, Marton

    Semiconductor nanoparticles (NP) are promising materials to build cheap and efficient solar cells. One of the key challenges in their utilization for solar energy conversion is the control of NP surfaces and ligand-NP interfaces. Recent experiments have shown that by carefully choosing the ligands terminating the NPs, one can tailor electronic and optical absorption properties of NP assemblies, along with their transport properties. By using density functional theory based methods, we investigated how the opto-electronic properties of lead chalcogenide NPs may be tuned by using diverse organic and inorganic ligands. We interpreted experiments, and we showed that an essential prerequisite to avoid detrimental trap states is to ensure charge balance at the ligand-NP interface, possibly with the help of hydrogen treatment Work supported by the Center for Advanced Solar Photophysics, an Energy Frontier Research Center funded by the US Department of Energy, Office of Science, Office of Basic Energy Sciences.

  2. Automated design of ligands to polypharmacological profiles

    PubMed Central

    Besnard, Jérémy; Ruda, Gian Filippo; Setola, Vincent; Abecassis, Keren; Rodriguiz, Ramona M.; Huang, Xi-Ping; Norval, Suzanne; Sassano, Maria F.; Shin, Antony I.; Webster, Lauren A.; Simeons, Frederick R.C.; Stojanovski, Laste; Prat, Annik; Seidah, Nabil G.; Constam, Daniel B.; Bickerton, G. Richard; Read, Kevin D.; Wetsel, William C.; Gilbert, Ian H.; Roth, Bryan L.; Hopkins, Andrew L.

    2012-01-01

    The clinical efficacy and safety of a drug is determined by its activity profile across multiple proteins in the proteome. However, designing drugs with a specific multi-target profile is both complex and difficult. Therefore methods to rationally design drugs a priori against profiles of multiple proteins would have immense value in drug discovery. We describe a new approach for the automated design of ligands against profiles of multiple drug targets. The method is demonstrated by the evolution of an approved acetylcholinesterase inhibitor drug into brain penetrable ligands with either specific polypharmacology or exquisite selectivity profiles for G-protein coupled receptors. Overall, 800 ligand-target predictions of prospectively designed ligands were tested experimentally, of which 75% were confirmed correct. We also demonstrate target engagement in vivo. The approach can be a useful source of drug leads where multi-target profiles are required to achieve either selectivity over other drug targets or a desired polypharmacology. PMID:23235874

  3. Ligand inducible assembly of a DNA tetrahedron.

    PubMed

    Dohno, Chikara; Atsumi, Hiroshi; Nakatani, Kazuhiko

    2011-03-28

    Here we show that a small synthetic ligand can be used as a key building component for DNA nanofabrication. Using naphthyridinecarbamate dimer (NCD) as a molecular glue for DNA hybridization, we demonstrate NCD-triggered formation of a DNA tetrahedron.

  4. Advanced Organic Ligands for Protecting Metal Nanoparticles

    NASA Astrophysics Data System (ADS)

    Yu, Jonathan Ka-Wing

    Organic monolayer protected metal nanoparticles have been utilized in many different fields such as catalysis, drug delivery, and sensor chemistry. However, these nanomaterials are prone to increase in size consequently losing its function at the nanoscale. The stability these nanoparticles have been a great interest of research. This thesis focuses on the synthesis of a novel cross-linkable ligand for the protection of metal nanoparticles. Chapter 1 reviews key concepts of nanoparticles, its usefulness in applications, some of the stabilizing strategies employed, and the scope of the thesis project. Chapter 2 describes the synthetic attempts and optimization of the novel cross-linkable ligand. In addition, its characterization data is also included. Section 2.8 also highlights another fully synthesized novel hydrophobic ligand. Chapter 3 contains the summary of the work and closing remarks. Future works is also included to describe the prospects of the synthesis of the novel ligand. Chapter 4 entails the experimental data and supplementary information.

  5. GW-501516 GlaxoSmithKline/Ligand.

    PubMed

    Pelton, Patricia

    2006-04-01

    GlaxoSmithKline and Ligand are developing GW-501516, a peroxisome proliferator-activator receptor-delta agonist for the potential treatment of dyslipidemia. Phase II clinical trials of this compound are ongoing.

  6. The Retinoid X Receptors and Their Ligands

    PubMed Central

    Dawson, Marcia I.; Xia, Zebin

    2014-01-01

    This chapter presents an overview of the current status of studies on the structural and molecular biology of the retinoid X receptor subtypes α, β, and γ (RXRs, NR2B1–3), their nuclear and cytoplasmic functions, post-transcriptional processing, and recently reported ligands. Points of interest are the different changes in the ligand-binding pocket induced by variously shaped agonists, the communication of the ligand–bound pocket with the coactivator binding surface and the heterodimerization interface, and recently identified ligands that are natural products, those that function as environmental toxins or drugs that had been originally designed to interact with other targets, as well as those that were deliberately designed as RXR-selective transcriptional agonists, synergists, or antagonists. Of these synthetic ligands, the general trend in design appears to be away from fully aromatic rigid structures to those containing partial elements of the flexible tetraene side chain of 9-cis-retinoic acid. PMID:22020178

  7. αvβ3 Integrin Boosts the Innate Immune Response Elicited in Epithelial Cells through Plasma Membrane and Endosomal Toll-Like Receptors

    PubMed Central

    Casiraghi, Costanza; Gianni, Tatiana

    2016-01-01

    We report that αvβ3 integrin strongly affects the innate immune response in epithelial cells. αvβ3 integrin greatly increased the response elicited via plasma membrane Toll-like receptors (TLRs) by herpes simplex virus or bacterial ligands. The endosomal TLR3, not the cytosolic sensor interferon gamma-inducible protein 16 (IFI16), was also boosted by αvβ3 integrin. The boosting was exerted specifically by αvβ3 integrin but not by αvβ6 or αvβ8 integrin. Current and previous work indicates that integrin-TLR cooperation occurs in epithelial and monocytic cells. The TLR response should be considered an integrin-TLR response. PMID:26842473

  8. Ex vivo genome-wide RNAi screening of the Drosophila Toll signaling pathway elicited by a larva-derived tissue extract.

    PubMed

    Kanoh, Hirotaka; Kuraishi, Takayuki; Tong, Li-Li; Watanabe, Ryo; Nagata, Shinji; Kurata, Shoichiro

    2015-11-13

    Damage-associated molecular patterns (DAMPs), so-called "danger signals," play important roles in host defense and pathophysiology in mammals and insects. In Drosophila, the Toll pathway confers damage responses during bacterial infection and improper cell-fate control. However, the intrinsic ligands and signaling mechanisms that potentiate innate immune responses remain unknown. Here, we demonstrate that a Drosophila larva-derived tissue extract strongly elicits Toll pathway activation via the Toll receptor. Using this extract, we performed ex vivo genome-wide RNAi screening in Drosophila cultured cells, and identified several signaling factors that are required for host defense and antimicrobial-peptide expression in Drosophila adults. These results suggest that our larva-derived tissue extract contains active ingredients that mediate Toll pathway activation, and the screening data will shed light on the mechanisms of damage-related Toll pathway signaling in Drosophila.

  9. Affinity Electrophoresis Using Ligands Attached To Polymers

    NASA Technical Reports Server (NTRS)

    Van Alstine, James M.; Snyder, Robert S.; Harris, J. M.; Brooks, D. E.

    1990-01-01

    In new technique, reduction of electrophoretic mobilities by addition of polyethylene glycol to ligands increases electrophoretic separabilities. In immuno-affinity electrophoresis, modification of ligands extends specificity of electrophoretic separation to particles having surface electric-charge structures otherwise making them electrophoretically inseparable. Modification of antibodies by polyethylene glycol greatly reduces ability to aggregate while enhancing ability to affect electrophoretic mobilities of cells. In hydrophobic-affinity electrophoresis, addition of polyethylene glycol reduces tendency toward aggregation of cells or macromolecules.

  10. Semiconductor Quantum Dots with Photoresponsive Ligands.

    PubMed

    Sansalone, Lorenzo; Tang, Sicheng; Zhang, Yang; Thapaliya, Ek Raj; Raymo, Françisco M; Garcia-Amorós, Jaume

    2016-10-01

    Photochromic or photocaged ligands can be anchored to the outer shell of semiconductor quantum dots in order to control the photophysical properties of these inorganic nanocrystals with optical stimulations. One of the two interconvertible states of the photoresponsive ligands can be designed to accept either an electron or energy from the excited quantum dots and quench their luminescence. Under these conditions, the reversible transformations of photochromic ligands or the irreversible cleavage of photocaged counterparts translates into the possibility to switch luminescence with external control. As an alternative to regulating the photophysics of a quantum dot via the photochemistry of its ligands, the photochemistry of the latter can be controlled by relying on the photophysics of the former. The transfer of excitation energy from a quantum dot to a photocaged ligand populates the excited state of the species adsorbed on the nanocrystal to induce a photochemical reaction. This mechanism, in conjunction with the large two-photon absorption cross section of quantum dots, can be exploited to release nitric oxide or to generate singlet oxygen under near-infrared irradiation. Thus, the combination of semiconductor quantum dots and photoresponsive ligands offers the opportunity to assemble nanostructured constructs with specific functions on the basis of electron or energy transfer processes. The photoswitchable luminescence and ability to photoinduce the release of reactive chemicals, associated with the resulting systems, can be particularly valuable in biomedical research and can, ultimately, lead to the realization of imaging probes for diagnostic applications as well as to therapeutic agents for the treatment of cancer.

  11. New bifunctional ligands for radioimmunoimaging and radioimmunotherapy

    SciTech Connect

    Brechbiel, M.W.

    1988-01-01

    The bifunctional EDTA ligand and two bifunctional DTPA ligands were synthesized by direct aminolysis of an amino acid ester followed by reduction, alkylation, and functional group modification to introduced bifunctionality. The reactive substituent chosen for protein conjugation was the isothiocyanate group. The generality of this approach was demonstrated with 9 different amino acids to produce the respective substituted diethylenetriamines. The remaining three bifunctional DTPA ligands were synthesized via classical peptide methodology producing a dipeptide amide which, after deprotection, was reduced to the triamine and alkylated to produce the ligand. Biodistribution studies of the ligands conjugated to monoclonal antibody B72.3 and labelled with In-111 revealed that superior retention of In-111 was attained and the dose to the liver was minimized when a full intact octadentate bifunctional DTPA chelate was used, e.g. DTPA > EDTA > DTTA (diethylenetritetraacetic acid from use of DTPA dianhydride (CA-DTPA)). The best scintigraphic images were obtained after 72 hours when a DTPA ligand was used to complex the In-111. Biodistribution studies using Yttrium-88 revealed that the disubstituted bifunctional DTPA was necessary to minimize the bone dose from the Yttrium while maintaining a high dose to the tumor.

  12. Interrupting autocrine ligand-receptor binding: comparison between receptor blockers and ligand decoys.

    PubMed Central

    Forsten, K E; Lauffenburger, D A

    1992-01-01

    Stimulation of cell behavioral functions by ligand/receptor binding can be accomplished in autocrine fashion, where cells secrete ligand capable of binding to receptors on their own surfaces. This proximal secretion of autocrine ligands near the surface receptors on the secreting cell suggests that control of these systems by inhibitors of receptor/ligand binding may be more difficult than for systems involving exogenous ligands. Hence, it is of interest to predict the conditions under which successful inhibition of cell receptor binding by the autocrine ligand can be expected. Previous theoretical work using a compartmentalized model for autocrine cells has elucidated the conditions under which addition of solution decoys for the autocrine ligand can interrupt cell receptor/ligand binding via competitive binding of the secreted molecules (Forsten, K. E., and D. A. Lauffenburger. 1992. Biophys. J. 61:1-12.) We now apply a similar modeling approach to examine the addition of solution blockers targeted against the cell receptor. Comparison of the two alternative inhibition strategies reveals that a significantly lower concentration of receptor blockers, compared to ligand decoys, will obtain a high degree of inhibition. The more direct interruption scheme characteristic of the receptor blockers may make them a preferred strategy when feasible. PMID:1330038

  13. Fully Flexible Docking of Medium Sized Ligand Libraries with RosettaLigand

    PubMed Central

    DeLuca, Samuel; Khar, Karen; Meiler, Jens

    2015-01-01

    RosettaLigand has been successfully used to predict binding poses in protein-small molecule complexes. However, the RosettaLigand docking protocol is comparatively slow in identifying an initial starting pose for the small molecule (ligand) making it unfeasible for use in virtual High Throughput Screening (vHTS). To overcome this limitation, we developed a new sampling approach for placing the ligand in the protein binding site during the initial ‘low-resolution’ docking step. It combines the translational and rotational adjustments to the ligand pose in a single transformation step. The new algorithm is both more accurate and more time-efficient. The docking success rate is improved by 10–15% in a benchmark set of 43 protein/ligand complexes, reducing the number of models that typically need to be generated from 1000 to 150. The average time to generate a model is reduced from 50 seconds to 10 seconds. As a result we observe an effective 30-fold speed increase, making RosettaLigand appropriate for docking medium sized ligand libraries. We demonstrate that this improved initial placement of the ligand is critical for successful prediction of an accurate binding position in the ‘high-resolution’ full atom refinement step. PMID:26207742

  14. Video elicitation interviews: a qualitative research method for investigating physician-patient interactions.

    PubMed

    Henry, Stephen G; Fetters, Michael D

    2012-01-01

    We describe the concept and method of video elicitation interviews and provide practical guidance for primary care researchers who want to use this qualitative method to investigate physician-patient interactions. During video elicitation interviews, researchers interview patients or physicians about a recent clinical interaction using a video recording of that interaction as an elicitation tool. Video elicitation is useful because it allows researchers to integrate data about the content of physician-patient interactions gained from video recordings with data about participants' associated thoughts, beliefs, and emotions gained from elicitation interviews. This method also facilitates investigation of specific events or moments during interactions. Video elicitation interviews are logistically demanding and time consuming, and they should be reserved for research questions that cannot be fully addressed using either standard interviews or video recordings in isolation. As many components of primary care fall into this category, high-quality video elicitation interviews can be an important method for understanding and improving physician-patient interactions in primary care.

  15. MIBE acts as antagonist ligand of both estrogen receptor α and GPER in breast cancer cells

    PubMed Central

    2012-01-01

    Introduction The multiple biological responses to estrogens are mainly mediated by the classical estrogen receptors ERα and ERβ, which act as ligand-activated transcription factors. ERα exerts a main role in the development of breast cancer; therefore, the ER antagonist tamoxifen has been widely used although its effectiveness is limited by de novo and acquired resistance. Recently, GPR30/GPER, a member of the seven-transmembrane G protein-coupled receptor family, has been implicated in mediating the effects of estrogens in various normal and cancer cells. In particular, GPER triggered gene expression and proliferative responses induced by estrogens and even ER antagonists in hormone-sensitive tumor cells. Likewise, additional ER ligands showed the ability to bind to GPER eliciting promiscuous and, in some cases, opposite actions through the two receptors. We synthesized a novel compound (ethyl 3-[5-(2-ethoxycarbonyl-1-methylvinyloxy)-1-methyl-1H-indol-3-yl]but-2-enoate), referred to as MIBE, and investigated its properties elicited through ERα and GPER in breast cancer cells. Methods Molecular modeling, binding experiments and functional assays were performed in order to evaluate the biological action exerted by MIBE through ERα and GPER in MCF7 and SkBr3 breast cancer cells. Results MIBE displayed the ability to act as an antagonist ligand for ERα and GPER as it elicited inhibitory effects on gene transcription and growth effects by binding to both receptors in breast cancer cells. Moreover, GPER was required for epidermal growth factor receptor (EGFR) and ERK activation by EGF as ascertained by using MIBE and performing gene silencing experiments. Conclusions Our findings provide novel insights on the functional cross-talk between GPER and EGFR signaling. Furthermore, the exclusive antagonistic activity exerted by MIBE on ERα and GPER could represent an innovative pharmacological approach targeting breast carcinomas which express one or both receptors at

  16. Time, the Forgotten Dimension of Ligand Binding Teaching

    ERIC Educational Resources Information Center

    Corzo, Javier

    2006-01-01

    Ligand binding is generally explained in terms of the equilibrium constant K[subscript d] for the protein-ligand complex dissociation. However, both theoretical considerations and experimental data point to the life span of the protein-ligand complex as an important, but generally overlooked, aspect of ligand binding by macromolecules. Short-lived…

  17. Hypertension and Life-Threatening Bleeding in Children with Relapsed Acute Myeloblastic Leukemia Treated with FLT3 Inhibitors.

    PubMed

    Yılmaz Karapınar, Deniz; Karadaş, Nihal; Önder Siviş, Zühal; Balkan, Can; Kavaklı, Kaan; Aydınok, Yeşim

    2015-09-01

    Experiences with new multikinase inhibitors are limited, especially in children. In this report we summarize our experience with 2 patients with relapsed acute myeloblastic leukemia (AML), one with FMS-like tyrosine kinase-3-internal tandem duplication mutation and the other with a single base mutation (D835Y). Both patients received sorafenib, one for 19 days and the other for 42 days, with clofarabine-including chemotherapy. One additionally received sunitinib for a total of 20 days. Both patients developed severe pancytopenia, hypertension, life-threatening bleedings from the gastrointestinal system, and, finally, intrapulmonary hemorrhage. Although both reached severe aplasia of the bone marrow without blastic infiltration, death occurred with neutropenic sepsis.

  18. Ligand clouds around protein clouds: a scenario of ligand binding with intrinsically disordered proteins.

    PubMed

    Jin, Fan; Yu, Chen; Lai, Luhua; Liu, Zhirong

    2013-01-01

    Intrinsically disordered proteins (IDPs) were found to be widely associated with human diseases and may serve as potential drug design targets. However, drug design targeting IDPs is still in the very early stages. Progress in drug design is usually achieved using experimental screening; however, the structural disorder of IDPs makes it difficult to characterize their interaction with ligands using experiments alone. To better understand the structure of IDPs and their interactions with small molecule ligands, we performed extensive simulations on the c-Myc₃₇₀₋₄₀₉ peptide and its binding to a reported small molecule inhibitor, ligand 10074-A4. We found that the conformational space of the apo c-Myc₃₇₀₋₄₀₉ peptide was rather dispersed and that the conformations of the peptide were stabilized mainly by charge interactions and hydrogen bonds. Under the binding of the ligand, c-Myc₃₇₀₋₄₀₉ remained disordered. The ligand was found to bind to c-Myc₃₇₀₋₄₀₉ at different sites along the chain and behaved like a 'ligand cloud'. In contrast to ligand binding to more rigid target proteins that usually results in a dominant bound structure, ligand binding to IDPs may better be described as ligand clouds around protein clouds. Nevertheless, the binding of the ligand and a non-ligand to the c-Myc₃₇₀₋₄₀₉ target could be clearly distinguished. The present study provides insights that will help improve rational drug design that targets IDPs.

  19. Posttranslational regulation of Fas ligand function

    PubMed Central

    Voss, Matthias; Lettau, Marcus; Paulsen, Maren; Janssen, Ottmar

    2008-01-01

    The TNF superfamily member Fas ligand acts as a prototypic death factor. Due to its ability to induce apoptosis in Fas (APO-1, CD95) expressing cells, Fas ligand participates in essential effector functions of the immune system. It is involved in natural killer cell- and T cell-mediated cytotoxicity, the establishment of immune privilege, and in termination of immune responses by induction of activation-induced cell death. In addition, Fas ligand-positive tumours may evade immune surveillance by killing Fas-positive tumour-infiltrating cells. Given these strong cytotoxic capabilities of Fas ligand, it is obvious that its function has to be strictly regulated to avoid uncontrolled damage. In hematopoietic cells, the death factor is stored in secretory lysosomes and is mobilised to the immunological synapse only upon activation. The selective sorting to and the release from this specific lysosomal compartment requires interactions of the Fas ligand cytosolic moiety, which mediates binding to various adapter proteins involved in trafficking and cytoskeletal reorganisation. In addition, Fas ligand surface expression is further regulated by posttranslational ectodomain shedding and subsequent regulated intramembrane proteolysis, releasing a soluble ectodomain cytokine into the extracellular space and an N-terminal fragment with a potential role in intracellular signalling processes. Moreover, other posttranslational modifications of the cytosolic domain, including phosphorylation and ubiquitylation, have been described to affect various aspects of Fas ligand biology. Since FasL is regarded as a potential target for immunotherapy, the further characterisation of its biological regulation and function will be of great importance for the development and evaluation of future therapeutic strategies. PMID:19114018

  20. Oxysterols are agonist ligands of RORγt and drive Th17 cell differentiation

    PubMed Central

    Soroosh, Pejman; Wu, Jiejun; Xue, Xiaohua; Song, Jiao; Sutton, Steven W.; Sablad, Marciano; Yu, Jingxue; Nelen, Marina I.; Liu, Xuejun; Castro, Glenda; Luna, Rosa; Crawford, Shelby; Banie, Homayon; Dandridge, Rose A.; Deng, Xiaohu; Bittner, Anton; Kuei, Chester; Tootoonchi, Mandana; Rozenkrants, Natasha; Herman, Krystal; Gao, Jingjin; Yang, Xia V.; Sachen, Kacey; Ngo, Karen; Fung-Leung, Wai-Ping; Nguyen, Steven; de Leon-Tabaldo, Aimee; Blevitt, Jonathan; Zhang, Yan; Cummings, Maxwell D.; Rao, Tadimeti; Mani, Neelakandha S.; Liu, Changlu; McKinnon, Murray; Milla, Marcos E.; Fourie, Anne M.; Sun, Siquan

    2014-01-01

    The RAR-related orphan receptor gamma t (RORγt) is a nuclear receptor required for generating IL-17–producing CD4+ Th17 T cells, which are essential in host defense and may play key pathogenic roles in autoimmune diseases. Oxysterols elicit profound effects on immune and inflammatory responses as well as on cholesterol and lipid metabolism. Here, we describe the identification of several naturally occurring oxysterols as RORγt agonists. The most potent and selective activator for RORγt is 7β, 27-dihydroxycholesterol (7β, 27-OHC). We show that these oxysterols reverse the inhibitory effect of an RORγt antagonist, ursolic acid, in RORγ- or RORγt-dependent cell-based reporter assays. These ligands bind directly to recombinant RORγ ligand binding domain (LBD), promote recruitment of a coactivator peptide, and reduce binding of a corepressor peptide to RORγ LBD. In primary cells, 7β, 27-OHC and 7α, 27-OHC enhance the differentiation of murine and human IL-17–producing Th17 cells in an RORγt-dependent manner. Importantly, we showed that Th17, but not Th1 cells, preferentially produce these two oxysterols. In vivo, administration of 7β, 27-OHC in mice enhanced IL-17 production. Mice deficient in CYP27A1, a key enzyme in generating these oxysterols, showed significant reduction of IL-17–producing cells, including CD4+ and γδ+ T cells, similar to the deficiency observed in RORγt knockout mice. Our results reveal a previously unknown mechanism for selected oxysterols as immune modulators and a direct role for CYP27A1 in generating these RORγt agonist ligands, which we propose as RORγt endogenous ligands, driving both innate and adaptive IL-17–dependent immune responses. PMID:25092323

  1. Multiple ligand simultaneous docking: orchestrated dancing of ligands in binding sites of protein.

    PubMed

    Li, Huameng; Li, Chenglong

    2010-07-30

    Present docking methodologies simulate only one single ligand at a time during docking process. In reality, the molecular recognition process always involves multiple molecular species. Typical protein-ligand interactions are, for example, substrate and cofactor in catalytic cycle; metal ion coordination together with ligand(s); and ligand binding with water molecules. To simulate the real molecular binding processes, we propose a novel multiple ligand simultaneous docking (MLSD) strategy, which can deal with all the above processes, vastly improving docking sampling and binding free energy scoring. The work also compares two search strategies: Lamarckian genetic algorithm and particle swarm optimization, which have respective advantages depending on the specific systems. The methodology proves robust through systematic testing against several diverse model systems: E. coli purine nucleoside phosphorylase (PNP) complex with two substrates, SHP2NSH2 complex with two peptides and Bcl-xL complex with ABT-737 fragments. In all cases, the final correct docking poses and relative binding free energies were obtained. In PNP case, the simulations also capture the binding intermediates and reveal the binding dynamics during the recognition processes, which are consistent with the proposed enzymatic mechanism. In the other two cases, conventional single-ligand docking fails due to energetic and dynamic coupling among ligands, whereas MLSD results in the correct binding modes. These three cases also represent potential applications in the areas of exploring enzymatic mechanism, interpreting noisy X-ray crystallographic maps, and aiding fragment-based drug design, respectively.

  2. Auditory Evoked Fields Elicited by Spectral, Temporal, and Spectral–Temporal Changes in Human Cerebral Cortex

    PubMed Central

    Okamoto, Hidehiko; Teismann, Henning; Kakigi, Ryusuke; Pantev, Christo

    2012-01-01

    Natural sounds contain complex spectral components, which are temporally modulated as time-varying signals. Recent studies have suggested that the auditory system encodes spectral and temporal sound information differently. However, it remains unresolved how the human brain processes sounds containing both spectral and temporal changes. In the present study, we investigated human auditory evoked responses elicited by spectral, temporal, and spectral–temporal sound changes by means of magnetoencephalography. The auditory evoked responses elicited by the spectral–temporal change were very similar to those elicited by the spectral change, but those elicited by the temporal change were delayed by 30–50 ms and differed from the others in morphology. The results suggest that human brain responses corresponding to spectral sound changes precede those corresponding to temporal sound changes, even when the spectral and temporal changes occur simultaneously. PMID:22593751

  3. Eyeglasses elicit effects similar to face-like perceptual expertise: evidence from the N170 response.

    PubMed

    Cao, Xiaohua; Yang, Qi; Hu, Fengpei

    2016-03-01

    Studies of event-related potentials show that the specific N170 response has become a stable electrophysiological hallmark of objects related to expertise in early perceptual processing. In the present study, we investigated whether eyeglasses can elicit N170 effects similar to those elicited by objects of expertise. Our results showed that the N170 response elicited by eyeglasses was larger than the response elicited by objects that do not generate perceptual expertise (e.g., houses). Importantly, we found that eyeglasses could produce a within-category N170 adaptation effect, similar to that produced in response to objects of expertise (e.g., faces). Our results have revealed for the first time that with a large amount of experience, eyeglasses could evoke the face-like N170 response, which suggested that eyeglasses may become an object of perceptual expertise to some human observers.

  4. Practical Guidance and Ethical Considerations for Studies Using Photo-Elicitation Interviews

    PubMed Central

    Bugos, Eva; Frasso, Rosemary; FitzGerald, Elizabeth; True, Gala; Adachi-Mejia, Anna M.

    2014-01-01

    Photo-elicitation is a qualitative interviewing technique that has gained popularity in recent years. It is the foundation for photovoice projects and is a tool well-suited for community-based participatory research. Photo-elicitation yields rich data, and interview participants say these interviews encourage community awareness and engagement. This article draws on 9 studies, conducted by researchers at 3 institutions (the University of Pennsylvania, the Philadelphia Veterans Affairs Medical Center, and the Geisel School of Medicine at Dartmouth) in partnership with community-based organizations and students, in which 303 participants completed photo-elicitation interviews. We offer 8 practical suggestions for overcoming challenges encountered during photo-elicitation research and for managing ethical concerns about the use of visual data in public health research. Our guidelines can inform study design, protocol development, and institutional review board approval. PMID:25357257

  5. ACTH acts via an anterior ventral third ventricular site to elicit grooming behavior.

    PubMed

    Dunn, A J; Hurd, R W

    1986-01-01

    Intracerebroventricular but not parenteral application of ACTH has been shown to elicit excessive grooming behavior in rats and mice. This behavior is elicited by administration of ACTH into the lateral, third, or fourth ventricles. Plugging of the cerebral aqueduct with cold cream fails to prevent grooming in response to lateral ventricle injection of ACTH. However, cold cream plugs in the third ventricle can prevent the subsequent induction of grooming behavior by lateral ventricle injection of ACTH, but only when the plugs are located in the anterior ventral third ventricle in the region of the organum vasculosum laminae terminalis (OVLT) and median eminence. These data suggest the anterior ventral third ventricle as the periventricular site of action of ACTH in eliciting excessive grooming, although it is possible that peptides taken up in this area are transported to other regions to elicit the behavioral response.

  6. Variation in accumulation of isoflavonoids in Phaseoleae seedlings elicited by Rhizopus.

    PubMed

    Aisyah, Siti; Gruppen, Harry; Andini, Silvia; Bettonvil, Monique; Severing, Edouard; Vincken, Jean-Paul

    2016-04-01

    Seeds from seven species of tribe Phaseoleae, i.e. Phaseolus, Vigna, Lablab and Psophocarpus, were investigated for inducibility of isoflavonoids by germination with or without subsequent elicitation with Rhizopus oryzae. Germination alone poorly induced isoflavonoid production (in the range of 0.2-0.7 mg representative compound equivalents (RCE)/g DW), whereas application of Rhizopus onto the seedlings increased the isoflavonoid content considerably (in the range of 0.5-3.3 mg RCE/g DW). The inducibility of different isoflavonoid subclasses in seedlings with Rhizopus varied per species. Isoflavones and isoflavanones were mainly found in elicited seedlings of Phaseolus, Vigna and Lablab, whereas pterocarpans were mainly observed in those of Psophocarpus. Despite their phylogenetic relatedness, the seeds of various species within Phaseoleae appeared to respond differently towards elicitation by Rhizopus during germination. The kind of molecules induced followed the phylogenetic relationship of the various species, but their amounts induced during germination, alone or combined with elicitation, did not.

  7. Practical guidance and ethical considerations for studies using photo-elicitation interviews.

    PubMed

    Bugos, Eva; Frasso, Rosemary; FitzGerald, Elizabeth; True, Gala; Adachi-Mejia, Anna M; Cannuscio, Carolyn

    2014-10-30

    Photo-elicitation is a qualitative interviewing technique that has gained popularity in recent years. It is the foundation for photovoice projects and is a tool well-suited for community-based participatory research. Photo-elicitation yields rich data, and interview participants say these interviews encourage community awareness and engagement. This article draws on 9 studies, conducted by researchers at 3 institutions (the University of Pennsylvania, the Philadelphia Veterans Affairs Medical Center, and the Geisel School of Medicine at Dartmouth) in partnership with community-based organizations and students, in which 303 participants completed photo-elicitation interviews. We offer 8 practical suggestions for overcoming challenges encountered during photo-elicitation research and for managing ethical concerns about the use of visual data in public health research. Our guidelines can inform study design, protocol development, and institutional review board approval.

  8. Implementation of the PR&PP methodology: the role of formal expert elicitations

    SciTech Connect

    Pilat, Joseph F

    2010-01-01

    The application of the methodology developed by the GenIV International Forum's (GIF's) Proliferation Resistance and Physical Protection (PR&PP) Working Group is an expert elicitation. Although the framework of the methodology is structured and systematic, it does not by itself constitute or require a formal elicitation. However, formal elicitation can be utilized in the PR&PP context to provide a systematic, credible and transparent qualitative analysis and develop input for quantitative analyses. This section provides an overview of expert elicitations, a discussion of the role formal expert elicitations can play in the PR&PP methodology, an outline of the formal expert elicitation process and a brief practical guide to conducting formal expert elicitations. Expert elicitation is a process utilizing knowledgeable people in cases, for example, when an assessment is needed but physically based data is absent or open to interpretation. More specifically, it can be used to: (1) predict future events; (2) provide estimates on new, rare, complex or poorly understood phenomena; (3) integrate or interpret existing information; or (4) determine what is currently known, how well it is known or what is worth learning in a field. Expert elicitation can be informal or formal. The informal application of expert judgment is frequently used. Although it can produce good results, it often provides demonstrably biased or otherwise flawed answers to problems. This along with the absence of transparency can result in a loss of confidence when experts speak on issues. More formal expert elicitation is a structured process that makes use of people knowledgeable in certain areas to make assessments. The reason for advocating formal use is that the quality and accuracy of expert judgment comes from the completeness of the expert's understanding of the phenomena and the process used to elicit and analyze the data. The use of a more formal process to obtain, lU1derstand and analyze expert

  9. Extending Hypothesis Testing of Edge Organizations Using Functional Magnetic Resonance Imaging (fMRI) During ELICIT

    DTIC Science & Technology

    2010-06-01

    task difficulty and response correctness on neural systems supporting fluid reasoning. Cognitive Neurodynamics 1 (1): 71-84. Kaplan, J.T., Iacoboni...dynamic influences on decision-making and trust during social interaction. ELICITing Behavior ELICIT is designed to explore social and cognitive ...a person’s own self-awareness in the game experience, (2) their cognitive processes of reasoning, and (3) the modulation of uncertainty that primes

  10. Evaluation of Antioxidant and Antibacterial Potentials of Nigella sativa L. Suspension Cultures under Elicitation

    PubMed Central

    Chaudhry, Hera; Fatima, Nida; Ahmad, Iffat Zareen

    2015-01-01

    Nigella sativa L. (family Ranunculaceae) is an annual herb of immense medicinal properties because of its major active components (i.e., thymoquinone (TQ), thymohydroquinone (THQ), and thymol (THY)). Plant tissue culture techniques like elicitation, Agrobacterium mediated transformation, hairy root culture, and so on, are applied for substantial metabolite production. This study enumerates the antibacterial and antioxidant potentials of N. sativa epicotyl suspension cultures under biotic and abiotic elicitation along with concentration optimization of the elicitors for enhanced TQ and THY production. Cultures under different concentrations of pectin and manganese chloride (MnCl2) elicitation (i.e., 5 mg/L, 10 mg/L, and 15 mg/L) showed that the control, MnCl2 10 mg/L, and pectin 15 mg/L suspension extracts greatly inhibited the growth of E. coli, S. typhimurium, and S. aureus (MIC against E. coli, i.e., 2.35 ± 0.8, 2.4 ± 0.2, and 2.46 ± 0.5, resp.). Elicitation decreased SOD enzyme activity whereas CAT enzyme activity increased remarkably under MnCl2 elicitation. MnCl2 10 mg/L and pectin 15 mg/L elicitation enhanced the DPPH radical inhibition ability, but ferric scavenging activity was comparable to the control. TQ and THY were quantified by LC-MS/MS in the cultures with high bioactive properties revealing maximum content under MnCl2 10 mg/L elicitation. Therefore, MnCl2 elicitation can be undertaken on large scale for sustainable metabolite production. PMID:26347883

  11. Design of a Hole Trapping Ligand

    DOE PAGES

    La Croix, Andrew D.; O’Hara, Andrew; Reid, Kemar R.; ...

    2017-01-18

    A new ligand that covalently attaches to the surface of colloidal CdSe/ CdS nanorods and can simultaneously chelate a molecular metal center is described. The dithiocarbamate$-$bipyridine ligand system facilitates hole transfer through energetic overlap at the inorganic$-$organic interface and conjugation through the organic ligand to a chelated metal center. Density functional theory calculations show that the coordination of the free ligand to a CdS surface causes the formation of two hybridized molecular states that lie in the band gap of CdS. The further chelation of Fe(II) to the bipyridine moiety causes the presence of seven midgap states. Hole transfer frommore » the CdS valence band to the midgap states is dipole allowed and occurs at a faster rate than what is experimentally known for the CdSe/CdS band-edge radiative recombination. In the case of the ligand bound with iron, a two-step process emerges that places the hole on the iron, again at rates much faster than band gap recombination. The system was experimentally assembled and characterized via UV$-$vis absorbance spectroscopy, fluorescence spectroscopy, time-resolved photoluminescence spectroscopy, and energy dispersive X-ray spectroscopy. Lastly, theoretically predicted red shifts in absorbance were observed experimentally, as well as the expected quench in photoluminescence and lifetimes in time-resolved photoluminescence« less

  12. Chelating ligands for nanocrystals' surface functionalization.

    PubMed

    Querner, Claudia; Reiss, Peter; Bleuse, Joël; Pron, Adam

    2004-09-22

    A new family of ligands for the surface functionalization of CdSe nanocrystals is proposed, namely alkyl or aryl derivatives of carbodithioic acids (R-C(S)SH). The main advantages of these new ligands are as follows: they nearly quantitatively exchange the initial surface ligands (TOPO) in very mild conditions; they significantly improve the resistance of nanocrystals against photooxidation because of their ability of strong chelate-type binding to metal atoms; their relatively simple preparation via Grignard intermediates facilitates the development of new bifunctional ligands containing, in addition to the anchoring carbodithioate group, a second function, which enables the grafting of molecules or macromolecules of interest on the nanocrystal surface. To give an example of this approach, we report, for the first time, the grafting of an electroactive oligomer from the polyaniline family-aniline tetramer-on CdSe nanocrystals after their functionalization with 4-formyldithiobenzoic acid. The grafting proceeds via a condensation reaction between the aldehyde group of the ligand and the terminal primary amine group of the tetramer. The resulting organic/inorganic hybrid exhibits complete extinction of the fluorescence of its constituents, indicating efficient charge or energy transfer between the organic and the inorganic semiconductors.

  13. Engineering death receptor ligands for cancer therapy.

    PubMed

    Wajant, Harald; Gerspach, Jeannette; Pfizenmaier, Klaus

    2013-05-28

    CD95, TNFR1, TRAILR1 and TRAILR2 belong to a subgroup of TNF receptors which is characterized by a conserved cell death-inducing protein domain that connects these receptors to the apoptotic machinery of the cell. Activation of death receptors in malignant cells attracts increasing attention as a principle to fight cancer. Besides agonistic antibodies the major way to stimulate death receptors is the use of their naturally occurring "death ligands" CD95L, TNF and TRAIL. However, dependent from the concept followed to develop a death ligand-based therapy various limiting aspects have to be taken into consideration on the way to a "bedside" usable drug. Problems arise in particular from the cell associated transmembrane nature of the death ligands, the poor serum half life of the soluble fragments derived from the transmembrane ligands, the ubiquitous expression of the death receptors and the existence of additional non-death receptors of the death ligands. Here, we summarize strategies how these limitations can be overcome by genetic engineering.

  14. A screening cascade to identify ERβ ligands

    PubMed Central

    Filgueira, Carly S.; Benod, Cindy; Lou, Xiaohua; Gunamalai, Prem S.; Villagomez, Rosa A.; Strom, Anders; Gustafsson, Jan-Åke; Berkenstam, Anders L.; Webb, Paul

    2014-01-01

    The establishment of effective high throughput screening cascades to identify nuclear receptor (NR) ligands that will trigger defined, therapeutically useful sets of NR activities is of considerable importance. Repositioning of existing approved drugs with known side effect profiles can provide advantages because de novo drug design suffers from high developmental failure rates and undesirable side effects which have dramatically increased costs. Ligands that target estrogen receptor β (ERβ) could be useful in a variety of diseases ranging from cancer to neurological to cardiovascular disorders. In this context, it is important to minimize cross-reactivity with ERα, which has been shown to trigger increased rates of several types of cancer. Because of high sequence similarities between the ligand binding domains of ERα and ERβ, preferentially targeting one subtype can prove challenging. Here, we describe a sequential ligand screening approach comprised of complementary in-house assays to identify small molecules that are selective for ERβ. Methods include differential scanning fluorimetry, fluorescence polarization and a GAL4 transactivation assay. We used this strategy to screen several commercially-available chemical libraries, identifying thirty ERβ binders that were examined for their selectivity for ERβ versus ERα, and tested the effects of selected ligands in a prostate cancer cell proliferation assay. We suggest that this approach could be used to rapidly identify candidates for drug repurposing. PMID:25422593

  15. Surface Ligand Mediated Plasmon Driven Photochemical Reactions.

    PubMed

    Kafle, Bijesh; Poveda, Marisa; Habteyes, Terefe G

    2017-02-07

    Contrary to the general expectation that surface ligands reduce the reactivity of surfaces by blocking the active sites, we present experimental evidence that surface ligands can in fact increase reactivity and induce important reaction pathways in plasmon-driven surface photochemistry. The remarkable effect of surface ligands is demonstrated by comparing the photochemistry of p-aminothiophenol (PATP) on resonant plasmonic gold nanorods (AuNRs) in the presence of citrate, hexadecyltrimethylammonium bromide (CTAB), and no surface ligands under visible light irradiation. The use of AuNRs with citrate and no surface ligand results in the usual azo-coupling reaction. In contrast, CTAB coated AuNRs oxidize PATP primarily to p-nitrothiophenol (PNTP). Strong correlation has been observed between the N-O and Au-Br vibration band intensities, suggesting that CTAB influences the reaction pathway through the Br- counterions that can minimize electron-hole recombination rate by reacting with hole, and hence increasing the concentration of hot electrons that drive the oxidation reaction.

  16. Controlling Gold Nanoclusters by Diphospine Ligands

    SciTech Connect

    Chen, Jing; Zhang, Qianfan; Bonaccorso, Timary A.; Williard, Paul G.; Wang, Lai S.

    2014-01-08

    We report the synthesis and structure determination of a new Au22 nanocluster coordinated by six bidentate diphosphine ligands: 1,8-bis(diphenylphosphino) octane (L8 for short). Single crystal x-ray crystallography and electrospray ionization mass spectrometry show that the cluster assembly is neutral and can be formulated as Au22(L8)6. The Au22 core consists of two Au11 units clipped together by four L8 ligands, while the additional two ligands coordinate to each Au11 unit in a bidentate fashion. Eight gold atoms at the interface of the two Au11 units are not coordinated by any ligands. Four short gold-gold distances (2.64?2.65 Å) are observed at the interface of the two Au11 clusters as a result of the clamping force of the four clipping ligands and strong electronic interactions. The eight uncoordinated surface gold atoms in the Au22(L8)6 nanocluster are unprecedented in atom-precise gold nanoparticles and can be considered as potential in-situ active sites for catalysis.

  17. Sliding tethered ligands add topological interactions to the toolbox of ligand-receptor design.

    PubMed

    Bauer, Martin; Kékicheff, Patrick; Iss, Jean; Fajolles, Christophe; Charitat, Thierry; Daillant, Jean; Marques, Carlos M

    2015-09-09

    Adhesion in the biological realm is mediated by specific lock-and-key interactions between ligand-receptor pairs. These complementary moieties are ubiquitously anchored to substrates by tethers that control the interaction range and the mobility of the ligands and receptors, thus tuning the kinetics and strength of the binding events. Here we add sliding anchoring to the toolbox of ligand-receptor design by developing a family of tethered ligands for which the spacer can slide at the anchoring point. Our results show that this additional sliding degree of freedom changes the nature of the adhesive contact by extending the spatial range over which binding may sustain a significant force. By introducing sliding tethered ligands with self-regulating length, this work paves the way for the development of versatile and reusable bio-adhesive substrates with potential applications for drug delivery and tissue engineering.

  18. A General Ligand Design for Gold Catalysis allowing Ligand-Directed Anti Nucleophilic Attack of Alkynes

    PubMed Central

    Wang, Yanzhao; Wang, Zhixun; Li, Yuxue; Wu, Gongde; Cao, Zheng; Zhang, Liming

    2014-01-01

    Most homogenous gold catalyses demand ≥0.5 mol % catalyst loading. Due to the high cost of gold, these reactions are unlikely to be applicable in medium or large scale applications. Here we disclose a novel ligand design based on the privileged biphenyl-2-phosphine framework that offers a potentially general approach to dramatically lowering catalyst loading. In this design, an amide group at the 3’ position of the ligand framework directs and promotes nucleophilic attack at the ligand gold complex-activated alkyne, which is unprecedented in homogeneous gold catalysis considering the spatial challenge of using ligand to reach antiapproaching nucleophile in a linear P-Au-alkyne centroid structure. With such a ligand, the gold(I) complex becomes highly efficient in catalyzing acid addition to alkynes, with a turnover number up to 99,000. Density functional theory calculations support the role of the amide moiety in directing the attack of carboxylic acid via hydrogen bonding. PMID:24704803

  19. Low zone tolerance requires ICAM-1 expression to limit contact hypersensitivity elicitation.

    PubMed

    Komura, Kazuhiro; Iwata, Yohei; Ogawa, Fumihide; Yoshizaki, Ayumi; Yamaoka, Toshifumi; Akiyama, Yuichiro; Hara, Toshihide; Hasegawa, Minoru; Fujimoto, Manabu; Sato, Shinichi

    2009-11-01

    Painting subsensitizing doses of contact sensitizers on skin (low-dose tolerization) induces antigen (Ag)-specific tolerance, known as low zone tolerance (LZT), which has been experimentally demonstrated by the inhibition of contact hypersensitivity (CHS). Although LZT resulted from the inhibition of the sensitization phase, the effects on the effector/elicitation phase remain unknown. L-selectin and ICAM-1 regulate leukocyte influx into inflamed tissues during the elicitation phase of CHS. LZT was investigated in mice lacking either L-selectin or ICAM-1 to evaluate the roles these leukocyte receptors play in LZT during the elicitation phase. Low-dose tolerization effectively suppressed CHS in wild-type and L-selectin-deficient mice, but not in ICAM-1-deficient mice. Low-dose-tolerized ICAM-1-deficient splenocytes effectively suppressed the elicitation phase in naive wild-type recipients. Sensitized ICAM-1-deficient splenocytes showed normal proliferative responses to the sensitizing Ag and generated normal CHS in wild-type recipients. Thus, ICAM-1 deficiency did not affect sensitization. LZT was associated with a lack of ICAM-1 upregulation after elicitation, suggesting a potentially mechanistic role for ICAM-1. The blockade of IL-10, a possible mediator of LZT, produced by hapten-specific suppressor cells, abrogated LZT and restored ICAM-1 upregulation. These results indicate that low-dose tolerization controls CHS by abrogating ICAM-1 upregulation during the elicitation phase.

  20. TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action.

    PubMed

    Gao, Yuan; Cao, Erhu; Julius, David; Cheng, Yifan

    2016-06-16

    When integral membrane proteins are visualized in detergents or other artificial systems, an important layer of information is lost regarding lipid interactions and their effects on protein structure. This is especially relevant to proteins for which lipids have both structural and regulatory roles. Here we demonstrate the power of combining electron cryo-microscopy with lipid nanodisc technology to ascertain the structure of the rat TRPV1 ion channel in a native bilayer environment. Using this approach, we determined the locations of annular and regulatory lipids and showed that specific phospholipid interactions enhance binding of a spider toxin to TRPV1 through formation of a tripartite complex. Furthermore, phosphatidylinositol lipids occupy the binding site for capsaicin and other vanilloid ligands, suggesting a mechanism whereby chemical or thermal stimuli elicit channel activation by promoting the release of bioactive lipids from a critical allosteric regulatory site.

  1. TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action

    PubMed Central

    Gao, Yuan; Cao, Erhu; Julius, David; Cheng, Yifan

    2016-01-01

    When integral membrane proteins are visualized in detergents or other artificial systems, an important layer of information is lost regarding lipid interactions and their effects on protein structure. This is especially relevant to proteins for which lipids play both structural and regulatory roles. Here, we demonstrate the power of combining electron cryo-microscopy with lipid nanodisc technology to ascertain the structure of the TRPV1 ion channel in a native bilayer environment. Using this approach, we determined the locations of annular and regulatory lipids and showed that specific phospholipid interactions enhance binding of a spider toxin to TRPV1 through formation of a tripartite complex. Furthermore, phosphatidylinositol lipids occupy the binding site for capsaicin and other vanilloid ligands, suggesting a mechanism whereby chemical or thermal stimuli elicit channel activation by promoting release of bioactive lipids from a critical allosteric regulatory site. PMID:27281200

  2. Cationic ruthenium alkylidene catalysts bearing phosphine ligands.

    PubMed

    Endo, Koji; Grubbs, Robert H

    2016-02-28

    The discovery of highly active catalysts and the success of ionic liquid immobilized systems have accelerated attention to a new class of cationic metathesis catalysts. We herein report the facile syntheses of cationic ruthenium catalysts bearing bulky phosphine ligands. Simple ligand exchange using silver(i) salts of non-coordinating or weakly coordinating anions provided either PPh3 or chelating Ph2P(CH2)nPPh2 (n = 2 or 3) ligated cationic catalysts. The structures of these newly reported catalysts feature unique geometries caused by ligation of the bulky phosphine ligands. Their activities and selectivities in standard metathesis reactions were also investigated. These cationic ruthenium alkylidene catalysts reported here showed moderate activity and very similar stereoselectivity when compared to the second generation ruthenium dichloride catalyst in ring-closing metathesis, cross metathesis, and ring-opening metathesis polymerization assays.

  3. Efficient chemoenzymatic synthesis of chiral pincer ligands.

    PubMed

    Felluga, Fulvia; Baratta, Walter; Fanfoni, Lidia; Pitacco, Giuliana; Rigo, Pierluigi; Benedetti, Fabio

    2009-05-01

    Chiral, nonracemic pincer ligands based on the 6-phenyl-2-aminomethylpyridine and 2-aminomethylbenzo[h]quinoline scaffolds were obtained by a chemoenzymatic approach starting from 2-pyridyl and 2-benzoquinolyl ethanone. In the enantiodifferentiating step, secondary alcohols of opposite absolute configuration were obtained by a baker's yeast reduction of the ketones and by lipase-mediated dynamic kinetic resolution of the racemic alcohols. Their transformation into homochiral 1-methyl-1-heteroarylethanamines occurred without loss of optical purity, giving access to pincer ligands used in enantioselective catalysis.

  4. Ligand Intermediates in Metal-Catalyzed Reactions

    SciTech Connect

    Gladysz, John A.

    1999-07-31

    The longest-running goal of this project has been the synthesis, isolation, and physical chemical characterization of homogeneous transition metal complexes containing ligand types believed to be intermediates in the metal-catalyzed conversion of CO/H{sub 2}, CO{sub 2}, CH{sub 4}, and similar raw materials to organic fuels, feedstocks, etc. In the current project period, complexes that contain unusual new types of C{sub x}(carbide) and C{sub x}O{sub y} (carbon oxide) ligands have been emphasized. A new program in homogeneous fluorous phase catalysis has been launched as described in the final report.

  5. Assessment of automatic ligand building in ARP/wARP

    PubMed Central

    Evrard, Guillaume X.; Langer, Gerrit G.; Perrakis, Anastassis; Lamzin, Victor S.

    2007-01-01

    The efficiency of the ligand-building module of ARP/wARP version 6.1 has been assessed through extensive tests on a large variety of protein–ligand complexes from the PDB, as available from the Uppsala Electron Density Server. Ligand building in ARP/wARP involves two main steps: automatic identification of the location of the ligand and the actual construction of its atomic model. The first step is most successful for large ligands. The second step, ligand construction, is more powerful with X-ray data at high resolution and ligands of small to medium size. Both steps are successful for ligands with low to moderate atomic displacement parameters. The results highlight the strengths and weaknesses of both the method of ligand building and the large-scale validation procedure and help to identify means of further improvement. PMID:17164533

  6. Assessment of automatic ligand building in ARP/wARP.

    PubMed

    Evrard, Guillaume X; Langer, Gerrit G; Perrakis, Anastassis; Lamzin, Victor S

    2007-01-01

    The efficiency of the ligand-building module of ARP/wARP version 6.1 has been assessed through extensive tests on a large variety of protein-ligand complexes from the PDB, as available from the Uppsala Electron Density Server. Ligand building in ARP/wARP involves two main steps: automatic identification of the location of the ligand and the actual construction of its atomic model. The first step is most successful for large ligands. The second step, ligand construction, is more powerful with X-ray data at high resolution and ligands of small to medium size. Both steps are successful for ligands with low to moderate atomic displacement parameters. The results highlight the strengths and weaknesses of both the method of ligand building and the large-scale validation procedure and help to identify means of further improvement.

  7. Ligand expansion in ligand-based virtual screening using relevance feedback

    NASA Astrophysics Data System (ADS)

    Abdo, Ammar; Saeed, Faisal; Hamza, Hentabli; Ahmed, Ali; Salim, Naomie

    2012-03-01

    Query expansion is the process of reformulating an original query to improve retrieval performance in information retrieval systems. Relevance feedback is one of the most useful query modification techniques in information retrieval systems. In this paper, we introduce query expansion into ligand-based virtual screening (LBVS) using the relevance feedback technique. In this approach, a few high-ranking molecules of unknown activity are filtered from the outputs of a Bayesian inference network based on a single ligand molecule to form a set of ligand molecules. This set of ligand molecules is used to form a new ligand molecule. Simulated virtual screening experiments with the MDL Drug Data Report and maximum unbiased validation data sets show that the use of ligand expansion provides a very simple way of improving the LBVS, especially when the active molecules being sought have a high degree of structural heterogeneity. However, the effectiveness of the ligand expansion is slightly less when structurally-homogeneous sets of actives are being sought.

  8. CLiBE: a database of computed ligand binding energy for ligand-receptor complexes.

    PubMed

    Chen, X; Ji, Z L; Zhi, D G; Chen, Y Z

    2002-11-01

    Consideration of binding competitiveness of a drug candidate against natural ligands and other drugs that bind to the same receptor site may facilitate the rational development of a candidate into a potent drug. A strategy that can be applied to computer-aided drug design is to evaluate ligand-receptor interaction energy or other scoring functions of a designed drug with that of the relevant ligands known to bind to the same binding site. As a tool to facilitate such a strategy, a database of ligand-receptor interaction energy is developed from known ligand-receptor 3D structural entries in the Protein Databank (PDB). The Energy is computed based on a molecular mechanics force field that has been used in the prediction of therapeutic and toxicity targets of drugs. This database also contains information about ligand function and other properties and it can be accessed at http://xin.cz3.nus.edu.sg/group/CLiBE.asp. The computed energy components may facilitate the probing of the mode of action and other profiles of binding. A number of computed energies of some PDB ligand-receptor complexes in this database are studied and compared to experimental binding affinity. A certain degree of correlation between the computed energy and experimental binding affinity is found, which suggests that the computed energy may be useful in facilitating a qualitative analysis of drug binding competitiveness.

  9. Agonists binding nicotinic receptors elicit specific channel-opening patterns at αγ and αδ sites

    PubMed Central

    Stock, Patrick; Ljaschenko, Dmitrij; Heckmann, Manfred; Dudel, Josef

    2014-01-01

    ‘Embryonic’ muscle-type nicotinic acetylcholine receptor channels (nAChRs) bind ligands at interfaces of α- and γ- or δ-subunits. αγ and αδ sites differ in affinity, but their contributions to opening the channel have remained elusive. We compared high-resolution patch clamp currents evoked by epibatidine (Ebd), carbamylcholine (CCh) and acetylcholine (ACh). Ebd binds with 75-fold higher affinity at αγ than at αδ sites, whereas CCh and ACh prefer αδ sites. Similar short (τO1), intermediate (τO2) and long (τO3) types of opening were observed with all three agonists. τO2 openings were maximally prevalent at low Ebd concentrations, binding at αγ sites. By contrast, τO1 openings appear to be generated at αδ sites. In addition, two types of burst appeared: short bursts of an average of 0.75 ms (τB1) that should arise from the αγ site, and long bursts of 12–25 ms (τB2) in duration arising from double liganded receptors. Limited by the temporal resolution, the closings within bursts were invariant at 3 μs. Corrected for missed closings, in the case of ACh the openings within long bursts lasted 170 μs and those in short bursts about 30 μs. Blocking αδ sites with α-conotoxin M1 (CTx) eliminated both τO1 and τB2 and left only τO2 and the short τB1 bursts, as expected. Furthermore we found desensitization when the receptors bound ACh only at the αγ site. When CTx was applied to ‘embryonic’ mouse endplates, monoquantal current rise times were increased, and amplitude and decay time constants were reduced, as expected. Thus the αγ and αδ sites of nAChRs elicit specific channel-opening patterns. PMID:24665094

  10. Optimizing electrostatic affinity in ligand-receptor binding: Theory, computation, and ligand properties

    NASA Astrophysics Data System (ADS)

    Kangas, Erik; Tidor, Bruce

    1998-11-01

    The design of a tight-binding molecular ligand involves a tradeoff between an unfavorable electrostatic desolvation penalty incurred when the ligand binds a receptor in aqueous solution and the generally favorable intermolecular interactions made in the bound state. Using continuum electrostatic models we have developed a theoretical framework for analyzing this problem and have shown that the ligand-charge distribution can be optimized to produce the most favorable balance of these opposing free energy contributions [L.-P. Lee and B. Tidor, J. Chem. Phys. 106, 8681 (1997)]. Herein the theoretical framework is extended and calculations are performed for a wide range of model receptors. We examine methods for computing optimal ligands (including cases where there is conformational change) and the resulting properties of optimized ligands. In particular, indicators are developed to aid in the determination of the deficiencies in a specific ligand or basis. A connection is established between the optimization problem here and a generalized image problem, from which an inverse-image basis set can be defined; this basis is shown to perform very well in optimization calculations. Furthermore, the optimized ligands are shown to have favorable electrostatic binding free energies (in contrast to many natural ligands), there is a strong correlation between the receptor desolvation penalty and the optimized binding free energy for fixed geometry, and the ligand and receptor cannot generally be mutually optimal. Additionally, we introduce the display of complementary desolvation and interaction potentials and the deviation of their relationship from ideal as a useful tool for judging effective complementarity. Scripts for computing and displaying these potentials with GRASP are available at http://mit.edu/tidor.

  11. [Kinetics of ligand binding to nucleic acids at random fillings].

    PubMed

    Arakelian, V B; Babaian, S Iu; Tairian, V I; Arakelian, A V; Parsadanian, M A; Vardevanian, P O

    2006-01-01

    Ligand binding with nucleic acids is described in frames of the theory of random processes. It is shown that the probabilistic description of binding of a ligand to nucleic acid allows one to describe not only the kinetics of changes in the number of bound ligands at arbitrary fillings but also to calculate stationary values of the number of bound ligands and its dispersion. A general analysis of absorption isotherms and the kinetics of ligand binding with nucleic acids allows one to determine the rate constants of formation and decomposition of the ligand-nucleic acid complex. A comparison of the results obtained with the case of low fillings is conducted.

  12. Solar energy conversion through ligand photodissociation

    SciTech Connect

    Hoffman, B.M.; Sima, P.D.

    1983-04-06

    A new technique for photochemical conversion of solar energy based on ligand photodissociation from metal complexes is examined. The concept is illustrated with a photogalvanic cell in which voltages are generated by photodissociation of CO from carbonylferroheme and with a cell in which the illuminated electrode is coated with an iron tetraphenylporphyrin.

  13. Identification of ligands for bacterial sensor proteins.

    PubMed

    Fernández, Matilde; Morel, Bertrand; Corral-Lugo, Andrés; Rico-Jiménez, Miriam; Martín-Mora, David; López-Farfán, Diana; Reyes-Darias, José Antonio; Matilla, Miguel A; Ortega, Álvaro; Krell, Tino

    2016-02-01

    Bacteria have evolved a variety of different signal transduction mechanisms. However, the cognate signal molecule for the very large amount of corresponding sensor proteins is unknown and their functional annotation represents a major bottleneck in the field of signal transduction. The knowledge of the signal molecule is an essential prerequisite to understand the signalling mechanisms. Recently, the identification of signal molecules by the high-throughput protein screening of commercially available ligand collections using differential scanning fluorimetry has shown promise to resolve this bottleneck. Based on the analysis of a significant number of different ligand binding domains (LBDs) in our laboratory, we identified two issues that need to be taken into account in the experimental design. Since a number of LBDs require the dimeric state for ligand recognition, it has to be assured that the protein analysed is indeed in the dimeric form. A number of other examples demonstrate that purified LBDs can contain bound ligand which prevents further binding. In such cases, the apo-form can be generated by denaturation and subsequent refolding. We are convinced that this approach will accelerate the functional annotation of sensor proteins which will help to understand regulatory circuits in bacteria.

  14. [Functional selectivity of opioid receptors ligands].

    PubMed

    Audet, Nicolas; Archer-Lahlou, Elodie; Richard-Lalonde, Mélissa; Piñeyro-Filpo, Graciela

    2010-01-01

    Opiates are the most effective analgesics available for the treatment of severe pain. However, their clinical use is restricted by unwanted side effects such as tolerance, physical dependence and respiratory depression. The strategy to develop new opiates with reduced side effects has mainly focused on the study and production of ligands that specifically bind to different opiate receptors subtypes. However, this strategy has not allowed the production of novel therapeutic ligands with a better side effects profile. Thus, other research strategies need to be explored. One which is receiving increasing attention is the possibility of exploiting ligand ability to stabilize different receptor conformations with distinct signalling profiles. This newly described property, termed functional selectivity, provides a potential means of directing the stimulus generated by an activated receptor towards a specific cellular response. Here we summarize evidence supporting the existence of ligand-specific active conformations for two opioid receptors subtypes (delta and mu), and analyze how functional selectivity may contribute in the production of longer lasting, better tolerated opiate analgesics. double dagger.

  15. Cooperative Ligand Binding to Linear Chain Molecules

    ERIC Educational Resources Information Center

    Applequist, Jon

    1977-01-01

    Summarizes the Ising model of ligand binding as it applies to cooperative binding to long chain molecules. Also presents some illustrations which help to visualize the connection between the interaction parameters and the shape of the binding isotherm. (Author/MR)

  16. [Central effects of ORL1 receptor ligands].

    PubMed

    Maslov, L N; Lishmanov, Iu B; Calo, G; Ma, L

    2003-01-01

    It has been discussed literature data on molecular structure of ORL1 receptor and its interaction with intracellular signal systems and neurotransmitters. Data on chemical structure of ORL1 receptor ligands and their central effects (nociception, locomotion, feeding, cognition) are presented.

  17. CXCR3 ligands in disease and therapy.

    PubMed

    Van Raemdonck, Katrien; Van den Steen, Philippe E; Liekens, Sandra; Van Damme, Jo; Struyf, Sofie

    2015-06-01

    Chemokines, binding their various G protein-coupled receptors, lead the way for leukocytes in health and inflammation. Yet chemokine receptor expression is not limited to leukocytes. Accordingly, chemokines are remarkably pleiotropic molecules involved in a range of physiological as well as pathological processes. For example, the CXCR3 chemokine receptor is expressed on activated T lymphocytes, dendritic cells and natural killer cells, but also fibroblasts and smooth muscle, epithelial and endothelial cells. In men, these cells express either CXCR3A, its splice variant CXCR3B or a balanced combination of both. The CXCR3 ligands, activating both receptor variants, include CXCL4, CXCL4L1, CXCL9, CXCL10 and CXCL11. Upon CXCR3A activation these ELR-negative CXC chemokines mediate chemotactic and proliferative responses, for example in leukocytes. In contrast, CXCR3B induces anti-proliferative and anti-migratory effects, as exemplified by angiostatic effects on endothelial cells. Taken together, the unusual and versatile characteristics of CXCR3 and its ligands form the basis for their pertinent involvement in a myriad of diseases. In this review, we discuss the presence and function of all CXCR3 ligands in various malignant, angiogenic, infectious, inflammatory and other disorders. By extension, we have also elaborated on the potential therapeutic applicability of CXCR3 ligand administration or blockade, as well as their additional value as predictive or prognostic biomarkers. This review illustrates the multifunctional, intriguing character of the various CXCR3-binding chemokines.

  18. Ligand iron catalysts for selective hydrogenation

    DOEpatents

    Casey, Charles P.; Guan, Hairong

    2010-11-16

    Disclosed are iron ligand catalysts for selective hydrogenation of aldehydes, ketones and imines. A catalyst such as dicarbonyl iron hydride hydroxycyclopentadiene) complex uses the OH on the five member ring and hydrogen linked to the iron to facilitate hydrogenation reactions, particularly in the presence of hydrogen gas.

  19. Cell movement elicited by epidermal growth factor receptor requires kinase and autophosphorylation but is separable from mitogenesis

    PubMed Central

    1994-01-01

    The EGF receptor (EGFR) upon activation signals increased cell movement. However, the domains within the receptor, and the pathway which trigger movement are undefined. We expressed EGFR mutants at physiologic levels in receptor-devoid NR6 cells to investigate this biologic response. The receptors possessed kinase activity and underwent autophosphorylation as predicted by primary amino acid sequence. EGF-induced cell motility was assessed in vitro by excess migration into an acellular area and colony scatter in the presence of saturating concentrations of EGF. Wild-type (WT)-EGFR signaled increased motility. However, replacing the conserved lysine721 with methionine resulted in a kinase-inactive receptor which did not elicit movement. Removal of the entire terminus by truncation (c'973) also abrogated ligand-induced motility. Thus, we concentrated on the carboxy- terminal domains. EGF-induced movement was seen with a less-truncated mutant (c'1000) that contained a single autophosphorylated tyrosine (tyrosine992). Other mutants, c'991 and c'1000F992, in which this tyrosine was removed did not signal motility. Fusion mutants which presented other autophosphorylated tyrosine domains also exhibited EGF- induced movement. These findings suggested that the presence of both an autophosphorylated tyrosine signaling domain and the kinase activity are necessary for this biologic response. All kinase-positive mutants signaled cell proliferation but only those that contained autophosphorylatable tyrosines induced movement. The motility responses mediated by these EGFR were identical in the presence or absence of mitomycin-C, at a dose (0.5 micrograms/ml) which completely inhibited cell proliferation. On the other side, D-actinomycin (50 ng/ml) blocked EGF-induced motility but did not affect thymidine incorporation. Thus, EGF-induced mitogenesis and cell motility are mediated through different pathways. PMID:8106552

  20. Functional Imaging of Human Vestibular Cortex Activity Elicited by Skull Tap and Auditory Tone Burst

    NASA Technical Reports Server (NTRS)

    Noohi, Fatemeh; Kinnaird, Catherine; Wood, Scott; Bloomberg, Jacob; Mulavara, Ajitkumar; Seidler, Rachael

    2014-01-01

    The aim of the current study was to characterize the brain activation in response to two modes of vestibular stimulation: skull tap and auditory tone burst. The auditory tone burst has been used in previous studies to elicit saccular Vestibular Evoked Myogenic Potentials (VEMP) (Colebatch & Halmagyi 1992; Colebatch et al. 1994). Some researchers have reported that airconducted skull tap elicits both saccular and utricle VEMPs, while being faster and less irritating for the subjects (Curthoys et al. 2009, Wackym et al., 2012). However, it is not clear whether the skull tap and auditory tone burst elicit the same pattern of cortical activity. Both forms of stimulation target the otolith response, which provides a measurement of vestibular function independent from semicircular canals. This is of high importance for studying the vestibular disorders related to otolith deficits. Previous imaging studies have documented activity in the anterior and posterior insula, superior temporal gyrus, inferior parietal lobule, pre and post central gyri, inferior frontal gyrus, and the anterior cingulate cortex in response to different modes of vestibular stimulation (Bottini et al., 1994; Dieterich et al., 2003; Emri et al., 2003; Schlindwein et al., 2008; Janzen et al., 2008). Here we hypothesized that the skull tap elicits the similar pattern of cortical activity as the auditory tone burst. Subjects put on a set of MR compatible skull tappers and headphones inside the 3T GE scanner, while lying in supine position, with eyes closed. All subjects received both forms of the stimulation, however, the order of stimulation with auditory tone burst and air-conducted skull tap was counterbalanced across subjects. Pneumatically powered skull tappers were placed bilaterally on the cheekbones. The vibration of the cheekbone was transmitted to the vestibular cortex, resulting in vestibular response (Halmagyi et al., 1995). Auditory tone bursts were also delivered for comparison. To validate

  1. The use of expert elicitation in environmental health impact assessment: a seven step procedure

    PubMed Central

    2010-01-01

    Background Environmental health impact assessments often have to deal with substantial uncertainties. Typically, the knowledge-base is limited with incomplete, or inconsistent evidence and missing or ambiguous data. Consulting experts can help to identify and address uncertainties. Methods Formal expert elicitation is a structured approach to systematically consult experts on uncertain issues. It is most often used to quantify ranges for poorly known parameters, but may also be useful to further develop qualitative issues such as definitions, assumptions or conceptual (causal) models. A thorough preparation and systematic design and execution of an expert elicitation process may increase the validity of its outcomes and transparency and trustworthiness of its conclusions. Various expert elicitation protocols and methods exist. However, these are often not universally applicable, and need customization to suite the needs of a specific study. In this paper, we set out to develop a widely applicable method for the use of expert elicitation in environmental health impact assessment. Results We present a practical yet flexible seven step procedure towards organising expert elicitation in the context of environmental health impact assessment, based on existing protocols. We describe how customization for specific applications is always necessary. In particular, three issues affect the choice of methods for a particular application: the types of uncertainties considered, the intended use of the elicited information, and the available resources. We outline how these three considerations guide choices regarding the design and execution of expert elicitation. We present signposts to sources where the issues are discussed in more depth to give the newcomer the insights needed to make the protocol work. The seven step procedure is illustrated using examples from earlier published elicitations in the field of environmental health research. Conclusions We conclude that, despite

  2. Rhizobium meliloti exopolysaccharide mutants elicit feedback regulation of nodule formation in alfalfa

    SciTech Connect

    Caetano-Anolles, G.; Lagares, A.; Bauer, W.D. )

    1990-02-01

    Nodule formation by wild-type Rhizobium meliloti is strongly suppressed in younger parts of alfalfa (Medicago sativum L.) root systems as a feedback response to development of the first nodules. Mutants of R. meliloti deficient in exopolysaccharide synthesis can induce the formation of organized nodular structures (pseudonodules) on alfalfa roots but are defective in their ability to invade and multiply within host tissues. The formation of empty pseudonodules by exo mutants was found to elicit a feedback suppression of nodule formation similar to that elicited by the wild-type bacteria. Inoculation of an exo mutant onto one side of a split-root system 24 hours before inoculation of the second side with wild-type cells suppressed wild-type nodule formation on the second side in proportion to the extent of pseudonodule formation by the exo mutants. The formation of pseudonodules is thus sufficient to elicit systemic feedback control of nodulation in the host root system: infection thread development and internal proliferation of the bacteria are not required for elicitation of feedback. Pseudonodule formation by the exo mutants was found to be strongly suppressed in split-root systems by prior inoculation on the opposite side with the wild type. Thus, feedback control elicited by the wild-type inhibits Rhizobium-induced redifferentiation of host root cells.

  3. Use (and abuse) of expert elicitation in support of decision making for public policy

    PubMed Central

    Morgan, M. Granger

    2014-01-01

    The elicitation of scientific and technical judgments from experts, in the form of subjective probability distributions, can be a valuable addition to other forms of evidence in support of public policy decision making. This paper explores when it is sensible to perform such elicitation and how that can best be done. A number of key issues are discussed, including topics on which there are, and are not, experts who have knowledge that provides a basis for making informed predictive judgments; the inadequacy of only using qualitative uncertainty language; the role of cognitive heuristics and of overconfidence; the choice of experts; the development, refinement, and iterative testing of elicitation protocols that are designed to help experts to consider systematically all relevant knowledge when they make their judgments; the treatment of uncertainty about model functional form; diversity of expert opinion; and when it does or does not make sense to combine judgments from different experts. Although it may be tempting to view expert elicitation as a low-cost, low-effort alternative to conducting serious research and analysis, it is neither. Rather, expert elicitation should build on and use the best available research and analysis and be undertaken only when, given those, the state of knowledge will remain insufficient to support timely informed assessment and decision making. PMID:24821779

  4. The capsaicin cough reflex in eczema patients with respiratory symptoms elicited by perfume.

    PubMed

    Elberling, Jesper; Dirksen, Asger; Johansen, Jeanne Duus; Mosbech, Holger

    2006-03-01

    Respiratory symptoms elicited by perfume are common in the population but have unclear pathophysiology. Increased capsaicin cough responsiveness has been associated with the symptoms, but it is unknown whether the site of the symptoms in the airways influences this association. The aim of this study was to investigate the association between the site of airway symptoms elicited by perfume and cough responsiveness to bronchial challenge with capsaicin. 21 eczema patients with respiratory symptoms elicited by perfume were compared with 21 healthy volunteers in a sex- and age-matched case control study. The participants completed a symptom questionnaire and underwent a bronchial challenge with capsaicin. Lower, but not upper, respiratory symptoms elicited by perfume were associated with increased capsaicin cough responsiveness. Having severe symptoms to perfume (n=11) did not relate to the site of the symptoms in the airways and was not associated with increased capsaicin cough responsiveness. In conclusion, respiratory symptoms elicited by perfume may reflect local hyperreactivity related to defensive reflexes in the airways, and measurements of the capsaicin cough reflex are relevant when patients with lower respiratory symptoms related to environmental perfume exposures are investigated.

  5. Enhanced scratching elicited by a pruritogen and an algogen in a mouse model of contact hypersensitivity.

    PubMed

    Fu, Kai; Qu, Lintao; Shimada, Steven G; Nie, Hong; LaMotte, Robert H

    2014-09-05

    Chemical pruritogens and algogens evoke primarily itch and pain, respectively, when administered to the skin of healthy human subjects. However, the dominant sensory quality elicited by an algesic chemical stimulus may change in patients with chronic itch where bradykinin, elicits itch in addition to pain. Here we tested whether normally pruritic and algesic chemicals evoked abnormal itch- or pain-like behaviors in the mouse after the development of contact hypersensitivity (CHS), an animal model of allergic contact dermatitis. Mice previously sensitized to a hapten (squaric acid dibutylester) applied to the abdomen, exhibited spontaneous itch-like scratching and pain-like wiping directed to the site on the cheek of the CHS elicited by a subsequent challenge with the same hapten. In comparison with responses of control mice, CHS mice exhibited a significant increase in the scratching evoked by bovine adrenal medulla 8-22, a peptide that elicits a histamine-independent itch, but did not alter the scratching to histamine. Bradykinin, an algogen that elicited only wiping in control mice, additionally evoked significant scratching in CHS mice. Thus, within an area of CHS, histamine-independent itch is enhanced and chemically evoked pain is accompanied by itch.

  6. Does alexithymia explain variation in cue-elicited craving reported by methamphetamine-dependent individuals?

    PubMed

    Saladin, Michael E; Santa Ana, Elizabeth J; LaRowe, Steven D; Simpson, Annie N; Tolliver, Bryan K; Price, Kimber L; McRae-Clark, Aimee L; Brady, Kathleen T

    2012-01-01

    Drug craving is an important motivational phenomenon among addicted individuals, and successful management of craving is essential to both the initiation and maintenance of abstinence. Although craving in response to drug cues is common in drug-dependent individuals, it is not universal. At the present time, it is not known why approximately 20-30% of all addicted persons fail to report appreciable craving in laboratory-based cue reactivity studies. This study examined the possibility that alexithymia, a personality attribute characterized by a difficulty identifying and describing emotions, may contribute to the impoverished cue-elicited craving experienced by some addicts. Specifically, we tested the hypothesis that alexithymia, as measured by the Toronto Alexithymia Scale (TAS), would be inversely related to the magnitude of cue-elicited craving obtained in a cue reactivity protocol. Forty methamphetamine-dependent individuals completed the TAS and provided craving ratings for methamphetamine after presentation of methamphetamine-associated cues. Thirteen participants (32%) reported no methamphetamine cue-elicited craving. Contrary to expectation, TAS factor 1 (a measure of difficulty identifying feelings) scores were positively associated with cue-elicited craving. Thus, the results suggest that increasing difficulty-identifying feelings may be associated with higher cue-elicited craving. Clinical implications for this finding are discussed.

  7. Ammonia formation by metal-ligand cooperative hydrogenolysis of a nitrido ligand

    NASA Astrophysics Data System (ADS)

    Askevold, Bjorn; Nieto, Jorge Torres; Tussupbayev, Samat; Diefenbach, Martin; Herdtweck, Eberhardt; Holthausen, Max C.; Schneider, Sven

    2011-07-01

    Bioinspired hydrogenation of N2 to ammonia at ambient conditions by stepwise nitrogen protonation/reduction with metal complexes in solution has experienced remarkable progress. In contrast, the highly desirable direct hydrogenation with H2 remains difficult. In analogy to the heterogeneously catalysed Haber-Bosch process, such a reaction is conceivable via metal-centred N2 splitting and unprecedented hydrogenolysis of the nitrido ligands to ammonia. We report the synthesis of a ruthenium(IV) nitrido complex. The high nucleophilicity of the nitrido ligand is demonstrated by unusual N-C coupling with π-acidic CO. Furthermore, the terminal nitrido ligand undergoes facile hydrogenolysis with H2 at ambient conditions to produce ammonia in high yield. Kinetic and quantum chemical examinations of this reaction suggest cooperative behaviour of a phosphorus-nitrogen-phosphorus pincer ligand in rate-determining heterolytic hydrogen splitting.

  8. Thermodynamic proton-ligand and metal-ligand stability constants of some drugs.

    PubMed

    Agrawal, Y K; Patel, D R

    1986-02-01

    The thermodynamic proton-ligand (pKa) and metal-ligand stability constants of clioquinol, clofibrate, nitrofurazone, and tetracycline with Cu2+, Zn2+, Mn2+, Mg2+, and Ca2+ have been determined at 35 degrees C in 50% ethanol-water media. An empirical pH correction for mixed-aqueous media has been applied. The metal-ligand stability constants were determined by following the Bjerrum Calvin titration technique as applied by Agrawal to mixed-aqueous solvents. The effect of the basicity of the ligand and the order of stability constants is discussed. The stability constants of the divalent metals follow the order: Cu2+ greater than Zn2+ greater than Mn2+ greater than Mg2+ greater than Ca2+ with all the drugs.

  9. Dissociation of Multisubunit Protein-Ligand Complexes in the Gas Phase. Evidence for Ligand Migration

    NASA Astrophysics Data System (ADS)

    Zhang, Yixuan; Deng, Lu; Kitova, Elena N.; Klassen, John S.

    2013-10-01

    The results of collision-induced dissociation (CID) experiments performed on gaseous protonated and deprotonated ions of complexes of cholera toxin B subunit homopentamer (CTB5) with the pentasaccharide (β-D-Gal p-(1→3)-β-D-Gal pNAc-(1→4)[α-D-Neu5Ac-(2→3)]-β-D-Gal p-(1→4)-β-D-Glc p (GM1)) and corresponding glycosphingolipid (β-D-Gal p-(1→3)-β-D-Gal pNAc-(1→4)[α-D-Neu5Ac-(2→3)]-β-D-Gal p-(1→4)-β-D-Glc p-Cer (GM1-Cer)) ligands, and the homotetramer streptavidin (S4) with biotin (B) and 1,2-dipalmitoyl- sn-glycero-3-phosphoethanolamine-N-(biotinyl) (Btl), are reported. The protonated (CTB5 + 5GM1)n+ ions dissociated predominantly by the loss of a single subunit, with the concomitant migration of ligand to another subunit. The simultaneous loss of ligand and subunit was observed as a minor pathway. In contrast, the deprotonated (CTB5 + 5GM1)n- ions dissociated preferentially by the loss of deprotonated ligand; the loss of ligand-bound and ligand-free subunit were minor pathways. The presence of ceramide (Cer) promoted ligand migration and the loss of subunit. The main dissociation pathway for the protonated and deprotonated (S4 + 4B)n+/- ions, as well as for deprotonated (S4 + 4Btl)n- ions, was loss of the ligand. However, subunit loss from the (S4 + 4B)n+ ions was observed as a minor pathway. The (S4 + 4Btl)n+ ions dissociated predominantly by the loss of free and ligand-bound subunit. The charge state of the complex and the collision energy were found to have little effect on the relative contribution of the different dissociation channels. Thermally-driven ligand migration between subunits was captured in the results of molecular dynamics simulations performed on protonated (CTB5 + 5GM1)15+ ions (with a range of charge configurations) at 800 K. Notably, the migration pathway was found to be highly dependent on the charge configuration of the ion. The main conclusion of this study is that the dissociation pathways of multisubunit protein-ligand

  10. Wnt ligands from the embryonic surface ectoderm regulate 'bimetallic strip' optic cup morphogenesis in mouse.

    PubMed

    Carpenter, April C; Smith, April N; Wagner, Heidi; Cohen-Tayar, Yamit; Rao, Sujata; Wallace, Valerie; Ashery-Padan, Ruth; Lang, Richard A

    2015-03-01

    The Wnt/β-catenin response pathway is central to many developmental processes. Here, we assessed the role of Wnt signaling in early eye development using the mouse as a model system. We showed that the surface ectoderm region that includes the lens placode expressed 12 out of 19 possible Wnt ligands. When these activities were suppressed by conditional deletion of wntless (Le-cre; Wls(fl/fl)) there were dramatic consequences that included a saucer-shaped optic cup, ventral coloboma, and a deficiency of periocular mesenchyme. This phenotype shared features with that produced when the Wnt/β-catenin pathway co-receptor Lrp6 is mutated or when retinoic acid (RA) signaling in the eye is compromised. Consistent with this, microarray and cell fate marker analysis identified a series of expression changes in genes known to be regulated by RA or by the Wnt/β-catenin pathway. Using pathway reporters, we showed that Wnt ligands from the surface ectoderm directly or indirectly elicit a Wnt/β-catenin response in retinal pigment epithelium (RPE) progenitors near the optic cup rim. In Le-cre; Wls(fl/fl) mice, the numbers of RPE cells are reduced and this can explain, using the principle of the bimetallic strip, the curvature of the optic cup. These data thus establish a novel hypothesis to explain how differential cell numbers in a bilayered epithelium can lead to shape change.

  11. Quantum.Ligand.Dock: protein–ligand docking with quantum entanglement refinement on a GPU system

    PubMed Central

    Kantardjiev, Alexander A.

    2012-01-01

    Quantum.Ligand.Dock (protein–ligand docking with graphic processing unit (GPU) quantum entanglement refinement on a GPU system) is an original modern method for in silico prediction of protein–ligand interactions via high-performance docking code. The main flavour of our approach is a combination of fast search with a special account for overlooked physical interactions. On the one hand, we take care of self-consistency and proton equilibria mutual effects of docking partners. On the other hand, Quantum.Ligand.Dock is the the only docking server offering such a subtle supplement to protein docking algorithms as quantum entanglement contributions. The motivation for development and proposition of the method to the community hinges upon two arguments—the fundamental importance of quantum entanglement contribution in molecular interaction and the realistic possibility to implement it by the availability of supercomputing power. The implementation of sophisticated quantum methods is made possible by parallelization at several bottlenecks on a GPU supercomputer. The high-performance implementation will be of use for large-scale virtual screening projects, structural bioinformatics, systems biology and fundamental research in understanding protein–ligand recognition. The design of the interface is focused on feasibility and ease of use. Protein and ligand molecule structures are supposed to be submitted as atomic coordinate files in PDB format. A customization section is offered for addition of user-specified charges, extra ionogenic groups with intrinsic pKa values or fixed ions. Final predicted complexes are ranked according to obtained scores and provided in PDB format as well as interactive visualization in a molecular viewer. Quantum.Ligand.Dock server can be accessed at http://87.116.85.141/LigandDock.html. PMID:22669908

  12. Quantum.Ligand.Dock: protein-ligand docking with quantum entanglement refinement on a GPU system.

    PubMed

    Kantardjiev, Alexander A

    2012-07-01

    Quantum.Ligand.Dock (protein-ligand docking with graphic processing unit (GPU) quantum entanglement refinement on a GPU system) is an original modern method for in silico prediction of protein-ligand interactions via high-performance docking code. The main flavour of our approach is a combination of fast search with a special account for overlooked physical interactions. On the one hand, we take care of self-consistency and proton equilibria mutual effects of docking partners. On the other hand, Quantum.Ligand.Dock is the the only docking server offering such a subtle supplement to protein docking algorithms as quantum entanglement contributions. The motivation for development and proposition of the method to the community hinges upon two arguments-the fundamental importance of quantum entanglement contribution in molecular interaction and the realistic possibility to implement it by the availability of supercomputing power. The implementation of sophisticated quantum methods is made possible by parallelization at several bottlenecks on a GPU supercomputer. The high-performance implementation will be of use for large-scale virtual screening projects, structural bioinformatics, systems biology and fundamental research in understanding protein-ligand recognition. The design of the interface is focused on feasibility and ease of use. Protein and ligand molecule structures are supposed to be submitted as atomic coordinate files in PDB format. A customization section is offered for addition of user-specified charges, extra ionogenic groups with intrinsic pK(a) values or fixed ions. Final predicted complexes are ranked according to obtained scores and provided in PDB format as well as interactive visualization in a molecular viewer. Quantum.Ligand.Dock server can be accessed at http://87.116.85.141/LigandDock.html.

  13. All-inorganic Germanium nanocrystal films by cationic ligand exchange

    DOE PAGES

    Wheeler, Lance M.; Nichols, Asa W.; Chernomordik, Boris D.; ...

    2016-01-21

    In this study, we introduce a new paradigm for group IV nanocrystal surface chemistry based on room temperature surface activation that enables ionic ligand exchange. Germanium nanocrystals synthesized in a gas-phase plasma reactor are functionalized with labile, cationic alkylammonium ligands rather than with traditional covalently bound groups. We employ Fourier transform infrared and 1H nuclear magnetic resonance spectroscopies to demonstrate the alkylammonium ligands are freely exchanged on the germanium nanocrystal surface with a variety of cationic ligands, including short inorganic ligands such as ammonium and alkali metal cations. This ionic ligand exchange chemistry is used to demonstrate enhanced transport inmore » germanium nanocrystal films following ligand exchange as well as the first photovoltaic device based on an all-inorganic germanium nanocrystal absorber layer cast from solution. This new ligand chemistry should accelerate progress in utilizing germanium and other group IV nanocrystals for optoelectronic applications.« less

  14. All-Inorganic Germanium Nanocrystal Films by Cationic Ligand Exchange.

    PubMed

    Wheeler, Lance M; Nichols, Asa W; Chernomordik, Boris D; Anderson, Nicholas C; Beard, Matthew C; Neale, Nathan R

    2016-03-09

    We introduce a new paradigm for group IV nanocrystal surface chemistry based on room temperature surface activation that enables ionic ligand exchange. Germanium nanocrystals synthesized in a gas-phase plasma reactor are functionalized with labile, cationic alkylammonium ligands rather than with traditional covalently bound groups. We employ Fourier transform infrared and (1)H nuclear magnetic resonance spectroscopies to demonstrate the alkylammonium ligands are freely exchanged on the germanium nanocrystal surface with a variety of cationic ligands, including short inorganic ligands such as ammonium and alkali metal cations. This ionic ligand exchange chemistry is used to demonstrate enhanced transport in germanium nanocrystal films following ligand exchange as well as the first photovoltaic device based on an all-inorganic germanium nanocrystal absorber layer cast from solution. This new ligand chemistry should accelerate progress in utilizing germanium and other group IV nanocrystals for optoelectronic applications.

  15. All-inorganic Germanium nanocrystal films by cationic ligand exchange

    SciTech Connect

    Wheeler, Lance M.; Nichols, Asa W.; Chernomordik, Boris D.; Anderson, Nicholas C.; Beard, Matthew C.; Neale, Nathan R.

    2016-01-21

    In this study, we introduce a new paradigm for group IV nanocrystal surface chemistry based on room temperature surface activation that enables ionic ligand exchange. Germanium nanocrystals synthesized in a gas-phase plasma reactor are functionalized with labile, cationic alkylammonium ligands rather than with traditional covalently bound groups. We employ Fourier transform infrared and 1H nuclear magnetic resonance spectroscopies to demonstrate the alkylammonium ligands are freely exchanged on the germanium nanocrystal surface with a variety of cationic ligands, including short inorganic ligands such as ammonium and alkali metal cations. This ionic ligand exchange chemistry is used to demonstrate enhanced transport in germanium nanocrystal films following ligand exchange as well as the first photovoltaic device based on an all-inorganic germanium nanocrystal absorber layer cast from solution. This new ligand chemistry should accelerate progress in utilizing germanium and other group IV nanocrystals for optoelectronic applications.

  16. Affinity Regulates Spatial Range of EGF Receptor Autocrine Ligand Binding

    SciTech Connect

    Dewitt, Ann; Iida, Tomoko; Lam, Ho-Yan; Hill, Virginia; Wiley, H S.; Lauffenburger, Douglas A.

    2002-08-08

    Proper spatial localization of EGFR signaling activated by autocrine ligands represents a critical factor in embryonic development as well as tissue organization and function, and ligand/receptor binding affinity is among the molecular and cellular properties suggested to play a role in governing this localization. The authors employ a computational model to predict how receptor-binding affinity affects local capture of autocrine ligand vis-a-vis escape to distal regions, and provide experimental test by constructing cell lines expressing EGFR along with either wild-type EGF or a low-affinity mutant, EGF{sup L47M}. The model predicts local capture of a lower affinity autocrine ligand to be less efficient when the ligand production rate is small relative to receptor appearance rate. The experimental data confirm this prediction, demonstrating that cells can use ligand/receptor binding affinity to regulate ligand spatial distribution when autocrine ligand production is limiting for receptor signaling.

  17. Fusion of ligand-coated nanoparticles with lipid bilayers: effect of ligand flexibility.

    PubMed

    Van Lehn, Reid C; Alexander-Katz, Alfredo

    2014-08-07

    Amphiphilic, monolayer-protected gold nanoparticles (AuNPs) have recently been shown to insert into and fuse with lipid bilayers, driven by the hydrophobic effect. The inserted transmembrane state is stabilized by the "snorkeling" of charged ligand end groups out of the bilayer interior. This snorkeling process is facilitated by the backbone flexibility of the alkanethiol ligands that comprise the monolayer. In this work, we show that fusion is favorable even in the absence of backbone flexibility by modeling the ligands as rigid rods. For rigid ligands, snorkeling is still accommodated by rotations of the ligand with respect to the grafting point, but the process incurs a more significant free energy penalty than if the backbone were fully flexible. We show that the rigid rod model predicts similar trends in the free energy change for insertion as the previous flexible model when the size of the AuNPs is varied. However, the rigidity of the ligand backbone reduces the overall magnitude of the free energy change compared to that of the flexible model. These results thus generalize previous findings to systems with hindered backbone flexibility due to either structural constraints or low temperature.

  18. 9-methoxycanthin-6-one production in elicited hairy roots culture of Eurycoma longifolia

    NASA Astrophysics Data System (ADS)

    Abdullah, Nazirah; Ismail, Ismanizan; Hassan, Nor Hasnida; Basherudin, Norlia

    2016-11-01

    Eurycoma longifolia (Tongkat Ali) is a highly sought after medicinal plant in Malaysia. Propagation of E. longifolia through tissue culture has been reported in order to cater the industry demands for planting and raw materials as well as for conservation purposes. E. longifolia hairy roots culture has been developed using Agrobacterium rhizogenes for the production of Tongkat Ali phytochemicals. Effects of three elicitors; methyl jasmonate, salicylic acid, and yeast extract at different concentrations were evaluated on the production of 9-methoxycanthin-6-one in E. longifolia hairy roots. The cultures were elicited at early exponential growth phase, followed by extraction of 9-methoxycanthin-6-one using methanol and HPLC analysis. Elicitation with methyl jasmonate at all concentrations increased 9-methoxycanthin-6-one up to 1-3 fold and treatment with (0.1 mM) was most efficient in enhancing 9-methoxycanthin-6-one production up to 3.902 mg/g dry weight after 7 days (168 hours) elicitation.

  19. Ecstasy and methamphetamine elicit action potential bursts via different mechanisms in a central snail neuron.

    PubMed

    Lin, Pei-Lin; Tsai, Ming-Cheng; Lu, Guan-Ling; Lu, Dah-Yuu; Chuang, Chieh-Min; Yang, Han-Yin; Huang, Shiang-Suo; Chen, Yi-Hung

    2010-01-01

    This study sought to determine the effects of (+) methamphetamine (METH) and its ring-substituted analog (+/-)3,4-methylenedioxymethamphetamine (MDMA; ecstasy) on electrophysiological behavior and their relationships to second messenger systems in an identifiable RP4 neuron of the African snail, Achatina fulica Ferussac. Extracellular application of MDMA at 1mM and METH at 3mM elicited action potential bursts that were not blocked after immersing the neurons in Ca(2+)-free solution. Notably, MDMA- (1mM) elicited action potential bursts were blocked by pretreatment with the protein kinase C (PKC) inhibitors chelerythrine (20 microM) and Ro 31-8220 (20 microM), but not by the PKA inhibitors KT-5720 (10 microM) and H89 (10 microM). The PKC activator phorbol 12,13-dibutyrate (PDBu; 3 microM), but not the PKA activator forskolin (50 microM), facilitated the induction of bursts elicited by MDMA at a lower concentration (0.3mM). In contrast, METH- (3mM) elicited action potential bursts were blocked by pretreatment with KT-5720 (10 microM) and H89 (10 microM), but not by chelerythrine (20 microM) and Ro 31-8220 (20 microM). Forskolin (50 microM), but not PDBu (3 microM) facilitated the induction of bursts elicited by METH at a lower concentration (1mM). Tetraethylammonium chloride (TEA), a blocker of the delayed rectifying K(+) current (I(KD)), did not elicit bursts at a concentration of 5mM but did facilitate the induction of action potential bursts elicited by both METH and MDMA. Voltage clamp studies revealed that both METH and MDMA decreased the TEA-sensitive I(KD) of the RP4 neuron. Forskolin (50 microM) or dibutyryl cAMP (1mM), a membrane-permeable cAMP analog, alone did not elicit action potential bursts. However, co-administration with forskolin (50 microM) and TEA (5mM) or co-administration with dibutyryl cAMP (1mM) and TEA (50mM) elicited action potential bursts in the presence of the PKC inhibitor chelerythrine (20 microM). Similarly, PDBu (10 microM) or phorbol

  20. Naturalistic observations of elicited expressive communication of children with autism: an analysis of teacher instructions.

    PubMed

    Chiang, Hsu-Min

    2009-03-01

    This study observed expressive communication of 17 Australian and 15 Taiwanese children with autism who were mute or had limited spoken language during 2 hour regular school routines and analyzed teacher instructions associated with elicited expressive communication. Results indicated: (a) the frequency of occurrence of elicited expressive communication was very low; (b) the incidence of elicited expressive communication was negatively correlated with autism severity; (c) verbal prompt and a combination of verbal prompt and modeling were the most common types of teacher instruction and the use of physical prompt was a rate event; (d) modeling and verbal prompt were positively correlated with speech and unaided augmentative and alternative communication (AAC) and a combination of verbal prompt and modeling was positively associated with aided AAC; and (e) modeling, verbal prompt, and a combination of modeling and verbal prompt were positively correlated with requesting function and commenting function was positively correlated with modeling and verbal prompt.

  1. Auditory modulation of wind-elicited walking behavior in the cricket Gryllus bimaculatus.

    PubMed

    Fukutomi, Matasaburo; Someya, Makoto; Ogawa, Hiroto

    2015-12-01

    Animals flexibly change their locomotion triggered by an identical stimulus depending on the environmental context and behavioral state. This indicates that additional sensory inputs in different modality from the stimulus triggering the escape response affect the neuronal circuit governing that behavior. However, how the spatio-temporal relationships between these two stimuli effect a behavioral change remains unknown. We studied this question, using crickets, which respond to a short air-puff by oriented walking activity mediated by the cercal sensory system. In addition, an acoustic stimulus, such as conspecific 'song' received by the tympanal organ, elicits a distinct oriented locomotion termed phonotaxis. In this study, we examined the cross-modal effects on wind-elicited walking when an acoustic stimulus was preceded by an air-puff and tested whether the auditory modulation depends on the coincidence of the direction of both stimuli. A preceding 10 kHz pure tone biased the wind-elicited walking in a backward direction and elevated a threshold of the wind-elicited response, whereas other movement parameters, including turn angle, reaction time, walking speed and distance were unaffected. The auditory modulations, however, did not depend on the coincidence of the stimulus directions. A preceding sound consistently altered the wind-elicited walking direction and response probability throughout the experimental sessions, meaning that the auditory modulation did not result from previous experience or associative learning. These results suggest that the cricket nervous system is able to integrate auditory and air-puff stimuli, and modulate the wind-elicited escape behavior depending on the acoustic context.

  2. Amino Acids in Nine Ligand-Prefer Ramachandran Regions.

    PubMed

    Cao, Chen; Wang, Lincong; Chen, Xiaoyang; Zou, Shuxue; Wang, Guishen; Xu, Shutan

    2015-01-01

    Several secondary structures, such as π-helix and left-handed helix, have been frequently identified at protein ligand-binding sites. A secondary structure is considered to be constrained to a specific region of dihedral angles. However, a comprehensive analysis of the correlation between main chain dihedral angles and ligand-binding sites has not been performed. We undertook an extensive analysis of the relationship between dihedral angles in proteins and their distance to ligand-binding sites, frequency of occurrence, molecular potential energy, amino acid composition, van der Waals contacts, and hydrogen bonds with ligands. The results showed that the values of dihedral angles have a strong preference for ligand-binding sites at certain regions in the Ramachandran plot. We discovered that amino acids preceding the ligand-prefer ϕ/ψ box residues are exposed more to solvents, whereas amino acids following ligand-prefer ϕ/ψ box residues form more hydrogen bonds and van der Waals contacts with ligands. Our method exhibited a similar performance compared with the program Ligsite-csc for both ligand-bound structures and ligand-free structures when just one ligand-binding site was predicted. These results should be useful for the prediction of protein ligand-binding sites and for analysing the relationship between structure and function.

  3. Design and Implementation of Tools to Increase User Control and Knowledge Elicitation in a Virtual Battlespace.

    DTIC Science & Technology

    1994-11-01

    S ELU.gTE, JAN 0 3 1994% OF ’Origi•,l oOntainzs colorplates: All DTIC reproduot.. ions will be in black and white- DESIGN AND IMPLEMENTATION OF TOOLS ...1I994 DESIGN AND IMPLEMENTATION OF TOOLS TO INCREASE USER CONTROL AND KNOWLEDGE ELICITATION IN A VIRTUAL BATTLESPACE _SFJ7 h- For THESIS •! -Jim J...AFIT/GCS/ENG/94-20 Design and Implementation of Tools to Increase User Control and Knowledge Elicitation in a Virtual Battlespace THESIS Presented to

  4. The Relation Between Molecular Weight of Antigen and Ability to Elicit Passive Cutaneous Anaphylaxis*

    PubMed Central

    Leskowitz, S.; Ovary, Z.

    1962-01-01

    Passive cutaneous anaphylaxis in the guinea pig has been studied with rabbit antibody to a series of antigens of differing molecular weight. The results indicated that at a given antibody level the weight of antigen needed to elicit a reaction increases with its molecular weight. Previous observations have been confirmed that the amount of antigen needed to elicit a reaction at a high level of antibody is less than that required at a lower level. The results suggest that extremely small amounts of small molecular weight antigens might be sufficient to produce anaphylactic symptoms in highly sensitive individuals. PMID:14464304

  5. Intensity versus type of affect elicited by dissociative cognitions in moderating acute pain.

    PubMed

    Stevens, M J; Rogers, R M

    1990-12-01

    75 undergraduates were trained to use cognitions that elicited either high or low pleasure, high or low fear, or received an expectancy manipulation. Groups high in pleasure showed greater tolerance for pressure pain than other treatment groups but did not differ from expectancy; differences were not found on discomfort ratings. Results were not attributable to differential compliance with instructions, perceived effectiveness of cognitions used, or the number of cognitions used. The percentage of time during stimulation that assigned cognitions were used appeared to mediate tolerance. Expectations for improved tolerance elicited by appealing cognitions and compelling placebos may also mediate tolerance.

  6. Expert Elicitation Methods in Quantifying the Consequences of Acoustic Disturbance from Offshore Renewable Energy Developments.

    PubMed

    Donovan, Carl; Harwood, John; King, Stephanie; Booth, Cormac; Caneco, Bruno; Walker, Cameron

    2016-01-01

    There are many developments for offshore renewable energy around the United Kingdom whose installation typically produces large amounts of far-reaching noise, potentially disturbing many marine mammals. The potential to affect the favorable conservation status of many species means extensive environmental impact assessment requirements for the licensing of such installation activities. Quantification of such complex risk problems is difficult and much of the key information is not readily available. Expert elicitation methods can be employed in such pressing cases. We describe the methodology used in an expert elicitation study conducted in the United Kingdom for combining expert opinions based on statistical distributions and copula-like methods.

  7. The interplay of elicitation and evaluation of trait-expressive behavior: Evidence in assessment center exercises.

    PubMed

    Lievens, Filip; Schollaert, Eveline; Keen, Gert

    2015-07-01

    In assessment centers (ACs), research on eliciting candidate behavior and evaluating candidate behavior have largely followed independent paths. This study integrates trait activation and trait rating models to posit hypotheses about the effects of behavior elicitation via situational cues on key assessor observation and rating variables. To test the hypotheses, a series of experimental and field studies are conducted. Only when trait-expressive behavior activation and evaluation models work in conjunction, increases in observability are coupled with increases in the interrater reliability, convergent validity, discriminant validity, and accuracy of AC ratings. Implications of these findings for AC theory and practice are formulated.

  8. Tritherapy (Spinalon)-Elicited Spinal Locomotor Network Activation: Phase I-IIa Clinical Trial in Spinal Cord-Injured Patients

    DTIC Science & Technology

    2013-10-01

    PROTOCOL SPIN-01 Tri- therapy (SPINALON)-elicited spinal locomotor network activation: Phase I-Ila clinicaltrials in spinalcord-injured patients Clinical...STUDY) described in the Protocol SPIN-01 (PROTOCOL) being entitled: "Tri- therapy (SPINALON)-elicited spinal locomotor network activation: Phase I...Report SC100155 SPIN-01 Tri- therapy (SPINALON)-elicited spinal locomotor network activation: Phase I-IIa clinical trials in spinal cord-injured

  9. Enhanced Ligand Sampling for Relative Protein–Ligand Binding Free Energy Calculations

    PubMed Central

    2016-01-01

    Free energy calculations are used to study how strongly potential drug molecules interact with their target receptors. The accuracy of these calculations depends on the accuracy of the molecular dynamics (MD) force field as well as proper sampling of the major conformations of each molecule. However, proper sampling of ligand conformations can be difficult when there are large barriers separating the major ligand conformations. An example of this is for ligands with an asymmetrically substituted phenyl ring, where the presence of protein loops hinders the proper sampling of the different ring conformations. These ring conformations become more difficult to sample when the size of the functional groups attached to the ring increases. The Adaptive Integration Method (AIM) has been developed, which adaptively changes the alchemical coupling parameter λ during the MD simulation so that conformations sampled at one λ can aid sampling at the other λ values. The Accelerated Adaptive Integration Method (AcclAIM) builds on AIM by lowering potential barriers for specific degrees of freedom at intermediate λ values. However, these methods may not work when there are very large barriers separating the major ligand conformations. In this work, we describe a modification to AIM that improves sampling of the different ring conformations, even when there is a very large barrier between them. This method combines AIM with conformational Monte Carlo sampling, giving improved convergence of ring populations and the resulting free energy. This method, called AIM/MC, is applied to study the relative binding free energy for a pair of ligands that bind to thrombin and a different pair of ligands that bind to aspartyl protease β-APP cleaving enzyme 1 (BACE1). These protein–ligand binding free energy calculations illustrate the improvements in conformational sampling and the convergence of the free energy compared to both AIM and AcclAIM. PMID:25906170

  10. Protein-ligand binding affinity determination by the waterLOGSY method: An optimised approach considering ligand rebinding

    PubMed Central

    Huang, Renjie; Bonnichon, Arnaud; Claridge, Timothy D. W.; Leung, Ivanhoe K. H.

    2017-01-01

    WaterLOGSY is a popular ligand-observed NMR technique to screen for protein-ligand interactions, yet when applied to measure dissociation constants (KD) through ligand titration, the results were found to be strongly dependent on sample conditions. Herein, we show that accurate KDs can be obtained by waterLOGSY with optimised experimental setup. PMID:28256624

  11. Protein-ligand binding affinity determination by the waterLOGSY method: An optimised approach considering ligand rebinding

    NASA Astrophysics Data System (ADS)

    Huang, Renjie; Bonnichon, Arnaud; Claridge, Timothy D. W.; Leung, Ivanhoe K. H.

    2017-03-01

    WaterLOGSY is a popular ligand-observed NMR technique to screen for protein-ligand interactions, yet when applied to measure dissociation constants (KD) through ligand titration, the results were found to be strongly dependent on sample conditions. Herein, we show that accurate KDs can be obtained by waterLOGSY with optimised experimental setup.

  12. Diamine Ligands in Copper-Catalyzed Reactions

    PubMed Central

    Surry, David S.

    2012-01-01

    The utility of copper-mediated cross-coupling reactions has been significantly increased by the development of mild reaction conditions and the ability to employ catalytic amounts of copper. The use of diamine-based ligands has been important in these advances and in this review we discuss these systems, including the choice of reaction conditions and applications in the synthesis of pharmaceuticals, natural products and designed materials. PMID:22384310

  13. Container molecules based on imine type ligands.

    PubMed

    Schulze, A Carina; Oppel, Iris M

    2012-01-01

    This chapter will give a short overview about container molecules, their synthesis and possible applications. The main focus is on those which are based on imine type ligands. These containers can be used for example for guest exchange, gas separation, as chemical sensors or for the stabilisation of white phosphorus under water. The described cages have wide openings or tightly closed ones. For one cage the reversible opening and closing is also described.

  14. galectin-3 ligand — EDRN Public Portal

    Cancer.gov

    Galectin-3 is an endogenous lectin that binds glycan epitopes of cell membrane and some extracellular glycoproteins such as integrins and laminin. Galectin-3 is involved in several biological activities including regulation of cellular cycle, modulation of adhesion and tumor progression and metastasis. Serum galectin-3 ligands have been shown to modulate the immune reaction against tumors and viruses and their level increases in sera of several neoplastic diseases.

  15. Selective oxoanion separation using a tripodal ligand

    DOEpatents

    Custelcean, Radu; Moyer, Bruce A.; Rajbanshi, Arbin

    2016-02-16

    The present invention relates to urea-functionalized crystalline capsules self-assembled by sodium or potassium cation coordination and by hydrogen-bonding water bridges to selectively encapsulate tetrahedral divalent oxoanions from highly competitive aqueous alkaline solutions and methods using this system for selective anion separations from industrial solutions. The method involves competitive crystallizations using a tripodal tris(urea) functionalized ligand and, in particular, provides a viable approach to sulfate separation from nuclear wastes.

  16. Efficient identification of murine M2 macrophage peptide targeting ligands by phage display and next-generation sequencing

    PubMed Central

    Liu, Gary W.; Livesay, Brynn R.; Kacherovsky, Nataly A.; Cieslewicz, Maryelise; Lutz, Emi; Waalkes, Adam; Jensen, Michael C.; Salipante, Stephen J.; Pun, Suzie H.

    2015-01-01

    Peptide ligands are used to increase the specificity of drug carriers to their target cells and to facilitate intracellular delivery. One method to identify such peptide ligands, phage display, enables high-throughput screening of peptide libraries for ligands binding to therapeutic targets of interest. However, conventional methods for identifying target binders in a library by Sanger sequencing are low-throughput, labor-intensive, and provide a limited perspective (< 0.01%) of the complete sequence space. Moreover, the small sample space can be dominated by non-specific, preferentially amplifying “parasitic sequences” and plastic-binding sequences, which may lead to the identification of false positives or exclude the identification of target-binding sequences. To overcome these challenges, we employed next-generation Illumina sequencing to couple high-throughput screening and high-throughput sequencing, enabling more comprehensive access to the phage display library sequence space. In this work, we define the hallmarks of binding sequences in next-generation sequencing data, and develop a method that identifies several target-binding phage clones for murine, alternatively-activated (M2) macrophages with a high (100%) success rate: sequences and binding motifs were reproducibly present across biological replicates; binding motifs were identified across multiple unique sequences; and an unselected, amplified library accurately filtered out parasitic sequences. In addition, we validate the Multiple Em for Motif Elicitation tool as an efficient and principled means of discovering binding sequences. PMID:26161996

  17. Search for α3β₂/₃γ2 subtype selective ligands that are stable on human liver microsomes.

    PubMed

    Namjoshi, Ojas A; Wang, Zhi-jian; Rallapalli, Sundari K; Johnson, Edward Merle; Johnson, Yun-Teng; Ng, Hanna; Ramerstorfer, Joachim; Varagic, Zdravko; Sieghart, Werner; Majumder, Samarpan; Roth, Bryan L; Rowlett, James K; Cook, James M

    2013-01-01

    Selective modulation of specific benzodiazepine receptor (BzR) gamma amino butyric acid-A (GABA(A)) receptor ion channels has been identified as an important method for separating out the variety of pharmacological effects elicited by BzR-related drugs. Importantly, it has been demonstrated that both α2β(2/3)γ2 (α2BzR) and α3BzR (and/or α2/α3) BzR subtype selective ligands exhibit anxiolytic effects with little or no sedation. Previously we have identified several such ligands; however, three of our parent ligands exhibited significant metabolic liability in rodents in the form of a labile ester group. Here eight analogs are reported which were designed to circumvent this liability by utilizing a rational replacement of the ester moiety based on medicinal chemistry precedents. In a metabolic stability study using human liver microsomes, four compounds were found to undergo slower metabolic transformation, as compared to their corresponding ester analogs. These compounds were also evaluated in in vitro efficacy assays. Additionally, bioisostere 11 was evaluated in a rodent model of anxiety. It exhibited anxiolytic activity at doses of 10 and 100mg/kg and was devoid of sedative properties.

  18. Systemic administration of a TLR7 ligand leads to transient immune incompetence due to peripheral-blood leukocyte depletion.

    PubMed

    Gunzer, Matthias; Riemann, Helge; Basoglu, Yasmin; Hillmer, Anja; Weishaupt, Carsten; Balkow, Sandra; Benninghoff, Bernd; Ernst, Beat; Steinert, Meike; Scholzen, Thomas; Sunderkötter, Cord; Grabbe, Stephan

    2005-10-01

    Toll-like receptor (TLR) ligands lead to the induction of proinflammatory cytokines and are potent enhancers of specific immune responses. We show here that a single systemic dose of R-848, a ligand for TLR7, potently enhanced hapten sensitization during the induction of contact hypersensitivity (CHS). However, R-848 administration also resulted in a rapid and almost complete depletion of leukocytes from the blood. This effect was transient and was associated with general induction of endothelial adhesiveness. In response to R-848, endothelial cells up-regulated adhesion molecules in vitro and in vivo and leukocytes exhibited increased rolling on endothelia in R-848-treated animals. Adhesion molecule induction appeared to be a direct effect, because endothelial cells expressed TLR7 in vitro and in vivo. After R-848 treatment, the tissue residence time of leukocytes was markedly prolonged in all major peripheral organs. The resulting transiently reduced availability of peripheral-blood leukocytes (PBLs) (TRAP) significantly inhibited otherwise potent CHS responses until the effector cells returned. Thus, although TLR7 ligands are effective adjuvants for the induction of cell-mediated immunity, they can transiently inhibit the elicitation of localized immune responses, possibly due to a systemic endothelial activation throughout the vasculature.

  19. Gated escaping of ligand out of protein

    NASA Astrophysics Data System (ADS)

    Sheu, Sheh-Yi; Yang, Dah-Yen

    2000-01-01

    We construct a new gating model and develop a new theory to study the escaping process of a ligand out of a spherical cavity with a puncture (or gate) on the surface. The gate undulation can be regulated by any time-dependent function and the motion of the ligand inside the spherical cavity is mapped into a two-dimensional entropy potential surface. Hence the driving force of our model is entropy only. For a static gate, the escaping process corresponds to climbing a two-dimensional entropy barrier. When the gate open angle is small, the escaping rate is proportional to the square of the opening angle. The prefactor of the escaping rate constant depends on the curvature of the entropy potential surface. For coherent gating, the survival time depends not only on the gate undulation frequency but also on how the initial state is defined. On the escaping from protein, our escaping rate shows it is qualitatively consistent with the experimental result of ligand recombination in myoglobin.

  20. RAGE and its ligands in retinal disease.

    PubMed

    Barile, Gaetano R; Schmidt, Ann M

    2007-12-01

    RAGE, the receptor for advanced glycation endproducts (AGEs), is a multiligand signal transduction receptor of the immunoglobulin superfamily of cell surface molecules that has been implicated in the pathogenesis of diabetic complications, neurodegenerative diseases, inflammatory disorders, and cancer. These diverse biologic disorders reflect the multiplicity of ligands capable of cellular interaction via RAGE that include, in addition to AGEs, amyloid-beta (Abeta) peptide, the S100/calgranulin family of proinflammatory cytokines, and amphoterin, a member of the High Mobility Group Box (HMGB) DNA-binding proteins. In the retina, RAGE expression is present in neural cells, the vasculature, and RPE cells, and it has also been detected in pathologic cellular retinal responses including epiretinal and neovascular membrane formation. Ligands for RAGE, in particular AGEs, have emerged as relevant to the pathogenesis of diabetic retinopathy and age-related macular disease. While the understanding of RAGE and its role in retinal dysfunction with aging, diabetes mellitus, and/or activation of pro-inflammatory pathways is less complete compared to other organ systems, increasing evidence indicates that RAGE can initiate and sustain significant cellular perturbations in the inner and outer retina. For these reasons, antagonism of RAGE interactions with its ligands may be a worthwhile therapeutic target in such seemingly disparate, visually threatening retinal diseases as diabetic retinopathy, age-related macular degeneration, and proliferative vitreoretinopathy.

  1. “PCB153-Elicited Hepatic Responses in the Immature, Ovariectomized C57BL/6 Mice: Comparative Toxicogenomic Effects of Dioxin and Non-Dioxin-Like Ligands”

    PubMed Central

    Kopec, Anna K.; Burgoon, Lyle D.; Ibrahim-Aibo, Daher; Mets, Bryan D.; Tashiro, Colleen; Potter, Dave; Sharratt, Bonnie; Harkema, Jack R.; Zacharewski, Timothy

    2010-01-01

    Polychlorinated biphenyls (PCBs) are ubiquitous contaminants found as complex mixtures of coplanar and non-coplanar congeners. The hepatic temporal and dose-dependent effects of the most abundant non-dioxin-like congener, 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB153), were examined in immature, ovariectomized C57BL/6 mice, and compared to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the prototypical aryl hydrocarbon receptor (AhR) ligand. Animals were gavaged once with 300 mg/kg PCB153 or sesame oil vehicle and sacrificed 4, 12, 24, 72 or 168 h post dose. In the dose-response study, mice were gavaged with 1, 3, 10, 30, 100 or 300 mg/kg PCB153 or sesame oil for 24 h. Significant increases in relative liver weights were induced with 300 mg/kg PCB153 between 24 and 168 h, accompanied by slight vacuolization and hepatocellular hypertrophy. The hepatic differential expression of 186 and 177 genes was detected using Agilent 4 x 44 K microarrays in the time course (|fold change|≥1.5, P1(t)≥0.999) and dose-response (|fold change|≥1.5, P1(t)≥0.985) studies, respectively. Comparative analysis with TCDD suggests that the differential gene expression elicited by PCB153 was not mediated by the AhR. Furthermore, constitutive androstane and pregnane X receptor (CAR/PXR) regulated genes including Cyp2b10, Cyp3a11, Ces2, Insig2 and Abcc3 were dose-dependently induced by PCB153. Collectively, these results suggest that the hepatocellular effects elicited by PCB153 are qualitatively and quantitatively different from TCDD and suggestive of CAR/PXR regulation. PMID:20005886

  2. Polyethylenimine-based siRNA nanocomplexes reprogram tumor-associated dendritic cells via TLR5 to elicit therapeutic antitumor immunity

    PubMed Central

    Cubillos-Ruiz, Juan R.; Engle, Xavier; Scarlett, Uciane K.; Martinez, Diana; Barber, Amorette; Elgueta, Raul; Wang, Li; Nesbeth, Yolanda; Durant, Yvon; Gewirtz, Andrew T.; Sentman, Charles L.; Kedl, Ross; Conejo-Garcia, Jose R.

    2009-01-01

    The success of clinically relevant immunotherapies requires reversing tumor-induced immunosuppression. Here we demonstrated that linear polyethylenimine-based (PEI-based) nanoparticles encapsulating siRNA were preferentially and avidly engulfed by regulatory DCs expressing CD11c and programmed cell death 1–ligand 1 (PD-L1) at ovarian cancer locations in mice. PEI-siRNA uptake transformed these DCs from immunosuppressive cells to efficient antigen-presenting cells that activated tumor-reactive lymphocytes and exerted direct tumoricidal activity, both in vivo and in situ. PEI triggered robust and selective TLR5 activation in vitro and elicited the production of hallmark TLR5-inducible cytokines in WT mice, but not in Tlr5–/– littermates. Thus, PEI is a TLR5 agonist that, to our knowledge, was not previously recognized. In addition, PEI-complexed nontargeting siRNA oligonucleotides stimulated TLR3 and TLR7. The nonspecific activation of multiple TLRs (specifically, TLR5 and TLR7) reversed the tolerogenic phenotype of human and mouse ovarian tumor–associated DCs. In ovarian carcinoma–bearing mice, this induced T cell–mediated tumor regression and prolonged survival in a manner dependent upon myeloid differentiation primary response gene 88 (MyD88; i.e., independent of TLR3). Furthermore, gene-specific siRNA-PEI nanocomplexes that silenced immunosuppressive molecules on mouse tumor-associated DCs elicited discernibly superior antitumor immunity and enhanced therapeutic effects compared with nontargeting siRNA-PEI nanocomplexes. Our results demonstrate that the intrinsic TLR5 and TLR7 stimulation of siRNA-PEI nanoparticles synergizes with the gene-specific silencing activity of siRNA to transform tumor-infiltrating regulatory DCs into DCs capable of promoting therapeutic antitumor immunity. PMID:19620771

  3. Presynaptic calcium stores contribute to nicotine-elicited potentiation of evoked synaptic transmission at CA3-CA1 connections in the neonatal rat hippocampus.

    PubMed

    Le Magueresse, Corentin; Cherubini, Enrico

    2007-01-01

    Nicotine acetylcholine (ACh) receptors (nAChRs) are ligand-gated ion channels that are widely expressed throughout the central nervous system. It is well established that presynaptic, alpha7-containing nAChRs modulate glutamate release in several brain areas, and that this modulation requires extracellular calcium. However, the intracellular mechanisms consecutive to nAChR opening are unclear. Recent studies have suggested a role for presynaptic calcium stores in the increase of neurotransmitter release following nAChR activation. Using the minimal stimulation protocol at low-probability Schaffer collateral synapses in acute hippocampal slices from neonatal rats, we show that nicotine acting on presynaptic alpha7 nAChRs persistently upregulates glutamate release. We tested the role of calcium stores in this potentiation. First, we examined the relationship between calcium stores and glutamate release. We found that bath application of SERCA pump inhibitors (cyclopiazonic acid and thapsigargin), as well as an agonist of ryanodine receptors (ryanodine 2 microM) increases the probability of glutamate release at CA3-CA1 synapses, decreases the coefficient of variation and the paired-pulse ratio, indicating that presynaptic activation of calcium-induced calcium release can modulate glutamatergic transmission. Next, we investigated whether blocking calcium release from internal stores could alter the effect of nicotine. Preincubation with thapsigargin (10 microM), cyclopiazonic acid (30 microM), or with a high (blocking) concentration of ryanodine (100 microM) for 30 min to 5 h failed to block the effect of nicotine. However, after preincubation in ryanodine, nicotine-elicited potentiation was significantly shortened. These results indicate that at immature Schaffer collateral-CA1 synapses, activation of presynaptic calcium stores is not necessary for but contributes to nicotine-elicited increase of neurotransmitter release.

  4. Modeling of the Aryl Hydrocarbon Receptor (AhR) ligand binding domain and its utility in virtual ligand screening to predict new AhR ligands

    PubMed Central

    Bisson, William; Koch, Daniel; O’Donnell, Edmond; Khalil, Sammy M.; Kerkvliet, Nancy; Tanguay, Robert; Abagyan, Ruben; Kolluri, Siva Kumar

    2012-01-01

    The Aryl Hydrocarbon Receptor (AhR) is a ligand-activated transcription factor; the AhR Per-AhR/Arnt-Sim (PAS) domain binds ligands. We developed homology models of the AhR PAS domain to characterize previously observed intra- and inter-species differences in ligand binding using Molecular Docking. In silico structure-based virtual ligand screening using our model resulted in the identification of pinocembrin and 5-hydroxy-7-methoxyflavone, which promoted nuclear translocation and transcriptional activation of AhR and AhR-dependent induction of endogenous target genes. PMID:19719119

  5. Assessment of automatic ligand building in ARP/wARP

    SciTech Connect

    Evrard, Guillaume X. Langer, Gerrit G.; Lamzin, Victor S.

    2007-01-01

    The performance of the ligand-building module of the ARP/wARP software suite is assessed through a large-scale test on known protein–ligand complexes. The results provide a detailed benchmark and guidelines for future improvements. The efficiency of the ligand-building module of ARP/wARP version 6.1 has been assessed through extensive tests on a large variety of protein–ligand complexes from the PDB, as available from the Uppsala Electron Density Server. Ligand building in ARP/wARP involves two main steps: automatic identification of the location of the ligand and the actual construction of its atomic model. The first step is most successful for large ligands. The second step, ligand construction, is more powerful with X-ray data at high resolution and ligands of small to medium size. Both steps are successful for ligands with low to moderate atomic displacement parameters. The results highlight the strengths and weaknesses of both the method of ligand building and the large-scale validation procedure and help to identify means of further improvement.

  6. The Recognition of Identical Ligands by Unrelated Proteins

    PubMed Central

    Barelier, Sarah; Sterling, Teague; O'Meara, Matthew J.; Shoichet, Brian K.

    2015-01-01

    The binding of drugs and reagents to off-targets is well-known. Whereas many off-targets are related to the primary target by sequence and fold, many ligands bind to unrelated pairs of proteins, and these are harder to anticipate. If the binding site in the off-target can be related to that of the primary target, this challenge resolves into aligning the two pockets. However, other cases are possible: the ligand might interact with entirely different residues and environments in the off-target, or wholly different ligand atoms may be implicated in the two complexes. To investigate these scenarios at atomic resolution, the structures of 59 ligands in 116 complexes (62 pairs in total), where the protein pairs were unrelated by fold but bound an identical ligand, were examined. In almost half of the pairs, the ligand interacted with unrelated residues in the two proteins (29 pairs), and in 14 of the pairs wholly different ligand moieties were implicated in each complex. Even in those 19 pairs of complexes that presented similar environments to the ligand, ligand superposition rarely resulted in the overlap of related residues. There appears to be no single pattern-matching “code” for identifying binding sites in unrelated proteins that bind identical ligands, though modeling suggests that there might be a limited number of different patterns that suffice to recognize different ligand functional groups. PMID:26421501

  7. Eliciting Children's Recall of Events: How Do Computers Compare with Humans?

    ERIC Educational Resources Information Center

    Powell, Martine B.; Wilson, J. Clare; Thomson, Donald M.

    2002-01-01

    Describes a study that investigated the usefulness of an interactive computer program in eliciting children's reports about an event. Compared results of interviews by computer with interviews with humans with children aged five through eight that showed little benefit in computers over face-to-face interviews. (Author/LRW)

  8. Comparison of Increasingly Detailed Elicitation Methods for the Assessment of Adverse Events in Pediatric Psychopharmacology.

    ERIC Educational Resources Information Center

    Greenhill, Laurence L.; Vitiello, Benedetto; Fisher, Prudence; Levine, Jerome; Davies, Mark; Abikoff, Howard; Chrisman, Allan K.; Chuang, Shirley; Findling, Robert L.; March, John; Scahill, Lawrence; Walkup, John; Riddle, Mark A.

    2004-01-01

    Objective: To improve the gathering of adverse events (AEs) in pediatric psychopharmacology by examining the value and acceptability of increasingly detailed elicitation methods. Method: Trained clinicians administered the Safety Monitoring Uniform Report Form (SMURF) to 59 parents and outpatients (mean age [+ or -] SD = 11.9 [+ or -] 3.2 years)…

  9. Immune response elicited by the oral administration of an intermediate strain of IBDV in chickens

    PubMed Central

    Carballeda, Juan Manuel; Zoth, Silvina Chimeno; Gómez, Evangelina; Lucero, María Soledad; Gravisaco, María José; Berinstein, Analía

    2014-01-01

    The immune response elicited by the oral inoculation of an intermediate strain of infectious bursal disease virus was studied in chickens. A strong over expression of IL-6, IL-8, IFNα and IFNγ was observed in bursa at 3 days post inoculation together with an increase in splenic NO2 release. An influx of T-lymphocytes was also detected. PMID:25763062

  10. Infection-derived lipids elicit a novel immune deficiency circuitry in arthropods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The insect Immune Deficiency (IMD) pathway resembles the tumor necrosis factor receptor network in mammals and senses diaminopimelic-type peptidoglycans present in Gram-negative bacteria. Whether unidentified chemical moieties elicit the IMD signaling cascade remains unknown. Here, we disclose thoug...

  11. Elicited Imitation as a Measure of Second Language Proficiency: A Narrative Review and Meta-Analysis

    ERIC Educational Resources Information Center

    Yan, Xun; Maeda, Yukiko; Lv, Jing; Ginther, April

    2016-01-01

    Elicited imitation (EI) has been widely used to examine second language (L2) proficiency and development and was an especially popular method in the 1970s and early 1980s. However, as the field embraced more communicative approaches to both instruction and assessment, the use of EI diminished, and the construct-related validity of EI scores as a…

  12. Deriving Childhood Temperament Measures from Emotion-Eliciting Behavioral Episodes: Scale Construction and Initial Validation

    ERIC Educational Resources Information Center

    Gagne, Jeffrey R.; Van Hulle, Carol A.; Aksan, Nazan; Essex, Marilyn J.; Goldsmith, H. Hill

    2011-01-01

    The authors describe the development and initial validation of a home-based version of the Laboratory Temperament Assessment Battery (Lab-TAB), which was designed to assess childhood temperament with a comprehensive series of emotion-eliciting behavioral episodes. This article provides researchers with general guidelines for assessing specific…

  13. Differential Neural Activity during Search of Specific and General Autobiographical Memories Elicited by Musical Cues

    ERIC Educational Resources Information Center

    Ford, Jaclyn Hennessey; Addis, Donna Rose; Giovanello, Kelly S.

    2011-01-01

    Previous neuroimaging studies that have examined autobiographical memory specificity have utilized retrieval cues associated with prior searches of the event, potentially changing the retrieval processes being investigated. In the current study, musical cues were used to naturally elicit memories from multiple levels of specificity (i.e., lifetime…

  14. Collage Life Story Elicitation Technique: A Representational Technique for Scaffolding Autobiographical Memories

    ERIC Educational Resources Information Center

    van Schalkwyk, Gertina J.

    2010-01-01

    A basic premise in narrative therapy and inquiry is that life story telling is a mechanism by which experiences are rendered meaningful within some form of structure. However, narrative inquiry has to take cognisance of difficulties ensuing from discursive practices for different populations when eliciting their life stories. In this article I…

  15. Does Technology Elicit Desired Behaviors in Emotionally Disturbed Students?: Perceptions of Elementary Educators

    ERIC Educational Resources Information Center

    Donnelly, Michael W.

    2016-01-01

    The purpose of this mixed methods study was to identify the perceptions of educators regarding the potential impact of technology as a motivator to elicit desirable behaviors within students that have been identified with an educational diagnosis of emotional disturbance at the elementary school level. A review of the literature focused on key…

  16. Elicited Response in the Pre-Kindergarten Setting: Good or Bad Idea?

    ERIC Educational Resources Information Center

    Montague, Nicole S.; Meza-Zaragosa, Elsa

    This paper argues in favor of dual language programs over typical bilingual education programs because in the dual language program the minority language has a status equal to the majority language. Through the use of the language experience approach, this study examines the role of elicited response from a small sample of English and Spanish…

  17. Fostering and Eliciting Emergent Literacy Skills in Potentially English Proficient Students.

    ERIC Educational Resources Information Center

    Beers, Elizabeth A.

    This report describes an early intervention program to elicit emergent literacy skills in Potentially English Proficient kindergarten students. The school is located in a suburb of a large city near a naval base. The problem, lack of literacy readiness skills, was documented with a battery of tests given prior to entrance in kindergarten and the…

  18. Using Model-Eliciting Activities as a Tool to Identify and Develop Mathematically Creative Students

    ERIC Educational Resources Information Center

    Coxbill, Emmy; Chamberlin, Scott A.; Weatherford, Jennifer

    2013-01-01

    Traditional classroom methods for identifying mathematically creative students have been inadequate. Identifying students who could potentially be mathematically creative is instrumental in the development of students and in meeting their affective and educational needs. One prospective identification tool is the use of model-eliciting activities…

  19. Model-Eliciting Activities as a Tool to Develop and Identify Creatively Gifted Mathematicians

    ERIC Educational Resources Information Center

    Chamberlin, Scott A.; Moon, Sidney M.

    2005-01-01

    This article addresses the use of Model-Eliciting Activities (MEAs) as a (curricular) tool to develop mathematical creativity and identify students who are creatively gifted in mathematics. The thesis of this article is that by using MEAs, gifted educators can: (a) provide students with opportunities to develop creative and applied mathematical…

  20. Sequential Immunization Elicits Broadly Neutralizing Anti-HIV-1 Antibodies in Ig Knockin Mice.

    PubMed

    Escolano, Amelia; Steichen, Jon M; Dosenovic, Pia; Kulp, Daniel W; Golijanin, Jovana; Sok, Devin; Freund, Natalia T; Gitlin, Alexander D; Oliveira, Thiago; Araki, Tatsuya; Lowe, Sarina; Chen, Spencer T; Heinemann, Jennifer; Yao, Kai-Hui; Georgeson, Erik; Saye-Francisco, Karen L; Gazumyan, Anna; Adachi, Yumiko; Kubitz, Michael; Burton, Dennis R; Schief, William R; Nussenzweig, Michel C

    2016-09-08

    A vaccine that elicits broadly neutralizing antibodies (bNAbs) against HIV-1 is likely to be protective, but this has not been achieved. To explore immunization regimens that might elicit bNAbs, we produced and immunized mice expressing the predicted germline PGT121, a bNAb specific for the V3-loop and surrounding glycans on the HIV-1 spike. Priming with an epitope-modified immunogen designed to activate germline antibody-expressing B cells, followed by ELISA-guided boosting with a sequence of directional immunogens, native-like trimers with decreasing epitope modification, elicited heterologous tier-2-neutralizing responses. In contrast, repeated immunization with the priming immunogen did not. Antibody cloning confirmed elicitation of high levels of somatic mutation and tier-2-neutralizing antibodies resembling the authentic human bNAb. Our data establish that sequential immunization with specifically designed immunogens can induce high levels of somatic mutation and shepherd antibody maturation to produce bNAbs from their inferred germline precursors.

  1. "Dare I Ask?": Eliciting Prior Knowledge and Its Implications for Teaching and Learning

    ERIC Educational Resources Information Center

    Dávila, Liv Thorstensson

    2015-01-01

    This article examines high school teachers' engagement of newcomer English learner students' prior knowledge. Three central research questions guided this study: 1) To what extent do teachers function as mediators of their students' prior knowledge? 2) What goes into teachers' thinking about how and when to elicit prior knowledge? and 3) How do…

  2. Use of expert judgment elicitation to estimate seismic vulnerability of selected building types

    USGS Publications Warehouse

    Jaiswal, K.S.; Aspinall, W.; Perkins, D.; Wald, D.; Porter, K.A.

    2012-01-01

    Pooling engineering input on earthquake building vulnerability through an expert judgment elicitation process requires careful deliberation. This article provides an overview of expert judgment procedures including the Delphi approach and the Cooke performance-based method to estimate the seismic vulnerability of a building category.

  3. Assessing Measurement Invariance for Spanish Sentence Repetition and Morphology Elicitation Tasks

    ERIC Educational Resources Information Center

    Kapantzoglou, Maria; Thompson, Marilyn S.; Gray, Shelley; Restrepo, M. Adelaida

    2016-01-01

    Purpose: The purpose of this study was to evaluate evidence supporting the construct validity of two grammatical tasks (sentence repetition, morphology elicitation) included in the Spanish Screener for Language Impairment in Children (Restrepo, Gorin, & Gray, 2013). We evaluated if the tasks measured the targeted grammatical skills in the same…

  4. A drug-paired taste cue elicits withdrawal and predicts cocaine self-administration.

    PubMed

    Nyland, Jennifer E; Grigson, Patricia S

    2013-03-01

    Addiction is a chronic disease where periods of abstinence are riddled with instances of craving, withdrawal, and eventual relapse to escalated drug use. Cues previously associated with drug use can have a deleterious effect on this cycle by precipitating withdrawal symptoms. Here we focus specifically on the relationship between avoidance of a drug-paired taste cue and the ability of the drug-paired cue to elicit withdrawal and, ultimately, drug seeking and taking. We used a rat model of drug addiction and naloxone-induced loss of body weight to test whether a taste cue elicits withdrawal in anticipation of drug availability. Experiment 1 investigated the ability of a taste cue to elicit signs of withdrawal when it predicted experimenter-administered morphine (15 mg/kg, i.p.). In Experiment 2, a saccharin taste cue was paired with the opportunity to actively self-administer cocaine (0.167 mg/infusion, i.v.). The results show that presentation of a morphine- or cocaine-paired taste cue is sufficient to elicit naloxone-induced withdrawal symptoms, and greater withdrawal predicts greater cocaine self-administration in rats.

  5. Eliciting, Identifying, Interpreting, and Responding to Students' Ideas: Teacher Candidates' Growth in Formative Assessment Practices

    ERIC Educational Resources Information Center

    Gotwals, Amelia Wenk; Birmingham, Daniel

    2016-01-01

    With the goal of helping teacher candidates become well-started beginners, it is important that methods courses in teacher education programs focus on high-leverage practices. Using responsive teaching practices, specifically eliciting, identifying, interpreting, and responding to students' science ideas (i.e., formative assessment), can be used…

  6. Differences between Spontaneous and Elicited Expressive Communication in Children with Autism

    ERIC Educational Resources Information Center

    Chiang, Hsu-Min

    2009-01-01

    The purpose of this study was to investigate the differences between spontaneous and elicited expressive communication in Australian and Taiwanese children with autism who were nonverbal or had limited speech. Thirty-four children with autism (17 Australian and 17 Taiwanese children) participated in this study. Each participant was observed for 2…

  7. Business Process Elicitation, Modeling, and Reengineering: Teaching and Learning with Simulated Environments

    ERIC Educational Resources Information Center

    Jeyaraj, Anand

    2010-01-01

    The design of enterprise information systems requires students to master technical skills for elicitation, modeling, and reengineering business processes as well as soft skills for information gathering and communication. These tacit skills and behaviors cannot be effectively taught students but rather experienced and learned by students. This…

  8. Elicitation and Reformulation and Their Relationship with Learner Repair in Dyadic Interaction

    ERIC Educational Resources Information Center

    Nassaji, Hossein

    2007-01-01

    This research investigates the usefulness of two major types of interactional feedback (elicitation and reformulation) in dyadic interaction. The focus is on the different ways in which each feedback type is provided and their relationship with learner repair. The participants were 42 adult intermediate English as a second language learners and…

  9. On a Method of Eliciting True/False Judgments from Young Children

    ERIC Educational Resources Information Center

    Lloyd, Peter; Donaldson, Margaret

    1976-01-01

    Experiments in eliciting true/false judgments from young children aged 3-5 used a "talking doll," a toy panda with a speaker installed. The procedure has been used in studies of language comprehension, communication skills, and free conversation experiments. (CHK)

  10. The Experimental State of Mind in Elicitation: Illustrations from Tonal Fieldwork

    ERIC Educational Resources Information Center

    Yu, Kristine M.

    2014-01-01

    This paper illustrates how an "experimental state of mind", i.e. principles of experimental design, can inform hypothesis generation and testing in structured fieldwork elicitation. The application of these principles is demonstrated with case studies in toneme discovery. Pike's classic toneme discovery procedure is shown to be a special…

  11. Linguistic Proficiency Assessment in Second Language Acquisition Research: The Elicited Imitation Task

    ERIC Educational Resources Information Center

    Gaillard, Stéphanie; Tremblay, Annie

    2016-01-01

    This study investigated the elicited imitation task (EIT) as a tool for measuring linguistic proficiency in a second/foreign (L2) language, focusing on French. Nonnative French speakers (n = 94) and native French speakers (n = 6) completed an EIT that included 50 sentences varying in length and complexity. Three raters evaluated productions on…

  12. Dilemmas in Continuing Professional Learning: Learning Inscribed in Frameworks or Elicited from Practice

    ERIC Educational Resources Information Center

    Reich, Ann; Rooney, Donna; Boud, David

    2015-01-01

    This paper explores a dilemma in continuing professional learning: the way learning is typically inscribed in continuing professional education (CPE) frameworks differs from that elicited from practice. It examines these differences in relation to both different underlying assumptions about learning and varying epistemological perspectives as well…

  13. Prospective Elementary Mathematics Teachers' Thought Processes on a Model Eliciting Activity

    ERIC Educational Resources Information Center

    Eraslan, Ali

    2012-01-01

    Mathematical model and modeling are one of the topics that have been intensively discussed in recent years. The purpose of this study is to examine prospective elementary mathematics teachers' thought processes on a model eliciting activity and reveal difficulties or blockages in the processes. The study includes forty-five seniors taking the…

  14. Early Morphological Productivity in Hungarian: Evidence from Sentence Repetition and Elicited Production

    ERIC Educational Resources Information Center

    Gabor, Balint; Lukacs, Agnes

    2012-01-01

    This paper investigates early productivity of morpheme use in Hungarian children aged between 2 ; 1 and 5 ; 3. Hungarian has a rich morphology which is the core marker of grammatical functions. A new method is introduced using the novel word paradigm in a sentence repetition task with masked inflections (i.e. a disguised elicited production task).…

  15. Keeping the Hands in Mind: What Elicited Pantomime Reveals about Language Structure

    ERIC Educational Resources Information Center

    Hall, Matthew L.

    2012-01-01

    This dissertation contains three studies that investigate whether attested patterns of constituent order distribution and change in the world's languages can be attributed, in part, to cognitive preferences for some constituent orders over others. To assess these preferences, seven experiments employed an "elicited pantomime" task.…

  16. Quantifying Aluminum Crystal Size Part 1: The Model-Eliciting Activity

    ERIC Educational Resources Information Center

    Diefes-Dux, Heidi A.; Hjalmarson, Margret; Zawojewski, Judith S.; Bowman, Keith

    2006-01-01

    Model-eliciting activities (MEA), specially designed client-drive, open-ended problems, have been implemented in a first-year engineering course and in secondary schools. The educational goals and settings are different, but the design of an MEA enables it to be versatile. This paper will introduce the reader to the principles that guide MEA…

  17. Exploring Technology as a Tool for Eliciting and Encouraging Beginning Teacher Reflection

    ERIC Educational Resources Information Center

    Romano, Molly; Schwartz, Jonathan

    2005-01-01

    As beginning teachers experience and process new information during their initial acts of teaching, reflection is an inherent part of the process. The following study was designed to explore technology as a tool for reflection by introducing first-year teachers to three technology tools designed to elicit and encourage their reflections on…

  18. Using Art Elicitation to Deliver and Evaluate a Grade 4 Climate Change Instructional Module

    ERIC Educational Resources Information Center

    Baker, Jillian; Loxton, Jason; Sherren, Kate

    2013-01-01

    We report the results of a climate change module delivered to 48 Grade 3/4 students in Nova Scotia, Canada. We tested for prior knowledge and evaluated interventional effectiveness using art elicitation. Common climate change misconceptions were demonstrated in their preintervention artwork, while postintervention artwork showed improved…

  19. Speech Error Elicitation and Co-Occurrence Restrictions in Two Ethiopian Semitic Languages

    ERIC Educational Resources Information Center

    Rose, Sharon; King, Lisa

    2007-01-01

    This article reports the results of speech error elicitation experiments investigating the role of two consonant co-occurrence restrictions in the productive grammar of speakers of two Ethiopian Semitic languages, Amharic and Chaha. Higher error rates were found with consonant combinations that violated co-occurrence constraints than with those…

  20. Comparing Elicited Imitation and Word Monitoring as Measures of Implicit Knowledge

    ERIC Educational Resources Information Center

    Suzuki, Yuichi; DeKeyser, Robert

    2015-01-01

    The present study challenges the validity of elicited imitation (EI) as a measure for implicit knowledge, investigating to what extent online error detection and subsequent sentence repetition draw on implicit knowledge. To assess online detection during listening, a word monitoring component was built into an EI task. Advanced-level Japanese L2…

  1. Eliciting and Developing Teachers' Conceptions of Random Processes in a Probability and Statistics Course

    ERIC Educational Resources Information Center

    Smith, Toni M.; Hjalmarson, Margret A.

    2013-01-01

    The purpose of this study is to examine prospective mathematics specialists' engagement in an instructional sequence designed to elicit and develop their understandings of random processes. The study was conducted with two different sections of a probability and statistics course for K-8 teachers. Thirty-two teachers participated. Video analyses…

  2. Elicitation Techniques: Getting People to Talk about Ideas They Don't Usually Talk About

    ERIC Educational Resources Information Center

    Barton, Keith C.

    2015-01-01

    Elicitation techniques are a category of research tasks that use visual, verbal, or written stimuli to encourage participants to talk about their ideas. These tasks are particularly useful for exploring topics that may be difficult to discuss in formal interviews, such as those that involve sensitive issues or rely on tacit knowledge. Elicitation…

  3. Sample Size for Measuring Grammaticality in Preschool Children From Picture-Elicited Language Samples

    PubMed Central

    Guo, Ling-Yu

    2015-01-01

    Purpose The purpose of this study was to investigate whether a shorter language sample elicited with fewer pictures (i.e., 7) would yield a percent grammatical utterances (PGU) score similar to that computed from a longer language sample elicited with 15 pictures for 3-year-old children. Method Language samples were elicited by asking forty 3-year-old children with varying language skills to talk about pictures in response to prompts. PGU scores were computed for each of two 7-picture sets and for the full set of 15 pictures. Results PGU scores for the two 7-picture sets did not differ significantly from, and were highly correlated with, PGU scores for the full set and with each other. Agreement for making pass–fail decisions between each 7-picture set and the full set and between the two 7-picture sets ranged from 80% to 100%. Conclusion The current study suggests that the PGU measure is robust enough that it can be computed on the basis of 7, at least in 3-year-old children whose language samples were elicited using similar procedures. PMID:25615691

  4. Children with Autism Respond Differently to Spontaneous, Elicited and Deferred Imitation

    ERIC Educational Resources Information Center

    Heimann, M.; Nordqvist, E.; Strid, K.; Connant Almrot, J.; Tjus, T.

    2016-01-01

    Background: Imitation, a key vehicle for both cognitive and social development, is often regarded as more difficult for children with autism spectrum disorders (ASD) than for children with Down syndrome (DS) or typically developing (TD) children. The current study investigates similarities and differences in observed elicited, spontaneous and…

  5. Emotion-Eliciting Qualities of Television Campaign Advertising as a Predictor of Voting Behavior.

    ERIC Educational Resources Information Center

    Masterson, John T.; Biggers, Thompson

    1986-01-01

    Relates political candidates' television advertising to voters' emotional reactions and voting behavior. Suggests that television campaign advertising elicits emotion along three continua: pleasure-displeasure, arousal-nonarousal, and dominance-submissiveness. Suggests that emotional response to such advertising is systematically related to voting…

  6. Effect of schedule of reinforcement on cue-elicited reinstatement of cocaine-seeking behavior.

    PubMed

    Acosta, Jazmin I; Thiel, Kenneth J; Sanabria, Federico; Browning, Jenny R; Neisewander, Janet L

    2008-03-01

    Cocaine-associated cues can elicit incentive motivational effects that drive cocaine-seeking behavior and contribute to relapse. The extinction/reinstatement model is commonly used to measure these effects in animals. This study examined the influence of training and testing schedules of reinforcement on cue-elicited reinstatement. Lever presses during training resulted in cues and cocaine (0.75 mg/kg/IV) on either continuous or partial reinforcement schedules [fixed ratio (FR) 1 or 11, variable ratio (VR) 5 or 11]. Animals then underwent extinction training, followed by a test for cue-elicited reinstatement of extinguished cocaine-seeking behavior by response-contingent cue presentations on either a continuous (FR 1) or a partial reinforcement schedule (FR 11). Partial reinforcement during training resulted in higher response rates during cue-elicited reinstatement relative to continuous reinforcement. In contrast, delivery of cues on a continuous reinforcement schedule during testing yielded higher response rates relative to delivery on a partial reinforcement schedule. Finally, the shift from a partial to a continuous reinforcement schedule across training and testing phases did not alter response rates. These findings provide important information for choosing parameters for reinstatement of drug-seeking behavior that would allow the most sensitive method to detect changes in response rate after an experimental manipulation.

  7. Feedback on Feedback: Eliciting Learners' Responses to Written Feedback through Student-Generated Screencasts

    ERIC Educational Resources Information Center

    Fernández-Toro, María; Furnborough, Concha

    2014-01-01

    Despite the potential benefits of assignment feedback, learners often fail to use it effectively. This study examines the ways in which adult distance learners engage with written feedback on one of their assignments. Participants were 10 undergraduates studying Spanish at the Open University, UK. Their responses to feedback were elicited by means…

  8. Production Deficiencies in Elicited Language but Not in the Spontaneous Verbalizations of Hyperactive Children.

    ERIC Educational Resources Information Center

    Zentall, Sydney S.

    1988-01-01

    Twenty-two hyperactive children were more spontaneously talkative than controls during transitions and nonverbal tasks (nonelicited conditions), but less talkative when asked to tell stories (elicited conditions). Findings suggest that minimal stimulus input precipitate excessive verbal activity from hyperactive children, while stories requiring…

  9. Using a Text-Based Verbal Protocol to Elicit Secondary English Teachers' Perspectives on New Literacies

    ERIC Educational Resources Information Center

    Lewis, Elizabeth C.; Chandler-Olcott, Kelly

    2012-01-01

    This article reports on the use of a text-based verbal protocol (Afflerbach, 2000) situated within individual interviews to elicit secondary English teachers' perspectives on new literacies (Albers & Harste, 2007; Coiro, Knobel, Lankshear, & Leu, 2008). Ranging significantly in their teaching experience and comfort with new media and…

  10. A Comparison of English Reading Passages for Elicitation of Speech Samples from Clinical Populations

    ERIC Educational Resources Information Center

    Powell, Thomas W.

    2006-01-01

    Oral reading passages are often used to elicit speech samples from clinical populations. Few objective guidelines exist, however, to guide one's selection from among the many existing passages. Therefore, this study was undertaken to describe phonetic, lexical, and structural characteristics of 15 oral reading passages. The passages differed…

  11. Improving the Communication Skills of IS Developers during Requirements Elicitation Using Experiential Learning

    ERIC Educational Resources Information Center

    Qurban, Mustafa H.; Austria, Richmond D.

    2009-01-01

    The improvement of communication skills among Information Systems (IS) developers can be considered as a strategy to mitigate the risk of project failure during IS design. This paper addresses issues on various communication barriers normally encountered during its requirements elicitation (RE) stage. This study aims to adopt experiential learning…

  12. Identification of Ligand-Receptor Interactions: Ligand Molecular Arrays, SPR and NMR Methodologies.

    PubMed

    Day, Christopher J; Hartley-Tassell, Lauren E; Korolik, Victoria

    2017-01-01

    Despite many years of research into bacterial chemotaxis, the only well characterized system to date is that of E. coli. Even for E. coli, the direct ligand binding had been fully characterized only for aspartate and serene receptors Tar and Tsr. In 30 years since, no other direct receptor-ligand interaction had been described for bacteria, until the characterization of the C. jejuni aspartate and multiligand receptors (Hartley-Tassell et al. Mol Microbiol 75:710-730, 2010). While signal transduction components of many sensory pathways have now been characterized, ligand-receptor interactions remain elusive due to paucity of high-throughput screening methods. Here, we describe the use of microarray screening we developed to identify ligands, surface plasmon resonance, and saturation transfer difference nuclear magnetic resonance (STD-NMR) we used to verify the hits and to determine the affinity constants of the interactions, allowing for more targeted verification of ligands with traditional chemotaxis and in vivo assays described in Chapter 13 .

  13. Bursts of potential elicited by d-amphetamine in central snail neuron: effect of sodium azide.

    PubMed

    Lin, Pei-Lin; Lu, Kuan-Ling; Lee, Ya-Ling; Chen, Yi-Hung; Chang, Yu-Chi; Chou, Hong-Nong; Tsai, Ming-Cheng

    2007-10-01

    Effects of sodium azide (NaN(3)) on spontaneously generated action potential and bursts of potential elicited by d-amphetamine (d-amphetamine-elicited BoP) were studied on the right parietal 4 (RP4) neuron of the snail Achatina fulica Ferussac in vitro. Sodium azide altered the spontaneous action potential of RP4 neuron in a concentration-dependent manner. In lower concentrations, neither NaN(3) (30, 100, 300 microM; 1 and 3 mM) nor d-amphetamine (135 microM) affect the resting membrane potential, amplitude and frequency of RP4 neurons, while in the higher concentrations NaN(3) (30 mM) did abolish the spontaneous action potential on RP4 neurons and depolarized the RP4 neurons reversibly. At lower concentration, NaN(3) (30 microM) facilitated the d-amphetamine-elicited BoP. The BoP elicited by NaN(3) (30 microM) and d-amphetamine (135 microM) were decreased following treatment with KT5720 (protein kinase A inhibitor), or intracellular injection of EGTA [ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid]. However, the BoP was not affected by applying U73122 (1-[6-[((17beta)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione) or neomycin (phospholipase inhibitors). Voltage clamp studies revealed that NaN(3) (30 microM) did not alter the total fast inwards currents (70 msec.) and the steady-state outwards currents (5 sec.). It appeared that the BoP elicited by NaN(3) (30 microM) and d-amphetamine (135 microM) was mainly due to protein kinase A-related messenger system and intracellular calcium. It is concluded that d-amphetamine-elicited BoP was not mainly due to inhibition of the function of mitochondria in the neuron while the function of mitochondria did alter the BoP elicited by amphetamine.

  14. Nanoparticle multivalency counterbalances the ligand affinity loss upon PEGylation.

    PubMed

    Hennig, Robert; Pollinger, Klaus; Veser, Anika; Breunig, Miriam; Goepferich, Achim

    2014-11-28

    The conjugation of receptor ligands to shielded nanoparticles is a widely used strategy to precisely control nanoparticle-cell interactions. However, it is often overlooked that a ligand's affinity can be severely impaired by its attachment to the polyethylene glycol (PEG) chains that are frequently used to protect colloids from serum protein adsorption. Using the model ligand EXP3174, a small-molecule antagonist for the angiotensin II receptor type 1 (AT1R), we investigated the ligand's affinity before and after its PEGylation and when attached to PEGylated nanoparticles. The PEGylated ligand displayed a 580-fold decreased receptor affinity compared to the native ligand. Due to their multivalency, the nanoparticles regained a low nanomolar receptor affinity, which is in the range of the affinity of the native ligand. Moreover, a four orders of magnitude higher concentration of free ligand was required to displace PEGylated nanoparticles carrying EXP3174 from the receptor. On average, one nanoparticle was decorated with 11.2 ligand molecules, which led to a multivalent enhancement factor of 22.5 compared to the monovalent PEGylated ligand. The targeted nanoparticles specifically bound the AT1R and showed no interaction to receptor negative cells. Our study shows that the attachment of a small-molecule ligand to a PEG chain can severely affect its receptor affinity. Concomitantly, when the ligand is tethered to nanoparticles, the immense avidity greatly increases the ligand-receptor interaction. Based on our results, we highly recommend the affinity testing of receptor ligands before and after PEGylation to identify potent molecules for active nanoparticle targeting.

  15. A chemogenomics view on protein-ligand spaces

    PubMed Central

    Strömbergsson, Helena; Kleywegt, Gerard J

    2009-01-01

    Background Chemogenomics is an emerging inter-disciplinary approach to drug discovery that combines traditional ligand-based approaches with biological information on drug targets and lies at the interface of chemistry, biology and informatics. The ultimate goal in chemogenomics is to understand molecular recognition between all possible ligands and all possible drug targets. Protein and ligand space have previously been studied as separate entities, but chemogenomics studies deal with large datasets that cover parts of the joint protein-ligand space. Since drug discovery has traditionally focused on ligand optimization, the chemical space has been studied extensively. The protein space has been studied to some extent, typically for the purpose of classification of proteins into functional and structural classes. Since chemogenomics deals not only with ligands but also with the macromolecules the ligands interact with, it is of interest to find means to explore, compare and visualize protein-ligand subspaces. Results Two chemogenomics protein-ligand interaction datasets were prepared for this study. The first dataset covers the known structural protein-ligand space, and includes all non-redundant protein-ligand interactions found in the worldwide Protein Data Bank (PDB). The second dataset contains all approved drugs and drug targets stored in the DrugBank database, and represents the approved drug-drug target space. To capture biological and physicochemical features of the chemogenomics datasets, sequence-based descriptors were computed for the proteins, and 0, 1 and 2 dimensional descriptors for the ligands. Principal component analysis (PCA) was used to analyze the multidimensional data and to create global models of protein-ligand space. The nearest neighbour method, computed using the principal components, was used to obtain a measure of overlap between the datasets. Conclusion In this study, we present an approach to visualize protein-ligand spaces from a

  16. Elicitation of broadly neutralizing influenza antibodies in animals with previous influenza exposure.

    PubMed

    Wei, Chih-Jen; Yassine, Hadi M; McTamney, Patrick M; Gall, Jason G D; Whittle, James R R; Boyington, Jeffrey C; Nabel, Gary J

    2012-08-15

    The immune system responds to influenza infection by producing neutralizing antibodies to the viral surface protein, hemagglutinin (HA), which regularly changes its antigenic structure. Antibodies that target the highly conserved stem region of HA neutralize diverse influenza viruses and can be elicited through vaccination in animals and humans. Efforts to develop universal influenza vaccines have focused on strategies to elicit such antibodies; however, the concern has been raised that previous influenza immunity may abrogate the induction of such broadly protective antibodies. We show here that prime-boost immunization can induce broadly neutralizing antibody responses in influenza-immune mice and ferrets that were previously infected or vaccinated. HA stem-directed antibodies were elicited in mice primed with a DNA vaccine and boosted with inactivated vaccine from H1N1 A/New Caledonia/20/1999 (1999 NC) HA regardless of preexposure. Similarly, gene-based vaccination with replication-defective adenovirus 28 (rAd28) and 5 (rAd5) vectors encoding 1999 NC HA elicited stem-directed neutralizing antibodies and conferred protection against unmatched 1934 and 2007 H1N1 virus challenge in influenza-immune ferrets. Indeed, previous exposure to certain strains could enhance immunogenicity: The strongest HA stem-directed immune response was observed in ferrets previously infected with a divergent 1934 H1N1 virus. These findings suggest that broadly neutralizing antibodies against the conserved stem region of HA can be elicited through vaccination despite previous influenza exposure, which supports the feasibility of developing stem-directed universal influenza vaccines for humans.

  17. Safety Risk Knowledge Elicitation in Support of Aeronautical R and D Portfolio Management: A Case Study

    NASA Technical Reports Server (NTRS)

    Shih, Ann T.; Ancel, Ersin; Jones, Sharon Monica; Reveley, Mary S.; Luxhoj, James T.

    2012-01-01

    Aviation is a problem domain characterized by a high level of system complexity and uncertainty. Safety risk analysis in such a domain is especially challenging given the multitude of operations and diverse stakeholders. The Federal Aviation Administration (FAA) projects that by 2025 air traffic will increase by more than 50 percent with 1.1 billion passengers a year and more than 85,000 flights every 24 hours contributing to further delays and congestion in the sky (Circelli, 2011). This increased system complexity necessitates the application of structured safety risk analysis methods to understand and eliminate where possible, reduce, and/or mitigate risk factors. The use of expert judgments for probabilistic safety analysis in such a complex domain is necessary especially when evaluating the projected impact of future technologies, capabilities, and procedures for which current operational data may be scarce. Management of an R&D product portfolio in such a dynamic domain needs a systematic process to elicit these expert judgments, process modeling results, perform sensitivity analyses, and efficiently communicate the modeling results to decision makers. In this paper a case study focusing on the application of an R&D portfolio of aeronautical products intended to mitigate aircraft Loss of Control (LOC) accidents is presented. In particular, the knowledge elicitation process with three subject matter experts who contributed to the safety risk model is emphasized. The application and refinement of a verbal-numerical scale for conditional probability elicitation in a Bayesian Belief Network (BBN) is discussed. The preliminary findings from this initial step of a three-part elicitation are important to project management practitioners as they illustrate the vital contribution of systematic knowledge elicitation in complex domains.

  18. Severity of dependence modulates smokers' neuronal cue reactivity and cigarette craving elicited by tobacco advertisement.

    PubMed

    Vollstädt-Klein, Sabine; Kobiella, Andrea; Bühler, Mira; Graf, Caroline; Fehr, Christoph; Mann, Karl; Smolka, Michael N

    2011-01-01

    Smoking-related cues elicit craving and mesocorticolimbic brain activation in smokers. Severity of nicotine dependence seems to moderate cue reactivity, but the direction and mechanisms of its influence remains unclear. Although tobacco control policies demand a ban on tobacco advertising, cue reactivity studies in smokers so far have not employed tobacco advertisement as experimental stimuli. We investigated whether tobacco advertisement elicits cue reactivity at a behavioral (subjective craving) and a neural level (using functional magnetic resonance imaging) in 22 smokers and 21 never-smokers. Moreover, we studied the influence of severity of dependence on cue reactivity. In smokers, tobacco advertisement elicited substantially more craving than control advertisement whereas never-smokers reported no cue induced craving. Surprisingly, neuronal cue reactivity did not differ between smokers and never-smokers. Moderately dependent smokers' craving increased over the course of the experiment, whereas highly dependent smokers' craving was unaffected. Moderately dependent smokers' brain activity elicited by tobacco advertisement was higher in the amygdala, hippocampus, putamen and thalamus compared with highly dependent smokers. Furthermore, limbic brain activation predicted picture recognition rates after the scanning session, even in never-smokers. Our findings show that tobacco advertisement elicits cigarette craving and neuronal cue reactivity primarily in moderately dependent smokers, indicating that they might be particularly responsive towards external smoking-related cues. On the other hand, neuronal cue reactivity and cigarette craving in highly dependent smokers is more likely triggered by internal cues such as withdrawal symptoms. Tobacco advertisement seems to likewise appeal to smokers and non-smokers, clarifying the potential danger especially for young non-smokers.

  19. Evaluation of small ligand-protein interaction by ligation reaction with DNA-modified ligand.

    PubMed

    Sugita, Rie; Mie, Masayasu; Funabashi, Hisakage; Kobatake, Eiry

    2010-01-01

    A method for the evaluation of interactions between protein and ligand using DNA-modified ligands, including signal enhancement of the DNA ligation reactions, is described. For proof of principle, a DNA probe modified by biotin was used. Two DNA probes were prepared with complementary sticky-ends. While one DNA probe was modified at the 5'-end of the sticky-end, the other was not modified. The probes could be ligated together by T4 DNA ligase along the strand without biotin modification. However, in the presence of streptavidin or anti-biotin Fab, the ligation reaction joining the two probes could not occur on either strand.

  20. Landscape of protein–small ligand binding modes

    PubMed Central

    Kinoshita, Kengo

    2016-01-01

    Abstract Elucidating the mechanisms of specific small‐molecule (ligand) recognition by proteins is a long‐standing conundrum. While the structures of these molecules, proteins and ligands, have been extensively studied, protein–ligand interactions, or binding modes, have not been comprehensively analyzed. Although methods for assessing similarities of binding site structures have been extensively developed, the methods for the computational treatment of binding modes have not been well established. Here, we developed a computational method for encoding the information about binding modes as graphs, and assessing their similarities. An all‐against‐all comparison of 20,040 protein–ligand complexes provided the landscape of the protein–ligand binding modes and its relationships with protein‐ and chemical spaces. While similar proteins in the same SCOP Family tend to bind relatively similar ligands with similar binding modes, the correlation between ligand and binding similarities was not very high (R 2 = 0.443). We found many pairs with novel relationships, in which two evolutionally distant proteins recognize dissimilar ligands by similar binding modes (757,474 pairs out of 200,790,780 pairs were categorized into this relationship, in our dataset). In addition, there were an abundance of pairs of homologous proteins binding to similar ligands with different binding modes (68,217 pairs). Our results showed that many interesting relationships between protein–ligand complexes are still hidden in the structure database, and our new method for assessing binding mode similarities is effective to find them. PMID:27327045