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Sample records for sensitizes androgen-independent human

  1. p38MAPK activation is involved in androgen-independent proliferation of human prostate cancer cells by regulating IL-6 secretion

    International Nuclear Information System (INIS)

    Shida, Yohei; Igawa, Tsukasa; Hakariya, Tomoaki; Sakai, Hideki; Kanetake, Hiroshi

    2007-01-01

    Increased levels of serum interleukin-6 (IL-6) are frequently observed in patients with advanced, hormone-refractory prostate cancer. However, the precise mechanism of IL-6 regulation is still largely unknown. Since prostate cancer gradually progresses to an androgen-independent state despite the stress caused by various therapeutic agents, we hypothesized the stress-activated protein kinases (SAPKs) involvement in androgen-independent growth or IL-6 secretion of prostate cancer cells. Using PC-3 and DU145 human prostate cancer cells, we analyzed the role of SAPKs in IL-6 mediated cell growth and found that the p38MAPK and JNK are involved in androgen-independent cancer cell growth. Furthermore, IL-6 secretion by PC-3 and DU145 cells was significantly suppressed by SAPKs inhibitor, especially by p38MAPK inhibitor SB203580, but not by JNK inhibitor SP600125 nor by MEK inhibitor, PD98059. These results raised the possibility that the IL-6 mediated androgen-independent proliferation of PC-3 and DU145 cells is regulated at least partly via SAPKs signaling pathway especially through p38MAPK activation

  2. Microwave mediated radiosynthesis of [F-18] FLT and its in-vitro study with androgen independent human prostate cancer cell line (PC-3)

    International Nuclear Information System (INIS)

    Ponde, D.E.; Dence, C.S.; Oyama, N.; Welch, M.J.

    2003-01-01

    The aim of this work was to improve the radiosynthesis of [F-18] FLT and to study its usefulness in monitoring change of proliferative activity of prostate cancer cells in the early phase of therapy. Method: Starting with anhydrothymidine, [F-18] FLT was synthesized by microwave mediated nucleophilic displacement by fluoride ion followed by acid hydrolysis in a synthesis time of just 55 minutes, which included Oasis solid phase and HPLC purification. The total radiochemical yield was 10-15% (at EOS), and the radiochemical purity was >99%. An in vitro study was carried out with androgen-independent human prostate cancer cell line PC-3. Two X 10e5 cells were seeded in 6 well plates with Ham's F-12K medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate supplemented with 10% heat activated FBS. One day later, PC-3 cells were at 50% confluent, the media was removed and the cells divided into two groups. In one group, cells were suspended in fresh media as above with 10% FBS, whereas in the other group cells were suspended in fresh media as above but without serum. Twenty-four hours later, [F-18] FLT was added to each flask (n=3). The cell-associated uptake of [F-18] FLT at 37 deg C was determined at 0, 1, 3, and 6 h after incubation. [F-18] FLT uptake in PC-3 cells decreased by 55% (from 9% to 4%) after 24h incubation with serum free media, indicating its potential usefulness to monitor cell proliferation in androgen-independent human prostate cancer. Studies to ascertain the uptake-mechanism are in the way. NIH grant HL13851

  3. Co-Targeting Prostate Cancer Epithelium and Bone Stroma by Human Osteonectin-Promoter-Mediated Suicide Gene Therapy Effectively Inhibits Androgen-Independent Prostate Cancer Growth.

    Directory of Open Access Journals (Sweden)

    Shian-Ying Sung

    Full Text Available Stromal-epithelial interaction has been shown to promote local tumor growth and distant metastasis. We sought to create a promising gene therapy approach that co-targets cancer and its supporting stromal cells for combating castration-resistant prostate tumors. Herein, we demonstrated that human osteonectin is overexpressed in the prostate cancer epithelium and tumor stroma in comparison with their normal counterpart. We designed a novel human osteonectin promoter (hON-522E containing positive transcriptional regulatory elements identified in both the promoter and exon 1 region of the human osteonectin gene. In vitro reporter assays revealed that the hON-522E promoter is highly active in androgen receptor negative and metastatic prostate cancer and bone stromal cells compared to androgen receptor-positive prostate cancer cells. Moreover, in vivo prostate-tumor-promoting activity of the hON-522E promoter was confirmed by intravenous administration of an adenoviral vector containing the hON-522E promoter-driven luciferase gene (Ad-522E-Luc into mice bearing orthotopic human prostate tumor xenografts. In addition, an adenoviral vector with the hON-522E-promoter-driven herpes simplex virus thymidine kinase gene (Ad-522E-TK was highly effective against the growth of androgen-independent human prostate cancer PC3M and bone stromal cell line in vitro and in pre-established PC3M tumors in vivo upon addition of the prodrug ganciclovir. Because of the heterogeneity of human prostate tumors, hON-522E promoter-mediated gene therapy has the potential for the treatment of hormone refractory and bone metastatic prostate cancers.

  4. The PPARγ ligand ciglitazone regulates androgen receptor activation differently in androgen-dependent versus androgen-independent human prostate cancer cells

    International Nuclear Information System (INIS)

    Moss, Patrice E.; Lyles, Besstina E.; Stewart, LaMonica V.

    2010-01-01

    The androgen receptor (AR) regulates growth and progression of androgen-dependent as well as androgen-independent prostate cancer cells. Peroxisome proliferator-activated receptor gamma (PPARγ) agonists have been reported to reduce AR activation in androgen-dependent LNCaP prostate cancer cells. To determine whether PPARγ ligands are equally effective at inhibiting AR activity in androgen-independent prostate cancer, we examined the effect of the PPARγ ligands ciglitazone and rosiglitazone on C4-2 cells, an androgen- independent derivative of the LNCaP cell line. Luciferase-based reporter assays and Western blot analysis demonstrated that PPARγ ligand reduced dihydrotestosterone (DHT)-induced increases in AR activity in LNCaP cells. However, in C4-2 cells, these compounds increased DHT-induced AR driven luciferase activity. In addition, ciglitazone did not significantly alter DHT-mediated increases in prostate specific antigen (PSA) protein or mRNA levels within C4-2 cells. siRNA-based experiments demonstrated that the ciglitazone-induced regulation of AR activity observed in C4-2 cells was dependent on the presence of PPARγ. Furthermore, overexpression of the AR corepressor cyclin D1 inhibited the ability of ciglitazone to induce AR luciferase activity in C4-2 cells. Thus, our data suggest that both PPARγ and cyclin D1 levels influence the ability of ciglitazone to differentially regulate AR signaling in androgen-independent C4-2 prostate cancer cells.

  5. Regulation of expression of Na+,K+-ATPase in androgen-dependent and androgen-independent prostate cancer

    NARCIS (Netherlands)

    L.J. Blok (Leen); G.T.G. Chang; M. Steenbeek-Slotboom (M.); W.M. van Weerden (Wytske); H.G. Swarts; J.J.H.H.M. de Pont (J. J H H M); G.J. van Steenbrugge (Gert Jan); A.O. Brinkmann (Albert)

    1999-01-01

    textabstractThe β1-subunit of Na+,K+-ATPase was isolated and identified as an androgen down-regulated gene. Expression was observed at high levels in androgen-independent as compared to androgen-dependent (responsive) human prostate cancer cell lines and xenografts when grown in the presence of

  6. Prostate cancer: molecular biology of early progression to androgen independence.

    Science.gov (United States)

    Sadar, M D; Hussain, M; Bruchovsky, N

    1999-12-01

    To improve the therapy for prostate cancer, it will be necessary to address the problems of progression to androgen independence and the process of metastatic spread of tumour. The complexity of the latter condition is likely to mitigate against the immediate development of relevant therapeutic approaches. However, the basis of androgen independence appears to be a problem of simpler dimensions and more amenable to treatment with current therapeutic technology. Since early tumour progression can be detected by an incomplete prostate-specific antigen (PSA) response to androgen withdrawal therapy, a study of the molecular biology of PSA gene regulation may well provide insight into new methods for preventing or delaying this problem. Mounting evidence suggests that ligand-independent activation of the androgen receptor may be one underlying mechanism of androgen independence. In the absence of androgen, a compensatory increase in the activity of cAMP-dependent protein kinase (PKA) enhances the ability of the androgen receptor to bind to the response elements regulating PSA gene expression. The activation of the androgen receptor through up-regulation of the PKA signal transduction pathway involves the amino-terminus of the androgen receptor, the function of which may be altered either by modifications such as phosphorylation, or through interactions with co-regulators or other proteins. Of therapeutic interest is the fact that this effect can be counteracted experimentally by the anti-androgen, bicalutamide, and clinically by several other similar agents. We speculate that the inhibition of PKA-activated androgen receptor might also be accomplished by decoy molecules that can bind to the relevant activated site on the amino-terminus or competitively interact with proteins recruited by the PKA pathway that are responsible for activating the receptor in the absence of androgen. Such molecules might include small mimetic substances or agents that can gain access to the

  7. Histological changes caused by meclofenamic acid in androgen independent prostate cancer tumors: evaluation in a mouse model

    Directory of Open Access Journals (Sweden)

    Iván Delgado-Enciso

    2015-10-01

    Full Text Available ABSTRACT Meclofenamic acid is a nonsteroidal anti-inflammatory drug that has shown therapeutic potential for different types of cancers, including androgen-independent prostate neoplasms. The antitumor effect of diverse nonsteroidal anti-inflammatory drugs has been shown to be accompanied by histological and molecular changes that are responsible for this beneficial effect. The objective of the present work was to analyze the histological changes caused by meclofenamic acid in androgen-independent prostate cancer. Tumors were created in a nude mouse model using PC3 cancerous human cells. Meclofenamic acid (10 mg/kg/day; experimental group, n=5 or saline solution (control group, n=5 was administered intraperitoneally for twenty days. Histological analysis was then carried out on the tumors, describing changes in the cellular architecture, fibrosis, and quantification of cellular proliferation and tumor vasculature. Meclofenamic acid causes histological changes that indicate less tumor aggression (less hypercellularity, fewer atypical mitoses, and fewer nuclear polymorphisms, an increase in fibrosis, and reduced cellular proliferation and tumor vascularity. Further studies are needed to evaluate the molecular changes that cause the beneficial and therapeutic effects of meclofenamic acid in androgen-independent prostate cancer.

  8. Neutral Endopeptidase Inhibits Neuropeptide Mediated Growth of Androgen-Independent Prostate Cancer

    National Research Council Canada - National Science Library

    Dai, Jie

    2000-01-01

    ...), a cell-surface peptidase which inactivates active peptides and reduces local concentrations of peptide available for receptor binding and signal transduction, in the growth inhibition of androgen-independent (Al) prostate cancer...

  9. Phase I trial of yttrium-90-labeled anti-prostate-specific membrane antigen monoclonal antibody J591 for androgen-independent prostate cancer.

    Science.gov (United States)

    Milowsky, Matthew I; Nanus, David M; Kostakoglu, Lale; Vallabhajosula, Shankar; Goldsmith, Stanley J; Bander, Neil H

    2004-07-01

    To determine the maximum-tolerated dose (MTD), toxicity, human antihuman antibody (HAHA) response, pharmacokinetics, organ dosimetry, targeting, and preliminary efficacy of yttrium-90-labeled anti-prostate-specific membrane antigen monoclonal antibody J591 ((90)Y-J591) in patients with androgen-independent prostate cancer (PC). Patients with androgen-independent PC and evidence of disease progression received indium-111-J591 for pharmacokinetic and biodistribution determinations followed 1 week later by (90)Y-J591 at five dose levels: 5, 10, 15, 17.5, and 20 mCi/m(2). Patients were eligible for up to three re-treatments if platelet and neutrophil recovery was satisfactory. Twenty-nine patients with androgen-independent PC received (90)Y-J591, four of whom were re-treated. Dose limiting toxicity (DLT) was seen at 20 mCi/m(2), with two patients experiencing thrombocytopenia with non-life-threatening bleeding episodes requiring platelet transfusions. The 17.5-mCi/m(2) dose level was determined to be the MTD. No re-treated patients experienced DLT. Nonhematologic toxicity was not dose limiting. Targeting of known sites of bone and soft tissue metastases was seen in the majority of patients. No HAHA response was seen. Antitumor activity was seen, with two patients experiencing 85% and 70% declines in prostate-specific antigen (PSA) levels lasting 8 and 8.6 months, respectively, before returning to baseline. Both patients had objective measurable disease responses. An additional six patients (21%) experienced PSA stabilization. The recommended dose for (90)Y-J591 is 17.5 mCi/m(2). Acceptable toxicity, excellent targeting of known sites of PC metastases, and biologic activity in patients with androgen-independent PC warrant further investigation of (90)Y-J591 in the treatment of patients with PC.

  10. Evolving perspectives of the role of novel agents in androgen-independent prostate cancer

    Directory of Open Access Journals (Sweden)

    Sujith Kalmadi

    2008-01-01

    Full Text Available Metastatic androgen-independent prostate cancer presents an intriguing clinical challenge, with a subtle interaction between hormone-responsive and refractory tumor cell elements. The treatment of advanced prostate carcinoma, which had remained stagnant for several decades following the understanding of the link between androgenic stimulation and carcinogenesis, has now started to make steady headway with chemotherapy and targeted approaches. Metastatic prostate cancer is almost always treated with initial androgen deprivation, in various forms. However, despite such treatment androgen-independent prostate cancer cells eventually emerge and progress to threaten life. The therapeutic objectives for treatment of metastatic prostate cancer are to maintain the quality of life and prolong survival. The out-dated nihilistic dogma of deferring chemotherapy until the most advanced stages in advanced prostate cancer is now falling by the wayside with the development of newer effective, tolerable agents.

  11. Hedgehog/Gli supports androgen signaling in androgen deprived and androgen independent prostate cancer cells

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    Shtutman Michael

    2010-04-01

    Full Text Available Abstract Background Castration resistant prostate cancer (CRPC develops as a consequence of hormone therapies used to deplete androgens in advanced prostate cancer patients. CRPC cells are able to grow in a low androgen environment and this is associated with anomalous activity of their endogenous androgen receptor (AR despite the low systemic androgen levels in the patients. Therefore, the reactivated tumor cell androgen signaling pathway is thought to provide a target for control of CRPC. Previously, we reported that Hedgehog (Hh signaling was conditionally activated by androgen deprivation in androgen sensitive prostate cancer cells and here we studied the potential for cross-talk between Hh and androgen signaling activities in androgen deprived and androgen independent (AI prostate cancer cells. Results Treatment of a variety of androgen-deprived or AI prostate cancer cells with the Hh inhibitor, cyclopamine, resulted in dose-dependent modulation of the expression of genes that are regulated by androgen. The effect of cyclopamine on endogenous androgen-regulated gene expression in androgen deprived and AI prostate cancer cells was consistent with the suppressive effects of cyclopamine on the expression of a reporter gene (luciferase from two different androgen-dependent promoters. Similarly, reduction of smoothened (Smo expression with siRNA co-suppressed expression of androgen-inducible KLK2 and KLK3 in androgen deprived cells without affecting the expression of androgen receptor (AR mRNA or protein. Cyclopamine also prevented the outgrowth of AI cells from androgen growth-dependent parental LNCaP cells and suppressed the growth of an overt AI-LNCaP variant whereas supplemental androgen (R1881 restored growth to the AI cells in the presence of cyclopamine. Conversely, overexpression of Gli1 or Gli2 in LNCaP cells enhanced AR-specific gene expression in the absence of androgen. Overexpressed Gli1/Gli2 also enabled parental LNCaP cells to

  12. Androgen-independent proliferation of LNCaP prostate cancer cells infected by xenotropic murine leukemia virus-related virus

    International Nuclear Information System (INIS)

    Kakoki, Katsura; Kamiyama, Haruka; Izumida, Mai; Yashima, Yuka; Hayashi, Hideki; Yamamoto, Naoki; Matsuyama, Toshifumi; Igawa, Tsukasa; Sakai, Hideki; Kubo, Yoshinao

    2014-01-01

    Highlights: • XMRV infection induces androgen-independent growth in LNCaP cells. • XMRV infection reduces expression of androgen receptor. • XMRV promotes appearance of androgen blocker-resistant prostate cancer cells. - Abstract: Xenotropic murine leukemia virus-related virus (XMRV) is a novel gammaretrovirus that was originally isolated from human prostate cancer. It is now believed that XMRV is not the etiologic agent of prostate cancer. An analysis of murine leukemia virus (MLV) infection in various human cell lines revealed that prostate cancer cell lines are preferentially infected by XMRV, and this suggested that XMRV infection may confer some sort of growth advantage to prostate cancer cell lines. To examine this hypothesis, androgen-dependent LNCaP cells were infected with XMRV and tested for changes in certain cell growth properties. We found that XMRV-infected LNCaP cells can proliferate in the absence of the androgen dihydrotestosterone. Moreover, androgen receptor expression is significantly reduced in XMRV-infected LNCaP cells. Such alterations were not observed in uninfected and amphotropic MLV-infected LNCaP cells. This finding explains why prostate cancer cell lines are preferentially infected with XMRV

  13. Activation of estrogen receptor beta (ERβ) regulates the expression of N-cadherin, E-cadherin and β-catenin in androgen-independent prostate cancer cells.

    Science.gov (United States)

    Silva, Rafael de Souza; Lombardi, Ana Paola G; de Souza, Deborah Simão; Vicente, Carolina M; Porto, Catarina S

    2018-03-01

    The aim of the present study was to investigate the impact of the activation of estrogen receptors on expression and localization of N-cadherin, E-cadherin and non-phosphorylated β-catenin in androgen-independent prostate cancer cells (PC-3 and DU-145) and in human post pubertal prostate epithelial cells (PNT1A). Expression of N-cadherin was detected in PNT1A and PC-3 cells, but not in DU-145 cells. E-cadherin was detected only in DU-145 cells and β-catenin was detected in all cells studied. N-cadherin and β-catenin were located preferentially in the cellular membrane of PNT1A cells and in the cytoplasm of PC-3 cells. E-cadherin and β-catenin were located preferentially in the cellular membrane of DU-145 cells. 17β-estradiol (E2) or the ERα-selective agonist PPT did not affect the content and localization of N-cadherin in PC-3 and PNT1A cells or E-cadherin in DU-145 cells. In PC-3 cells, ERβ-selective agonist DPN decreased the expression of N-cadherin. DPN-induced downregulation of N-cadherin was blocked by pretreatment with the ERβ-selective antagonist (PHTPP), indicating that ERβ1 is the upstream receptor regulating the expression of N-cadherin. In DU-145 cells, the activation of ERβ1 by DPN increased the expression of E-cadherin. Taken together, these results suggest that activation of ERβ1 is required to maintain an epithelial phenotype in PC-3 and DU-145 cells. The activation of ERβ1 also increased the expression of β-catenin in cytoplasm of PC-3 and in the cellular membrane of DU-145 cells. In conclusion, our results indicate differential expression and localization of N-cadherin, E-cadherin and β-catenin in androgen-independent prostate cancer cells. The reduction of N-cadherin content by activation of ERβ, exclusively observed in androgen-independent prostate cancer cells (PC-3), may be related to the activation of signaling pathways, such as the release of β-catenin into the cytoplasm, translocation of β-catenin to the nucleus and

  14. Natural proteasome inhibitor celastrol suppresses androgen-independent prostate cancer progression by modulating apoptotic proteins and NF-kappaB.

    Directory of Open Access Journals (Sweden)

    Yao Dai

    Full Text Available Celastrol is a natural proteasome inhibitor that exhibits promising anti-tumor effects in human malignancies, especially the androgen-independent prostate cancer (AIPC with constitutive NF-κB activation. Celastrol induces apoptosis by means of proteasome inhibition and suppresses prostate tumor growth. However, the detailed mechanism of action remains elusive. In the current study, we aim to test the hypothesis that celastrol suppresses AIPC progression via inhibiting the constitutive NF-κB activity as well as modulating the Bcl-2 family proteins.We examined the efficacy of celastrol both in vitro and in vivo, and evaluated the role of NF-κB in celastrol-mediated AIPC regression. We found that celastrol inhibited cell proliferation in all three AIPC cell lines (PC-3, DU145 and CL1, with IC₅₀ in the range of 1-2 µM. Celastrol also suppressed cell migration and invasion. Celastrol significantly induced apoptosis as evidenced by increased sub-G1 population, caspase activation and PARP cleavage. Moreover, celastrol promoted cleavage of the anti-apoptotic protein Mcl-1 and activated the pro-apoptotic protein Noxa. In addition, celastrol rapidly blocked cytosolic IκBα degradation and nuclear translocation of RelA. Likewise, celastrol inhibited the expression of multiple NF-κB target genes that are involved in proliferation, invasion and anti-apoptosis. Celastrol suppressed AIPC tumor progression by inhibiting proliferation, increasing apoptosis and decreasing angiogenesis, in PC-3 xenograft model in nude mouse. Furthermore, increased cellular IκBα and inhibited expression of various NF-κB target genes were observed in tumor tissues.Our data suggest that, via targeting the proteasome, celastrol suppresses proliferation, invasion and angiogenesis by inducing the apoptotic machinery and attenuating constitutive NF-κB activity in AIPC both in vitro and in vivo. Celastrol as an active ingredient of traditional herbal medicine could thus be

  15. Ferruginol suppresses survival signaling pathways in androgen-independent human prostate cancer cells

    NARCIS (Netherlands)

    de Jesus, Marcelo Bispo; Zambuzzi, Willian Fernando; Ruela de Sousa, Roberta Regina; Areche, Carlos; Santos de Souza, Ana Carolina; Aoyama, Hiroshi; Schmeda-Hirschmann, Guillermo; Rodriguez, Jaime A.; Monteiro de Souza Brito, Alba Regina; Peppelenbosch, Maikel P.; den Hertog, Jeroen; de Paula, Eneida; Ferreira, Carmen Verissima

    Ferruginol, a bioactive compound isolated from a Chilean tree (Podocarpaceae), attracts attention as a consequence of its pharmacological properties, which include anti-fungal, anti-bacterial, cardioprotective, anti-oxidative, anti-plasmodial and anti-ulcerogenic actions. Nevertheless, the molecular

  16. Ferruginol suppresses survival signaling pathways in androgen-independent human prostate cancer cells.

    NARCIS (Netherlands)

    Bispo de Jesus, M.; Zambuzzi, W.F.; Ruela de Sousa, R.R.; Areche, C.; Santos de Souza, A.C.; Aoyama, H.; Schmeda-Hirschmann, G.; Rodriguez, J.A.; Monteiro de Souza Brito, A.R.; Peppelenbosch, M.P.; den Hertog, J.; de Paula, E.; Ferreira, C.V.

    2008-01-01

    Ferruginol, a bioactive compound isolated from a Chilean tree (Podocarpaceae), attracts attention as a consequence of its pharmacological properties, which include anti-fungal, anti-bacterial, cardioprotective, anti-oxidative, anti-plasmodial and anti-ulcerogenic actions. Nevertheless, the molecular

  17. The Role of AKT in Androgen-Independent Progression of Human Prostate Cancer

    Science.gov (United States)

    2005-02-01

    androgen ablation (2). However, there has been a growing appreciation that most patients treated by androgen ablation ultimately relapse to more aggressive...activation. (c) Bicistronic vector, iAkt,., containing M-FRB12 and F3-APH.Akt separated by the poliovirus IRES, functions more efficiently than iAkt...characterized IRES from poliovirus . Following specific antibodies against Akt S473 and T308 sites as electroporation of bicistronic vectors into Jurkat

  18. Wnt signaling promotes androgen-independent prostate cancer cell proliferation through up-regulation of the hippo pathway effector YAP.

    Science.gov (United States)

    Seo, Won Ik; Park, Seoyoung; Gwak, Jungsug; Ju, Bong Gun; Chung, Jae Il; Kang, Pil Moon; Oh, Sangtaek

    2017-05-13

    Aberrant up-regulation of Wnt/β-catenin signaling is associated with the development and progression of prostate cancer, but the underlying mechanism is unclear. Here we show that in the absence of androgens, the Wnt/β-catenin pathway activates AR-mediated transcription through up-regulation of the Hippo pathway effector Yes-associated protein (YAP). Wnt3a-conditioned medium (Wnt3a-CM) promotes the growth of LNCaP cells and increases AR and YAP protein levels. Moreover, Wnt3a-CM induces the nuclear translocation of YAP and the AR, but not β-catenin, thereby activating the expression of AR- and YAP-dependent genes, in an androgen-independent manner. In addition, depletion of YAP with small interfering RNA (siRNA) prevented Wnt3a-CM-mediated up-regulation of AR-dependent gene expression. Thus, our findings provide mechanistic insight into the proposed cross-talk between the Wnt/β-catenin and Hippo pathways in androgen-independent prostate cancer development. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Inhibition of Androgen-Independent Prostate Cancer by Estrogenic Compounds Is Associated with Increased Expression of Immune-Related Genes

    Directory of Open Access Journals (Sweden)

    Ilsa M. Coleman

    2006-10-01

    Full Text Available The clinical utility of estrogens for treating prostate cancer (CaP was established in the 1940s by Huggins. The classic model of the anti-CaP activity of estrogens postulates an indirect mechanism involving the suppression of androgen production. However, clinical, preclinical studies have shown that estrogens exert growth-inhibitory effects on CaP under low-androgen conditions, suggesting additional modes whereby estrogens affect CaP cells and/or the microenvironment. Here we have investigated the activity of 17β estradiol (E2 against androgen-independent CaP, identified molecular alterations in tumors exposed to E2. E2 treatment inhibited the growth of all four androgen-independent CaP xenografts studied (LuCaP 35V, LuCaP 23.1AI, LuCaP 49, LuCaP 58 in castrated male mice. The molecular basis of growth suppression was studied by cDNA microarray analysis, which indicated that multiple pathways are altered by E2 treatment. Of particular interest are changes in transcripts encoding proteins that mediate immune responses, regulate androgen receptor signaling. In conclusion, our data show that estrogens have powerful inhibitory effects on CaP in vivo in androgendepleted environments, suggest novel mechanisms of estrogen-mediated antitumor activity. These results indicate that incorporating estrogens into CaP treatment protocols could enhance therapeutic efficacy even in cases of advanced disease.

  20. Androgen receptor signaling is required for androgen-sensitive human prostate cancer cell proliferation and survival

    Directory of Open Access Journals (Sweden)

    Day Wanda V

    2005-04-01

    Full Text Available Abstract Background Androgens and androgen receptors (AR regulate normal prostate development and growth. They also are involved in pathological development of prostatic diseases, including benign prostatic hyperplasia (BPH and prostate cancer (PCa. Antiandrogen therapy for PCa, in conjunction with chemical or surgical castration, offers initial positive responses and leads to massive prostate cell death. However, cancer cells later appear as androgen-independent PCa. To investigate the role of AR in prostate cell proliferation and survival, we introduced a vector-based small interfering RNA (siRNA. This siRNA targeted 5'-untranslated region of AR mRNA for extended suppression of AR expression in androgen-sensitive human prostate LNCaP cells. Results The siRNA design successfully suppressed endogenous AR expression, as revealed by western blotting and immunofluorescence staining in LNCaP cells. LNCaP cells did not proliferate in the absence of AR and underwent apoptosis, based on elevated phospho-Histone H2B expression and higher number of apoptotic body as compared to control cells. Conclusion We demonstrated that AR is vital for prostate cell proliferation and survival in this androgen-sensitive prostate cell line. These results further strengthen the hypothesis that AR can be a therapeutic target for treating androgen-sensitive stages of PCa. Unlike antiandorgens, however, siRNA targeting AR provides a direct inactivation of AR function through the suppression of AR protein expression.

  1. Androgen-independent effects of Serenoa repens extract (Prostasan®) on prostatic epithelial cell proliferation and inflammation

    DEFF Research Database (Denmark)

    Iglesias-Gato, Diego; Carsten, Tober; Vesterlund, Mattias

    2012-01-01

    . Prostasan® inhibited epidermal growth factor (EGF) and lipopolysaccharide (LPS) induced proliferation of the prostatic epithelial, androgen independent cell line PC-3. At effective concentrations of 50 µg/mL, Prostasan® partly displaced EGF from EGF receptor (EGFR) but fully blocked EGF-induced cell...... proliferation of PC-3 cells. Similarly, Prostasan® inhibited LPS-induced proliferation of PC-3 cells without affecting LPS activation of the NFĸB pathway via toll-like receptor-4 (TLR-4). Additionally, Prostasan® reduced the constitutive secretion of monocyte chemotactic protein-1 (MCP-1), the LPS......-induced secretion of IL-12 and inhibited MCP-1 and granulocyte-macrophage colony-stimulating factor (GM-CSF) production in the presence of LPS on PC-3 cells. Taken together, our results suggest that S. repens extracts, in addition to other reported effects on BPH development and prostatitis, inhibits EGF...

  2. Phenotypic Plasticity, Bet-Hedging, and Androgen Independence in Prostate Cancer: Role of Non-Genetic Heterogeneity

    Directory of Open Access Journals (Sweden)

    Mohit Kumar Jolly

    2018-03-01

    Full Text Available It is well known that genetic mutations can drive drug resistance and lead to tumor relapse. Here, we focus on alternate mechanisms—those without mutations, such as phenotypic plasticity and stochastic cell-to-cell variability that can also evade drug attacks by giving rise to drug-tolerant persisters. The phenomenon of persistence has been well-studied in bacteria and has also recently garnered attention in cancer. We draw a parallel between bacterial persistence and resistance against androgen deprivation therapy in prostate cancer (PCa, the primary standard care for metastatic disease. We illustrate how phenotypic plasticity and consequent mutation-independent or non-genetic heterogeneity possibly driven by protein conformational dynamics can stochastically give rise to androgen independence in PCa, and suggest that dynamic phenotypic plasticity should be considered in devising therapeutic dosing strategies designed to treat and manage PCa.

  3. Identification of an anabolic selective androgen receptor modulator that actively induces death of androgen-independent prostate cancer cells.

    Science.gov (United States)

    Schmidt, Azriel; Meissner, Robert S; Gentile, Michael A; Chisamore, Michael J; Opas, Evan E; Scafonas, Angela; Cusick, Tara E; Gambone, Carlo; Pennypacker, Brenda; Hodor, Paul; Perkins, James J; Bai, Chang; Ferraro, Damien; Bettoun, David J; Wilkinson, Hilary A; Alves, Stephen E; Flores, Osvaldo; Ray, William J

    2014-09-01

    Prostate cancer (PCa) initially responds to inhibition of androgen receptor (AR) signaling, but inevitably progresses to hormone ablation-resistant disease. Much effort is focused on optimizing this androgen deprivation strategy by improving hormone depletion and AR antagonism. However we found that bicalutamide, a clinically used antiandrogen, actually resembles a selective AR modulator (SARM), as it partially regulates 24% of endogenously 5α-dihydrotestosterone (DHT)-responsive genes in AR(+) MDA-MB-453 breast cancer cells. These data suggested that passive blocking of all AR functions is not required for PCa therapy. Hence, we adopted an active strategy that calls for the development of novel SARMs, which induce a unique gene expression profile that is intolerable to PCa cells. Therefore, we screened 3000 SARMs for the ability to arrest the androgen-independent growth of AR(+) 22Rv1 and LNCaP PCa cells but not AR(-) PC3 or DU145 cells. We identified only one such compound; the 4-aza-steroid, MK-4541, a potent and selective SARM. MK-4541 induces caspase-3 activity and cell death in both androgen-independent, AR(+) PCa cell lines but spares AR(-) cells or AR(+) non-PCa cells. This activity correlates with its promoter context- and cell-type dependent transcriptional effects. In rats, MK-4541 inhibits the trophic effects of DHT on the prostate, but not the levator ani muscle, and triggers an anabolic response in the periosteal compartment of bone. Therefore, MK-4541 has the potential to effectively manage prostatic hypertrophic diseases owing to its antitumor SARM-like mechanism, while simultaneously maintaining the anabolic benefits of natural androgens. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Increased expression of heparin binding EGF (HB-EGF), amphiregulin, TGF alpha and epiregulin in androgen-independent prostate cancer cell lines.

    DEFF Research Database (Denmark)

    Tørring, Niels; Sørensen, Boe Sandahl; Nexø, Ebba

    2000-01-01

    BACKGROUND: The proliferation of androgen-independent prostate cancer cell lines has previously been shown to be influenced by an autocrine loop of the epidermal growth factor (EGF) system. This observation has alerted us to study the expression of ligands and receptors from the EGF-system in pro...

  5. Androgen and taxol cause cell type-specific alterations of centrosome and DNA organization in androgen-responsive LNCaP and androgen-independent DU145 prostate cancer cells

    Science.gov (United States)

    Schatten, H.; Ripple, M.; Balczon, R.; Weindruch, R.; Chakrabarti, A.; Taylor, M.; Hueser, C. N.

    2000-01-01

    We investigated the effects of androgen and taxol on the androgen-responsive LNCaP and androgen-independent DU145 prostate cancer cell lines. Cells were treated for 48 and 72 h with 0.05-1 nM of the synthetic androgen R1881 and with 100 nM taxol. Treatment of LNCaP cells with 0.05 nM R1881 led to increased cell proliferation, whereas treatment with 1 nM R1881 resulted in inhibited cell division, DNA cycle arrest, and altered centrosome organization. After treatment with 1 nM R1881, chromatin became clustered, nuclear envelopes convoluted, and mitochondria accumulated around the nucleus. Immunofluorescence microscopy with antibodies to centrosomes showed altered centrosome structure. Although centrosomes were closely associated with the nucleus in untreated cells, they dispersed into the cytoplasm after treatment with 1 nM R1881. Microtubules were only faintly detected in 1 nM R1881-treated LNCaP cells. The effects of taxol included microtubule bundling and altered mitochondria morphology, but not DNA organization. As expected, the androgen-independent prostate cancer cell line DU145 was not affected by R1881. Treatment with taxol resulted in bundling of microtubules in both cell lines. Additional taxol effects were seen in DU145 cells with micronucleation of DNA, an indication of apoptosis. Simultaneous treatment with R1881 and taxol had no additional effects on LNCaP or DU145 cells. These results suggest that LNCaP and DU145 prostate cancer cells show differences not only in androgen responsiveness but in sensitivity to taxol as well. Copyright 2000 Wiley-Liss, Inc.

  6. Preparation of nanobubbles carrying androgen receptor siRNA and their inhibitory effects on androgen-independent prostate cancer when combined with ultrasonic irradiation.

    Directory of Open Access Journals (Sweden)

    Luofu Wang

    Full Text Available OBJECTIVE: The objective of this study was to investigate nanobubbles carrying androgen receptor (AR siRNA and their in vitro and in vivo anti-tumor effects, when combined with ultrasonic irradiation, on androgen-independent prostate cancer (AIPC. MATERIALS AND METHODS: Nanobubbles carrying AR siRNA were prepared using poly-L-lysine and electrostatic adsorption methods. Using C4-2 cell activity as a testing index, the optimal irradiation parameters (including the nanobubble number/cell number ratio, mechanical index [MI], and irradiation time were determined and used for transfection of three human prostate cancer cell lines (C4-2, LNCaP, and PC-3 cells. The AR expression levels were investigated with RT-PCR and Western blot analysis. Additionally, the effects of the nanobubbles and control microbubbles named SonoVue were assessed via imaging in a C4-2 xenograft model. Finally, the growth and AR expression of seven groups of tumor tissues were assessed using the C4-2 xenograft mouse model. RESULTS: The nanobubbles had an average diameter of 609.5±15.6 nm and could effectively bind to AR siRNA. Under the optimized conditions of a nanobubble number/cell number ratio of 100∶1, an MI of 1.2, and an irradiation time of 2 min, the highest transfection rates in C4-2, LNCaP, and PC-3 cells were 67.4%, 74.0%, and 63.96%, respectively. In the C4-2 and LNCaP cells, treatment with these binding nanobubbles plus ultrasonic irradiation significantly inhibited cell growth and resulted in the suppression of AR mRNA and protein expression. Additionally, contrast-enhanced ultrasound showed that the nanobubbles achieved stronger signals than the SonoVue control in the central hypovascular area of the tumors. Finally, the anti-tumor effect of these nanobubbles plus ultrasonic irradiation was most significant in the xenograft tumor model compared with the other groups. CONCLUSION: Nanobubbles carrying AR siRNA could be potentially used as gene vectors in

  7. Human sensitization to Ganoderma antigen.

    Science.gov (United States)

    Tarlo, S M; Bell, B; Srinivasan, J; Dolovich, J; Hargreave, F E

    1979-07-01

    Continuous air sampling with a Hirst volumetric spore trap over 3 yr has identified basidiospores of Ganoderma applanatum, a bracket fungus, as the most numerous fungal spores in two southern Ontario locations. The particle size is small and the calculated total spore mass approximates that of the spores of Cladosporium and Alternaria. Extracts of Ganoderma applanatum bracket fungus and spores in w/v, 1:10 concentration were prepared after collection of samples of the fungus from local woods. Skin prick tests with the extracts were performed in 294 consecutive children and adults attending two chest/allergy clinics. Of these patients, 182 (61.9%) reacted to 1 or more of the common inhalant allergen extracts and 24 (8.2%) reacted to Ganoderma antigen. There was no consistent relationship between reactivity to Ganoderma antigen and any of the common inhaled allergens. IgE-dependent sensitization to Ganoderma was confirmed by the radioallergosorbent test (RAST). Rabbit antisera to Ganoderma antigen preparations did not appear to cross-react with preparations of the various clinically important allergens. The findings indicate that Ganoderma antigen is commonly encountered, can induce human sensitization, and has unique antigenicity among common allergens of clinical importance.

  8. Withaferin A Induces Cell Death Selectively in Androgen-Independent Prostate Cancer Cells but Not in Normal Fibroblast Cells.

    Directory of Open Access Journals (Sweden)

    Yukihiro Nishikawa

    Full Text Available Withaferin A (WA, a major bioactive component of the Indian herb Withania somnifera, induces cell death (apoptosis/necrosis in multiple types of tumor cells, but the molecular mechanism underlying this cytotoxicity remains elusive. We report here that 2 μM WA induced cell death selectively in androgen-insensitive PC-3 and DU-145 prostate adenocarcinoma cells, whereas its toxicity was less severe in androgen-sensitive LNCaP prostate adenocarcinoma cells and normal human fibroblasts (TIG-1 and KD. WA also killed PC-3 cells in spheroid-forming medium. DNA microarray analysis revealed that WA significantly increased mRNA levels of c-Fos and 11 heat-shock proteins (HSPs in PC-3 and DU-145, but not in LNCaP and TIG-1. Western analysis revealed increased expression of c-Fos and reduced expression of the anti-apoptotic protein c-FLIP(L. Expression of HSPs such as HSPA6 and Hsp70 was conspicuously elevated; however, because siRNA-mediated depletion of HSF-1, an HSP-inducing transcription factor, reduced PC-3 cell viability, it is likely that these heat-shock genes were involved in protecting against cell death. Moreover, WA induced generation of reactive oxygen species (ROS in PC-3 and DU-145, but not in normal fibroblasts. Immunocytochemistry and immuno-electron microscopy revealed that WA disrupted the vimentin cytoskeleton, possibly inducing the ROS generation, c-Fos expression and c-FLIP(L suppression. These observations suggest that multiple events followed by disruption of the vimentin cytoskeleton play pivotal roles in WA-mediated cell death.

  9. Transfected poly(I:C) activates different dsRNA receptors, leading to apoptosis or immunoadjuvant response in androgen-independent prostate cancer cells.

    Science.gov (United States)

    Palchetti, Sara; Starace, Donatella; De Cesaris, Paola; Filippini, Antonio; Ziparo, Elio; Riccioli, Anna

    2015-02-27

    Despite the effectiveness of surgery or radiation therapy for the treatment of early-stage prostate cancer (PCa), there is currently no effective strategy for late-stage disease. New therapeutic targets are emerging; in particular, dsRNA receptors Toll-like receptor 3 (TLR3) and cytosolic helicases expressed by cancer cells, once activated, exert a pro-apoptotic effect in different tumors. We previously demonstrated that the synthetic analog of dsRNA poly(I:C) induces apoptosis in the androgen-dependent PCa cell line LNCaP in a TLR3-dependent fashion, whereas only a weak apoptotic effect is observed in the more aggressive and androgen-independent PCa cells PC3 and DU145. In this paper, we characterize the receptors and the signaling pathways involved in the remarkable apoptosis induced by poly(I:C) transfected by Lipofectamine (in-poly(I:C)) compared with the 12-fold higher free poly(I:C) concentration in PC3 and DU145 cells. By using genetic inhibition of different poly(I:C) receptors, we demonstrate the crucial role of TLR3 and Src in in-poly(I:C)-induced apoptosis. Therefore, we show that the increased in-poly(I:C) apoptotic efficacy is due to a higher binding of endosomal TLR3. On the other hand, we show that in-poly(I:C) binding to cytosolic receptors MDA5 and RIG-I triggers IRF3-mediated signaling, leading uniquely to the up-regulation of IFN-β, which likely in turn induces increased TLR3, MDA5, and RIG-I proteins. In summary, in-poly(I:C) activates two distinct antitumor pathways in PC3 and DU145 cells: one mediated by the TLR3/Src/STAT1 axis, leading to apoptosis, and the other one mediated by MDA5/RIG-I/IRF3, leading to immunoadjuvant IFN-β expression. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Human sensitivity to vertical self-motion.

    Science.gov (United States)

    Nesti, Alessandro; Barnett-Cowan, Michael; Macneilage, Paul R; Bülthoff, Heinrich H

    2014-01-01

    Perceiving vertical self-motion is crucial for maintaining balance as well as for controlling an aircraft. Whereas heave absolute thresholds have been exhaustively studied, little work has been done in investigating how vertical sensitivity depends on motion intensity (i.e., differential thresholds). Here we measure human sensitivity for 1-Hz sinusoidal accelerations for 10 participants in darkness. Absolute and differential thresholds are measured for upward and downward translations independently at 5 different peak amplitudes ranging from 0 to 2 m/s(2). Overall vertical differential thresholds are higher than horizontal differential thresholds found in the literature. Psychometric functions are fit in linear and logarithmic space, with goodness of fit being similar in both cases. Differential thresholds are higher for upward as compared to downward motion and increase with stimulus intensity following a trend best described by two power laws. The power laws' exponents of 0.60 and 0.42 for upward and downward motion, respectively, deviate from Weber's Law in that thresholds increase less than expected at high stimulus intensity. We speculate that increased sensitivity at high accelerations and greater sensitivity to downward than upward self-motion may reflect adaptations to avoid falling.

  11. Auditory brainstem's sensitivity to human voices.

    Science.gov (United States)

    Nan, Yun; Skoe, Erika; Nicol, Trent; Kraus, Nina

    2015-03-01

    Differentiating between voices is a basic social skill humans acquire early in life. The current study aimed to understand the subcortical mechanisms of voice processing by focusing on the two most important acoustical voice features: the fundamental frequency (F0) and harmonics. We measured frequency following responses in a group of young adults to a naturally produced speech syllable under two linguistic contexts: same-syllable and multiple-syllable. Compared to the same-syllable context, the multiple-syllable context contained more speech cues to aid voice processing. We analyzed the magnitude of the response to the F0 and harmonics between same-talker and multiple-talker conditions within each linguistic context. Results establish that the human auditory brainstem is sensitive to different talkers as shown by enhanced harmonic responses under the multiple-talker compared to the same-talker condition, when the stimulus stream contained multiple syllables. This study thus provides the first electrophysiological evidence of the auditory brainstem's sensitivity to human voices. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Cyproterone acetate enhances TRAIL-induced androgen-independent prostate cancer cell apoptosis via up-regulation of death receptor 5.

    Science.gov (United States)

    Chen, Linjie; Wolff, Dennis W; Xie, Yan; Lin, Ming-Fong; Tu, Yaping

    2017-03-07

    Virtually all prostate cancer deaths occur due to obtaining the castration-resistant phenotype after prostate cancer cells escaped from apoptosis and/or growth suppression initially induced by androgen receptor blockade. TNF-related apoptosis-inducing ligand (TRAIL) was an attractive cancer therapeutic agent due to its minimal toxicity to normal cells and remarkable apoptotic activity in tumor cells. However, most localized cancers including prostate cancer are resistant to TRAIL-induced apoptosis, thereby creating a therapeutic challenge of inducing TRAIL sensitivity in cancer cells. Herein the effects of cyproterone acetate, an antiandrogen steroid, on the TRAIL-induced apoptosis of androgen receptor-negative prostate cancer cells are reported. Cell apoptosis was assessed by both annexin V/propidium iodide labeling and poly (ADP-ribose) polymerase cleavage assays. Gene and protein expression changes were determined by quantitative real-time PCR and western blot assays. The effect of cyproterone acetate on gene promoter activity was determined by luciferase reporter assay. Cyproterone acetate but not AR antagonist bicalutamide dramatically increased the susceptibility of androgen receptor-negative human prostate cancer PC-3 and DU145 cells to TRAIL-induced apoptosis but no effects on immortalized human prostate stromal PS30 cells and human embryonic kidney HEK293 cells. Further investigation of the TRAIL-induced apoptosis pathway revealed that cyproterone acetate exerted its effect by selectively increasing death receptor 5 (DR5) mRNA and protein expression. Cyproterone acetate treatment also increased DR5 gene promoter activity, which could be abolished by mutation of a consensus binding domain of transcription factor CCAAT-enhancer-binding protein homologous protein (CHOP) in the DR5 gene promoter. Cyproterone acetate increases CHOP expression in a concentration and time-dependent manner and endoplasmic reticulum stress reducer 4-phenylbutyrate could block

  13. Temporal sensitivity. [time dependent human perception of visual stimuli

    Science.gov (United States)

    Watson, Andrew B.

    1986-01-01

    Human visual temporal sensitivity is examined. The stimuli used to measure temporal sensitivity are described and the linear systems theory is reviewed in terms of temporal sensitivity. A working model which represents temporal sensitivity is proposed. The visibility of a number of temporal wave forms, sinusoids, rectangular pulses, and pulse pairs, is analyzed. The relation between spatial and temporal effects is studied. Temporal variations induced by image motion and the effects of light adaptation on temporal sensitivity are considered.

  14. Proteasomal degradation of sphingosine kinase 1 and inhibition of dihydroceramide desaturase by the sphingosine kinase inhibitors, SKi or ABC294640, induces growth arrest in androgen-independent LNCaP-AI prostate cancer cells.

    Science.gov (United States)

    McNaughton, Melissa; Pitman, Melissa; Pitson, Stuart M; Pyne, Nigel J; Pyne, Susan

    2016-03-29

    Sphingosine kinases (two isoforms termed SK1 and SK2) catalyse the formation of the bioactive lipid sphingosine 1-phosphate. We demonstrate here that the SK2 inhibitor, ABC294640 (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide) or the SK1/SK2 inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole)) induce the proteasomal degradation of SK1a (Mr = 42 kDa) and inhibit DNA synthesis in androgen-independent LNCaP-AI prostate cancer cells. These effects are recapitulated by the dihydroceramide desaturase (Des1) inhibitor, fenretinide. Moreover, SKi or ABC294640 reduce Des1 activity in Jurkat cells and ABC294640 induces the proteasomal degradation of Des1 (Mr = 38 kDa) in LNCaP-AI prostate cancer cells. Furthermore, SKi or ABC294640 or fenretinide increase the expression of the senescence markers, p53 and p21 in LNCaP-AI prostate cancer cells. The siRNA knockdown of SK1 or SK2 failed to increase p53 and p21 expression, but the former did reduce DNA synthesis in LNCaP-AI prostate cancer cells. Moreover, N-acetylcysteine (reactive oxygen species scavenger) blocked the SK inhibitor-induced increase in p21 and p53 expression but had no effect on the proteasomal degradation of SK1a. In addition, siRNA knockdown of Des1 increased p53 expression while a combination of Des1/SK1 siRNA increased the expression of p21. Therefore, Des1 and SK1 participate in regulating LNCaP-AI prostate cancer cell growth and this involves p53/p21-dependent and -independent pathways. Therefore, we propose targeting androgen-independent prostate cancer cells with compounds that affect Des1/SK1 to modulate both de novo and sphingolipid rheostat pathways in order to induce growth arrest.

  15. Oncolytic targeting of androgen-sensitive prostate tumor by the respiratory syncytial virus (RSV: consequences of deficient interferon-dependent antiviral defense

    Directory of Open Access Journals (Sweden)

    Hubbard Gene B

    2011-01-01

    Full Text Available Abstract Background Oncolytic virotherapy for cancer treatment utilizes viruses for selective infection and death of cancer cells without any adverse effect on normal cells. We previously reported that the human respiratory syncytial virus (RSV is a novel oncolytic virus against androgen-independent PC-3 human prostate cancer cells. The present study extends the result to androgen-dependent prostate cancer, and explores the underlying mechanism that triggers RSV-induced oncolysis of prostate cancer cells. Methods The oncolytic effect of RSV on androgen-sensitive LNCaP human prostate cancer cells and on androgen-independent RM1 murine prostate cancer cells was studied in vitro in culture and in vivo in a xenograft or allograft tumor model. In vitro, cell viability, infectivity and apoptosis were monitored by MTT assay, viral plaque assay and annexin V staining, respectively. In vivo studies involved virus administration to prostate tumors grown in immune compromised nude mice and in syngeneic immune competent C57BL/6J mice. Anti-tumorogenic oncolytic activity was monitored by measuring tumor volume, imaging bioluminescent tumors in live animals and performing histopathological analysis and TUNEL assay with tumors Results We show that RSV imposes a potent oncolytic effect on LNCaP prostate cancer cells. RSV infectivity was markedly higher in LNCaP cells compared to the non-tumorigenic RWPE-1 human prostate cells. The enhanced viral burden led to LNCaP cell apoptosis and growth inhibition of LNCaP xenograft tumors in nude mice. A functional host immune response did not interfere with RSV-induced oncolysis, since growth of xenograft tumors in syngeneic C57BL/6J mice from murine RM1 cells was inhibited upon RSV administration. LNCaP cells failed to activate the type-I interferon (IFNα/β-induced transcription factor STAT-1, which is required for antiviral gene expression, although these cells could produce IFN in response to RSV infection. The

  16. Central sensitization in humans: assessment and pharmacology.

    Science.gov (United States)

    Arendt-Nielsen, Lars

    2015-01-01

    It is evident that chronic pain can modify the excitability of central nervous system which imposes a specific challenge for the management and for the development of new analgesics. The central manifestations can be difficult to quantify using standard clinical examination procedures, but quantitative sensory testing (QST) may help to quantify the degree and extend of the central reorganization and effect of pharmacological interventions. Furthermore, QST may help in optimizing the development programs for new drugs.Specific translational mechanistic QST tools have been developed to quantify different aspects of central sensitization in pain patients such as threshold ratios, provoked hyperalgesia/allodynia, temporal summation (wind-up like pain), after sensation, spatial summation, reflex receptive fields, descending pain modulation, offset analgesia, and referred pain areas. As most of the drug development programs in the area of pain management have not been very successful, the pharmaceutical industry has started to utilize the complementary knowledge obtained from QST profiling. Linking patients QST profile with drug efficacy profile may provide the fundamentals for developing individualized, targeted pain management programs in the future. Linking QST-assessed pain mechanisms with treatment outcome provides new valuable information in drug development and for optimizing the management regimes for chronic pain.

  17. Enhancement of Bleomycin Sensitivity in Human Lung Cancer Cell ...

    African Journals Online (AJOL)

    Enhancement of Bleomycin Sensitivity in Human Lung Cancer Cell Line using Centella asiatica Leaf Extract. Yang Wu, Shi Gao, Tan Yuan. Abstract. Purpose: To demonstrate the effectiveness of Centella asiatica aqueous extract in augmenting the cytotoxic effect of bleomycin in the adenocarcinoma human alveolar basal ...

  18. Sensitivity of risk parameters to human errors for a PWR

    International Nuclear Information System (INIS)

    Samanta, P.; Hall, R.E.; Kerr, W.

    1980-01-01

    Sensitivities of the risk parameters, emergency safety system unavailabilities, accident sequence probabilities, release category probabilities and core melt probability were investigated for changes in the human error rates within the general methodological framework of the Reactor Safety Study for a Pressurized Water Reactor (PWR). Impact of individual human errors were assessed both in terms of their structural importance to core melt and reliability importance on core melt probability. The Human Error Sensitivity Assessment of a PWR (HESAP) computer code was written for the purpose of this study

  19. Mithramycin A induces apoptosis by regulating the mTOR/Mcl-1/tBid pathway in androgen-independent prostate cancer cells

    Science.gov (United States)

    Choi, Eun-Sun; Chung, Taeho; Kim, Jun-Sung; Lee, Hakmo; Kwon, Ki Han; Cho, Nam-Pyo; Cho, Sung-Dae

    2013-01-01

    Mithramycin A (Mith) is an aureolic acid-type polyketide produced by various soil bacteria of the genus Streptomyces. Mith inhibits myeloid cell leukemia-1 (Mcl-1) to induce apoptosis in prostate cancer, but the molecular mechanism underlying this process has not been fully elucidated. The aim of this study was therefore to investigate the detailed molecular mechanism related to Mith-induced apoptosis in prostate cancer cells. Mith decreased the phosphorylation of mammalian target of rapamycin (mTOR) in both cell lines overexpressing phospho-mTOR compared to RWPE-1 human normal prostate epithelial cells. Mith significantly induced truncated Bid (tBid) and siRNA-mediated knock-down of Mcl-1 increased tBid protein levels. Moreover, Mith also inhibited the phosphorylation of mTOR on serine 2448 and Mcl-1, and increased tBid protein in prostate tumors in athymic nude mice bearing DU145 cells as xenografts. Thus, Mith acts as an effective tumor growth inhibitor in prostate cancer cells through the mTOR/Mcl-1/tBid signaling pathway. PMID:24062605

  20. A specific and sensitive radioimmunoassay for human choriogonadotropin

    International Nuclear Information System (INIS)

    Muralidhar, K.; Chaudhuri, G.; Lippes, J.; Bahl, O.P.

    1983-01-01

    A specific and sensitive radioimmunoassay for human choriogonadotropin (hCG) has been developed using rabbit antiserum to chemical analogs of beta subunit of human chorionic gonadotropin prepared by controlled reduction and S-alkylation of its disulfied linkages. The assay was highly specific for hCG as the binding of [ 125 1]-hCG to the antibody was not affected by standard human lutropin, by human male serum, postpartum serum from women, serum from post-menopausal women and human menopausal gonadotropin (Pergonal). The assay was highly sensitive, the minimal detection limit in terms of highly purified hCG (L-129, 12.500 IU/mg) being 1 ng/ml or 0.2 ng/tube (or 12 mlU/ml in terms of WHO 2nd international reference preparation of hCG). Using this assay we were unable to detect any immunoreactive hCG in human tissues like lung, liver and colon. The high specificity, sensitivity, accuracy and reproducibility of the assay make this a highly desirable radioimmunoassay for human choriogonadotropin. (orig.)

  1. A non-human primate model for gluten sensitivity.

    Directory of Open Access Journals (Sweden)

    Michael T Bethune

    2008-02-01

    Full Text Available Gluten sensitivity is widespread among humans. For example, in celiac disease patients, an inflammatory response to dietary gluten leads to enteropathy, malabsorption, circulating antibodies against gluten and transglutaminase 2, and clinical symptoms such as diarrhea. There is a growing need in fundamental and translational research for animal models that exhibit aspects of human gluten sensitivity.Using ELISA-based antibody assays, we screened a population of captive rhesus macaques with chronic diarrhea of non-infectious origin to estimate the incidence of gluten sensitivity. A selected animal with elevated anti-gliadin antibodies and a matched control were extensively studied through alternating periods of gluten-free diet and gluten challenge. Blinded clinical and histological evaluations were conducted to seek evidence for gluten sensitivity.When fed with a gluten-containing diet, gluten-sensitive macaques showed signs and symptoms of celiac disease including chronic diarrhea, malabsorptive steatorrhea, intestinal lesions and anti-gliadin antibodies. A gluten-free diet reversed these clinical, histological and serological features, while reintroduction of dietary gluten caused rapid relapse.Gluten-sensitive rhesus macaques may be an attractive resource for investigating both the pathogenesis and the treatment of celiac disease.

  2. Radiation sensitivity of human lung cancer cell lines

    International Nuclear Information System (INIS)

    Carmichael, J.; Degraff, W.G.; Gamson, J.; Russo, G.; Mitchell, J.B.; Gazdar, A.F.; Minna, J.D.; Levitt, M.L.

    1989-01-01

    X-Ray survival curves were determined using a panel of 17 human lung cancer cell lines, with emphasis on non-small cell lung cancer (NSCLC). In contrast to classic small cell lung cancer (SCLC) cell lines, NSCLC cell lines were generally less sensitive to radiation as evidenced by higher radiation survival curve extrapolation numbers, surviving fraction values following a 2Gy dose (SF2) and the mean inactivation dose values (D) values. The spectrum of in vitro radiation responses observed was similar to that expected in clinical practice, although mesothelioma was unexpectedly sensitive in vitro. Differences in radiosensitivity were best distinguished by comparison of SF2 values. Some NSCLC lines were relatively sensitive, and in view of this demonstrable variability in radiation sensitivity, the SF2 value may be useful for in vitro predictive assay testing of clinical specimens. (author)

  3. Differential sensitivity of Chironomus and human hemoglobin to gamma radiation

    International Nuclear Information System (INIS)

    Gaikwad, Pallavi S.; Panicker, Lata; Mohole, Madhura; Sawant, Sangeeta; Mukhopadhyaya, Rita; Nath, Bimalendu B.

    2016-01-01

    Chironomus ramosus is known to tolerate high doses of gamma radiation exposure. Larvae of this insect possess more than 95% of hemoglobin (Hb) in its circulatory hemolymph. This is a comparative study to see effect of gamma radiation on Hb of Chironomus and humans, two evolutionarily diverse organisms one having extracellular and the other intracellular Hb respectively. Stability and integrity of Chironomus and human Hb to gamma radiation was compared using biophysical techniques like Dynamic Light Scattering (DLS), UV-visible spectroscopy, fluorescence spectrometry and CD spectroscopy after exposure of whole larvae, larval hemolymph, human peripheral blood, purified Chironomus and human Hb. Sequence- and structure-based bioinformatics methods were used to analyze the sequence and structural similarities or differences in the heme pockets of respective Hbs. Resistivity of Chironomus Hb to gamma radiation is remarkably higher than human Hb. Human Hb exhibited loss of heme iron at a relatively low dose of gamma radiation exposure as compared to Chironomus Hb. Unlike human Hb, the heme pocket of Chironomus Hb is rich in aromatic amino acids. Higher hydophobicity around heme pocket confers stability of Chironomus Hb compared to human Hb. Previously reported gamma radiation tolerance of Chironomus can be largely attributed to its evolutionarily ancient form of extracellular Hb as evident from the present study. -- Highlights: •Comparison of radiation tolerant Chironomus Hb and radiation sensitive Human Hb. •Amino acid composition of midge and human heme confer differential hydrophobicity. •Heme pocket of evolutionarily ancient midge Hb provide gamma radiation resistivity.

  4. Differential sensitivity of Chironomus and human hemoglobin to gamma radiation

    Energy Technology Data Exchange (ETDEWEB)

    Gaikwad, Pallavi S. [Stress Biology Research Laboratory, Department of Zoology, Savitribai Phule University, Pune, 411007 (India); Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, 400085 (India); Panicker, Lata [Solid State Physics Division, Bhabha Atomic Research Centre, Trombay, Mumbai, 400085 (India); Mohole, Madhura; Sawant, Sangeeta [Bioinformatics Center, Savitribai Phule Pune University, Pune, 411007 (India); Mukhopadhyaya, Rita [Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, 400085 (India); Nath, Bimalendu B., E-mail: bbnath@gmail.com [Stress Biology Research Laboratory, Department of Zoology, Savitribai Phule University, Pune, 411007 (India)

    2016-08-05

    Chironomus ramosus is known to tolerate high doses of gamma radiation exposure. Larvae of this insect possess more than 95% of hemoglobin (Hb) in its circulatory hemolymph. This is a comparative study to see effect of gamma radiation on Hb of Chironomus and humans, two evolutionarily diverse organisms one having extracellular and the other intracellular Hb respectively. Stability and integrity of Chironomus and human Hb to gamma radiation was compared using biophysical techniques like Dynamic Light Scattering (DLS), UV-visible spectroscopy, fluorescence spectrometry and CD spectroscopy after exposure of whole larvae, larval hemolymph, human peripheral blood, purified Chironomus and human Hb. Sequence- and structure-based bioinformatics methods were used to analyze the sequence and structural similarities or differences in the heme pockets of respective Hbs. Resistivity of Chironomus Hb to gamma radiation is remarkably higher than human Hb. Human Hb exhibited loss of heme iron at a relatively low dose of gamma radiation exposure as compared to Chironomus Hb. Unlike human Hb, the heme pocket of Chironomus Hb is rich in aromatic amino acids. Higher hydophobicity around heme pocket confers stability of Chironomus Hb compared to human Hb. Previously reported gamma radiation tolerance of Chironomus can be largely attributed to its evolutionarily ancient form of extracellular Hb as evident from the present study. -- Highlights: •Comparison of radiation tolerant Chironomus Hb and radiation sensitive Human Hb. •Amino acid composition of midge and human heme confer differential hydrophobicity. •Heme pocket of evolutionarily ancient midge Hb provide gamma radiation resistivity.

  5. Identification of failure sequences sensitive to human error

    International Nuclear Information System (INIS)

    1987-06-01

    This report prepared by the participants of the technical committee meeting on ''Identification of Failure Sequences Sensitive to Human Error'' addresses the subjects discussed during the meeting and the conclusions reached by the committee. Chapter 1 reviews the INSAG recommendations and the main elements of the IAEA Programme in the area of human element. In Chapter 2 the role of human actions in nuclear power plants safety from insights of operational experience is reviewed. Chapter 3 is concerned with the relationship between probabilistic safety assessment and human performance associated with severe accident sequences. Chapter 4 addresses the role of simulators in view of training for accident conditions. Chapter 5 presents the conclusions and future trends. The seven papers presented by members of this technical committee are also included in this technical document. A separate abstract was prepared for each of these papers

  6. Sensitivity analysis techniques for models of human behavior.

    Energy Technology Data Exchange (ETDEWEB)

    Bier, Asmeret Brooke

    2010-09-01

    Human and social modeling has emerged as an important research area at Sandia National Laboratories due to its potential to improve national defense-related decision-making in the presence of uncertainty. To learn about which sensitivity analysis techniques are most suitable for models of human behavior, different promising methods were applied to an example model, tested, and compared. The example model simulates cognitive, behavioral, and social processes and interactions, and involves substantial nonlinearity, uncertainty, and variability. Results showed that some sensitivity analysis methods create similar results, and can thus be considered redundant. However, other methods, such as global methods that consider interactions between inputs, can generate insight not gained from traditional methods.

  7. Sensitive detection of viral transcripts in human tumor transcriptomes.

    Directory of Open Access Journals (Sweden)

    Sven-Eric Schelhorn

    Full Text Available In excess of 12% of human cancer incidents have a viral cofactor. Epidemiological studies of idiopathic human cancers indicate that additional tumor viruses remain to be discovered. Recent advances in sequencing technology have enabled systematic screenings of human tumor transcriptomes for viral transcripts. However, technical problems such as low abundances of viral transcripts in large volumes of sequencing data, viral sequence divergence, and homology between viral and human factors significantly confound identification of tumor viruses. We have developed a novel computational approach for detecting viral transcripts in human cancers that takes the aforementioned confounding factors into account and is applicable to a wide variety of viruses and tumors. We apply the approach to conducting the first systematic search for viruses in neuroblastoma, the most common cancer in infancy. The diverse clinical progression of this disease as well as related epidemiological and virological findings are highly suggestive of a pathogenic cofactor. However, a viral etiology of neuroblastoma is currently contested. We mapped 14 transcriptomes of neuroblastoma as well as positive and negative controls to the human and all known viral genomes in order to detect both known and unknown viruses. Analysis of controls, comparisons with related methods, and statistical estimates demonstrate the high sensitivity of our approach. Detailed investigation of putative viral transcripts within neuroblastoma samples did not provide evidence for the existence of any known human viruses. Likewise, de-novo assembly and analysis of chimeric transcripts did not result in expression signatures associated with novel human pathogens. While confounding factors such as sample dilution or viral clearance in progressed tumors may mask viral cofactors in the data, in principle, this is rendered less likely by the high sensitivity of our approach and the number of biological replicates

  8. Zebra finches are sensitive to prosodic features of human speech.

    Science.gov (United States)

    Spierings, Michelle J; ten Cate, Carel

    2014-07-22

    Variation in pitch, amplitude and rhythm adds crucial paralinguistic information to human speech. Such prosodic cues can reveal information about the meaning or emphasis of a sentence or the emotional state of the speaker. To examine the hypothesis that sensitivity to prosodic cues is language independent and not human specific, we tested prosody perception in a controlled experiment with zebra finches. Using a go/no-go procedure, subjects were trained to discriminate between speech syllables arranged in XYXY patterns with prosodic stress on the first syllable and XXYY patterns with prosodic stress on the final syllable. To systematically determine the salience of the various prosodic cues (pitch, duration and amplitude) to the zebra finches, they were subjected to five tests with different combinations of these cues. The zebra finches generalized the prosodic pattern to sequences that consisted of new syllables and used prosodic features over structural ones to discriminate between stimuli. This strong sensitivity to the prosodic pattern was maintained when only a single prosodic cue was available. The change in pitch was treated as more salient than changes in the other prosodic features. These results show that zebra finches are sensitive to the same prosodic cues known to affect human speech perception. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

  9. Salivary Proteome Patterns Affecting Human Salt Taste Sensitivity.

    Science.gov (United States)

    Stolle, Theresa; Grondinger, Freya; Dunkel, Andreas; Meng, Chen; Médard, Guillaume; Kuster, Bernhard; Hofmann, Thomas

    2017-10-25

    To investigate the role of perireceptor events in inter-individual variability in salt taste sensitivity, 31 volunteers were monitored in their detection functions for sodium chloride (NaCl) and classified into sensitive (0.6-1.7 mmol/L), medium-sensitive (1.8-6.9 mmol/L), and nonsensitive (7.0-11.2 mmol/L) subjects. Chemosensory intervention of NaCl-sensitive (S + ) and nonsensitive (S - ) panellists with potassium chloride, ammonium chloride, and sodium gluconate showed the salt taste sensitivity to be specific for NaCl. As no significant differences were found between S + and S - subjects in salivary sodium and protein content, salivary proteome differences and their stimulus-induced dynamic changes were analyzed by tryptic digestion, iTRAQ labeling, and liquid chromatography-tandem mass spectrometry analysis. Differences in the salivary proteome between S + and S - subjects were found primarily in resting saliva and were largely independent of the dynamic alterations observed upon salt stimulation. Gene ontology enrichment analysis of key proteins, i.e., immunoglobulin heavy constant y1, myeloblastin, cathepsin G, and kallikrein, revealed significantly increased serine-type endopeptidase activity for the S + group, while the S - group exhibited augmented cysteine-type endopeptidase inhibitor activity by increased abundances in lipocalin-1 and cystatin-D, -S, and -SN, respectively. As proteases have been suggested to facilitate transepithelial sodium transport by cleaving the y-subunit of the epithelial sodium channel (ENaC) and protease inhibitors have been shown to reduce ENaC-mediated sodium transport, the differentially modulated proteolytic activity patterns observed in vivo for S + and S - subjects show evidence of them playing a crucial role in affecting human NaCl sensitivity.

  10. Highly sensitive radioimmunoassay for chorionic gonadotropin in human urine

    International Nuclear Information System (INIS)

    Ayala, A.R.; Nisula, B.C.; Chen, H.C.; Hodgen, G.D.; Ross, G.T.

    1978-01-01

    The value of RIAs that measure hCG levels in human urine has been limited principally because of cross-reactivity with human LH. Recently, antisera generated to antigenic determinants on the intact hCGβ subunit and its carboxyl-terminal peptide have been shown to exhibit substantially reduced human LH cross-reactivity. To take maximal advantage of these antisera and to minimize interference by nonspecific substances in urine, a procedure for extracting and concentrating hCG from 24-h urine samples was developed. The procedure involves preparation of a standard kaolin-acetone urine concentrate and adsorption of the hCG in the concentrate to Concanavalin A covalently linked to agarose for purification and subsequent RIA. In urine samples obtained from patients with gestational trophoblastic disease, there was a direct correlation between hCG levels measured by RIA and those estimated by mouse uterine weight bioassy. In individual subjects, hCG levels were determined in serum and urine obtained the same day. When hCG was clearly detectable in the serum at levels greater than 1 ng/ml, the quantity of hCG measured in the urine concentrate exceeded 500 ng/24 h. The concentrates prepared from the urine of normal persons contained an hCG-like glycoprotein substance with antigenic determinants similar to those of the carboxyl-terminal peptide of hCGβ. As the range of hCG immunoreactivity measured in the urine concentrates of normal subjects was 6 to 52 ng/24 h, specific and sensitive detection of urinary hCG could be accomplished in patients whose sera contained hCG undetectable by conventional RIA. Partial purification and concentration of urinary hCG by this procedure with subsequent RIA provides a sensitive and reliable method for detecting hCG in urine

  11. A rapid, sensitive and specific radioimmunoassay for human chorionic gonadotrophin

    International Nuclear Information System (INIS)

    Kardana, A.; Bagshawe, K.D.

    1976-01-01

    A specific, quantitative assay for human chorionic gonadotrophin (hCG) in plasma has been developed using an hCG-β antiserum and an assay procedure lasting four hours. It employs a double antibody method and in a procedure prior to assay the two antibodies are pre-incubated in bulk and then stored in ampoules in lyophilised form ready for use. The assay procedure is a dis-equilibrium (sequential saturation) system in which the sample or unlabelled antigen is first incubated with the pre-incubated antisera followed by the addition of the labelled antigen and a further incubation. The antigen-antibody complex is separated from free antigen by vacuum filtration on glass fibre discs. The assay sensitivity is 100-200 picograms hCG/ml (0.68 mIU/ml) and is uninfluenced by normal concentrations of luteinising hormone in the sample

  12. Cost-Sensitive Bayesian Control Policy in Human Active Sensing

    Directory of Open Access Journals (Sweden)

    Sheeraz eAhmad

    2014-12-01

    Full Text Available An important but poorly understood aspect of sensory processing is the role of active sensing, the use of self-motion such as eye or head movements to focus sensing resources on the most rewarding or informative aspects of the sensory environment. Here, we present behavioral data from a visual search experiment, as well as a Bayesian model of within-trial dynamics of sensory processing and eye movements. Within this Bayes-optimal inference and control framework, which we call C-DAC (Context-Dependent Active Controller, various types of behavioral costs, such as temporal delay, response error, and sensor repositioning cost, are explicitly minimized. This contrasts with previously proposed algorithms that optimize abstract statistical objectives such as anticipated information gain (Infomax (Butko and Movellan, 2010 and one-step look-ahead accuracy (greedy MAP (Najemnik and Geisler, 2005. We find that C-DAC captures human visual search dynamics better than previous models, in particular a certain form of confirmation bias apparent in the way human subjects utilize prior knowledge about the spatial distribution of the search target to improve search speed and accuracy. We also examine several computationally efficient approximations to C-DAC that may present biologically more plausible accounts of the neural computations underlying active sensing, as well as practical tools for solving active sensing problems in engineering applications. To summarize, this paper makes several key contributions: human visual search behavioral data, a context-sensitive Bayesian active sensing model, a comparative study between different models of human active sensing, and a family of efficient approximations to the optimal model.

  13. Influence of heredity on human sensitivity to environmental chemicals

    Energy Technology Data Exchange (ETDEWEB)

    Weber, W.W. [Univ. of Michigan, Ann Arbor, MI (United States)

    1995-12-31

    Hereditary peculiarities in individual responses to environmental chemicals are a common occurrence in human populations. Genetic variation in glutathione S-transferase, CYP1A2, N-acetyltransferase, and paraoxonase exemplify the relationship of metabolic variation to individual susceptibility to cancer and other toxicants of environmental origin. Heritable receptor protein variants, a subset of proteins of enormous pharmacogenetic, potential that have not thus far been extensively explored form the pharmacogenetic standpoint, and also considered. Examples of interest that are considered include receptor variants associated with retinoic acid resistance in acute promyelocytic leukemia, with paradoxical responses to antiandrogens in prostate cancer, and with retinitis pigmentosa. Additional heritable protein variants of pharmacogenetic interest that result in antibiotic-induced deafness, glucocorticoid-remediable aldosteronism and hypertension, the long-QT syndrome, and beryllium-induced lung disease are also discussed. These traits demonstrate how knowledge of the molecular basis and mechanism of the variant response may contribute to its prevention in sensitive persons as well as to improved therapy for genetically conditioned disorders that arise form environmental chemicals. 99 refs.

  14. Comparison of risk sensitivity to human errors in the Oconee and LaSalle PRAs

    International Nuclear Information System (INIS)

    Wong, S.; Higgins, J.

    1991-01-01

    This paper describes the comparative analyses of plant risk sensitivity to human errors in the Oconee and La Salle Probabilistic Risk Assessment (PRAs). These analyses were performed to determine the reasons for the observed differences in the sensitivity of core melt frequency (CMF) to changes in human error probabilities (HEPs). Plant-specific design features, PRA methods, and the level of detail and assumptions in the human error modeling were evaluated to assess their influence risk estimates and sensitivities

  15. Machine Learning Approaches for Predicting Human Skin Sensitization Hazard

    Science.gov (United States)

    One of ICCVAM’s top priorities is the development and evaluation of non-animal approaches to identify potential skin sensitizers. The complexity of biological events necessary for a substance to elicit a skin sensitization reaction suggests that no single in chemico, in vit...

  16. Capsaicin-induced central sensitization evokes segmental increases in trigger point sensitivity in humans.

    Science.gov (United States)

    Srbely, John Z; Dickey, James P; Bent, Leah R; Lee, David; Lowerison, Mark

    2010-07-01

    This study investigated whether inducing central sensitization evokes segmental increases in trigger point pressure sensitivity. We evoked central sensitization at the C(5) segment and validated its presence via mechanical cutaneous sensitivity (brush allodynia) testing. Trigger point pressure sensitivity was quantified using the pain pressure threshold (PPT) value. A 50 cm(2) area of the C(5) dermatome at the right lateral elbow was pretreated with 45 degrees heat for 10 minutes. Test subjects (n = 20) then received topical capsaicin cream (0.075%; Medicis, Toronto, Canada) to the C(5) dermatome, whereas control subjects (n = 20) received a topical placebo cream (Biotherm Massage, Montreal, Canada). PPT readings were recorded from the infraspinatus (C(5,6)) and gluteus medius (L(4,5)S(1)) trigger points at zero (pre-intervention), 10, 20, and 30 minutes after intervention; all PPT readings were normalized to pre-intervention (baseline) values. The difference between the PPT readings at the 2 trigger point sites represents the direct influence of segmental mechanisms on the trigger point sensitivity at the infraspinatus site (PPT(seg)). Test subjects demonstrated statistically significant increases in Total Allodynia scores and significant decreases in PPT(seg) at 10, 20, and 30 minutes after application, when compared with control subjects. These results demonstrate that increases in central sensitization evoke increases in trigger point pressure sensitivity in segmentally related muscles. Myofascial pain is the most common form of musculoskeletal pain. Myofascial trigger points play an important role in the clinical manifestation of myofascial pain syndrome. Elucidating the role of central sensitization in the pathophysiology of trigger points is fundamental to developing optimal strategies in the management of myofascial pain syndrome.

  17. Annatto Tocotrienol Induces a Cytotoxic Effect on Human Prostate Cancer PC3 Cells via the Simultaneous Inhibition of Src and Stat3.

    Science.gov (United States)

    Sugahara, Ryosuke; Sato, Ayami; Uchida, Asuka; Shiozawa, Shinya; Sato, Chiaki; Virgona, Nantiga; Yano, Tomohiro

    2015-01-01

    Prostate cancer is one of the most frequently occurring cancers and often acquires the potential of androgen-independent growth as a malignant phenotype. Androgen-independent prostate cancer has severe chemoresistance towards conventional chemotherapeutic agents, so a new treatment approach is required for curing such prostate cancer. In this context, the present study was undertaken to check if annatto tocotrienol (main component δ-tocotrienol) could suppress cell growth in human prostate cancer (PC3, androgen-independent type) cells via the inhibition of Src and Stat3. The tocotrienol showed cytotoxic effects on PC3 cells in a dose-dependent manner, and the effect depended on G1 arrest in the cell cycle and subsequent induction of apoptosis. In a cytotoxic dose, the tocotrienol suppressed cellular growth via the simultaneous inhibition of Src and Stat3. Similarly, the treatment combination of both Src and Stat3 inhibitors induced cytotoxic effects in PC3 cells in an additive manner compared to each by itself. With respect to cell cycle regulation and the induction of apoptosis, the combination treatment showed a similar effect to that of the tocotrienol treatment. These results suggest that annatto tocotrienol effectively induces cytotoxicity in androgen-independent prostate cancer cells via the suppression of Src and Stat3.

  18. Caffeine markedly sensitizes human mesothelioma cell lines to pemetrexed

    Science.gov (United States)

    Min, Sang Hee; Goldman, I. David; Zhao, Rongbao

    2013-01-01

    Pemetrexed is a new generation antifolate approved for the treatment of mesothelioma and non-small cell lung cancer. Caffeine is known to augment radiation or chemotherapeutic drug-induced cell killing. The current study addresses the impact of caffeine on the activity of pemetrexed in mesothelioma cell lines. Caffeine enhanced pemetrexed activity in all four mesothelioma cell lines tested (H2052, H2373, H28 and MSTO-211H). Caffeine sensitized H2052 cells in a dose- and schedule-dependent manner, and was associated with a markedly decreased clonogenic survival. Caffeine sensitization occurred only in cells subjected to pulse, but not continuous, exposure to pemetrexed. Similar pemetrexed sensitization was also observed with the clinically better tolerated caffeine analog, theobromine. Pemetrexed sensitization by caffeine was associated with an increase in pemetrexed-induced phosphorylation of ataxia-telangiectasia-mutated (ATM) and Chk1. These data indicate that caffeine and its analog, theobromine, may be a useful approach to enhance pemetrexed-based chemotherapy. PMID:17594092

  19. Sensitivity to musical structure in the human brain

    Science.gov (United States)

    McDermott, Josh H.; Norman-Haignere, Sam; Kanwisher, Nancy

    2012-01-01

    Evidence from brain-damaged patients suggests that regions in the temporal lobes, distinct from those engaged in lower-level auditory analysis, process the pitch and rhythmic structure in music. In contrast, neuroimaging studies targeting the representation of music structure have primarily implicated regions in the inferior frontal cortices. Combining individual-subject fMRI analyses with a scrambling method that manipulated musical structure, we provide evidence of brain regions sensitive to musical structure bilaterally in the temporal lobes, thus reconciling the neuroimaging and patient findings. We further show that these regions are sensitive to the scrambling of both pitch and rhythmic structure but are insensitive to high-level linguistic structure. Our results suggest the existence of brain regions with representations of musical structure that are distinct from high-level linguistic representations and lower-level acoustic representations. These regions provide targets for future research investigating possible neural specialization for music or its associated mental processes. PMID:23019005

  20. Is there a sensitive period in human incest avoidance?

    Science.gov (United States)

    Luo, Liqun

    2011-06-24

    Many studies support the proposition that early cosocialization with opposite-sex children has the effect of inhibiting later mutual sexual attraction, but the existence of a period in the life cycle in which individuals are sensitive to the effect of early cosocialization has been a matter of controversy. Drawing on earlier traditional psychological research, and on more recent work guided by parental investment theory, we hypothesized that only for maternal perinatal association (MPA)-absent males a less-than- around-three-years age difference with the sister can predict stronger aversion to sibling incest. The results corroborated the hypothesis. The results can be interpreted as support for the existence of a sensitive period as well as for the potent role of MPA. Cross-cultural comparative studies were called on to further test the hypothesis.

  1. Is There a Sensitive Period in Human Incest Avoidance?

    Directory of Open Access Journals (Sweden)

    Liqun Luo

    2011-04-01

    Full Text Available Many studies support the proposition that early cosocialization with opposite-sex children has the effect of inhibiting later mutual sexual attraction, but the existence of a period in the life cycle in which individuals are sensitive to the effect of early cosocialization has been a matter of controversy. Drawing on earlier traditional psychological research, and on more recent work guided by parental investment theory, we hypothesized that only for maternal perinatal association (MPA-absent males a less-than-around-three-years age difference with the sister can predict stronger aversion to sibling incest. The results corroborated the hypothesis. The results can be interpreted as support for the existence of a sensitive period as well as for the potent role of MPA. Cross-cultural comparative studies were called on to further test the hypothesis.

  2. Optimizing human activity patterns using global sensitivity analysis.

    Science.gov (United States)

    Fairchild, Geoffrey; Hickmann, Kyle S; Mniszewski, Susan M; Del Valle, Sara Y; Hyman, James M

    2014-12-01

    Implementing realistic activity patterns for a population is crucial for modeling, for example, disease spread, supply and demand, and disaster response. Using the dynamic activity simulation engine, DASim, we generate schedules for a population that capture regular (e.g., working, eating, and sleeping) and irregular activities (e.g., shopping or going to the doctor). We use the sample entropy (SampEn) statistic to quantify a schedule's regularity for a population. We show how to tune an activity's regularity by adjusting SampEn, thereby making it possible to realistically design activities when creating a schedule. The tuning process sets up a computationally intractable high-dimensional optimization problem. To reduce the computational demand, we use Bayesian Gaussian process regression to compute global sensitivity indices and identify the parameters that have the greatest effect on the variance of SampEn. We use the harmony search (HS) global optimization algorithm to locate global optima. Our results show that HS combined with global sensitivity analysis can efficiently tune the SampEn statistic with few search iterations. We demonstrate how global sensitivity analysis can guide statistical emulation and global optimization algorithms to efficiently tune activities and generate realistic activity patterns. Though our tuning methods are applied to dynamic activity schedule generation, they are general and represent a significant step in the direction of automated tuning and optimization of high-dimensional computer simulations.

  3. Human gut microbes impact host serum metabolome and insulin sensitivity

    DEFF Research Database (Denmark)

    Pedersen, Helle Krogh; Gudmundsdottir, Valborg; Nielsen, Henrik Bjørn

    2016-01-01

    Insulin resistance is a forerunner state of ischaemic cardiovascular disease and type 2 diabetes. Here we show how the human gut microbiome impacts the serum metabolome and associates with insulin resistance in 277 non-diabetic Danish individuals. The serum metabolome of insulin-resistant individ......Insulin resistance is a forerunner state of ischaemic cardiovascular disease and type 2 diabetes. Here we show how the human gut microbiome impacts the serum metabolome and associates with insulin resistance in 277 non-diabetic Danish individuals. The serum metabolome of insulin...

  4. Human error in strabismus surgery: Quantification with a sensitivity analysis

    NARCIS (Netherlands)

    S. Schutte (Sander); J.R. Polling (Jan Roelof); F.C.T. van der Helm (Frans); H.J. Simonsz (Huib)

    2009-01-01

    textabstractBackground: Reoperations are frequently necessary in strabismus surgery. The goal of this study was to analyze human-error related factors that introduce variability in the results of strabismus surgery in a systematic fashion. Methods: We identified the primary factors that influence

  5. Human error in strabismus surgery : Quantification with a sensitivity analysis

    NARCIS (Netherlands)

    Schutte, S.; Polling, J.R.; Van der Helm, F.C.T.; Simonsz, H.J.

    2008-01-01

    Background- Reoperations are frequently necessary in strabismus surgery. The goal of this study was to analyze human-error related factors that introduce variability in the results of strabismus surgery in a systematic fashion. Methods- We identified the primary factors that influence the outcome of

  6. Oxygen-Sensitive K+ Channels Modulate Human Chorionic Gonadotropin Secretion from Human Placental Trophoblast.

    Science.gov (United States)

    Díaz, Paula; Sibley, Colin P; Greenwood, Susan L

    2016-01-01

    Human chorionic gonadotropin (hCG) is a key autocrine/paracrine regulator of placental syncytiotrophoblast, the transport epithelium of the human placenta. Syncytiotrophoblast hCG secretion is modulated by the partial pressure of oxygen (pO2), reactive oxygen species (ROS) and potassium (K+) channels. Here we test the hypothesis that K+ channels mediate the effects of pO2 and ROS on hCG secretion. Placental villous explants from normal term pregnancies were cultured for 6 days at 6% (normoxia), 21% (hyperoxia) or 1% (hypoxia) pO2. On days 3-5, explants were treated with 5mM 4-aminopyridine (4-AP) or tetraethylammonium (TEA), blockers of pO2-sensitive voltage-gated K+ (KV) channels, or ROS (10-1000μM H2O2). hCG secretion and lactate dehydrogenase (LDH) release, a marker of necrosis, were determined daily. At day 6, hCG and LDH were measured in tissue lysate and 86Rb (K+) efflux assessed to estimate syncytiotrophoblast K+ permeability. hCG secretion and 86Rb efflux were significantly greater in explants maintained in 21% pO2 than normoxia. 4-AP/TEA inhibited hCG secretion to a greater extent at 21% than 6% and 1% pO2, and reduced 86Rb efflux at 21% but not 6% pO2. LDH release and tissue LDH/hCG were similar in 6%, 21% and 1% pO2 and unaffected by 4-AP/TEA. H2O2 stimulated 86Rb efflux and hCG secretion at normoxia but decreased 86Rb efflux, without affecting hCG secretion, at 21% pO2. 4-AP/TEA-sensitive K+ channels participate in pO2-sensitive hCG secretion from syncytiotrophoblast. ROS effects on both hCG secretion and 86Rb efflux are pO2-dependent but causal links between the two remain to be established.

  7. Oxygen-Sensitive K+ Channels Modulate Human Chorionic Gonadotropin Secretion from Human Placental Trophoblast

    Science.gov (United States)

    Díaz, Paula; Sibley, Colin P.; Greenwood, Susan L.

    2016-01-01

    Human chorionic gonadotropin (hCG) is a key autocrine/paracrine regulator of placental syncytiotrophoblast, the transport epithelium of the human placenta. Syncytiotrophoblast hCG secretion is modulated by the partial pressure of oxygen (pO2), reactive oxygen species (ROS) and potassium (K+) channels. Here we test the hypothesis that K+ channels mediate the effects of pO2 and ROS on hCG secretion. Placental villous explants from normal term pregnancies were cultured for 6 days at 6% (normoxia), 21% (hyperoxia) or 1% (hypoxia) pO2. On days 3–5, explants were treated with 5mM 4-aminopyridine (4-AP) or tetraethylammonium (TEA), blockers of pO2-sensitive voltage-gated K+ (KV) channels, or ROS (10–1000μM H2O2). hCG secretion and lactate dehydrogenase (LDH) release, a marker of necrosis, were determined daily. At day 6, hCG and LDH were measured in tissue lysate and 86Rb (K+) efflux assessed to estimate syncytiotrophoblast K+ permeability. hCG secretion and 86Rb efflux were significantly greater in explants maintained in 21% pO2 than normoxia. 4-AP/TEA inhibited hCG secretion to a greater extent at 21% than 6% and 1% pO2, and reduced 86Rb efflux at 21% but not 6% pO2. LDH release and tissue LDH/hCG were similar in 6%, 21% and 1% pO2 and unaffected by 4-AP/TEA. H2O2 stimulated 86Rb efflux and hCG secretion at normoxia but decreased 86Rb efflux, without affecting hCG secretion, at 21% pO2. 4-AP/TEA-sensitive K+ channels participate in pO2-sensitive hCG secretion from syncytiotrophoblast. ROS effects on both hCG secretion and 86Rb efflux are pO2-dependent but causal links between the two remain to be established. PMID:26863525

  8. Effect of ethanol of the radiation sensitivity of human hemoglobin

    International Nuclear Information System (INIS)

    Szweda-Lewandowska, Z.; Puchala, M.

    1981-01-01

    Radiation sensitivity of oxy-, deoxy-, and methemoglobin (HbOs, Hbbj, and MetHb) in water solutions containing 0.2 M ethanol and in ethanol-free solutions was compared. Radiation sensitivity was estimated on the basis of changes in absorbance at the Soret band (a = 430 nm for deoxyhemoglobin), changes in the absorbance ration Avqv/Avwt determined after conversion of irradiated preparations to methemoglobin, and changes in the value of parameters describing the reaction of hemoglobin oxygenation. The protection coefficient p of hemoglobin by ethanol (ratio of a change in the absence of ethanol to that in its presence) calculated from changes in absorbance at the Soret band equaled about 1.5 at a 4-Mrad dose in all bases except MetHb irradiated in air for which p was much higher (about 3.2). The protection coefficient p' calculated from Dtx values for changes in Avchemically bondv/Avwt equaled 2.2 for HbOs, and 2.8 for MetHb for preparations irradiated in air; p' = 1.7 for Hbbj and 1.8 for MetHb for preparations irradiated under argon. On the basis of these results, the role of /sup ./OH radicals and oxygen in the radiation damage of hemoglobin is discussed

  9. NHERF1 Enhances Cisplatin Sensitivity in Human Cervical Cancer Cells

    Science.gov (United States)

    Tao, Tao; Yang, Xiaomei; Qin, Qiong; Shi, Wen; Wang, Qiqi; Yang, Ying; He, Junqi

    2017-01-01

    Cervical cancer is one of the most common female malignancies, and cisplatin-based chemotherapy is routinely utilized in locally advanced cervical cancer patients. However, resistance has been the major limitation. In this study, we found that Na+/H+ Exchanger Regulatory Factor 1 (NHERF1) was downregulated in cisplatin-resistant cells. Analysis based on a cervical cancer dataset from The Cancer Genome Atlas (TCGA) showed association of NHERF1 expression with disease-free survival of patients received cisplatin treatment. NHERF1 overexpression inhibited proliferation and enhanced apoptosis in cisplatin-resistant HeLa cells, whereas NHERF1 knockdown had inverse effects. While parental HeLa cells were more resistant to cisplatin after NHERF1 knockdown, NHERF1 overexpression in CaSki cells promoted cisplatin sensitivity. Overexpression and knockdown studies also showed that NHERF1 significantly inhibited AKT and extracellular signal–regulated kinase (ERK) signaling pathways in cisplatin-resistant cells. Taken together, our results provide the first evidence that NHERF1 can sensitize cisplatin-refractory cervical cancer cells. This study may help to increase understanding of the molecular mechanisms underlying cisplatin resistance in tumors. PMID:28085111

  10. NHERF1 Enhances Cisplatin Sensitivity in Human Cervical Cancer Cells.

    Science.gov (United States)

    Tao, Tao; Yang, Xiaomei; Qin, Qiong; Shi, Wen; Wang, Qiqi; Yang, Ying; He, Junqi

    2017-01-12

    Cervical cancer is one of the most common female malignancies, and cisplatin-based chemotherapy is routinely utilized in locally advanced cervical cancer patients. However, resistance has been the major limitation. In this study, we found that Na⁺/H⁺ Exchanger Regulatory Factor 1 (NHERF1) was downregulated in cisplatin-resistant cells. Analysis based on a cervical cancer dataset from The Cancer Genome Atlas (TCGA) showed association of NHERF1 expression with disease-free survival of patients received cisplatin treatment. NHERF1 overexpression inhibited proliferation and enhanced apoptosis in cisplatin-resistant HeLa cells, whereas NHERF1 knockdown had inverse effects. While parental HeLa cells were more resistant to cisplatin after NHERF1 knockdown, NHERF1 overexpression in CaSki cells promoted cisplatin sensitivity. Overexpression and knockdown studies also showed that NHERF1 significantly inhibited AKT and extracellular signal-regulated kinase (ERK) signaling pathways in cisplatin-resistant cells. Taken together, our results provide the first evidence that NHERF1 can sensitize cisplatin-refractory cervical cancer cells. This study may help to increase understanding of the molecular mechanisms underlying cisplatin resistance in tumors.

  11. Are human peripheral nerves sensitive to X-ray imaging?

    Directory of Open Access Journals (Sweden)

    Jonas Francisco Scopel

    Full Text Available Diagnostic imaging techniques play an important role in assessing the exact location, cause, and extent of a nerve lesion, thus allowing clinicians to diagnose and manage more effectively a variety of pathological conditions, such as entrapment syndromes, traumatic injuries, and space-occupying lesions. Ultrasound and nuclear magnetic resonance imaging are becoming useful methods for this purpose, but they still lack spatial resolution. In this regard, recent phase contrast x-ray imaging experiments of peripheral nerve allowed the visualization of each nerve fiber surrounded by its myelin sheath as clearly as optical microscopy. In the present study, we attempted to produce high-resolution x-ray phase contrast images of a human sciatic nerve by using synchrotron radiation propagation-based imaging. The images showed high contrast and high spatial resolution, allowing clear identification of each fascicle structure and surrounding connective tissue. The outstanding result is the detection of such structures by phase contrast x-ray tomography of a thick human sciatic nerve section. This may further enable the identification of diverse pathological patterns, such as Wallerian degeneration, hypertrophic neuropathy, inflammatory infiltration, leprosy neuropathy and amyloid deposits. To the best of our knowledge, this is the first successful phase contrast x-ray imaging experiment of a human peripheral nerve sample. Our long-term goal is to develop peripheral nerve imaging methods that could supersede biopsy procedures.

  12. d -Limonene sensitizes docetaxel-induced cytotoxicity in human prostate cancer cells: Generation of reactive oxygen species and induction of apoptosis

    Directory of Open Access Journals (Sweden)

    Rabi Thangaiyan

    2009-01-01

    Full Text Available Background: Clinical trials have shown that docetaxel combined with other novel agents can improve the survival of androgen-independent prostate cancer patients. d -Limonene, a non-nutrient dietary component, has been found to inhibit various cancer cell growths without toxicity. We sought to characterize whether a non-toxic dose of d -limonene may enhance tumor response to docetaxel in an in vitro model of metastatic prostate cancer. Materials and Methods: Human prostate carcinoma DU-145 and normal prostate epithelial PZ-HPV-7 cells were treated with various concentrations of d -limonene, docetaxel or a combination of both, and cell viability was determined by MTT assay. Intracellular reactive oxygen species (ROS, reduced glutathione (GSH and caspase activity were measured. Apoptosis and apoptosis-related proteins were studied by enzyme-linked immunosorbent assay and Western blotting, respectively. Results: d -Limonene and docetaxel in combination significantly enhanced the cytotoxicity to DU-145 cells than PZ-HPV-7 cells. Exposure of DU-145 cells to a combined d -limonene and docetaxel resulted in higher ROS generation, depletion of GSH, accompanied by increased caspase activity than docetaxel alone. It also triggered a series of effects involving cytochrome c , cleavages of caspase-9, 3 and poly (ADP-ribose polymerase, and a shift in Bad:Bcl-xL ratio in favor of apoptosis. Apoptotic effect was significantly blocked on pretreatment with N -acetylcystein, indicating that antitumor effect is initiated by ROS generation, and caspase cascades contribute to the cell death. Conclusion: Our results show, for the first time, that d -limonene enhanced the antitumor effect of docetaxel against prostate cancer cells without being toxic to normal prostate epithelial cells. The combined beneficial effect could be through the modulation of proteins involved in mitochondrial pathway of apoptosis. d -Limonene could be used as a potent non-toxic agent to

  13. Sensitivity-enhanced 13C MR spectroscopy of the human brain at 3 Tesla.

    NARCIS (Netherlands)

    Klomp, D.W.J.; Renema, W.K.J.; Graaf, M. van der; Galan, B.E. de; Kentgens, A.P.M.; Heerschap, A.

    2006-01-01

    A new coil design for sensitivity-enhanced 13C MR spectroscopy (MRS) of the human brain is presented. The design includes a quadrature transmit/receive head coil optimized for 13C MR sensitivity. Loss-less blocking circuits inside the coil conductors allow this coil to be used inside a homogeneous

  14. Sensitivity-enhanced C-13 MR spectroscopy of the human brain at 3 Tesla

    NARCIS (Netherlands)

    Klomp, D.W.J.; Renema, W.K.J.; Graaf, M. van der; Galan, B.E. de; Kentgens, A.P.M.; Heerschap, A.

    2006-01-01

    A new coil design for sensitivity-enhanced C-13 MR spectroscopy (MRS) of the human brain is presented. The design includes a quadrature transmit/receive head coil optimized for C-13 MR sensitivity. Loss-less blocking circuits inside the coil conductors allow this coil to be used inside a homogeneous

  15. Evaluation of human skin tests for potential dermal irritant and contact sensitizing products: a position paper

    NARCIS (Netherlands)

    Loveren H van; Jong WH de; Garssen J; LPI

    1998-01-01

    Prediction of human cutaneous irritation and sensitization in view of hazard identification has primarily relied on the use of laboratory animals. Such studies in laboratory animals have been very instrumental in the detection of potential contact sensitizing agents. There are however many

  16. Human gut microbes impact host serum metabolome and insulin sensitivity.

    Science.gov (United States)

    Pedersen, Helle Krogh; Gudmundsdottir, Valborg; Nielsen, Henrik Bjørn; Hyotylainen, Tuulia; Nielsen, Trine; Jensen, Benjamin A H; Forslund, Kristoffer; Hildebrand, Falk; Prifti, Edi; Falony, Gwen; Le Chatelier, Emmanuelle; Levenez, Florence; Doré, Joel; Mattila, Ismo; Plichta, Damian R; Pöhö, Päivi; Hellgren, Lars I; Arumugam, Manimozhiyan; Sunagawa, Shinichi; Vieira-Silva, Sara; Jørgensen, Torben; Holm, Jacob Bak; Trošt, Kajetan; Kristiansen, Karsten; Brix, Susanne; Raes, Jeroen; Wang, Jun; Hansen, Torben; Bork, Peer; Brunak, Søren; Oresic, Matej; Ehrlich, S Dusko; Pedersen, Oluf

    2016-07-21

    Insulin resistance is a forerunner state of ischaemic cardiovascular disease and type 2 diabetes. Here we show how the human gut microbiome impacts the serum metabolome and associates with insulin resistance in 277 non-diabetic Danish individuals. The serum metabolome of insulin-resistant individuals is characterized by increased levels of branched-chain amino acids (BCAAs), which correlate with a gut microbiome that has an enriched biosynthetic potential for BCAAs and is deprived of genes encoding bacterial inward transporters for these amino acids. Prevotella copri and Bacteroides vulgatus are identified as the main species driving the association between biosynthesis of BCAAs and insulin resistance, and in mice we demonstrate that P. copri can induce insulin resistance, aggravate glucose intolerance and augment circulating levels of BCAAs. Our findings suggest that microbial targets may have the potential to diminish insulin resistance and reduce the incidence of common metabolic and cardiovascular disorders.

  17. A Sensitive Period: Bioethics, Human Rights, and Child Development.

    Science.gov (United States)

    Denburg, Avram

    2015-06-11

    This paper explores complementarities between bioethics and human rights in the ethical analysis of early childhood development (ECD) policies. It is argued that conceptual synergies arising from the integration of these fields are considerable, if underexplored, and best illumined through application to specific domains of health policy. ECD represents an especially germane case study: it is characterized by rapidly evolving science whose normative implications are complex, emergent, and understudied, yet whose societal impacts are wide-ranging. The paper first charts the disciplinary evolution of bioethics, demonstrating its gradual social turn: from the individual to collective, from the medical to the societal. It then reviews points of theoretical confluence between bioethics and human rights, to assess the value and feasibility of their joint application to health policy analysis. Finally, it maps these complementarities onto issues provoked by the epigenetics of ECD, in the hopes that both the policy domain and the analysis of theoretical synergies are enriched. It finds that the distinctly relational and emergent nature of ECD science and policy demands novel forms of normative inquiry. Only an ethical approach supple enough to adapt to emergent questions, examine issues from varied theoretical perspectives, and assimilate insights across traditional disciplinary bounds will prove sufficient to the task. Copyright 2015 Denburg. This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

  18. Perceiving Group Behavior: Sensitive Ensemble Coding Mechanisms for Biological Motion of Human Crowds

    Science.gov (United States)

    Sweeny, Timothy D.; Haroz, Steve; Whitney, David

    2013-01-01

    Many species, including humans, display group behavior. Thus, perceiving crowds may be important for social interaction and survival. Here, we provide the first evidence that humans use ensemble-coding mechanisms to perceive the behavior of a crowd of people with surprisingly high sensitivity. Observers estimated the headings of briefly presented…

  19. Sensitivity to ionizing radiation and chemotherapeutic agents in gemcitabine-resistant human tumor cell lines

    NARCIS (Netherlands)

    van Bree, Chris; Castro Kreder, Natasja; Loves, Willem J. P.; Franken, Nicolaas A. P.; Peters, Godefridus J.; Haveman, Jaap

    2002-01-01

    Purpose: To determine cross-resistance to anti-tumor treatments in 2',2'difluorodeoxycytidine (dFdC, gemcitabine)-resistant human tumor cells. Methods and Materials: Human lung carcinoma cells SW-1573 (SWp) were made resistant to dFdC (SWg). Sensitivity to cisplatin (cDDP), paclitaxel,

  20. Vibration sensitivity of human muscle spindles and Golgi tendon organs.

    Science.gov (United States)

    Fallon, James B; Macefield, Vaughan G

    2007-07-01

    The responses of the various muscle receptors to vibration are more complicated than a naïve categorization into stretch (muscle spindle primary ending), length (muscle spindle secondary endings), and tension (Golgi tendon organs) receptors. To emphasize the similarity of responses to small length changes, we recorded from 58 individual muscle afferents subserving receptors in the ankle or toe dorsiflexors of awake human subjects (32 primary endings, 20 secondary endings, and six Golgi tendon organs). Transverse sinusoidal vibration was applied to the distal tendon of the receptor-bearing muscle, while subjects either remained completely relaxed or maintained a weak isometric contraction of the appropriate muscle. In relaxed muscle, few units responded in a 1:1 manner to vibration, and there was no evidence of a preferred frequency of activation. In active muscle the response profiles of all three receptor types overlapped, with no significant difference in threshold between receptor types. These results emphasize that when intramuscular tension increases during a voluntary contraction, Golgi tendon organs and muscle spindle secondary endings, not just muscle spindle primary endings, can effectively encode small imposed length changes.

  1. Hyperbaric oxygen therapy attenuates central sensitization induced by a thermal injury in humans

    DEFF Research Database (Denmark)

    Rasmussen, V M; Borgen, A E; Jansen, E C

    2015-01-01

    BACKGROUND: Hyperbaric oxygen (HBO2 ) treatment has in animal experiments demonstrated antinociceptive effects. It was hypothesized that these effects would attenuate secondary hyperalgesia areas (SHAs), an expression of central sensitization, after a first-degree thermal injury in humans. METHOD......, compared with control. These new and original findings in humans corroborate animal experimental data. The thermal injury model may give impetus to future human neurophysiological studies exploring the central effects of hyperbaric oxygen treatment....

  2. Tuning and sensitivity of the human vestibular system to low-frequency vibration.

    Science.gov (United States)

    Todd, Neil P McAngus; Rosengren, Sally M; Colebatch, James G

    2008-10-17

    Mechanoreceptive hair-cells of the vertebrate inner ear have a remarkable sensitivity to displacement, whether excited by sound, whole-body acceleration or substrate-borne vibration. In response to seismic or substrate-borne vibration, thresholds for vestibular afferent fibre activation have been reported in anamniotes (fish and frogs) in the range -120 to -90 dB re 1g. In this article, we demonstrate for the first time that the human vestibular system is also extremely sensitive to low-frequency and infrasound vibrations by making use of a new technique for measuring vestibular activation, via the vestibulo-ocular reflex (VOR). We found a highly tuned response to whole-head vibration in the transmastoid plane with a best frequency of about 100 Hz. At the best frequency we obtained VOR responses at intensities of less than -70 dB re 1g, which was 15 dB lower than the threshold of hearing for bone-conducted sound in humans at this frequency. Given the likely synaptic attenuation of the VOR pathway, human receptor sensitivity is probably an order of magnitude lower, thus approaching the seismic sensitivity of the frog ear. These results extend our knowledge of vibration-sensitivity of vestibular afferents but also are remarkable as they indicate that the seismic sensitivity of the human vestibular system exceeds that of the cochlea for low-frequencies.

  3. Lack of influence of GTP cyclohydrolase gene (GCH1 variations on pain sensitivity in humans

    Directory of Open Access Journals (Sweden)

    Dionne Raymond A

    2007-03-01

    Full Text Available Abstract Objectives To assess the effect of variations in GTP cyclohydrolase gene (GCH1 on pain sensitivity in humans. Methods Thermal and cold pain sensitivity were evaluated in a cohort of 735 healthy volunteers. Among this cohort, the clinical pain responses of 221 subjects after the surgical removal of impacted third molars were evaluated. Genotyping was done for 38 single nucleotide polymorphisms (SNPs whose heterozygosity > 0.2 in GCH1. Influence of the genetic variations including SNPs and haplotypes on pain sensitivity were analyzed. Results Minor allele frequencies and linkage disequilibrium show significant differences in European Americans, African Americans, Hispanic Americans and Asian Americans. Association analyses in European Americans do not replicate the previously reported important influence of GCH1 variations on pain sensitivity. Conclusion Considering population stratification, previously reported associations between GCH1 genetic variations and pain sensitivity appear weak or negligible in this well characterized model of pain.

  4. Early endotoxemia increases peripheral and hepatic insulin sensitivity in healthy humans

    NARCIS (Netherlands)

    van der Crabben, Saskia N.; Blümer, Regje M. E.; Stegenga, Michiel E.; Ackermans, Mariëtte T.; Endert, Erik; Tanck, Michael W. T.; Serlie, Mireille J.; van der Poll, Tom; Sauerwein, Hans P.

    2009-01-01

    Context: Sepsis-induced hypoglycemia is a well known, but rare, event of unknown origin. Objective: To obtain insight into the mechanism of sepsis-induced hypoglycemia, focusing on glucose kinetics and insulin sensitivity measured with stable isotopes by using the model of human endotoxemia. Design:

  5. Specific alleles of bitter receptor genes influence human sensitivity to the bitterness of aloin and saccharin.

    Science.gov (United States)

    Pronin, Alexey N; Xu, Hong; Tang, Huixian; Zhang, Lan; Li, Qing; Li, Xiaodong

    2007-08-21

    Variation in human taste is a well-known phenomenon. However, little is known about the molecular basis for it. Bitter taste in humans is believed to be mediated by a family of 25 G protein-coupled receptors (hT2Rs, or TAS2Rs). Despite recent progress in the functional expression of hT2Rs in vitro, up until now, hT2R38, a receptor for phenylthiocarbamide (PTC), was the only gene directly linked to variations in human bitter taste. Here we report that polymorphism in two hT2R genes results in different receptor activities and different taste sensitivities to three bitter molecules. The hT2R43 gene allele, which encodes a protein with tryptophan in position 35, makes people very sensitive to the bitterness of the natural plant compounds aloin and aristolochic acid. People who do not possess this allele do not taste these compounds at low concentrations. The same hT2R43 gene allele makes people more sensitive to the bitterness of an artificial sweetener, saccharin. In addition, a closely related gene's (hT2R44's) allele also makes people more sensitive to the bitterness of saccharin. We also demonstrated that some people do not possess certain hT2R genes, contributing to taste variation between individuals. Our findings thus reveal new examples of variations in human taste and provide a molecular basis for them.

  6. Tumor necrosis factor alpha selectively sensitizes human immunodeficiency virus-infected cells to heat and radiation

    International Nuclear Information System (INIS)

    Wong, G.H.; McHugh, T.; Weber, R.; Goeddel, D.V.

    1991-01-01

    We report here that infection of the human T-cell line HUT-78 with human immunodeficiency virus (HIV) increases its sensitivity to heat and radiation toxicity. A possible explanation for this result may be the reduced expression of manganous superoxide dismutase (MnSOD) in HIV-infected cells compared to uninfected cells. Tumor necrosis factor alpha (TNF-alpha) further sensitizes HIV-infected cells but not uninfected cells to heat and radiation. This is consistent with the ability of TNF-alpha to induce the expression of MnSOD in uninfected but not in HIV-infected cells. HIV-infected HUT-78 cell lines engineered to overexpress MnSOD are more resistant to heat and radiation than HIV-infected cells that do not overexpress MnSOD. However, treatment with TNF-alpha still sensitizes these cells to heat and radiation

  7. Androgen Metabolism in Progression to Androgen-Independent Prostate Cancer

    Science.gov (United States)

    2011-06-01

    Neutrophils 1 0 0 Fatigue 22 1 1 Constitutional, other 2 0 0 INR 0 0 1 Rash/desquamation 1 0 0 Hot flashes 3 0 0 Anorexia 5 0 0 Dehydration 0 1 0 Nausea 11 4...and dutasteride (0.5 mg/d). Dose modifications for toxicity were specified. Patients were evaluated every 4 weeks, with history , physical examination...treatment at 5 weeks for pain management and 1 pa- tient stopped at 8 months for nerve root compression. Toxicity. Toxicities were reported among all patients

  8. Culture-sensitive neural substrates of human cognition: a transcultural neuroimaging approach.

    Science.gov (United States)

    Han, Shihui; Northoff, Georg

    2008-08-01

    Our brains and minds are shaped by our experiences, which mainly occur in the context of the culture in which we develop and live. Although psychologists have provided abundant evidence for diversity of human cognition and behaviour across cultures, the question of whether the neural correlates of human cognition are also culture-dependent is often not considered by neuroscientists. However, recent transcultural neuroimaging studies have demonstrated that one's cultural background can influence the neural activity that underlies both high- and low-level cognitive functions. The findings provide a novel approach by which to distinguish culture-sensitive from culture-invariant neural mechanisms of human cognition.

  9. Effect of training on insulin sensitivity of glucose uptake and lipolysis in human adipose tissue

    DEFF Research Database (Denmark)

    Stallknecht, B; Larsen, J J; Mikines, K J

    2000-01-01

    Training increases insulin sensitivity of both whole body and muscle in humans. To investigate whether training also increases insulin sensitivity of adipose tissue, we performed a three-step hyperinsulinemic, euglycemic clamp in eight endurance-trained (T) and eight sedentary (S) young men...... (glucose only). Adipose tissue blood flow was measured by (133)Xe washout. In the basal state, adipose tissue blood flow tended to be higher in T compared with S subjects, and in both groups blood flow was constant during the clamp. The change from basal in arterial-interstitial glucose concentration......-time: T, 44 +/- 9 min (n = 7); S, 102 +/- 23 min (n = 5); P training enhances insulin sensitivity of glucose uptake in subcutaneous adipose tissue and in skeletal muscle. Furthermore, interstitial glycerol data suggest that training also increases insulin sensitivity of lipolysis...

  10. Comparative sensitivity of human and rat neural cultures to chemical-induced inhibition of neurite outgrowth

    International Nuclear Information System (INIS)

    Harrill, Joshua A.; Freudenrich, Theresa M.; Robinette, Brian L.; Mundy, William R.

    2011-01-01

    There is a need for rapid, efficient and cost-effective alternatives to traditional in vivo developmental neurotoxicity testing. In vitro cell culture models can recapitulate many of the key cellular processes of nervous system development, including neurite outgrowth, and may be used as screening tools to identify potential developmental neurotoxicants. The present study compared primary rat cortical cultures and human embryonic stem cell-derived neural cultures in terms of: 1) reproducibility of high content image analysis based neurite outgrowth measurements, 2) dynamic range of neurite outgrowth measurements and 3) sensitivity to chemicals which have been shown to inhibit neurite outgrowth. There was a large increase in neurite outgrowth between 2 and 24 h in both rat and human cultures. Image analysis data collected across multiple cultures demonstrated that neurite outgrowth measurements in rat cortical cultures were more reproducible and had higher dynamic range as compared to human neural cultures. Human neural cultures were more sensitive than rat cortical cultures to chemicals previously shown to inhibit neurite outgrowth. Parallel analysis of morphological (neurite count, neurite length) and cytotoxicity (neurons per field) measurements were used to detect selective effects on neurite outgrowth. All chemicals which inhibited neurite outgrowth in rat cortical cultures did so at concentrations which did not concurrently affect the number of neurons per field, indicating selective effects on neurite outgrowth. In contrast, more than half the chemicals which inhibited neurite outgrowth in human neural cultures did so at concentrations which concurrently decreased the number of neurons per field, indicating that effects on neurite outgrowth were secondary to cytotoxicity. Overall, these data demonstrate that the culture models performed differently in terms of reproducibility, dynamic range and sensitivity to neurite outgrowth inhibitors. While human neural

  11. Distinct sulfonation activities in resveratrol-sensitive and resveratrol-insensitive human glioblastoma cells.

    Science.gov (United States)

    Sun, Zheng; Li, Hong; Shu, Xiao-Hong; Shi, Hui; Chen, Xiao-Yan; Kong, Qing-You; Wu, Mo-Li; Liu, Jia

    2012-07-01

    Glioblastoma multiforme (GBM) cells show different responses to resveratrol, for unknown reasons. Our data from human medulloblastoma cells and primary cultures of rat brain cells revealed an inverse correlation of sulfonation activity with resveratrol sensitivities, providing a clue to the underlying mechanisms of the variable sensitivities of GBM cells to resveratrol. In this study, we found that U251 cells were sensitive and LN229 cells were insensitive to resveratrol. Thus, these two cell lines were taken as comparable models for elucidating the influence of sulfonation activities on resveratrol sensitivity. HPLC showed identical resveratrol metabolic patterns in both cell lines. LC/MS and high-resolution mass MS analyses further demonstrated that resveratrol monosulfate generated by sulfotransferases (SULTs) was the major metabolite of human GBM cells. The levels of brain-associated SULT (SULT1A1, SULT1C2, and SULT4A1) expression in U251 cells were lower than those in LN229 cells, suggesting the inverse relationship of SULT-mediated sulfonation activity with high intracellular resveratrol bioavailability and resveratrol sensitivity of human GBM cells. Furthermore, immunohistochemical staining revealed reductions in expression of the three brain-associated SULTs in 72.8%, 47.5% and 66.3% of astrocytomas, respectively. Therefore, the levels of brain-associated SULTs and sulfonation activity mediated by them could be important parameters for evaluating the potential response of human GBM cells to resveratrol, and may have value in the personalized treatment of GBMs with resveratrol. © 2012 The Authors Journal compilation © 2012 FEBS.

  12. Higher sensory processing sensitivity, introversion and ectomorphism: New biomarkers for human creativity in developing rural areas.

    Science.gov (United States)

    Rizzo-Sierra, Carlos V; Leon-S, Martha E; Leon-Sarmiento, Fidias E

    2012-05-01

    The highly sensitive trait present in animals, has also been proposed as a human neurobiological trait. People having such trait can process larger amounts of sensory information than usual, making it an excellent attribute that allows to pick up subtle environmental details and cues. Furthermore, this trait correlates to some sort of giftedness such as higher perception, inventiveness, imagination and creativity. We present evidences that support the existance of key neural connectivity between the mentioned trait, higher sensory processing sensitivity, introversion, ectomorphism and creativity. The neurobiological and behavioral implications that these biomarkers have in people living in developing rural areas are discussed as well.

  13. Endogenous Opioid-Masked Latent Pain Sensitization: Studies from Mouse to Human.

    Directory of Open Access Journals (Sweden)

    Manuel P Pereira

    Full Text Available Following the resolution of a severe inflammatory injury in rodents, administration of mu-opioid receptor inverse agonists leads to reinstatement of pain hypersensitivity. The mechanisms underlying this form of latent pain sensitization (LS likely contribute to the development of chronic pain, but LS has not yet been demonstrated in humans. Using a C57BL/6 mouse model of cutaneous mild heat injury (MHI we demonstrated a dose-dependent reinstatement of pain sensitization, assessed as primary (P < 0.001 and secondary hyperalgesia (P < 0.001 by naloxone (0.3–10 mg/kg, 168 hrs after the induction of MHI. Forward-translating the dose data to a human MHI model (n = 12 we could show that LS does indeed occur after naloxone 2 mg/kg, 168 hrs after a MHI. Our previous unsuccessful efforts to demonstrate unmasking of LS in humans are thus likely explained by an insufficient naloxone dose (0.021 mg/kg. However, while LS was consistently demonstrated in 21/24 mice, LS was only seen in 4/12 subjects. This difference is likely due to selection bias since the C57BL/6 mouse strain exhibits markedly enhanced pain sensitivity in assays of acute thermal nociception. Future exploratory studies in humans should prioritize inclusion of “high-sensitizers” prone to develop LS and use post-surgical models to elucidate markers of vulnerability to chronic postsurgical pain.EudraCT 2012-005663-27.

  14. Isolation of uv-sensitive variants of human FL cells by a viral suicide method

    International Nuclear Information System (INIS)

    Shiomi, T.; Sato, K.

    1979-01-01

    A new method (viral suicide method) for the isolation of uv-sensitive mutants is described. Colonies of mutagenized human FL cells were infected with uv-irradiated Herpes simplex viruses and surviving ones which seemed to be deficient in host cell reactivation (HCR) were examined for their uv sensitivity. Nineteen of 238 clones examined were sensitive to uv irradiation at the time of the isolation. After recloning, four of these clones have been studied and two (UVS-1 and UVS-2) of them are stable in their uv sensitivity for 4 months in culture. uv sensitivity of UVS-1, UVS-2, and the parental FL cells are as follows: the extrapolation numbers (n) are 2.2, 2.1, and 1.8 and mean lethal doses (DO) are 2.9, 3.7, and 7.8 J/m 2 for UVS-1, UVS-2, and the parental FL cells, respectively. They are no more sensitive than FL cells to x-irradiation. The ability of HCR in UVS-2 cells is apparently lower than that in FL cells, whereas UVS-1 cells are the same as FL cells in the ability

  15. Advances in radiation biology: Relative radiation sensitivities of human organ systems. Volume 12

    Energy Technology Data Exchange (ETDEWEB)

    Lett, J.T.; Altman, K.I.; Ehmann, U.K.; Cox, A.B.

    1987-01-01

    This volume is a thematically focused issue of Advances in Radiation Biology. The topic surveyed is relative radiosensitivity of human organ systems. Topics considered include relative radiosensitivities of the thymus, spleen, and lymphohemopoietic systems; relative radiosensitivities of the small and large intestine; relative rediosensitivities of the oral cavity, larynx, pharynx, and esophagus; relative radiation sensitivity of the integumentary system; dose response of the epidermal; microvascular, and dermal populations; relative radiosensitivity of the human lung; relative radiosensitivity of fetal tissues; and tolerance of the central and peripheral nervous system to therapeutic irradiation.

  16. Identification of human immunodeficiency virus subtypes with distinct patterns of sensitivity to serum neutralization.

    OpenAIRE

    Cheng-Mayer, C; Homsy, J; Evans, L A; Levy, J A

    1988-01-01

    The human immunodeficiency virus (HIV) type 1 displays a high degree of genetic variation, especially in the glycoprotein (gp120) domain of the envelope gene. To determine whether this genomic heterogeneity leads to the expression of independent HIV subtypes, 12 sera from HIV type 1 antibody-positive individuals were tested for their ability to neutralize 20 HIV isolates of various origins. Four distinct HIV subtypes with different sensitivity to serum neutralization were identified. These re...

  17. Sensitivity field distributions for segmental bioelectrical impedance analysis based on real human anatomy

    Science.gov (United States)

    Danilov, A. A.; Kramarenko, V. K.; Nikolaev, D. V.; Rudnev, S. G.; Salamatova, V. Yu; Smirnov, A. V.; Vassilevski, Yu V.

    2013-04-01

    In this work, an adaptive unstructured tetrahedral mesh generation technology is applied for simulation of segmental bioimpedance measurements using high-resolution whole-body model of the Visible Human Project man. Sensitivity field distributions for a conventional tetrapolar, as well as eight- and ten-electrode measurement configurations are obtained. Based on the ten-electrode configuration, we suggest an algorithm for monitoring changes in the upper lung area.

  18. Neural prediction errors reveal a risk-sensitive reinforcement-learning process in the human brain.

    Science.gov (United States)

    Niv, Yael; Edlund, Jeffrey A; Dayan, Peter; O'Doherty, John P

    2012-01-11

    Humans and animals are exquisitely, though idiosyncratically, sensitive to risk or variance in the outcomes of their actions. Economic, psychological, and neural aspects of this are well studied when information about risk is provided explicitly. However, we must normally learn about outcomes from experience, through trial and error. Traditional models of such reinforcement learning focus on learning about the mean reward value of cues and ignore higher order moments such as variance. We used fMRI to test whether the neural correlates of human reinforcement learning are sensitive to experienced risk. Our analysis focused on anatomically delineated regions of a priori interest in the nucleus accumbens, where blood oxygenation level-dependent (BOLD) signals have been suggested as correlating with quantities derived from reinforcement learning. We first provide unbiased evidence that the raw BOLD signal in these regions corresponds closely to a reward prediction error. We then derive from this signal the learned values of cues that predict rewards of equal mean but different variance and show that these values are indeed modulated by experienced risk. Moreover, a close neurometric-psychometric coupling exists between the fluctuations of the experience-based evaluations of risky options that we measured neurally and the fluctuations in behavioral risk aversion. This suggests that risk sensitivity is integral to human learning, illuminating economic models of choice, neuroscientific models of affective learning, and the workings of the underlying neural mechanisms.

  19. Spatial sensitivities of human health risk to intercontinental and high-altitude pollution

    Science.gov (United States)

    Koo, Jamin; Wang, Qiqi; Henze, Daven K.; Waitz, Ian A.; Barrett, Steven R. H.

    2013-06-01

    We perform the first long-term (>1 year) continuous adjoint simulations with a global atmospheric chemistry-transport model focusing on population exposure to fine particulate matter (PM2.5) and associated risk of early death. Sensitivities relevant to intercontinental and high-altitude PM pollution are calculated with particular application to aircraft emissions. Specifically, the sensitivities of premature mortality risk in different regions to NOx, SOx, CO, VOC and primary PM2.5 emissions as a function of location are computed. We apply the resultant sensitivity matrices to aircraft emissions, finding that NOx emissions are responsible for 93% of population exposure to aircraft-attributable PM2.5. Aircraft NOx accounts for all of aircraft-attributable nitrate exposure (as expected) and 53% of aircraft-attributable sulfate exposure due to the strong "oxidative coupling" between aircraft NOx emissions and non-aviation SO2 emissions in terms of sulfate formation. Of the health risk-weighted human PM2.5 exposure attributable to aviation, 73% occurs in Asia, followed by 18% in Europe. 95% of the air quality impacts of aircraft emissions in the US are incurred outside the US. We also assess the impact of uncertainty or changes in (non-aviation) ammonia emissions on aviation-attributable PM2.5 exposure by calculating second-order sensitivities. We note the potential application of the sensitivity matrices as a rapid policy analysis tool in aviation environmental policy contexts.

  20. Cisplatin and low dose rate irradiation in cisplatin resistant and sensitive human glioma cells

    International Nuclear Information System (INIS)

    Wilkins, David E.; Cheng, E. Ng; Raaphorst, G. Peter

    1996-01-01

    Purpose: Human glioma cell lines resistant (U373MG CP ) and sensitive (U373MG) to cisplatin were used to evaluate the effect of cisplatin as a sensitizer to low dose rate irradiation (LDRI). Methods and Materials: A cisplatin resistant glioma cell line U373MG CP was developed by chronic exposure of parental U373MG cells to cisplatin. Plateau phase cells were treated with cisplatin, high dose rate (HDR) irradiation (1.12 Gy/min), LDRI (0.0088 Gy/min), or cisplatin concurrent with LDRI. Cell survival was determined by the colony forming assay. Results: Both cell lines showed increased resistance to radiation at LDR compared with HDR, with Dose Modifying Factors (DMF at 10% survival level) of 1.7 for U373MG and 2.5 for U373MG CP . The increased LDR sparing effect in the cisplatin resistant U373MG CP cells indicates increased repair proficiency. The resistant cell line showed a fourfold increase in resistance to cisplatin cytotoxicity at the 10% survival level compared with the parental U373MG cells. Cisplatin enhanced the response of both cell lines to LDRI. The DMFs were 1.2, 1.2, and 1.7, respectively, for the sensitive U373MG cell line given 1 μg/ml, and the resistant cell line given 3 or 6 μg/ml cisplatin treatments concurrent with LDRI. Conclusions: These data show that cisplatin can be an effective sensitizer to LDRI in both cisplatin resistant and sensitive glioma cell lines. However, in the resistant cell line, higher concentrations of cisplatin were necessary to achieve the same level of sensitization as in the sensitive cell line

  1. Hyperbaric oxygen therapy attenuates central sensitization induced by a thermal injury in humans.

    Science.gov (United States)

    Rasmussen, V M; Borgen, A E; Jansen, E C; Rotbøll Nielsen, P H; Werner, M U

    2015-07-01

    Hyperbaric oxygen (HBO2 ) treatment has in animal experiments demonstrated antinociceptive effects. It was hypothesized that these effects would attenuate secondary hyperalgesia areas (SHAs), an expression of central sensitization, after a first-degree thermal injury in humans. Seventeen healthy volunteers were examined during two sessions using a randomized crossover design. Volunteers were studied during control conditions (ambient pressure, FI O2  = 0.21) and during HBO2 (2.4 standard atmosphere, FI O2  = 1.0, 90 min) conditions in a pressure chamber. Quantitative sensory testing, including assessment of SHAs was performed. A statistically significant overall attenuation of SHAs was seen during the HBO2 sessions compared with the control-sessions (P = 0.011). In the eight volunteers starting with the HBO2 session, no difference in SHAs compared with control was demonstrated. However, in the nine volunteers starting with the control session, a statistical significant attenuation of SHAs was demonstrated in the HBO2 session (P = 0.004). The results indicate that HBO2 therapy in humans attenuates central sensitization induced by a thermal skin injury, compared with control. These new and original findings in humans corroborate animal experimental data. The thermal injury model may give impetus to future human neurophysiological studies exploring the central effects of hyperbaric oxygen treatment. © 2015 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

  2. S100A10 protein expression is associated with oxaliplatin sensitivity in human colorectal cancer cells

    Directory of Open Access Journals (Sweden)

    Suzuki Sayo

    2011-12-01

    Full Text Available Abstract Background Individual responses to oxaliplatin (L-OHP-based chemotherapy remain unpredictable. The objective of our study was to find candidate protein markers for tumor sensitivity to L-OHP from intracellular proteins of human colorectal cancer (CRC cell lines. We performed expression difference mapping (EDM analysis of whole cell lysates from 11 human CRC cell lines with different sensitivities to L-OHP by using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS, and identified a candidate protein by liquid chromatography/mass spectrometry ion trap time-of-flight (LCMS-IT-TOF. Results Of the qualified mass peaks obtained by EDM analysis, 41 proteins were differentially expressed in 11 human colorectal cancer cell lines. Among these proteins, the peak intensity of 11.1 kDa protein was strongly correlated with the L-OHP sensitivity (50% inhibitory concentrations (P R2 = 0.80. We identified this protein as Protein S100-A10 (S100A10 by MS/MS ion search using LCMS-IT-TOF. We verified its differential expression and the correlation between S100A10 protein expression levels in drug-untreated CRC cells and their L-OHP sensitivities by Western blot analyses. In addition, S100A10 protein expression levels were not correlated with sensitivity to 5-fluorouracil, suggesting that S100A10 is more specific to L-OHP than to 5-fluorouracil in CRC cells. S100A10 was detected in cell culture supernatant, suggesting secretion out of cells. Conclusions By proteomic approaches including SELDI technology, we have demonstrated that intracellular S100A10 protein expression levels in drug-untreated CRC cells differ according to cell lines and are significantly correlated with sensitivity of CRC cells to L-OHP exposure. Our findings provide a new clue to searching predictive markers of the response to L-OHP, suggesting that S100A10 is expected to be one of the candidate protein markers.

  3. Assessment of metal sensitizer potency with the reconstructed human epidermis IL-18 assay.

    Science.gov (United States)

    Gibbs, Susan; Kosten, Ilona; Veldhuizen, Rosalien; Spiekstra, Sander; Corsini, Emanuela; Roggen, Erwin; Rustemeyer, Thomas; Feilzer, Albert J; Kleverlaan, Cees J

    2018-01-15

    According to the new EU Medical Devices (MDR) legislation coming into effect in 2017, manufactures will have to comply with higher standards of quality and safety for medical devices in order to meet common safety concerns regarding such products. Metal alloys are extensively used in dentistry and medicine (e.g. orthopedic surgery and cardiology) even though clinical experience suggests that many metals are sensitizers. The aim of this study was to further test the applicability domain of the in vitro reconstructed human epidermis (RhE) IL-18 assay developed to identify contact allergens and in doing so: i) determine whether different metal salts, representing leachables from metal alloys used in medical devices, could be correctly labelled and classified; and ii) assess the ability of different salts for the same metal to penetrate the skin stratum corneum. Twenty eight chemicals including 15 metal salts were topically exposed to RhE. Nickel, chrome, gold, palladium were each tested in two different salt forms, and titanium in 4 different salt forms. Metal salts were labelled (YES/NO) as sensitizer if a threshold of more than 5 fold IL18 release was reached. The in vitro estimation of expected sensitization induction level (potency) was assessed by interpolating in vitro EC50 and IL-18 SI2 with LLNA EC3 and human NOEL values from standard reference curves generated using DNCB (extreme) and benzocaine (weak). Metal salts, in contrast to other chemical sensitizers and with the exception of potassium dichromate (VI) and cobalt (II) chloride, were not identified as contact allergens since they only induced a small or no increase in IL-18 production. This finding was not related to a lack of stratum corneum skin penetration since EC50 values (decrease in metabolic activity; MTT assay) were obtained after topical RhE exposure to 8 of the 15 metal salts. For nickel, gold and palladium salts, differences in EC50 values between two salts for the same metal could not be

  4. Sensitivity to an Illusion of Sound Location in Human Auditory Cortex

    Directory of Open Access Journals (Sweden)

    Nathan C. Higgins

    2017-05-01

    Full Text Available Human listeners place greater weight on the beginning of a sound compared to the middle or end when determining sound location, creating an auditory illusion known as the Franssen effect. Here, we exploited that effect to test whether human auditory cortex (AC represents the physical vs. perceived spatial features of a sound. We used functional magnetic resonance imaging (fMRI to measure AC responses to sounds that varied in perceived location due to interaural level differences (ILD applied to sound onsets or to the full sound duration. Analysis of hemodynamic responses in AC revealed sensitivity to ILD in both full-cue (veridical and onset-only (illusory lateralized stimuli. Classification analysis revealed regional differences in the sensitivity to onset-only ILDs, where better classification was observed in posterior compared to primary AC. That is, restricting the ILD to sound onset—which alters the physical but not the perceptual nature of the spatial cue—did not eliminate cortical sensitivity to that cue. These results suggest that perceptual representations of auditory space emerge or are refined in higher-order AC regions, supporting the stable perception of auditory space in noisy or reverberant environments and forming the basis of illusions such as the Franssen effect.

  5. Females are sensitive to unpleasant human emotions regardless of the emotional context of photographs.

    Science.gov (United States)

    Kato, Ryousuke; Takeda, Yuji

    2017-06-09

    Previous studies have demonstrated that females exhibit higher sensitivity than males to the emotional state of a person in a photograph. The present study examined whether such females' sensitivity to human emotions could be observed even when the background emotional contexts were incongruent with facial expressions. The late positive potential (LPP) was measured while 19-female and 15-male participants viewed a photograph of a face with varied emotional expressions (pleasant, neutral, or unpleasant) superimposed on a background photograph with varied valences (pleasant, neutral, or unpleasant). The results showed that unpleasant background photographs elicited a larger LPP compared to pleasant and neutral background photographs in both female and male participants. In contrast, a larger LPP for the unpleasant face photographs was observed only in female participants. Furthermore, the effect of face photographs did not interact with the effect of background photographs. These results suggest that females are sensitive to human emotions regardless of the emotional context. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. DNA-mediated strand displacement facilitates sensitive electronic detection of antibodies in human serums.

    Science.gov (United States)

    Dou, Baoting; Yang, Jianmei; Shi, Kai; Yuan, Ruo; Xiang, Yun

    2016-09-15

    We describe here the development of a sensitive and convenient electronic sensor for the detection of antibodies in human serums. The sensor is constructed by self-assembly formation of a mixed monolayer containing the small molecule epitope conjugated double stranded DNA probes on gold electrode. The target antibody binds the epitope on the dsDNA probe and lowers the melting temperature of the duplex, which facilitates the displacement of the antibody-linked strand of the duplex probe by an invading methylene blue-tagged single stranded DNA (MB-ssDNA) through the strand displacement reaction and leads to the capture of many MB-ssDNA on the sensor surface. Subsequent electrochemical oxidation of the methylene blue labels results in amplified current response for sensitive monitoring of the antibodies. The antibody assay conditions are optimized and the sensor exhibits a linear range between 1.0 and 25.0nM with a detection limit of 0.67nM for the target antibody. The sensor is also selective and can be employed to detect the target antibodies in human serum samples. With the advantages of using small molecule epitope as the antibody recognition element over traditional antigen, the versatile manipulability of the DNA probes and the unique properties of the electrochemical transduction technique, the developed sensor thus hold great potential for simple and sensitive detection of different antibodies and other proteins in real samples. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Non-human biota dose assessment. Sensitivity analysis and knowledge quality assessment

    International Nuclear Information System (INIS)

    Smith, K.; Robinson, C.; Jackson, D.; La Cruz, I. de; Zinger, I.; Avila, R.

    2010-10-01

    This report provides a summary of a programme of work, commissioned within the BIOPROTA collaborative forum, to assess the quantitative and qualitative elements of uncertainty associated with biota dose assessment of potential impacts of long-term releases from geological disposal facilities (GDF). Quantitative and qualitative aspects of uncertainty were determined through sensitivity and knowledge quality assessments, respectively. Both assessments focused on default assessment parameters within the ERICA assessment approach. The sensitivity analysis was conducted within the EIKOS sensitivity analysis software tool and was run in both generic and test case modes. The knowledge quality assessment involved development of a questionnaire around the ERICA assessment approach, which was distributed to a range of experts in the fields of non-human biota dose assessment and radioactive waste disposal assessments. Combined, these assessments enabled critical model features and parameters that are both sensitive (i.e. have a large influence on model output) and of low knowledge quality to be identified for each of the three test cases. The output of this project is intended to provide information on those parameters that may need to be considered in more detail for prospective site-specific biota dose assessments for GDFs. Such information should help users to enhance the quality of their assessments and build greater confidence in the results. (orig.)

  8. Lansoprazole induces sensitivity to suboptimal doses of paclitaxel in human melanoma.

    Science.gov (United States)

    Azzarito, Tommaso; Venturi, Giulietta; Cesolini, Albino; Fais, Stefano

    2015-01-28

    Tumor acidity is now considered an important determinant of drug-resistance and tumor progression, and anti-acidic approaches, such as Proton Pump inhibitors (PPIs), have demonstrated promising antitumor and chemo-sensitizing efficacy. The main purpose of the present study was to evaluate the possible PPI-induced sensitization of human melanoma cells to Paclitaxel (PTX). Our results show that PTX and the PPI Lansoprazole (LAN) combination was extremely efficient against metastatic melanoma cells, as compared to the single treatments, both in vitro and in vivo. We also showed that acidity plays an important role on the anti-tumor activity of these drugs, being detrimental for PTX activity, while crucial for the synergistic effect of PTX following pretreatment with LAN, due to its nature of pro-drug needing protonation for a full activation. We obtained straightforward results in a human melanoma xenograft model combining well tolerated LAN doses with suboptimal and poorly toxic doses of PTX. With this study we provide a clear evidence that the PPI LAN may be included in new combined therapy of human melanoma together with low doses of PTX. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  9. Imprisonment and women's health: concerns about gender sensitivity, human rights and public health.

    Science.gov (United States)

    van den Bergh, Brenda J; Gatherer, Alex; Fraser, Andrew; Moller, Lars

    2011-09-01

    The health of prisoners is among the poorest of any population group and the apparent inequalities pose both a challenge and an opportunity for country health systems. The high rates of imprisonment in many countries, the resulting overcrowding, characteristics of prison populations and the disproportionate prevalence of health problems in prison should make prison health a matter of public health importance.Women prisoners constitute a minority within all prison systems and their special health needs are frequently neglected. The urgent need to review current services is clear from research, expert opinion and experience from countries worldwide. Current provision of health care to imprisoned women fails to meet their needs and is, in too many cases, far short of what is required by human rights and international recommendations. The evidence includes a lack of gender sensitivity in policies and practices in prisons, violations of women's human rights and failure to accept that imprisoned women have more and different health-care needs compared with male prisoners, often related to reproductive health issues, mental health problems, drug dependencies and histories of violence and abuse. Additional needs stem from their frequent status as a mother and usually the primary carer for her children.National governments, policy-makers and prison management need to address gender insensitivity and social injustice in prisons. There are immediate steps which could be taken to deal with public health neglect, abuses of human rights and failures in gender sensitivity.

  10. Highly Sensitive FPW-Based Microsystem for Rapid Detection of Tetrahydrocannabinol in Human Urine.

    Science.gov (United States)

    Lan, Je-Wei; Hsieh, Chia-Hsu; Huang, I-Yu; Lin, Yu-Cheng; Tsai, Tsung-Yi; Wang, Chua-Chin

    2017-11-29

    This paper presents a highly sensitive flexural plate-wave (FPW)-based microsystem for rapid detection of tetrahydrocannabinol (THC) in human urine. First, a circular-type interdigital transducer (IDT) was integrated with a circular-type silicon-grooved reflective grating structure (RGS) to reduce insertion loss. Then, with lower insertion loss (-38.758 dB), the FPW device was used to develop a novel THC biosensor, and the results reveal that this FPW-THC biosensor has low detection limit (1.5625 ng/mL) and high mass-sensitivity (126.67 cm²/g). Finally, this biosensor was integrated with field-programmable gate array (FPGA) board and discrete components for prototyping a FPW readout system, whose maximum error was 12.378 kHz to ensure that the linearity of detection up to R-square is equal to 0.9992.

  11. Highly Sensitive FPW-Based Microsystem for Rapid Detection of Tetrahydrocannabinol in Human Urine

    Directory of Open Access Journals (Sweden)

    Je-Wei Lan

    2017-11-01

    Full Text Available This paper presents a highly sensitive flexural plate-wave (FPW-based microsystem for rapid detection of tetrahydrocannabinol (THC in human urine. First, a circular-type interdigital transducer (IDT was integrated with a circular-type silicon-grooved reflective grating structure (RGS to reduce insertion loss. Then, with lower insertion loss (−38.758 dB, the FPW device was used to develop a novel THC biosensor, and the results reveal that this FPW-THC biosensor has low detection limit (1.5625 ng/mL and high mass-sensitivity (126.67 cm2/g. Finally, this biosensor was integrated with field-programmable gate array (FPGA board and discrete components for prototyping a FPW readout system, whose maximum error was 12.378 kHz to ensure that the linearity of detection up to R-square is equal to 0.9992.

  12. MicroRNA-203 Modulates the Radiation Sensitivity of Human Malignant Glioma Cells

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Ji Hyun [Department of Radiation Oncology, Graduate School of Medicine, Seoul National University, Seoul (Korea, Republic of); Hwang, Yeo Hyun; Lee, David J.; Kim, Dan Hyo; Park, Ji Min [Medical Science Research Institute, Seoul National University Bundang Hospital, Kyeonggido (Korea, Republic of); Wu, Hong-Gyun [Department of Radiation Oncology, Graduate School of Medicine, Seoul National University, Seoul (Korea, Republic of); Kim, In Ah, E-mail: inah228@snu.ac.kr [Department of Radiation Oncology, Graduate School of Medicine, Seoul National University, Seoul (Korea, Republic of); Medical Science Research Institute, Seoul National University Bundang Hospital, Kyeonggido (Korea, Republic of); Cancer Research Institute, Seoul National University, Seoul (Korea, Republic of)

    2016-02-01

    Purpose: We investigated whether miR-203 could modulate the radiation sensitivity of glioblastoma (GBM) cells and which target gene(s) could be involved. Methods and Materials: Three human malignant glioma (MG) cell lines and normal human astrocytes were transfected with control microRNA, pre-miR-203, or antisense miR-203. Real-time PCR (RT-PCR), clonogenic assays, immunofluorescence, and invasion/migration assays were performed. To predict the target(s), bioinformatics analyses using microRNA target databases were performed. Results: Overexpression of miR-203 increased the radiation sensitivity of all 3 human MG cell lines and prolonged radiation-induced γ-H2AX foci formation. Bioinformatics analyses suggested that miR-203 could be involved in post-transcriptional control of DNA repair, PI3K/AKT, SRC, and JAK/STAT3 and the vascular signaling pathway. Western blot analysis validated the fact that miR-203 downregulated ATM, RAD51, SRC, PLD2, PI3K-AKT, JAK-STAT3, VEGF, HIF-1α, and MMP2. Overexpression of miR-203 inhibited invasion and migration potentials, downregulated SLUG and Vimentin, and upregulated Claudin-1 and ZO1. Conclusions: These data demonstrate that miR-203 potentially controls DNA damage repair via the PI3K/AKT and JAK/STAT3 pathways and may collectively contribute to the modulation of radiation sensitivity in MG cells by inhibiting DNA damage repair, prosurvival signaling, and epithelium-mesenchyme transition. Taken together, these findings demonstrate that miR-203 could be a target for overcoming the radiation resistance of GBM.

  13. The Arctic Alzheimer mutation enhances sensitivity to toxic stress in human neuroblastoma cells

    DEFF Research Database (Denmark)

    Sennvik, Kristina; Nilsberth, Camilla; Stenh, Charlotte

    2002-01-01

    The E693G (Arctic) mutation of the amyloid precursor protein was recently found to lead to early-onset Alzheimer's disease in a Swedish family. In the present study, we report that the Arctic mutation decreases cell viability in human neuroblastoma cells. The cell viability, as measured by the MTT...... their secretion of beta-secretase cleaved amyloid precursor protein. The enhanced sensitivity to toxic stress in cells with the Arctic mutation most likely contributes to the pathogenic pathway leading to Alzheimer's disease....

  14. The Human Milk Protein-Lipid Complex HAMLET Sensitizes Bacterial Pathogens to Traditional Antimicrobial Agents

    Science.gov (United States)

    Marks, Laura R.; Clementi, Emily A.; Hakansson, Anders P.

    2012-01-01

    The fight against antibiotic resistance is one of the most significant challenges to public health of our time. The inevitable development of resistance following the introduction of novel antibiotics has led to an urgent need for the development of new antibacterial drugs with new mechanisms of action that are not susceptible to existing resistance mechanisms. One such compound is HAMLET, a natural complex from human milk that kills Streptococcus pneumoniae (the pneumococcus) using a mechanism different from common antibiotics and is immune to resistance-development. In this study we show that sublethal concentrations of HAMLET potentiate the effect of common antibiotics (penicillins, macrolides, and aminoglycosides) against pneumococci. Using MIC assays and short-time killing assays we dramatically reduced the concentrations of antibiotics needed to kill pneumococci, especially for antibiotic-resistant strains that in the presence of HAMLET fell into the clinically sensitive range. Using a biofilm model in vitro and nasopharyngeal colonization in vivo, a combination of HAMLET and antibiotics completely eradicated both biofilms and colonization in mice of both antibiotic-sensitive and resistant strains, something each agent alone was unable to do. HAMLET-potentiation of antibiotics was partially due to increased accessibility of antibiotics to the bacteria, but relied more on calcium import and kinase activation, the same activation pathway HAMLET uses when killing pneumococci by itself. Finally, the sensitizing effect was not confined to species sensitive to HAMLET. The HAMLET-resistant respiratory species Acinetobacter baumanii and Moraxella catarrhalis were all sensitized to various classes of antibiotics in the presence of HAMLET, activating the same mechanism as in pneumococci. Combined these results suggest the presence of a conserved HAMLET-activated pathway that circumvents antibiotic resistance in bacteria. The ability to activate this pathway may extend

  15. STUDIES ON THE ANTIBODIES IN RABBIT ANTISERA RESPONSIBLE FOR SENSITIZATION OF HUMAN SKIN

    Science.gov (United States)

    Vaughan, John H.; Kabat, Elvin A.

    1953-01-01

    The capacity of rabbit anti-egg albumin sera to sensitize human skin has been studied. It has been shown that passive transfer by these sera is completely unrelated to the egg albumin-anti-egg albumin system, as demonstrated by a failure of passive transfer by some antisera containing ample anti-egg albumin and persistence of passive transfer in other antisera from which all anti-egg albumin had been removed by precipitation with homologous antigen. Three preparations of non-precipitating anti-egg albumin have been shown to have sensitizing capacities which bear no relation to their non-precipitating anti-egg albumin contents. From a portion of one of these the non-precipitating anti-egg albumin was removed without impairing its sensitizing ability, while in another portion obliteration of the sensitizing capacity was accomplished without reducing the anti-egg albumin. Evidence is presented to show that there are at least two possible antibodies in anti-egg albumin sera which are capable of inducing skin sensitivity and that they are antibodies against egg white impurities in crystalline egg albumin other than anti-conalbumin, anti-ovomucoid, and anti-lysozyme. The usefulness of a suitable quantitative precipitin technic for the analysis for antibodies against antigen impurities and for their selective absorption from sera is illustrated. The principle governing the procedure is described. The technic allows for the determination of a given trace antibody by working with such small concentrations of its purified specific antigen that whatever other antigen-antibody compounds are formed simultaneously with that to be determined will be below their solubility levels and consequently will not contribute appreciably to the precipitate. PMID:13069639

  16. Localization and function of ATP-sensitive potassium channels in human skeletal muscle

    DEFF Research Database (Denmark)

    Nielsen, Jens Jung; Kristensen, Michael; Hellsten, Ylva

    2003-01-01

    The present study investigated the localization of ATP-sensitive K+ (KATP) channels in human skeletal muscle and the functional importance of these channels for human muscle K+ distribution at rest and during muscle activity. Membrane fractionation based on the giant vesicle technique...... or the sucrose-gradient technique in combination with Western blotting demonstrated that the KATP channels are mainly located in the sarcolemma. This localization was confirmed by immunohistochemical measurements. With the microdialysis technique, it was demonstrated that local application of the KATP channel...... inhibitor glibenclamide reduced (P approximately 4.5 to 4.0 mM, whereas the concentration in the control leg remained constant. Glibenclamide had no effect on the interstitial K+ accumulation during knee-extensor exercise at a power output of 60 W. In contrast...

  17. Decoding of faces and face components in face-sensitive human visual cortex

    Directory of Open Access Journals (Sweden)

    David F Nichols

    2010-07-01

    Full Text Available A great challenge to the field of visual neuroscience is to understand how faces are encoded and represented within the human brain. Here we show evidence from functional magnetic resonance imaging (fMRI for spatially distributed processing of the whole face and its components in face-sensitive human visual cortex. We used multi-class linear pattern classifiers constructed with a leave-one-scan-out verification procedure to discriminate brain activation patterns elicited by whole faces, the internal features alone, and the external head outline alone. Furthermore, our results suggest that whole faces are represented disproportionately in the fusiform cortex (FFA whereas the building blocks of faces are represented disproportionately in occipitotemporal cortex (OFA. Faces and face components may therefore be organized with functional clustering within both the FFA and OFA, but with specialization for face components in the OFA and the whole face in the FFA.

  18. Sensitive and Flexible Polymeric Strain Sensor for Accurate Human Motion Monitoring.

    Science.gov (United States)

    Khan, Hassan; Razmjou, Amir; Ebrahimi Warkiani, Majid; Kottapalli, Ajay; Asadnia, Mohsen

    2018-02-01

    Flexible electronic devices offer the capability to integrate and adapt with human body. These devices are mountable on surfaces with various shapes, which allow us to attach them to clothes or directly onto the body. This paper suggests a facile fabrication strategy via electrospinning to develop a stretchable, and sensitive poly (vinylidene fluoride) nanofibrous strain sensor for human motion monitoring. A complete characterization on the single PVDF nano fiber has been performed. The charge generated by PVDF electrospun strain sensor changes was employed as a parameter to control the finger motion of the robotic arm. As a proof of concept, we developed a smart glove with five sensors integrated into it to detect the fingers motion and transfer it to a robotic hand. Our results shows that the proposed strain sensors are able to detect tiny motion of fingers and successfully run the robotic hand.

  19. Sensitive and Flexible Polymeric Strain Sensor for Accurate Human Motion Monitoring

    Directory of Open Access Journals (Sweden)

    Hassan Khan

    2018-02-01

    Full Text Available Flexible electronic devices offer the capability to integrate and adapt with human body. These devices are mountable on surfaces with various shapes, which allow us to attach them to clothes or directly onto the body. This paper suggests a facile fabrication strategy via electrospinning to develop a stretchable, and sensitive poly (vinylidene fluoride nanofibrous strain sensor for human motion monitoring. A complete characterization on the single PVDF nano fiber has been performed. The charge generated by PVDF electrospun strain sensor changes was employed as a parameter to control the finger motion of the robotic arm. As a proof of concept, we developed a smart glove with five sensors integrated into it to detect the fingers motion and transfer it to a robotic hand. Our results shows that the proposed strain sensors are able to detect tiny motion of fingers and successfully run the robotic hand.

  20. Suppression of Mcl-1 via RNA interference sensitizes human hepatocellular carcinoma cells towards apoptosis induction

    International Nuclear Information System (INIS)

    Schulze-Bergkamen, Henning; Fleischer, Binje; Schuchmann, Marcus; Weber, Achim; Weinmann, Arndt; Krammer, Peter H; Galle, Peter R

    2006-01-01

    Hepatocelluar carcinoma (HCC) is one of the most common cancers worldwide and a major cause of cancer-related mortality. HCC is highly resistant to currently available chemotherapeutic drugs. Defects in apoptosis signaling contribute to this resistance. Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 protein family which interferes with mitochondrial activation. In a previous study we have shown that Mcl-1 is highly expressed in tissues of human HCC. In this study, we manipulated expression of the Mcl-1 protein in HCC cells by RNA interference and analyzed its impact on apoptosis sensitivity of HCC cells in vitro. RNA interference was performed by transfecting siRNA to specifically knock down Mcl-1 expression in HCC cells. Mcl-1 expression was measured by quantitative real-time PCR and Western blot. Induction of apoptosis and caspase activity after treatment with chemotherapeutic drugs and different targeted therapies were measured by flow cytometry and fluorometric analysis, respectively. Here we demonstrate that Mcl-1 expressing HCC cell lines show low sensitivity towards treatment with a panel of chemotherapeutic drugs. However, treatment with the anthracycline derivative epirubicin resulted in comparatively high apoptosis rates in HCC cells. Inhibition of the kinase PI3K significantly increased apoptosis induction by chemotherapy. RNA interference efficiently downregulated Mcl-1 expression in HCC cells. Mcl-1 downregulation sensitized HCC cells to different chemotherapeutic agents. Sensitization was accompanied by profound activation of caspase-3 and -9. In addition, Mcl-1 downregulation also increased apoptosis rates after treatment with PI3K inhibitors and, to a lower extent, after treatment with mTOR, Raf I and VEGF/PDGF kinase inhibitors. TRAIL-induced apoptosis did not markedly respond to Mcl-1 knockdown. Additionally, knockdown of Mcl-1 efficiently enhanced apoptosis sensitivity towards combined treatment modalities: Mcl-1

  1. UV-sensitive photoreceptor protein OPN5 in humans and mice.

    Science.gov (United States)

    Kojima, Daisuke; Mori, Suguru; Torii, Masaki; Wada, Akimori; Morishita, Rika; Fukada, Yoshitaka

    2011-01-01

    A variety of animal species utilize the ultraviolet (UV) component of sunlight as their environmental cues, whereas physiological roles of UV photoreception in mammals, especially in human beings, remain open questions. Here we report that mouse neuropsin (OPN5) encoded by the Opn5 gene exhibited an absorption maximum (λmax) at 380 nm when reconstituted with 11-cis-retinal. Upon UV-light illumination, OPN5 was converted to a blue-absorbing photoproduct (λmax 470 nm), which was stable in the dark and reverted to the UV-absorbing state by the subsequent orange light illumination, indicating its bistable nature. Human OPN5 also had an absorption maximum at 380 nm with spectral properties similar to mouse OPN5, revealing that OPN5 is the first and hitherto unknown human opsin with peak sensitivity in the UV region. OPN5 was capable of activating heterotrimeric G protein Gi in a UV-dependent manner. Immuno-blotting analyses of mouse tissue extracts identified the retina, the brain and, unexpectedly, the outer ears as the major sites of OPN5 expression. In the tissue sections of mice, OPN5 immuno-reactivities were detected in a subset of non-rod/non-cone retinal neurons as well as in the epidermal and muscle cells of the outer ears. Most of these OPN5-immuno-reactivities in mice were co-localized with positive signals for the alpha-subunit of Gi. These results demonstrate the first example of UV photoreceptor in human beings and strongly suggest that OPN5 triggers a UV-sensitive Gi-mediated signaling pathway in the mammalian tissues.

  2. Human platelet antigen antibody induction in uncomplicated pregnancy is associated with HLA sensitization.

    Science.gov (United States)

    Reiher, Viktoria S A; Hönger, Gideon; Infanti, Laura; Passweg, Jakob R; Hösli, Irene; Frey, Beat M; Gassner, Christoph; Meyer, Stefan; Buser, Andreas S; Holbro, Andreas; Schaub, Stefan

    2017-05-01

    Alloimmunization against human platelet antigens (HPAs) during pregnancy is rare but can lead to severe bleeding disorders, such as fetal and neonatal alloimmune thrombocytopenia. In a cohort of 241 uncomplicated pregnancies, we investigated the immunogenicity of HPA mismatches and correlated HLA sensitization with HPA antibody formation. HPA antibodies were measured with a Luminex-based multiplex assay. HPA mismatches were observed in 109 of 241 pregnancies (45%), but child-specific HPA antibodies were only found in two of 109 cases (2%), indicating a low immunogenicity. Only nine of 241 women (4%) had detectable HPA antibodies. HLA sensitization was identified as a strong and independent predictor for HPA antibody formation (hazard ratio, 10.2; 95% confidence interval, 1.8-193; p = 0.006), whereas the number of pregnancies was not. Our observational data indicated a low immunogenicity of HPA and suggest that a broader immune response-inferred by HLA sensitization-is probably associated with HPA antibody induction. © 2017 AABB.

  3. Dynamic modulation of thymidylate synthase gene expression and fluorouracil sensitivity in human colorectal cancer cells.

    Directory of Open Access Journals (Sweden)

    Kentaro Wakasa

    Full Text Available Biomarkers have revolutionized cancer chemotherapy. However, many biomarker candidates are still in debate. In addition to clinical studies, a priori experimental approaches are needed. Thymidylate synthase (TS expression is a long-standing candidate as a biomarker for 5-fluorouracil (5-FU treatment of cancer patients. Using the Tet-OFF system and a human colorectal cancer cell line, DLD-1, we first constructed an in vitro system in which TS expression is dynamically controllable. Quantitative assays have elucidated that TS expression in the transformant was widely modulated, and that the dynamic range covered 15-fold of the basal level. 5-FU sensitivity of the transformant cells significantly increased in response to downregulated TS expression, although being not examined in the full dynamic range because of the doxycycline toxicity. Intriguingly, our in vitro data suggest that there is a linear relationship between TS expression and the 5-FU sensitivity in cells. Data obtained in a mouse model using transformant xenografts were highly parallel to those obtained in vitro. Thus, our in vitro and in vivo observations suggest that TS expression is a determinant of 5-FU sensitivity in cells, at least in this specific genetic background, and, therefore, support the possibility of TS expression as a biomarker for 5-FU-based cancer chemotherapy.

  4. Hydrogen-bond network and pH sensitivity in human transthyretin

    Energy Technology Data Exchange (ETDEWEB)

    Yokoyama, Takeshi, E-mail: tyokoya3@pha.u-toyama.ac.jp; Mizuguchi, Mineyuki; Nabeshima, Yuko [University of Toyama, 2630 Sugitani, Toyama 930-0914 (Japan); Kusaka, Katsuhiro; Yamada, Taro [Ibaraki University, 162-1 Shirakata, Tokai, Ibaraki 319-1106 (Japan); Hosoya, Takaaki [Ibaraki University, 162-1 Shirakata, Tokai, Ibaraki 319-1106 (Japan); Ibaraki University, 4-12-1 Naka-Narusawa, Hitachi, Ibaraki 316-8511 (Japan); Ohhara, Takashi [Comprehensive Research Organization for Science and Society, 162-1 Shirakata, Tokai, Ibaraki 319-1106 (Japan); Kurihara, Kazuo [Japan Atomic Energy Agency, 2-4 Shirakata, Tokai, Ibaraki 319-1195 (Japan); Tanaka, Ichiro [Ibaraki University, 162-1 Shirakata, Tokai, Ibaraki 319-1106 (Japan); Ibaraki University, 4-12-1 Naka-Narusawa, Hitachi, Ibaraki 316-8511 (Japan); Niimura, Nobuo [Ibaraki University, 162-1 Shirakata, Tokai, Ibaraki 319-1106 (Japan)

    2013-11-01

    The neutron crystal structure of human transthyretin is presented. Transthyretin (TTR) is a tetrameric protein. TTR misfolding and aggregation are associated with human amyloid diseases. Dissociation of the TTR tetramer is believed to be the rate-limiting step in the amyloid fibril formation cascade. Low pH is known to promote dissociation into monomer and the formation of amyloid fibrils. In order to reveal the molecular mechanisms underlying pH sensitivity and structural stabilities of TTR, neutron diffraction studies were conducted using the IBARAKI Biological Crystal Diffractometer with the time-of-flight method. Crystals for the neutron diffraction experiments were grown up to 2.5 mm{sup 3} for four months. The neutron crystal structure solved at 2.0 Å revealed the protonation states of His88 and the detailed hydrogen-bond network depending on the protonation states of His88. This hydrogen-bond network is involved in monomer–monomer and dimer–dimer interactions, suggesting that the double protonation of His88 by acidification breaks the hydrogen-bond network and causes the destabilization of the TTR tetramer. Structural comparison with the X-ray crystal structure at acidic pH identified the three amino acid residues responsible for the pH sensitivity of TTR. Our neutron model provides insights into the molecular stability related to amyloidosis.

  5. Evaluation of cloned cells, animal model, and ATRA sensitivity of human testicular yolk sac tumor

    Directory of Open Access Journals (Sweden)

    Zhao Junfeng

    2012-03-01

    Full Text Available Abstract The testicular yolk sac tumor (TYST is the most common neoplasm originated from germ cells differentiated abnormally, a major part of pediatric malignant testicular tumors. The present study aimed at developing and validating the in vitro and vivo models of TYST and evaluating the sensitivity of TYST to treatments, by cloning human TYST cells and investigating the histology, ultra-structure, growth kinetics and expression of specific proteins of cloned cells. We found biological characteristics of cloned TYST cells were similar to the yolk sac tumor and differentiated from the columnar to glandular-like or goblet cells-like cells. Chromosomes for tumor identification in each passage met nature of the primary tumor. TYST cells were more sensitive to all-trans-retinoic acid which had significantly inhibitory effects on cell proliferation. Cisplatin induced apoptosis of TYST cells through the activation of p53 expression and down-regulation of Bcl- expression. Thus, we believe that cloned TYST cells and the animal model developed here are useful to understand the molecular mechanism of TYST cells and develop potential therapies for human TYST.

  6. Sensitivity to ionizing radiation and chemotherapeutic agents in gemcitabine-resistant human tumor cell lines

    International Nuclear Information System (INIS)

    Bree, Chris van; Kreder, Natasja Castro; Loves, Willem J.P.; Franken, Nicolaas A.P.; Peters, Godefridus J.; Haveman, Jaap

    2002-01-01

    Purpose: To determine cross-resistance to anti-tumor treatments in 2',2'difluorodeoxycytidine (dFdC, gemcitabine)-resistant human tumor cells. Methods and Materials: Human lung carcinoma cells SW-1573 (SWp) were made resistant to dFdC (SWg). Sensitivity to cisplatin (cDDP), paclitaxel, 5-fluorouracil (5-FU), methotrexate (MTX), cytarabine (ara-C), and dFdC was measured by a proliferation assay. Radiosensitivity and radioenhancement by dFdC of this cell panel and the human ovarian carcinoma cell line A2780 and its dFdC-resistant variant AG6000 were determined by clonogenic assay. Bivariate flowcytometry was performed to study cell cycle changes. Results: In the SWg, a complete deoxycytidine kinase (dCK) deficiency was found on mRNA and protein level. This was accompanied by a 10-fold decrease in dCK activity which resulted in the >1000-fold resistance to dFdC. Sensitivity to other anti-tumor drugs was not altered, except for ara-C (>100-fold resistance). Radiosensitivity was not altered in the dFdC-resistant cell lines SWg and AG6000. High concentrations (50-100 μM dFdC) induced radioenhancement in the dFdC-resistant cell lines similar to the radioenhancement obtained at lower concentrations (10 nM dFdC) in the parental lines. An early S-phase arrest was found in all cell lines after dFdC treatment where radioenhancement was achieved. Conclusions: In the dFdC-resistant lung tumor cell line SWg, the deficiency in dCK is related to the resistance to dFdC and ara-C. No cross-resistance was observed to other anti-tumor drugs used for the treatment in lung cancer. Sensitivity to ionizing radiation was not altered in two different dFdC-resistant cell lines. Resistance to dFdC does not eliminate the ability of dFdC to sensitize cells to radiation

  7. Dietary Sodium Restriction Decreases Insulin Secretion Without Affecting Insulin Sensitivity in Humans

    Science.gov (United States)

    Byrne, Loretta M.; Yu, Chang; Wang, Thomas J.; Brown, Nancy J.

    2014-01-01

    Context: Interruption of the renin-angiotensin-aldosterone system prevents incident diabetes in high-risk individuals, although the mechanism remains unclear. Objective: To test the hypothesis that activation of the endogenous renin-angiotensin-aldosterone system or exogenous aldosterone impairs insulin secretion in humans. Design: We conducted a randomized, blinded crossover study of aldosterone vs vehicle and compared the effects of a low-sodium versus a high-sodium diet. Setting: Academic clinical research center. Participants: Healthy, nondiabetic, normotensive volunteers. Interventions: Infusion of exogenous aldosterone (0.7 μg/kg/h for 12.5 h) or vehicle during low or high sodium intake. Low sodium (20 mmol/d; n = 12) vs high sodium (160 mmol/d; n = 17) intake for 5–7 days. Main Outcome Measures: Change in acute insulin secretory response assessed during hyperglycemic clamps while in sodium balance during a low-sodium vs high-sodium diet during aldosterone vs vehicle. Results: A low-sodium diet increased endogenous aldosterone and plasma renin activity, and acute glucose-stimulated insulin (−16.0 ± 5.6%; P = .007) and C-peptide responses (−21.8 ± 8.4%; P = .014) were decreased, whereas the insulin sensitivity index was unchanged (−1.0 ± 10.7%; P = .98). Aldosterone infusion did not affect the acute insulin response (+1.8 ± 4.8%; P = .72) or insulin sensitivity index (+2.0 ± 8.8%; P = .78). Systolic blood pressure and serum potassium were similar during low and high sodium intake and during aldosterone infusion. Conclusions: Low dietary sodium intake reduces insulin secretion in humans, independent of insulin sensitivity. PMID:25029426

  8. Adaptive fusion of human visual sensitive features for surveillance video summarization.

    Science.gov (United States)

    Salehin, Md Musfequs; Paul, Manoranjan

    2017-05-01

    Surveillance video cameras capture large amounts of continuous video streams every day. To analyze or investigate any significant events, it is a laborious and boring job to identify these events from the huge video data if it is done manually. Existing approaches sometimes neglect key frames with significant visual contents and/or select some unimportant frames with low/no activity. To solve this problem, in this paper, a video summarization technique is proposed by combining three multimodal human visual sensitive features, such as foreground objects, motion information, and visual saliency. In a video stream, foreground objects are one of the most important pieces of a video as they contain more detailed information and play a major role in important events. Moreover, motion is another stimulus of a video that significantly attracts human visual attention. To obtain this, motion information is calculated in the spatial domain as well as the frequency domain. Spatial motion information can select object motion accurately; however, it is sensitive to illumination changes. On the other hand, frequency motion information is robust to illumination change, although it is easily affected by noise. Therefore, motion information in both the spatial and the frequency domains is employed. Furthermore, the visual attention cue is a sensitive feature to measure the indication of a user's attraction label for determining key frames. As these features individually cannot perform very well, they are combined to obtain better results. For this purpose, an adaptive linear weighted fusion scheme is proposed to combine the features to rank video frames for summarization. Experimental results reveal that the proposed method outperforms the state-of-the-art methods.

  9. Development of nanobody-based flow injection chemiluminescence immunoassay for sensitive detection of human prealbumin.

    Science.gov (United States)

    Ma, Lei; Sun, Yanyan; Kang, Xuejun; Wan, Yakun

    2014-11-15

    Nanobodies, derived from camelid heavy-chain antibodies, have novel and impactful applications in clinical diagnostics. Our objective is to develop a nanobody-based chemiluminescence immunoassay for sensitive detection of human prealbumin (PA). In this context, a phage display nanobody library is constructed via immunizing dromedary camel with human prealbumin. Three nanobodies have been identified by five successive bio-panning steps. Based on their high expression level and good affinity, two out of three are chosen for further study. Magnetic beads (MBs) were functionalized with PEI by acylamide bond formed between the carboxyl group on the surface of the MB. Then, an anti-PA nanobody (Nb1) can be effectively immobilized onto the surface of the functionalized MB using glutaradehyde as the link. The modified MBs with Nb1 can specifically capture the target PA and reacted with silica nanoparticles with co-immobilized HRP and anti-PA nanobody (Nb2). The concentration of PA was detected by flow injection chemiluminescence. When using MB/PEI as the carrier of anti-PA Nb1, the CL signal significantly increased to 4-fold compared with the signal using MB without PEI modification. The CL signal was further amplified to 5-fold when Si/Nb2 was used as the signal probe. Under optimized conditions, the present immunoassay exhibited a wide quantitative range from 0.05 to 1000 μg L(-1) with a detection limit of 0.01 μg L(-1). The sensitivity of the proposed immunoassay offers great promises in providing a sensitive, specific, time saving, and potential method for detecting PA in clinical settings. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Rapid and sensitive immunomagnetic-electrochemiluminescent detection of p53 antibodies in human serum.

    Science.gov (United States)

    Yan, Guihong; Xing, Da; Tan, Shici; Chen, Qun

    2004-05-01

    The mutation of tumor suppressor p53 gene is common in malignant tumor. p53 antibodies are products of immunoresponse against abnormal p53 protein. It has been found that p53 antibodies are of importance in tumor's diagnosis, prognosis and relapse monitoring. However, current method for detecting p53 antibodies, i.e. enzyme-linked immunosorbent assay (ELISA), requires a long time with multiple steps, and the assay is only semi-quantitative. In this work, a protocol for quantitative detection of p53 antibodies in human serum using immunomagnetic electrochemiluminescence (IM-ECL) was devoloped. The immunoassay format consisted of a three antibody sandwich in which a biotinylated capture antibody, was banded with the commercial p53 protein. A detector antibody was added to bind the p53 protein at another site. Then, secondary antibody, labeled with ruthenium(II) tris-bipyridal, was added and, when bound to the bead immunocompiex, generated light in the presence of an excess of tripropylamine. The light was detected and measured by the analyzer made by us. Our experimental results indicate that the sensitivity of this assay was 10 pg of p53 antibodies per ml of reference serum (normal human serum). A stable calibration curve with a wide dynamic range was established. The calibration curve was linear from 0.01 to 1000 ng/ml, thus, making quantitation possible. An immunologic prozone effect was observed above 1000 ng p53 antibodies per milliliter of serum. Serum samples from lung and nasopharyngeal carcinoma patients were tested using the IM-ECL assay. The positive rate of p53 antibodies were 28.6% in lung carcinoma and 8.33% in nasopharyngeal carcinoma, respectively. p53 antibody concentration in the carcerous human sera were quantified from the calibration curve. In the case of lung carcinoma, a trend was found that a higher p53 antibody concentration in the serum was likely linked to a higher stage of the cancer. This trend was not found in nasopharyngeal carcinoma

  11. Ultra-Sensitive HIV-1 Latency Viral Outgrowth Assays Using Humanized Mice

    Directory of Open Access Journals (Sweden)

    Kimberly Schmitt

    2018-03-01

    Full Text Available In the current quest for a complete cure for HIV/AIDS, highly sensitive HIV-1 latency detection methods are critical to verify full viral eradication. Until now, the in vitro quantitative viral outgrowth assays (qVOA have been the gold standard for assessing latent HIV-1 viral burden. However, these assays have been inadequate in detecting the presence of ultralow levels of latent virus in a number of patients who were initially thought to have been cured, but eventually showed viral rebound. In this context, new approaches utilizing in vivo mouse-based VOAs are promising. In the murine VOA (mVOA, large numbers of CD4+ T cells or PBMC from aviremic subjects are xenografted into immunodeficient NSG mice, whereas in the humanized mouse-based VOA (hmVOA patient CD4+ T cell samples are injected into BLT or hu-hematopoetic stem cells (hu-HSC humanized mice. While latent virus could be recovered in both of these systems, the hmVOA provides higher sensitivity than the mVOA using a fewer number of input cells. In contrast to the mVOA, the hmVOA provides a broader spectrum of highly susceptible HIV-1 target cells and enables newly engrafted cells to home into preformed human lymphoid organs where they can infect cells in situ after viral activation. Hu-mice also allow for both xenograft- and allograft-driven cell expansions with less severe GvH providing a longer time frame for potential viral outgrowth from cells with a delayed latent viral activation. Based on these advantages, the hmVOA has great potential in playing an important role in HIV-1 latency and cure research.

  12. Ultra-Sensitive HIV-1 Latency Viral Outgrowth Assays Using Humanized Mice.

    Science.gov (United States)

    Schmitt, Kimberly; Akkina, Ramesh

    2018-01-01

    In the current quest for a complete cure for HIV/AIDS, highly sensitive HIV-1 latency detection methods are critical to verify full viral eradication. Until now, the in vitro quantitative viral outgrowth assays (qVOA) have been the gold standard for assessing latent HIV-1 viral burden. However, these assays have been inadequate in detecting the presence of ultralow levels of latent virus in a number of patients who were initially thought to have been cured, but eventually showed viral rebound. In this context, new approaches utilizing in vivo mouse-based VOAs are promising. In the murine VOA (mVOA), large numbers of CD4 + T cells or PBMC from aviremic subjects are xenografted into immunodeficient NSG mice, whereas in the humanized mouse-based VOA (hmVOA) patient CD4 + T cell samples are injected into BLT or hu-hematopoetic stem cells (hu-HSC) humanized mice. While latent virus could be recovered in both of these systems, the hmVOA provides higher sensitivity than the mVOA using a fewer number of input cells. In contrast to the mVOA, the hmVOA provides a broader spectrum of highly susceptible HIV-1 target cells and enables newly engrafted cells to home into preformed human lymphoid organs where they can infect cells in situ after viral activation. Hu-mice also allow for both xenograft- and allograft-driven cell expansions with less severe GvH providing a longer time frame for potential viral outgrowth from cells with a delayed latent viral activation. Based on these advantages, the hmVOA has great potential in playing an important role in HIV-1 latency and cure research.

  13. Sensitive liquid chromatography-tandem mass spectrometry method for quantification of hydrochlorothiazide in human plasma.

    Science.gov (United States)

    Ramakrishna, N V S; Vishwottam, K N; Manoj, S; Koteshwara, M; Wishu, S; Varma, D P

    2005-12-01

    A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of hydrochlorothiazide (I), a common diuretic and anti-hypertensive agent. The analyte and internal standard, tamsulosin (II) were extracted by liquid-liquid extraction with diethyl ether-dichloromethane (70:30, v/v) using a Glas-Col Multi-Pulse Vortexer. The chromatographic separation was performed on a reversed-phase column (Waters symmetry C18) with a mobile phase of 10 mm ammonium acetate-methanol (15:85, v/v). The protonated analyte was quantitated in negative ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions m/z 296.1 solidus in circle 205.0 and m/z 407.2 solidus in circle 184.9 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 0.5-200 ng/mL for hydrochlorothiazide in human plasma. The lower limit of quantitation was 500 pg/mL, with a relative standard deviation of less than 9%. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A run time of 2.5 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies. (c) 2005 John Wiley & Sons, Ltd.

  14. Do dogs (Canis lupus familiaris) make counterproductive choices because they are sensitive to human ostensive cues?

    Science.gov (United States)

    Marshall-Pescini, Sarah; Passalacqua, Chiara; Miletto Petrazzini, Maria Elena; Valsecchi, Paola; Prato-Previde, Emanuela

    2012-01-01

    Dogs appear to be sensitive to human ostensive communicative cues in a variety of situations, however there is still a measure of controversy as to the way in which these cues influence human-dog interactions. There is evidence for instance that dogs can be led into making evaluation errors in a quantity discrimination task, for example losing their preference for a larger food quantity if a human shows a preference for a smaller one, yet there is, so far, no explanation for this phenomenon. Using a modified version of this task, in the current study we investigated whether non-social, social or communicative cues (alone or in combination) cause dogs to go against their preference for the larger food quantity. Results show that dogs' evaluation errors are indeed caused by a social bias, but, somewhat contrary to previous studies, they highlight the potent effect of stimulus enhancement (handling the target) in influencing the dogs' response. A mild influence on the dog's behaviour was found only when different ostensive cues (and no handling of the target) were used in combination, suggesting their cumulative effect. The discussion addresses possible motives for discrepancies with previous studies suggesting that both the intentionality and the directionality of the action may be important in causing dogs' social biases.

  15. Antibiotic sensitivity pattern of indigenous lactobacilli isolated from curd and human milk samples.

    Science.gov (United States)

    Sharma, Chetan; Gulati, Sachin; Thakur, Nishchal; Singh, Brij Pal; Gupta, Sanjolly; Kaur, Simranpreet; Mishra, Santosh Kumar; Puniya, Anil Kumar; Gill, Jatinder Pal Singh; Panwar, Harsh

    2017-05-01

    The gut microbiota plays a vital role in host well-being and lactic acid bacteria (LAB) have gained an overwhelming attention as health promoter. This perception has evolved from traditional dairy products to a money-spinning market of probiotics. The safety of probiotics is coupled to their intended use and LAB may act as pool of antimicrobial resistance genes that could be transferred to pathogens, either in food matrix or in gastrointestinal tract, which could be detrimental to host. This study evaluated the antibiotic susceptibility patterns of LAB isolated from curd (20) and human milk (11) samples. Antibiotic susceptibility was determined against 26 common antibiotics, following reference disc diffusion assay. A varied response in terms of susceptibility and resistance towards antibiotics was recorded. Among curd isolates, D7 (Lactobacillus plantarum) was the most resistant followed by D4, D8, D10 and D25. Among human milk isolates, HM-1 (L. casei) showed the highest resistance profile. All LAB isolates displayed high susceptibility pattern towards imipenem and meropenem. In general, high resistivity was exhibited by human milk isolates. The present study showed that antibiotic resistance is widespread among different lactobacilli, which may pose a food safety concern. Therefore, antibiotic sensitivity should be considered as a vital tool for safety assessment of probiotics.

  16. Do dogs (Canis lupus familiaris make counterproductive choices because they are sensitive to human ostensive cues?

    Directory of Open Access Journals (Sweden)

    Sarah Marshall-Pescini

    Full Text Available Dogs appear to be sensitive to human ostensive communicative cues in a variety of situations, however there is still a measure of controversy as to the way in which these cues influence human-dog interactions. There is evidence for instance that dogs can be led into making evaluation errors in a quantity discrimination task, for example losing their preference for a larger food quantity if a human shows a preference for a smaller one, yet there is, so far, no explanation for this phenomenon. Using a modified version of this task, in the current study we investigated whether non-social, social or communicative cues (alone or in combination cause dogs to go against their preference for the larger food quantity. Results show that dogs' evaluation errors are indeed caused by a social bias, but, somewhat contrary to previous studies, they highlight the potent effect of stimulus enhancement (handling the target in influencing the dogs' response. A mild influence on the dog's behaviour was found only when different ostensive cues (and no handling of the target were used in combination, suggesting their cumulative effect. The discussion addresses possible motives for discrepancies with previous studies suggesting that both the intentionality and the directionality of the action may be important in causing dogs' social biases.

  17. Acquisition and processing method for human sensorial, sensitive, motory and phonatory circuits reaction times

    International Nuclear Information System (INIS)

    Doche, Claude

    1972-01-01

    This work describes a storage and acquisition device and a method for human sensorial and sensitive motory and phonatory reaction times. The considered circuits are those made with the visual, auditory and sensory receptor organs and the motory or phonatory effector organs. The anatomo-physiological localization of these circuits allows us to appreciate the possibilities of the central nervous system for different angles. The experimental population is made of normal and pathological individuals (individuals having tumoral or vascular, localized or diffused cerebral lesions or parkinsonian individuals). The parameter processing method is based on the multivariate analysis results and allows us to position each individual compared to a normal individual and to appreciate the weight of each circuit in this positioning. Clinical exploitation results give to this method a prognosis and therapeutic interest. It seems though untimely to talk about its diagnosis value. (author) [fr

  18. Calcineurin inhibitors acutely improve insulin sensitivity without affecting insulin secretion in healthy human volunteers

    DEFF Research Database (Denmark)

    Øzbay, Aygen; Møller, Niels; Juhl, Claus

    2012-01-01

    WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: New onset diabetes after transplantation is related to treatment with immunosuppressive medications. Clinical studies have shown that risk of new onset diabetes is greater with tacrolimus compared with ciclosporin. The diabetogenicity of ciclosporin...... of NODAT remains unclear. We sought to compare the impact of CsA and Tac on glucose metabolism in human subjects. METHODS: Ten healthy men underwent 5 h infusions of CsA, Tac and saline in a randomized, double-blind, crossover study. During infusion glucose metabolism was investigated using following.......047), whereas first phase and pulsatile insulin secretion were unaffected. Coinciding with the CNI induced improved insulin sensitivity, glucose oxidation rates increased, while insulin clearance rates decreased, only non-significantly. Tac singularly lowered hsCRP concentrations, otherwise no changes were...

  19. The Human Influence on Global Warming: Sensitivity to AMOC and OHE

    Science.gov (United States)

    Salawitch, R. J.; Hope, A. P.; Mascioli, N. R.; Canty, T. P.

    2015-12-01

    We use an empirical model of global climate to quantify the role of human activity on global mean surface temperature (GMST), with particular attention to the treatment of Atlantic Meridional Overturning Circulation (AMOC) and the export of heat from the atmosphere to the world's oceans. The study examines changes in GMST from 1860 to present with particular attention focused on two time periods: 1998 to 2012, the time of the so-called global warming hiatus and 1979 to 2010, a three decade span for which researchers have attempted to quantify the human influence on GMST. We will show that the existence of the global warming hiatus depends on which dataset is used to define GMST: in our model framework there is little or no global warming hiatus upon use of either the recent NOAA record for GMST (Karl et al., 2015) or the use of a revision to the CRU4 dataset suggested by Cowtan and Way (2014). Next, we show that humans have been responsible for 0.13±0.06°C/decade rise in GMST during the past three decades (1979 to 2010), considerably less than the rise in GMST attributed to humans over this same time period (0.17±0.01°C/decade) by Foster and Rahmstorf (2011). We suggest this prior study obtained an erroneously high value due to their neglect of the influence of variations in the strength of the AMOC on global climate. Finally, we'll provide projections of GMST over the next four decades based on the rise in greenhouse gases (GHGs) given in the four Representative Concentration Pathway scenarios of IPCC AR5. These projections include detailed quantitative assessments of the sensitivity of global warming to the efficiency of ocean heat export, resulting in a probability distribution function of future GMST for each RCP scenario.

  20. Prospective analysis of human leukocyte functional tests reveals metal sensitivity in patients with hip implant

    Science.gov (United States)

    2013-01-01

    Background The aim of the study was to examine the reactivity of peripheral human leukocytes to various metal ions prior and following hip replacement in order to investigate implant-induced metal sensitivity. Methods Three patient groups were set up: (1) individuals without implants and no history of metal allergy (7 cases), (2) individuals without implants and known history of metal allergy (7 cases), and (3) patients undergoing cementless hip replacement (40 cases). Blood samples were taken in groups 1 and 2 at three different occasions; in group 3, prior and 3, 6, 12, 24, and 36 months after surgery. Peripheral leukocytes were separated and left either untreated or challenged with Ti, NiCl2, CoCl2, CrCl3, and phytohemagglutinin. Cell proliferation, cytokine release, and leukocyte migration inhibition assays were performed. Metal-induced reactivity was considered when all three assays showed significant change. Skin patch tests were also carried out. Results Both skin patch tests and leukocyte functional tests were negative in group 1, and both were positive in group 2. In group 3, after 6 months, 12% of the patients showed reactivity to the tested metals except for NiCl2. Following the 36-month period, 18% of group three became sensitive to metals (including all the earlier 12%). In contrast, patch tests were negative at each time point in group 3. Conclusions Orthopedic implant material may induce metal reactivity after implantation in a manner where susceptibility is yet to be elucidated. Leukocyte triple assay technique might be a useful tool to test implant material-related sensitivity. PMID:23680415

  1. Butyrate down regulates BCL-XL and sensitizes human fibroblasts to radiation and chemotherapy induced apoptosis

    International Nuclear Information System (INIS)

    Chung, Diana H.; Ljungman, Mats; Zhang Fenfen; Chen Feng; McLaughlin, William P.

    1997-01-01

    Purpose/Objective: Butyrate is a short chain fatty acid that has been implicated in the induction of cell cycle arrest, cell differentiation and apoptosis. The purpose of this study was to determine if butyrate treatment sensitizes cells to radiation or chemotherapy induced apoptosis. Materials and Methods: Normal neonatal human diploid fibroblasts were used throughout this study. Apoptosis was scored and quantified using three different methods. First, cell morphology using propidium iodide and fluorescence microscopy was used to qualitatively determine apoptosis and to quantify the percentage of cells undergoing apoptosis. Second, apoptosis induced DNA degradation was scored by quantifying the amount of cells appearing in a sub-G1 peak using fixed and PI-stained cells and flow cytometry. Third, apoptosis-induced DNA degradation was examined by using an assay involving direct lysis of cells in the wells of agarose gels followed by conventional gel electrophoresis. Western blotting was used to quantify the cellular levels of the apoptosis regulators, Bcl-2, Bcl-XL and Bax. Results: Human diploid fibroblasts, which were resistant to radiation induced apoptosis, were found to undergo massive apoptosis when radiation was combined with butyrate treatment. Sensitization was obtained when butyrate was added before or after radiation although the combination of both pre and post-treatment was the most effective. Butyrate was also found to enhance UV light and cisplatin-induced apoptosis. These findings correlated with a reduction of the apoptosis antagonist Bcl-XL. Bcl-XL levels significantly dropped in a time and dose dependent manner. In addition, butyrate effectively blocked UV-induced accumulation of p53. Conclusion: Our results suggest that butyrate may be an attractive agent to use in combination with radiation or chemotherapy to lower the apoptotic threshold of tumor cells, regardless of the p53 status of the tumor cells

  2. Sensitive and rapid RT-qPCR quantification of pathogenic Candida species in human blood.

    Science.gov (United States)

    Ogata, Kiyohito; Matsuda, Kazunori; Tsuji, Hirokazu; Nomoto, Koji

    2015-10-01

    For accurate diagnosis and appropriate treatment of candidiasis, we developed a highly sensitive quantitative RT-PCR (RT-qPCR) system for five Candida species that have been reported to be the major causes of bloodstream fungal infection (Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, and Candida krusei), together with a system for all pathogenic Candida species. Cells of each fungal species spiked into human peripheral blood (PB) were specifically detected at a lower detection limit of 10(0) cell/1 mL PB by this system using the newly developed specific primer sets targeting 18S or 26S rRNA of the five Candida species, together with the existing group primer set. The total count of the five Candida spp. as the sum of those obtained by using the five species primer sets was equivalent to the count obtained by using the group primer set, indicating that the group set covered the major five Candida spp. in human blood with the same degree of accuracy as the species primer sets. The RT-qPCR counts of the Candida species were in good agreement with CFU counts obtained by their culture on CHROMagar™, with a lower detection limit of 10(0)cell/mL of PB. Candida rRNA molecules were stably stored for at least 7 days at 4°C by keeping the blood specimens in an RNA stabilizing reagent. These results strongly suggest that this sensitive system is useful for accurate and rapid diagnosis of Candida bloodstream infections. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Reduced sensitivity to sooner reward during intertemporal decision-making following insula damage in humans

    Directory of Open Access Journals (Sweden)

    Manuela eSellitto

    2016-01-01

    Full Text Available During intertemporal choice, humans tend to prefer small-sooner rewards over larger-delayed rewards, reflecting temporal discounting (TD of delayed outcomes. Functional neuroimaging evidence has implicated the insular cortex in time-sensitive decisions, yet it is not clear whether activity in this brain region is crucial for, or merely associated with, TD behaviour. Here, patients with damage to the insula (Insular patients, control patients with lesions outside the insula, and healthy individuals chose between smaller-sooner and larger-later monetary rewards. Insular patients were less sensitive to sooner rewards than were the control groups, exhibiting reduced TD. A Voxel-based Lesion-Symptom Mapping (VLSM analysis confirmed a statistically significant association between insular damage and reduced TD. These results indicate that the insular cortex is crucial for intertemporal choice. We suggest that he insula may be necessary to anticipate the bodily/emotional effects of receiving rewards at different delays, influencing the computation of their incentive value. Devoid of such input, insular patients’ choices would be governed by a heuristic of quantity, allowing patients to wait for larger options.

  4. Hydrogen-bond network and pH sensitivity in human transthyretin.

    Science.gov (United States)

    Yokoyama, Takeshi; Mizuguchi, Mineyuki; Nabeshima, Yuko; Kusaka, Katsuhiro; Yamada, Taro; Hosoya, Takaaki; Ohhara, Takashi; Kurihara, Kazuo; Tanaka, Ichiro; Niimura, Nobuo

    2013-11-01

    Transthyretin (TTR) is a tetrameric protein. TTR misfolding and aggregation are associated with human amyloid diseases. Dissociation of the TTR tetramer is believed to be the rate-limiting step in the amyloid fibril formation cascade. Low pH is known to promote dissociation into monomer and the formation of amyloid fibrils. In order to reveal the molecular mechanisms underlying pH sensitivity and structural stabilities of TTR, neutron diffraction studies were conducted using the IBARAKI Biological Crystal Diffractometer with the time-of-flight method. Crystals for the neutron diffraction experiments were grown up to 2.5 mm(3) for four months. The neutron crystal structure solved at 2.0 Å revealed the protonation states of His88 and the detailed hydrogen-bond network depending on the protonation states of His88. This hydrogen-bond network is involved in monomer-monomer and dimer-dimer interactions, suggesting that the double protonation of His88 by acidification breaks the hydrogen-bond network and causes the destabilization of the TTR tetramer. Structural comparison with the X-ray crystal structure at acidic pH identified the three amino acid residues responsible for the pH sensitivity of TTR. Our neutron model provides insights into the molecular stability related to amyloidosis.

  5. Inhibition of SRC-3 enhances sensitivity of human cancer cells to histone deacetylase inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Zou, Zhengzhi, E-mail: zouzhengzhi@m.scnu.edu.cn [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510000 (China); Luo, Xiaoyong [Department of Oncology, The Affiliated Luoyang Central Hospital of Zhengzhou University, Luoyang 471000 (China); Nie, Peipei [KingMed Diagnostics and KingMed School of Laboratory Medicine, Guangzhou Medical University, Guangzhou 510000 (China); Wu, Baoyan; Zhang, Tao; Wei, Yanchun [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510000 (China); Wang, Wenyi [Xiamen Cancer Center, Department of Medical Oncology, The First Affiliated Hospital of Xiamen University, Xiamen 361000 (China); Geng, Guojun; Jiang, Jie [Xiamen Cancer Center, Department of Thoracic Surgery, The First Affiliated Hospital of Xiamen University, Xiamen 361000 (China); Mi, Yanjun, E-mail: myjgj_77@163.com [Xiamen Cancer Center, Department of Medical Oncology, The First Affiliated Hospital of Xiamen University, Xiamen 361000 (China)

    2016-09-09

    SRC-3 is widely expressed in multiple tumor types and involved in cancer cell proliferation and apoptosis. Histone deacetylase (HDAC) inhibitors are promising antitumor drugs. However, the poor efficacy of HDAC inhibitors in solid tumors has restricted its further clinical application. Here, we reported the novel finding that depletion of SRC-3 enhanced sensitivity of breast and lung cancer cells to HDAC inhibitors (SAHA and romidepsin). In contrast, overexpression of SRC-3 decreased SAHA-induced cancer cell apoptosis. Furthermore, we found that SRC-3 inhibitor bufalin increased cancer cell apoptosis induced by HDAC inhibitors. The combination of bufalin and SAHA was particular efficient in attenuating AKT activation and reducing Bcl-2 levels. Taken together, these accumulating data might guide development of new breast and lung cancer therapies. - Highlights: • Depletion of SRC-3 enhanced sensitivity of breast and lung cancer cells to HDAC inhibitors. • Overexpression of SRC-3 enhanced cancer cell resistance to HDAC inhibitors. • SRC-3 inhibitor bufalin increased cancer cell apoptosis induced by HDAC inhibitors. • Bufalin synergized with HDAC inhibitor attenuated AKT activation and reduced Bcl-2 levels in human cancer cell.

  6. Application of multiplex PCR for Rapid and sensitive detection of human papillomaviruses in cervical cancer.

    Science.gov (United States)

    Zandnia, Fateme; Doosti, Abbas; Mokhtari-Farsani, Abbas; Kardi, Mohammad Taghi; Movafagh, Abolfazl

    2016-01-01

    Reffering to an increase in cervical cancer in the recent years, rapid, sensitive and economical detection of human papillomaviruses (HPVs) as causative agents of cervical cancer is important. The traditional methods for the detection of HPVs in cervical cancer, such as pap smear, suffer from limitation and PCR has a potential to overcome the limitaitons. The purpose of present research work was to identify the five important strains of HPV (16, 18, 31, 33 and 45) simultaneously by Multiplex PCR application. Study was done on 100 cervical lesions of women. DNA was extracted from specimens by a genomic DNA purification kit. A 5-plex PCR was developed for the simultaneous detection of major HPV. Five pair of new primers was designed for detection of HPV 16, 18, 31, 33 and 45 by Multiplex PCR. Among the 100 evaluated samples, 82 were found positive to HPVs. In the meantime the highest rate of infection was for HPV 16. Also 30 of HPV positive samples had infections with two or more HPV types. Multiplex PCR assay used in present study can provide a rapid, sensitive and economical method for detection of viral infections and is applicable to small volumes of vaginal samples.

  7. Glucose Metabolism of Human Prostate Cancer Mouse Xenografts

    Directory of Open Access Journals (Sweden)

    Hossein Jadvar

    2005-04-01

    Full Text Available We hypothesized that the glucose metabolism of prostate cancer is modulated by androgen. We performed in vivo biodistribution and imaging studies of [F-18] fluorodeoxyglucose (FDG accumulation in androgen-sensitive (CWR-22 and androgen-independent (PC-3 human prostate cancer xenografts implanted in castrated and noncastrated male athymic mice. The growth pattern of the CWR-22 tumor was best approximated by an exponential function (tumor size in mm3 = 14.913 e0.108 × days, R2 = .96, n = 5. The growth pattern of the PC-3 tumor was best approximated by a quadratic function (tumor size in mm3 = 0.3511 × days2 + 49.418 × day −753.33, R2 = .96, n = 3. The FDG accumulation in the CWR-22 tumor implanted in the castrated mice was significantly lower, by an average of 55%, in comparison to that implanted in the noncastrated host (1.27 vs. 2.83, respectively, p < .05. The 3-week maximal standardized uptake value (SUVmax was 0.99 ± 0.43 (mean ± SD for CWR-22 and 1.21 ± 0.32 for PC-3, respectively. The 5-week SUVmax was 1.22 ± 0.08 for CWR-22 and 1.35 ± 0.17 for PC-3, respectively. The background muscle SUVmax was 0.53 ± 0.11. Glucose metabolism was higher in the PC-3 tumor than in the CWR-22 tumor at both the 3-week (by 18% and the 5-week (by 9.6% micro-PET imaging sessions. Our results support the notions that FDG PET may be useful in the imaging evaluation of response to androgen ablation therapy and in the early prediction of hormone refractoriness in men with metastatic prostate cancer.

  8. Treatment of system dependencies and human interactions in PRA studies: a review and sensitivity study

    International Nuclear Information System (INIS)

    Orvis, D.D.; Joksimovich, V.; Worledge, D.H.

    1985-01-01

    The Electric Power Research Institute sponsored the review and comparison of five PRA studies: Arkansas Nuclear One - Unit 1, Big Rock Point, Grand Gulf, Limerick, and Zion - Unit 1. The review has been conducted in two phases. The Phase I review may be characterized as a qualitative look into many aspects of a PRA study. The Phase II review was performed to quantify the extent that differences in analytical techniques or key assumptions in these areas affect the differences in study results. In each of the PRA studies reviewed, the general descriptions of analytical approaches and descriptions of the analyses of event tree, fault tree and human interaction analyses that affected the dominant core damage sequences were reviewed. When these descriptions aroused interest because of seeming inconsistencies within the study or with other studies, they were pursued in some depth. The approaches or assumptions were contrasted to similar elements from other studies, and sensitivity analyses were performed in many cases to test the significance of results to the analytical models or assumptions. Inferences were drawn from the results regarding significance of the item to plant-specific results and, where possible, were generalized to other PRAs. This paper describes the results of the review of system dependencies and human interactions

  9. Human bronchial epithelial cells express PAR-2 with different sensitivity to thermolysin.

    Science.gov (United States)

    Ubl, Joachim J; Grishina, Zoryana V; Sukhomlin, Tatiana K; Welte, Tobias; Sedehizade, Fariba; Reiser, Georg

    2002-06-01

    Protease-activated receptor-2 (PAR-2) plays a role in inflammatory reactions in airway physiology. Proteases cleaving the extracellular NH(2) terminus of receptors activate or inactivate PAR, thus possessing a therapeutic potential. Using RT-PCR and immunocytochemistry, we show PAR-2 in human airway epithelial cell lines human bronchial epithelial (HBE) and A549. Functional expression of PAR-2 was confirmed by Ca(2+) imaging studies using the receptor agonist protease trypsin. The effect was abolished by soybean trypsin inhibitor and mimicked by the specific PAR-2 peptide agonist SLIGKV. Amplitude and duration of PAR-2-elicited Ca(2+) response in HBE and A549 cells depend on concentration and time of agonist superfusion. The response is partially pertussis toxin (PTX) insensitive, abolished by the phospholipase C inhibitor U-73122, and diminished by the inositol 1,4,5-trisphosphate receptor antagonist 2-aminoethoxydiphenyl borate. Cathepsin G altered neither the resting Ca(2+) level nor PAR-2-elicited Ca(2+) response. Thermolysin, a prototypic bacterial metalloprotease, induced a dose-dependent Ca(2+) response in HBE, but not A549, cells. In both cell lines, thermolysin abolished the response to a subsequent trypsin challenge but not to SLIGKV. Thus different epithelial cell types express different PAR-2 with identical responses to physiological stimuli (trypsin, SLIGKV) but different sensitivity to modifying proteases, such as thermolysin.

  10. Human Gut Bacteria Are Sensitive to Melatonin and Express Endogenous Circadian Rhythmicity.

    Directory of Open Access Journals (Sweden)

    Jiffin K Paulose

    Full Text Available Circadian rhythms are fundamental properties of most eukaryotes, but evidence of biological clocks that drive these rhythms in prokaryotes has been restricted to Cyanobacteria. In vertebrates, the gastrointestinal system expresses circadian patterns of gene expression, motility and secretion in vivo and in vitro, and recent studies suggest that the enteric microbiome is regulated by the host's circadian clock. However, it is not clear how the host's clock regulates the microbiome. Here, we demonstrate at least one species of commensal bacterium from the human gastrointestinal system, Enterobacter aerogenes, is sensitive to the neurohormone melatonin, which is secreted into the gastrointestinal lumen, and expresses circadian patterns of swarming and motility. Melatonin specifically increases the magnitude of swarming in cultures of E. aerogenes, but not in Escherichia coli or Klebsiella pneumoniae. The swarming appears to occur daily, and transformation of E. aerogenes with a flagellar motor-protein driven lux plasmid confirms a temperature-compensated circadian rhythm of luciferase activity, which is synchronized in the presence of melatonin. Altogether, these data demonstrate a circadian clock in a non-cyanobacterial prokaryote and suggest the human circadian system may regulate its microbiome through the entrainment of bacterial clocks.

  11. Highly sensitive analysis of sterol profiles in human serum by LC-ESI-MS/MS.

    Science.gov (United States)

    Honda, Akira; Yamashita, Kouwa; Miyazaki, Hiroshi; Shirai, Mutsumi; Ikegami, Tadashi; Xu, Guorong; Numazawa, Mitsuteru; Hara, Takashi; Matsuzaki, Yasushi

    2008-09-01

    We have developed a highly sensitive and specific method for the analysis of serum sterol profiles. Sterols in 1 mul of dried serum were derivatized into picolinyl esters (3beta-picolinate) and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using the electrospray ionization (ESI) mode. In addition to cholesterol, 19 cholesterol precursors, cholestanol, campesterol, sitosterol, and sitostanol were identified simultaneously. Quantitative analyses for the picolinyl esters of 11 available sterols were performed, and detection limits were found to be less than 1 pg on-column. Reproducibilities and recoveries of 8 noncholesterol sterols were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements by this method were calculated to be 1.6% to 8.2% and 2.5% to 16.5%, respectively. The recovery experiments were performed using 1 mul aliquots of normal human serum spiked with 1 ng to 6 ng of sterols, and recoveries of the sterols ranged from 88.1% to 102.5% with a mean recovery of 98.1%. The present method provides reliable and reproducible results for the identification and quantification of neutral sterols, especially in small volumes of blood samples, which is useful for serological diagnosis of inherited disorders in cholesterol metabolism and for noninvasive evaluation of cholesterol biosynthesis and absorption in humans.

  12. Sensitivity of human embryonic stem cells to different conditions during cryopreservation.

    Science.gov (United States)

    Xu, Yanqing; Zhang, Liang; Xu, Jiandong; Wei, Yuping; Xu, Xia

    2015-12-01

    Low cell recovery rate of human embryonic stem cells (hESCs) resulting from cryopreservation damages leads to the difficulty in their successful commercialization of clinical applications. Hence in this study, sensitivity of human embryonic stem cells (hESCs) to different cooling rates, ice seeding and cryoprotective agent (CPA) types was compared and cell viability and recovery after cryopreservation under different cooling conditions were assessed. Both extracellular and intracellular ice formation were observed. Reactive oxidative species (ROS) accumulation of hESCs was determined. Cryopreservation of hESCs at 1 °C/min with the ice seeding and at the theoretically predicted optimal cooling rate (TPOCR) led to lower level of intracellular ROS, and prevented irregular and big ice clump formation compared with cryopreservation at 1 °C/min. This strategy further resulted in a significant increase in the hESC recovery when glycerol and 1,2-propanediol were used as the CPAs, but no increase for Me2SO. hESCs after cryopreservation under all the tested conditions still maintained their pluripotency. Our results provide guidance for improving the hESC cryopreservation recovery through the combination of CPA type, cooling rate and ice seeding. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Human body temperature and new approaches to constructing temperature-sensitive bacterial vaccines.

    Science.gov (United States)

    White, Matthew D; Bosio, Catharine M; Duplantis, Barry N; Nano, Francis E

    2011-09-01

    Many of the live human and animal vaccines that are currently in use are attenuated by virtue of their temperature-sensitive (TS) replication. These vaccines are able to function because they can take advantage of sites in mammalian bodies that are cooler than the core temperature, where TS vaccines fail to replicate. In this article, we discuss the distribution of temperature in the human body, and relate how the temperature differential can be exploited for designing and using TS vaccines. We also examine how one of the coolest organs of the body, the skin, contains antigen-processing cells that can be targeted to provoke the desired immune response from a TS vaccine. We describe traditional approaches to making TS vaccines, and highlight new information and technologies that are being used to create a new generation of engineered TS vaccines. We pay particular attention to the recently described technology of substituting essential genes from Arctic bacteria for their homologues in mammalian pathogens as a way of creating TS vaccines.

  14. Androgen-Sensitized Apoptosis of HPr-1AR Human Prostate Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Congcong Chen

    Full Text Available Androgen receptor (AR signaling is crucial to the development and homeostasis of the prostate gland, and its dysregulation mediates common prostate pathologies. The mechanisms whereby AR regulates growth suppression and differentiation of luminal epithelial cells in the prostate gland and proliferation of malignant versions of these cells have been investigated in human and rodent adult prostate. However, the cellular stress response of human prostate epithelial cells is not well understood, though it is central to prostate health and pathology. Here, we report that androgen sensitizes HPr-1AR and RWPE-AR human prostate epithelial cells to cell stress agents and apoptotic cell death. Although 5α-dihydrotestosterone (DHT treatment alone did not induce cell death, co-treatment of HPr-1AR cells with DHT and an apoptosis inducer, such as staurosporine (STS, TNFt, or hydrogen peroxide, synergistically increased cell death in comparison to treatment with each apoptosis inducer by itself. We found that the synergy between DHT and apoptosis inducer led to activation of the intrinsic/mitochondrial apoptotic pathway, which is supported by robust cleavage activation of caspase-9 and caspase-3. Further, the dramatic depolarization of the mitochondrial membrane potential that we observed upon co-treatment with DHT and STS is consistent with increased mitochondrial outer membrane permeabilization (MOMP in the pro-apoptotic mechanism. Interestingly, the synergy between DHT and apoptosis inducer was abolished by AR antagonists and inhibitors of transcription and protein synthesis, suggesting that AR mediates pro-apoptotic synergy through transcriptional regulation of MOMP genes. Expression analysis revealed that pro-apoptotic genes (BCL2L11/BIM and AIFM2 were DHT-induced, whereas pro-survival genes (BCL2L1/BCL-XL and MCL1 were DHT-repressed. Hence, we propose that the net effect of these AR-mediated expression changes shifts the balance of BCL2-family proteins

  15. Contrasting effects of ultraviolet-A and ultraviolet B exposure on induction of contact sensitivity in human skin

    DEFF Research Database (Denmark)

    Skov, Lone; Hansen, Henrik; Barker, J. N.

    1997-01-01

    Ultraviolet-B (UVB), in addition to direct effects on DNA, induces immunological changes in the skin that predispose to the development of skin cancer. Whether ultraviolet-A (UVA) induces similar changes is unknown. This effect can be investigated in humans in vivo using epicutaneous antigens...... as a model of tumour antigens. Volunteers (n = 46) were randomly assigned to received no sensitization, sensitization with the allergen diphenylcyclopropenone (DPCP) on non-UV-exposed normal skin, or sensitization with DPCP on skin exposed to three minimal erythema doses (MED) of either UVA or UVB radiation...... the immunization rate compared with sensitization on non-irradiated skin (P radiation did not result in a decreased immunization rate compared with non-irradiated skin. These results indicate that in humans erythemagenic...

  16. Development of a sensitive and specific epitope-blocking ELISA for universal detection of antibodies to human enterovirus 71 strains.

    Directory of Open Access Journals (Sweden)

    Fang He

    Full Text Available BACKGROUND: Human Enterovirus 71 (EV71 is a common cause of hand, foot and mouth disease (HFMD in young children. It is often associated with severe neurological diseases and mortalities in recent outbreaks across the Asia Pacific region. Currently, there is no efficient universal antibody test available to detect EV71 infections. METHODOLOGY/PRINCIPAL FINDING: In the present study, an epitope-blocking ELISA was developed to detect specific antibodies to human EV71 viruses in human or animal sera. The assay relies on a novel monoclonal antibody (Mab 1C6 that specifically binds to capsid proteins in whole EV71 viruses without any cross reaction to any EV71 capsid protein expressed alone. The sensitivity and specificity of the epitope-blocking ELISA for EV71 was evaluated and compared to microneutralization using immunized animal sera to multiple virus genotypes of EV71 and coxsackieviruses. Further, 200 serum sample from human individuals who were potentially infected with EV71 viruses were tested in both the blocking ELISA and microneutralization. Results indicated that antibodies to EV71 were readily detected in immunized animals or human sera by the epitope blocking ELISA whereas specimens with antibodies to other enteroviruses yielded negative results. This assay is not only simpler to perform but also shows higher sensitivity and specificity as compared to microneutralization. CONCLUSION: The epitope-blocking ELISA based on a unique Mab 1C6 provided highly sensitive and 100% specific detection of antibodies to human EV71 viruses in human sera.

  17. Does the A-not-B error in adult pet dogs indicate sensitivity to human communication?

    Science.gov (United States)

    Kis, Anna; Topál, József; Gácsi, Márta; Range, Friederike; Huber, Ludwig; Miklósi, Adám; Virányi, Zsófia

    2012-07-01

    Recent dog-infant comparisons have indicated that the experimenter's communicative signals in object hide-and-search tasks increase the probability of perseverative (A-not-B) errors in both species (Topál et al. 2009). These behaviourally similar results, however, might reflect different mechanisms in dogs and in children. Similar errors may occur if the motor response of retrieving the object during the A trials cannot be inhibited in the B trials or if the experimenter's movements and signals toward the A hiding place in the B trials ('sham-baiting') distract the dogs' attention. In order to test these hypotheses, we tested dogs similarly to Topál et al. (2009) but eliminated the motor search in the A trials and 'sham-baiting' in the B trials. We found that neither an inability to inhibit previously rewarded motor response nor insufficiencies in their working memory and/or attention skills can explain dogs' erroneous choices. Further, we replicated the finding that dogs have a strong tendency to commit the A-not-B error after ostensive-communicative hiding and demonstrated the crucial effect of socio-communicative cues as the A-not-B error diminishes when location B is ostensively enhanced. These findings further support the hypothesis that the dogs' A-not-B error may reflect a special sensitivity to human communicative cues. Such object-hiding and search tasks provide a typical case for how susceptibility to human social signals could (mis)lead domestic dogs.

  18. Proteasome Dysfunction Associated to Oxidative Stress and Proteotoxicity in Adipocytes Compromises Insulin Sensitivity in Human Obesity

    Science.gov (United States)

    Díaz-Ruiz, Alberto; Guzmán-Ruiz, Rocío; Moreno, Natalia R.; García-Rios, Antonio; Delgado-Casado, Nieves; Membrives, Antonio; Túnez, Isaac; El Bekay, Rajaa; Fernández-Real, José M.; Tovar, Sulay; Diéguez, Carlos; Tinahones, Francisco J.; Vázquez-Martínez, Rafael; López-Miranda, José

    2015-01-01

    Abstract Aims: Obesity is characterized by a low-grade systemic inflammatory state and adipose tissue (AT) dysfunction, which predispose individuals to the development of insulin resistance (IR) and metabolic disease. However, a subset of obese individuals, referred to as metabolically healthy obese (MHO) individuals, are protected from obesity-associated metabolic abnormalities. Here, we aim at identifying molecular factors and pathways in adipocytes that are responsible for the progression from the insulin-sensitive to the insulin-resistant, metabolically unhealthy obese (MUHO) phenotype. Results: Proteomic analysis of paired samples of adipocytes from subcutaneous (SC) and omental (OM) human AT revealed that both types of cells are altered in the MUHO state. Specifically, the glutathione redox cycle and other antioxidant defense systems as well as the protein-folding machinery were dysregulated and endoplasmic reticulum stress was increased in adipocytes from IR subjects. Moreover, proteasome activity was also compromised in adipocytes of MUHO individuals, which was associated with enhanced accumulation of oxidized and ubiquitinated proteins in these cells. Proteasome activity was also impaired in adipocytes of diet-induced obese mice and in 3T3-L1 adipocytes exposed to palmitate. In line with these data, proteasome inhibition significantly impaired insulin signaling in 3T3-L1 adipocytes. Innovation: This study provides the first evidence of the occurrence of protein homeostasis deregulation in adipocytes in human obesity, which, together with oxidative damage, interferes with insulin signaling in these cells. Conclusion: Our results suggest that proteasomal dysfunction and impaired proteostasis in adipocytes, resulting from protein oxidation and/or misfolding, constitute major pathogenic mechanisms in the development of IR in obesity. Antioxid. Redox Signal. 23, 597–612. PMID:25714483

  19. Metabolic profiling of the human response to a glucose challenge reveals distinct axes of insulin sensitivity

    Science.gov (United States)

    Shaham, Oded; Wei, Ru; Wang, Thomas J; Ricciardi, Catherine; Lewis, Gregory D; Vasan, Ramachandran S; Carr, Steven A; Thadhani, Ravi; Gerszten, Robert E; Mootha, Vamsi K

    2008-01-01

    Glucose ingestion after an overnight fast triggers an insulin-dependent, homeostatic program that is altered in diabetes. The full spectrum of biochemical changes associated with this transition is currently unknown. We have developed a mass spectrometry-based strategy to simultaneously measure 191 metabolites following glucose ingestion. In two groups of healthy individuals (n=22 and 25), 18 plasma metabolites changed reproducibly, including bile acids, urea cycle intermediates, and purine degradation products, none of which were previously linked to glucose homeostasis. The metabolite dynamics also revealed insulin's known actions along four key axes—proteolysis, lipolysis, ketogenesis, and glycolysis—reflecting a switch from catabolism to anabolism. In pre-diabetics (n=25), we observed a blunted response in all four axes that correlated with insulin resistance. Multivariate analysis revealed that declines in glycerol and leucine/isoleucine (markers of lipolysis and proteolysis, respectively) jointly provide the strongest predictor of insulin sensitivity. This observation indicates that some humans are selectively resistant to insulin's suppression of proteolysis, whereas others, to insulin's suppression of lipolysis. Our findings lay the groundwork for using metabolic profiling to define an individual's 'insulin response profile', which could have value in predicting diabetes, its complications, and in guiding therapy. PMID:18682704

  20. Human muscle spindle sensitivity reflects the balance of activity between antagonistic muscles.

    Science.gov (United States)

    Dimitriou, Michael

    2014-10-08

    Muscle spindles are commonly considered as stretch receptors encoding movement, but the functional consequence of their efferent control has remained unclear. The "α-γ coactivation" hypothesis states that activity in a muscle is positively related to the output of its spindle afferents. However, in addition to the above, possible reciprocal inhibition of spindle controllers entails a negative relationship between contractile activity in one muscle and spindle afferent output from its antagonist. By recording spindle afferent responses from alert humans using microneurography, I show that spindle output does reflect antagonistic muscle balance. Specifically, regardless of identical kinematic profiles across active finger movements, stretch of the loaded antagonist muscle (i.e., extensor) was accompanied by increased afferent firing rates from this muscle compared with the baseline case of no constant external load. In contrast, spindle firing rates from the stretching antagonist were lowest when the agonist muscle powering movement (i.e., flexor) acted against an additional resistive load. Stepwise regressions confirmed that instantaneous velocity, extensor, and flexor muscle activity had a significant effect on spindle afferent responses, with flexor activity having a negative effect. Therefore, the results indicate that, as consequence of their efferent control, spindle sensitivity (gain) to muscle stretch reflects the balance of activity between antagonistic muscles rather than only the activity of the spindle-bearing muscle. Copyright © 2014 the authors 0270-6474/14/3413644-12$15.00/0.

  1. In vivo imaging of human burn injuries with polarization-sensitive optical coherence tomography

    Science.gov (United States)

    Kim, Ki Hean; Pierce, Mark C.; Maguluri, Gopi; Park, B. Hyle; Yoon, Sang June; Lydon, Martha; Sheridan, Robert; de Boer, Johannes F.

    2012-06-01

    The accurate determination of burn depth is critical in the clinical management of burn wounds. Polarization-sensitive optical coherence tomography (PS-OCT) has been proposed as a potentially non-invasive method for determining burn depth by measuring thermally induced changes in the structure and birefringence of skin, and has been investigated in pre-clinical burn studies with animal models and ex vivo human skin. In this study, we applied PS-OCT to the in-vivo imaging of two pediatric burn patients. Deep and superficial burned skins along with contralateral controls were imaged in 3D. The imaging size was 8 mm×6 mm×2 mm in width, length, and depth in the air respectively, and the imaging time was approximately 6 s per volume. Superficially burned skins exhibited the same layered structure as the contralateral controls, but more visible vasculature and reduced birefringence compared to the contralateral controls. In contrast, a deeply burned skin showed loss of the layered structure, almost absent vasculature, and smaller birefringence compared to superficial burns. This study suggested the vasculature and birefringence as parameters for characterizing burn wounds.

  2. Sensitivity Analysis of Hybrid Propulsion Transportation System for Human Mars Expeditions

    Science.gov (United States)

    Chai, Patrick R.; Joyce, Ryan T.; Kessler, Paul D.; Merrill, Raymond G.; Qu, Min

    2017-01-01

    The National Aeronautics and Space Administration continues to develop and refine various transportation options to successfully field a human Mars campaign. One of these transportation options is the Hybrid Transportation System which utilizes both solar electric propulsion and chemical propulsion. The Hybrid propulsion system utilizes chemical propulsion to perform high thrust maneuvers, where the delta-V is most optimal when ap- plied to save time and to leverage the Oberth effect. It then utilizes solar electric propulsion to augment the chemical burns throughout the interplanetary trajectory. This eliminates the need for the development of two separate vehicles for crew and cargo missions. Previous studies considered single point designs of the architecture, with fixed payload mass and propulsion system performance parameters. As the architecture matures, it is inevitable that the payload mass and the performance of the propulsion system will change. It is desirable to understand how these changes will impact the in-space transportation system's mass and power requirements. This study presents an in-depth sensitivity analysis of the Hybrid crew transportation system to payload mass growth and solar electric propulsion performance. This analysis is used to identify the breakpoints of the current architecture and to inform future architecture and campaign design decisions.

  3. Sensitivity of PCR assays for murine gammaretroviruses and mouse contamination in human blood samples.

    Directory of Open Access Journals (Sweden)

    Li Ling Lee

    Full Text Available Gammaretroviruses related to murine leukemia virus (MLV have variously been reported to be present or absent in blood from chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME patients and healthy controls. Using subjects from New York State, we have investigated by PCR methods whether MLV-related sequences can be identified in nucleic acids isolated from whole blood or from peripheral blood mononuclear cells (PBMCs or following PBMC culture. We have also passaged the prostate cancer cell line LNCaP following incubation with plasma from patients and controls and assayed nucleic acids for viral sequences. We have used 15 sets of primers that can effectively amplify conserved regions of murine endogenous and exogenous retrovirus sequences. We demonstrate that our PCR assays for MLV-related gag sequences and for mouse DNA contamination are extremely sensitive. While we have identified MLV-like gag sequences following PCR on human DNA preparations, we are unable to conclude that these sequences originated in the blood samples.

  4. Coupled multiview autoencoders with locality sensitivity for three-dimensional human pose estimation

    Science.gov (United States)

    Yu, Jialin; Sun, Jifeng; Luo, Shasha; Duan, Bichao

    2017-09-01

    Estimating three-dimensional (3D) human poses from a single camera is usually implemented by searching pose candidates with image descriptors. Existing methods usually suppose that the mapping from feature space to pose space is linear, but in fact, their mapping relationship is highly nonlinear, which heavily degrades the performance of 3D pose estimation. We propose a method to recover 3D pose from a silhouette image. It is based on the multiview feature embedding (MFE) and the locality-sensitive autoencoders (LSAEs). On the one hand, we first depict the manifold regularized sparse low-rank approximation for MFE and then the input image is characterized by a fused feature descriptor. On the other hand, both the fused feature and its corresponding 3D pose are separately encoded by LSAEs. A two-layer back-propagation neural network is trained by parameter fine-tuning and then used to map the encoded 2D features to encoded 3D poses. Our LSAE ensures a good preservation of the local topology of data points. Experimental results demonstrate the effectiveness of our proposed method.

  5. Assessing the sensitivity of human skin hyperspectral responses to increasing anemia severity levels

    Science.gov (United States)

    Baranoski, Gladimir V. G.; Dey, Ankita; Chen, Tenn F.

    2015-09-01

    Anemia is a prevalent medical condition that seriously affects millions of people all over the world. In many regions, not only its initial detection but also its monitoring are hindered by limited access to laboratory facilities. This situation has motivated the development of a wide range of optical devices and procedures to assist physicians in these tasks. Although noticeable progress has been achieved in this area, the search for reliable, low-cost, and risk-free solutions still continues, and the strengthening of the knowledge base about this disorder and its effects is essential for the success of these initiatives. We contribute to these efforts by closely examining the sensitivity of human skin hyperspectral responses (within and outside the visible region of the light spectrum) to reduced hemoglobin concentrations associated with increasing anemia severity levels. This investigation, which involves skin specimens with distinct biophysical and morphological characteristics, is supported by controlled in silico experiments performed using a predictive light transport model and measured data reported in the biomedical literature. We also propose a noninvasive procedure to be employed in the monitoring of this condition at the point-of-care.

  6. Upregulation of SOX9 inhibits the growth of human and mouse melanomas and restores their sensitivity to retinoic acid.

    Science.gov (United States)

    Passeron, Thierry; Valencia, Julio C; Namiki, Takeshi; Vieira, Wilfred D; Passeron, Hélène; Miyamura, Yoshinori; Hearing, Vincent J

    2009-04-01

    Treatments for primary and metastatic melanomas are rarely effective. Even therapeutics such as retinoic acid (RA) that are successfully used to treat several other forms of cancer are ineffective. Recent evidence indicates that the antiproliferative effects of RA are mediated by the transcription factor SOX9 in human cancer cell lines. As we have previously shown that SOX9 is expressed in normal melanocytes, here we investigated SOX9 expression and function in human melanomas. Although SOX9 was expressed in normal human skin, it was increasingly downregulated as melanocytes progressed to the premalignant and then the malignant and metastatic states. Overexpression of SOX9 in both human and mouse melanoma cell lines induced cell cycle arrest by increasing p21 transcription and restored sensitivity to RA by downregulating expression of PRAME, a melanoma antigen. Furthermore, SOX9 overexpression in melanoma cell lines inhibited tumorigenicity both in mice and in a human ex vivo model of melanoma. Treatment of melanoma cell lines with PGD2 increased SOX9 expression and restored sensitivity to RA. Thus, combined treatment with PGD2 and RA substantially decreased tumor growth in human ex vivo and mouse in vivo models of melanoma. The results of our experiments targeting SOX9 provide insight into the pathophysiology of melanoma. Further, the effects of SOX9 on melanoma cell proliferation and RA sensitivity suggest the encouraging possibility of a noncytotoxic approach to the treatment of melanoma.

  7. Thymidine kinase deficient human cells have increased UV sensitivity in their capacity to support herpes simplex virus but normal UV sensitivity for colony formation

    International Nuclear Information System (INIS)

    Rainbow, A.J.

    1989-01-01

    A thymidine kinase deficient (tk - ) and two thymidine kinase proficient (tk + ) human cell lines were compared for UV sensitivity using colony-forming ability as well as their capacity to support the plaque formation of herpes simplex type 1 (HSV-1).The tk - line (143 cells) was a derivative of one of the tk + lines (R970-5), whereas the other tk + line (AC4 cells) was a derivative of the 143 cells obtained by transfection with purified sheared HSV-2 DNA encoding the viral tk gene. 143, R970-5 and AC4 cells showed a similar UV sensitivity for colony-forming ability. In contrast, the capacity to support HSV-1 plaque formation immediately (within 1 h) afte UV-irradiation was reduced to a greater extent in the 143 cells compared to the R970-5 and AC4 cells. Capacity curves for plaque formation of the HSV-1: KOS wild-type (tk + ) strain were similar to those for the HSV-1: PTK3B mutant (tk - ) strain were similar to those for the HSV-1: PTK3B mutant (tk - ) strain in the 3 cell strains, indicating that the viral tk gene does not influence the ability of HSV-1 to form plaques in UV-irradiated compared to unirradiated human cells. Cellular capacity for HSV-1 plaque formation was found to recover in both tk - and tk + cells for cultures infected 24 h after UV-irradiation. These results suggest that repair of UV-damaged DNA takes place to a similar extent in both tk - and tk + human cells, but the kinetics of repair are initially slower in tk - compared to tk + human cells. (author). 33 refs.; 3 figs.; 1 tab

  8. Proteasome inhibitor MG132 sensitizes HPV-positive human cervical cancer cells to rhTRAIL-induced apoptosis

    NARCIS (Netherlands)

    Hougardy, BMT; Maduro, JH; van der Zee, AGJ; de Groot, DJA; van den Heuvel, FAJ; de Vries, EGE; de Jong, S

    2006-01-01

    In cervical carcinogenesis, the p53 tumor suppressor pathway is disrupted by HPV (human papilloma virus) E6 oncogene expression. E6 targets p53 for rapid proteasome-mediated degradation. We therefore investigated whether proteasome inhibition by MG132 could restore wild-type p53 levels and sensitize

  9. Sensitive Cell-Based Assay for Determination of Human Immunodeficiency Virus Type 1 Coreceptor Tropism

    Science.gov (United States)

    Weber, Jan; Vazquez, Ana C.; Winner, Dane; Gibson, Richard M.; Rhea, Ariel M.; Rose, Justine D.; Wylie, Doug; Henry, Kenneth; Wright, Alison; King, Kevin; Archer, John; Poveda, Eva; Soriano, Vicente; Robertson, David L.; Olivo, Paul D.; Arts, Eric J.

    2013-01-01

    CCR5 antagonists are a powerful new class of antiretroviral drugs that require a companion assay to evaluate the presence of CXCR4-tropic (non-R5) viruses prior to use in human immunodeficiency virus (HIV)-infected individuals. In this study, we have developed, characterized, verified, and prevalidated a novel phenotypic test to determine HIV-1 coreceptor tropism (VERITROP) based on a sensitive cell-to-cell fusion assay. A proprietary vector was constructed containing a near-full-length HIV-1 genome with the yeast uracil biosynthesis (URA3) gene replacing the HIV-1 env coding sequence. Patient-derived HIV-1 PCR products were introduced by homologous recombination using an innovative yeast-based cloning strategy. The env-expressing vectors were then used in a cell-to-cell fusion assay to determine the presence of R5 and/or non-R5 HIV-1 variants within the viral population. Results were compared with (i) the original version of Trofile (Monogram Biosciences, San Francisco, CA), (ii) population sequencing, and (iii) 454 pyrosequencing, with the genotypic data analyzed using several bioinformatics tools, i.e., the 11/24/25 rule, Geno2Pheno (2% to 5.75%, 3.5%, or 10% false-positive rate [FPR]), and webPSSM. VERITROP consistently detected minority non-R5 variants from clinical specimens, with an analytical sensitivity of 0.3%, with viral loads of ≥1,000 copies/ml, and from B and non-B subtypes. In a pilot study, a 73.7% (56/76) concordance was observed with the original Trofile assay, with 19 of the 20 discordant results corresponding to non-R5 variants detected using VERITROP and not by the original Trofile assay. The degree of concordance of VERITROP and Trofile with population and deep sequencing results depended on the algorithm used to determine HIV-1 coreceptor tropism. Overall, VERITROP showed better concordance with deep sequencing/Geno2Pheno at a 0.3% detection threshold (67%), whereas Trofile matched better with population sequencing (79%). However, 454

  10. Highly sensitive detection of human IgG using a novel bio-barcode assay combined with DNA chip technology

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Zhenbao [Central South University, School of Pharmaceutical Sciences (China); Zhou, Bo, E-mail: zhoubo1771@163.com [The Affiliated Zhongda Hospital of Southeast University, Department of Gerontology (China); Wang, Haiqing; Lu, Feng; Liu, Tianjun; Song, Cunxian; Leng, Xigang, E-mail: lengxigyky@163.com [Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College (China)

    2013-09-15

    A simple and ultrasensitive detection of human IgG based on signal amplification using a novel bio-barcode assay and DNA chip technology was developed. The sensing platform was a sandwich system made up of antibody-modified magnetic microparticles (Ab-MMPs)/human IgG/Cy3-labeled single-stranded DNA and antibody-modified gold nanoparticles (Cy3-ssDNA-Ab-AuNPs). The MMPs (2.5 {mu}m in diameter) modified with mouse anti-human IgG monoclonal-antibodies could capture human IgG and further be separated and enriched via a magnetic field. The AuNPs (13 nm in diameter) conjugated with goat anti-human IgG polyclonal-antibodies and Cy3-ssDNA could further combine with the human IgG/Ab-MMP complex. The Cy3-ssDNA on AuNPs was then released by TCEP to hybridize with the DNA chip, thus generating a detectable signal by the fluorescence intensity of Cy3. In order to improve detection sensitivity, a three-level cascaded signal amplification was developed: (1) The MMP enrichment as the first-level; (2) Large quantities of Cy3-ssDNA on AuNPs as the second-level; (3) The Cy3-ssDNA conjugate with DNA chip as the third-level. The highly sensitive technique showed an increased response of the fluorescence intensity to the increased concentration of human IgG through a detection range from 1 pg mL{sup -1} to 10 ng mL{sup -1}. This sensing technique could not only improve the detection sensitivity for the low concentration of human IgG but also present a robust and efficient signal amplification model. The detection method has good stability, specificity, and reproducibility and could be applied in the detection of human IgG in the real samples.

  11. An ancestral haplotype of the human PERIOD2 gene associates with reduced sensitivity to light-induced melatonin suppression.

    Directory of Open Access Journals (Sweden)

    Tokiho Akiyama

    Full Text Available Humans show various responses to the environmental stimulus in individual levels as "physiological variations." However, it has been unclear if these are caused by genetic variations. In this study, we examined the association between the physiological variation of response to light-stimulus and genetic polymorphisms. We collected physiological data from 43 subjects, including light-induced melatonin suppression, and performed haplotype analyses on the clock genes, PER2 and PER3, exhibiting geographical differentiation of allele frequencies. Among the haplotypes of PER3, no significant difference in light sensitivity was found. However, three common haplotypes of PER2 accounted for more than 96% of the chromosomes in subjects, and 1 of those 3 had a significantly low-sensitive response to light-stimulus (P < 0.05. The homozygote of the low-sensitive PER2 haplotype showed significantly lower percentages of melatonin suppression (P < 0.05, and the heterozygotes of the haplotypes varied their ratios, indicating that the physiological variation for light-sensitivity is evidently related to the PER2 polymorphism. Compared with global haplotype frequencies, the haplotype with a low-sensitive response was more frequent in Africans than in non-Africans, and came to the root in the phylogenetic tree, suggesting that the low light-sensitive haplotype is the ancestral type, whereas the other haplotypes with high sensitivity to light are the derived types. Hence, we speculate that the high light-sensitive haplotypes have spread throughout the world after the Out-of-Africa migration of modern humans.

  12. Sensitive spectrophotometric determination of metoclopramide hydrochloride in dosage forms and spiked human urine using vanillin

    Directory of Open Access Journals (Sweden)

    O. Zenita Devi

    2016-09-01

    Full Text Available A new spectrophotometric method which is simple, sensitive, selective and rapid is described for the determination of metoclopramide hydrochloride (MCP in bulk drug and in dosage forms using vanillin as the chromogenic agent. The method is based on the condensation reaction between primary aromatic amine group present in MCP with aromatic aldehyde, vanillin to produce an intense yellow colored product. The resulting Schiff’s base shows an absorption maximum at 410 nm and the reaction product is stable for more than one day. The reaction was carried out in acetic acid and perchloric acid medium. Beer’s law was obeyed in the concentration range 1.5–15.0 μg ml−1 MCP with a molar absorptivity of 1.89 × 104 l mol−1 cm−1. The limit of detection (LOD and limit of quantification (LOQ were found to be 0.51 and 1.55 μg ml−1, respectively. The method was statistically evaluated by calculating percent relative error (% RE for accuracy and percent relative standard deviation (% RSD for precision, and was applied successfully to the determination of MCP in tablets, in injection and also in spiked human urine. No interference was observed from common additives found in pharmaceutical preparations. The results obtained by the proposed method were validated statistically by comparing the results with those of the reference method by applying the Student’s t-test and F-test. The accuracy and reliability of the method were further ascertained by performing recovery tests via standard-addition technique.

  13. Effect of low frequency modulated microwave exposure on human EEG: individual sensitivity.

    Science.gov (United States)

    Hinrikus, Hiie; Bachmann, Maie; Lass, Jaanus; Karai, Deniss; Tuulik, Viiu

    2008-10-01

    The aim of this study was to evaluate the effect of modulated microwave exposure on human EEG of individual subjects. The experiments were carried out on four different groups of healthy volunteers. The 450 MHz microwave radiation modulated at 7 Hz (first group, 19 subjects), 14 and 21 Hz (second group, 13 subjects), 40 and 70 Hz (third group, 15 subjects), 217 and 1000 Hz (fourth group, 19 subjects) frequencies was applied. The field power density at the scalp was 0.16 mW/cm(2). The calculated spatial peak SAR averaged over 1 g was 0.303 W/kg. Ten cycles of the exposure (1 min off and 1 min on) at fixed modulation frequencies were applied. All subjects completed the experimental protocols with exposure and sham. The exposed and sham-exposed subjects were randomly assigned. A computer also randomly assigned the succession of modulation frequencies. Our results showed that microwave exposure increased the EEG energy. Relative changes in the EEG beta1 power in P3-P4 channels were selected for evaluation of individual sensitivity. The rate of subjects significantly affected is similar in all groups except for the 1000 Hz group: in first group 3 subjects (16%) at 7 Hz modulation; in second group 4 subjects (31%) at 14 Hz modulation and 3 subjects (23%) at 21 Hz modulation; in third group 3 subjects (20%) at 40 Hz and 2 subjects (13%) at 70 Hz modulation; in fourth group 3 subjects (16%) at 217 Hz and 0 subjects at 1000 Hz modulation frequency.

  14. Sensitive microculture method for isolation of human immunodeficiency virus type 1 from blood leukocytes.

    Science.gov (United States)

    Erice, A; Sannerud, K J; Leske, V L; Aeppli, D; Balfour, H H

    1992-02-01

    A study was conducted to compare our standard culture with a new microculture procedure for isolation of human immunodeficiency virus type 1 (HIV-1) from blood leukocytes. A total of 137 blood specimens from 102 HIV-1 antibody-positive individuals (52 were asymptomatic, 31 were symptomatic, and 19 had AIDS) were cultured in a microculture system in which 10(6) of the patients' peripheral blood mononuclear cells (PBMC) were cocultured with 10(6) phytohemagglutinin (PHA)-stimulated PBMC from an HIV-1 antibody-negative blood donor in 1.2 ml of culture medium. Results were compared with those of a historical control group of 139 standard HIV-1 cultures from 108 HIV-1 antibody-positive subjects (58 were asymptomatic, 36 were symptomatic, and 14 had AIDS). For standard cultures, 10 x 10(6) of the patients' PBMC were cocultured with 5 x 10(6) PHA-stimulated PBMC from an HIV-1 antibody-negative blood donor in 15 ml of culture medium. HIV-1 was isolated in 128 (93%) microcultures and 133 (96%) standard cultures. Both methods identified more than 75% of the positive cultures within 7 days and 100% of the positive cultures within 14 days. The isolation rates for HIV-1 in microcultures compared with standard cultures were 91 versus 93% (specimens from asymptomatic individuals), 93 versus 96% (specimens from symptomatic individuals), and 97 versus 100% (specimens from patients with AIDS). The median time to positivity for both culture methods was 7 days, and this correlated significantly with symptoms and CD4+ cell counts. The microculture method is a sensitive and less expensive system for isolation of HIV-1 from PBMC of HIV-1 antibody-positive individuals, and we recommend it as the culture method of choice, especially for children and patients with AIDS and severe anemia or leukopenia whose blood volume is an important consideration.

  15. Insulin sensitivity is independent of lipid binding protein trafficking at the plasma membrane in human skeletal muscle

    DEFF Research Database (Denmark)

    Jordy, Andreas Børsting; Serup, Annette Karen; Karstoft, Kristian

    2014-01-01

    The aim of the present study was to investigate lipid-induced regulation of lipid binding proteins in human skeletal muscle and the impact hereof on insulin sensitivity. Eleven healthy male subjects underwent a 3-day hyper-caloric and high-fat diet regime. Muscle biopsies were taken before......-regulated by increased fatty acid availability. This suggests a time dependency in the up-regulation of FAT/CD36 and FABPpm protein during high availability of plasma fatty acids. Furthermore, we did not detect FATP1 and FATP4 protein in giant sarcolemmal vesicles obtained from human skeletal muscle. In conclusion......, this study shows that a short-term lipid-load increases mRNA content of key lipid handling proteins in human muscle. However, decreased insulin sensitivity after high-fat diet is not accompanied with relocation of FAT/CD36 or FABPpm protein to the sarcolemma. Finally, FATP1 and FATP4 protein could...

  16. [Variability of the sensitivity of human lymphocytes to the antiproliferative action of alkylating agents].

    Science.gov (United States)

    Veremko, L N; Telegin, L Iu; Pevnitskii, L A

    1983-05-01

    A study was made of variability of the sensitivity of peripheral blood lymphocytes from different donors to an antiproliferative action of cyclophosphamide and thiophosphamide. A similar degree of the sensitivity was revealed to alkylating agents differing in the action mode, with this degree being independent of the "stimulation index" magnitude.

  17. Inhibition of c-Kit signaling is associated with reduced heat and cold pain sensitivity in humans.

    Science.gov (United States)

    Ceko, Marta; Milenkovic, Nevena; le Coutre, Philipp; Westermann, Jörg; Lewin, Gary R

    2014-07-01

    The tyrosine kinase receptor c-Kit is critically involved in the modulation of nociceptive sensitivity in mice. Ablation of the c-Kit gene results in hyposensitivity to thermal pain, whereas activation of c-Kit produces hypersensitivity to noxious heat, without altering sensitivity to innocuous mechanical stimuli. In this study, we investigated the role of c-Kit signaling in human pain perception. We hypothesized that subjects treated with Imatinib or Nilotinib, potent inhibitors of tyrosine kinases including c-Kit but also Abl1, PDFGFRα, and PDFGFRβ, that are used to treat chronic myeloid leukemia (CML), would experience changes in thermal pain sensitivity. We examined 31 asymptomatic CML patients (14 male and 17 female) receiving Imatinib/Nilotinib treatment and compared them to 39 age- and sex-matched healthy controls (12 male and 27 female). We used cutaneous heat and cold stimulation to test normal and noxious thermal sensitivity, and a grating orientation task to assess tactile acuity. Thermal pain thresholds were significantly increased in the Imatinib/Nilotinib-treated group, whereas innocuous thermal and tactile thresholds were unchanged compared to those in the control group. In conclusion, our findings suggest that the biological effects of c-Kit inhibition are comparable in mice and humans in that c-Kit activity is required to regulate thermal pain sensitivity but does not affect innocuous thermal and mechanical sensation. The effect on experimental heat pain observed in our study is comparable to those of several common analgesics; thus modulation of the c-Kit pathway can be used to specifically modulate noxious heat and cold sensitivity in humans. Copyright © 2014 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

  18. Nasal insulin changes peripheral insulin sensitivity simultaneously with altered activity in homeostatic and reward-related human brain regions.

    Science.gov (United States)

    Heni, M; Kullmann, S; Ketterer, C; Guthoff, M; Linder, K; Wagner, R; Stingl, K T; Veit, R; Staiger, H; Häring, H-U; Preissl, H; Fritsche, A

    2012-06-01

    Impaired insulin sensitivity is a major factor leading to type 2 diabetes. Animal studies suggest that the brain is involved in the regulation of insulin sensitivity. We investigated whether insulin action in the human brain regulates peripheral insulin sensitivity and examined which brain areas are involved. Insulin and placebo were given intranasally. Plasma glucose, insulin and C-peptide were measured in 103 participants at 0, 30 and 60 min. A subgroup (n = 12) was also studied with functional MRI, and blood sampling at 0, 30 and 120 min. For each time-point, the HOMA of insulin resistance (HOMA-IR) was calculated as an inverse estimate of peripheral insulin sensitivity. Plasma insulin increased and subsequently decreased. This excursion was accompanied by slightly decreased plasma glucose, resulting in an initially increased HOMA-IR. At 1 h after insulin spray, the HOMA-IR subsequently decreased and remained lower up to 120 min. An increase in hypothalamic activity was observed, which correlated with the increased HOMA-IR at 30 min post-spray. Activity in the putamen, right insula and orbitofrontal cortex correlated with the decreased HOMA-IR at 120 min post-spray. Central insulin action in specific brain areas, including the hypothalamus, may time-dependently regulate peripheral insulin sensitivity. This introduces a potential novel mechanism for the regulation of peripheral insulin sensitivity and underlines the importance of cerebral insulin action for the whole organism.

  19. Accounting for human variability and sensitivity in setting standards for electromagnetic fields.

    Science.gov (United States)

    Bailey, William H; Erdreich, Linda S

    2007-06-01

    Biological sensitivity and variability are key issues for risk assessment and standard setting. Variability encompasses general inter-individual variations in population responses, while sensitivity relates to unusual or extreme responses based on genetic, congenital, medical, or environmental conditions. For risk assessment and standard setting, these factors affect estimates of thresholds for effects and dose-response relationships and inform efforts to protect the more sensitive members of the population, not just the typical or average person. While issues of variability and sensitivity can be addressed by experimental and clinical studies of electromagnetic fields, investigators have paid little attention to these important issues. This paper provides examples that illustrate how default assumptions regarding variability can be incorporated into estimates of 60-Hz magnetic field exposures with no risk of cardiac stimulation and how population thresholds and variability of peripheral nerve stimulation responses at 60-Hz can be estimated from studies of pulsed gradient magnetic fields in magnetic resonance imaging studies. In the setting of standards for radiofrequency exposures, the International Commission for Non-Ionizing Radiation Protection uses inter-individual differences in thermal sensitivity as one of the considerations in the development of "safety factors." However, neither the range of sensitivity nor the sufficiency or excess of the 10-fold and the additional 5-fold safety factors have been assessed quantitatively. Data on the range of responses between median and sensitive individuals regarding heat stress and cognitive function should be evaluated to inform a reassessment of these safety factors and to identify data gaps.

  20. Properties of pressure-sensitive adhesive tapes with soft adhesives to human skin and their mechanism.

    Science.gov (United States)

    Tokumura, Fumio; Homma, Takeyasu; Tomiya, Toshiki; Kobayashi, Yuko; Matsuda, Tetsuaki

    2007-05-01

    The use of soft adhesives in the manufacture of pressure-sensitive adhesive tapes has recently increased. The dermal peeling force of adhesive tapes with soft adhesives was studied. Four kinds of adhesive tapes with adhesives of different softness were made, by adding varying amounts of isopropyl myristate as a softener. The tapes were applied on the flexor side of the forearm of six healthy male volunteers. The dermal peeling force, the amount of stripped corneocytes, the level of pain when the tapes were removed and the degree of penetration of adhesives into the sulcus cutis (skin furrows) were evaluated at 1 and 24 h after application of the tapes. Furthermore, a skin model panel (a sulcus cutis and crista cutis model panel) and a crista cutis model panel were constructed from a general stainless-steel panel, and the peeling force of the tapes against the model panels was measured. As the softness of adhesives increased, the peeling force against a general stainless-steel panel with a flat surface decreased, although the peeling force against human skin did not significantly change. The amount of stripped corneocytes on the removed tapes and the level of pain when the tapes were removed decreased with the increase in softness of the adhesives. These results suggest that adhesive tapes with soft adhesives that contain isopropyl myristate as a softener are suitable for the skin. Furthermore, the degree of penetration of adhesive into the sulcus cutis increased as the softness of adhesives increased. Upon evaluation of the peeling force against the model panels, as the softness of adhesives increased, there was a slight decrease in the peeling force against the skin model panel, while there was a remarkable decrease in the peeling force against the crista cutis model panel. These results suggest that the lack of change in the dermal peeling force as the softness of adhesives increased was caused by penetration of soft adhesive into the sulcus cutis, and that the

  1. A conceptual framework for predicting temperate ecosystem sensitivity to human impacts on fire regimes

    Science.gov (United States)

    D. B. McWethy; P. E. Higuera; C. Whitlock; T. T. Veblen; D. M. J. S. Bowman; G. J. Cary; S. G. Haberle; R. E. Keane; B. D. Maxwell; M. S. McGlone; G. L. W. Perry; J. M. Wilmshurst

    2013-01-01

    The increased incidence of large fires around much of the world in recent decades raises questions about human and non-human drivers of fire and the likelihood of increased fire activity in the future. The purpose of this paper is to outline a conceptual framework for examining where human-set fires and feedbacks are likely to be most pronounced in temperate forests...

  2. Effects of Lactobacillus acidophilus NCFM on insulin sensitivity and the systemic inflammatory response in human subjects

    DEFF Research Database (Denmark)

    Andreasen, Anne Sofie; Larsen, Nadja; Pedersen-Skovsgaard, Theis

    2010-01-01

    According to animal studies, intake of probiotic bacteria may improve glucose homeostasis. We hypothesised that probiotic bacteria improve insulin sensitivity by attenuating systemic inflammation. Therefore, the effects of oral supplementation with the probiotic bacterium Lactobacillus acidophilus...

  3. Central sensitization in spinal cord injured humans assessed by reflex receptive fields

    DEFF Research Database (Denmark)

    Biurrun Manresa, José Alberto; Finnerup, Nanna Susanne Brix; Johannesen, Inger Lauge

    2014-01-01

    OBJECTIVE: To investigate the effects of central sensitization, elicited by intramuscular injection of capsaicin, by comparing the reflex receptive fields (RRF) of spinally-intact volunteers and spinal cord injured volunteers that present presensitized spinal nociceptive mechanisms. METHODS: Fift...

  4. Syringic acid from Tamarix aucheriana possesses antimitogenic and chemo-sensitizing activities in human colorectal cancer cells.

    Science.gov (United States)

    Abaza, Mohamed-Salah; Al-Attiyah, Raja'a; Bhardwaj, Radhika; Abbadi, Ghaneim; Koyippally, Mathew; Afzal, Mohammad

    2013-09-01

    For its variety of biological activities, Tamarix aucheriana (Decne.) Baum. (Tamaricaceae) has an extensive history as a traditional Arab medicine. Antimitogenic and chemo-sensitizing activities of syringic acid (SA) were studied against human colorectal cancer. Chromatographic and spectral data were used for the isolation and identification of SA. MTT, flow cytometry, in vitro invasion and angiogenesis assays, fluoremetry, ELISA and Real Time qPCR were used to test antimitogenic and chemo-sensitizing activities of SA, cell cycle, apoptosis, proteasome and NFκB-DNA-binding activities, cancer cell invasion and angiogenesis, and expression of cell cycle/apoptosis-related genes. SA showed a time- and dose-dependent (IC₅₀ = 0.95-1.2 mg mL⁻¹) antimitogenic effect against cancer cells with little cytotoxicity on normal fibroblasts (≤20%). SA-altered cell cycle (S/G2-M or G1/G2-M phases) in a time-dependent manner, induced apoptosis, inhibited DNA-binding activity of NFκB (p ≤ 0.0001), chymotrypsin-like/PGPH (peptidyl-glutamyl peptide-hydrolyzing) (p ≤ 0.0001) and the trypsin-like (p ≤ 0.002) activities of 26S proteasome and angiogenesis. SA also differentially sensitized cancer cells to standard chemotherapies with a marked increase in their sensitivity to camptothecin (500-fold), 5FU (20,000-fold), doxorubicin (210-fold), taxol (3134-fold), vinblastine (1000-fold), vincristine (130-fold) and amsacrine (107-fold) compared to standard drugs alone. SA exerted its chemotherapeutic and chemo-sensitizing effects through an array of mechanisms including cell-cycle arrest, apoptosis induction, inhibition of cell proliferation, cell migration, angiogenesis, NFκB DNA-binding and proteasome activities. These results demonstrate the potential of SA as an antimitogenic and chemo-sensitizing agent for human colorectal cancer.

  5. Impact of sensitivity of human leucocyte antigen antibody detection by Luminex technology on graft loss at 1 year

    OpenAIRE

    Szatmary, Peter; Jones, James; Hammad, Abdul; Middleton, Derek

    2013-01-01

    Background The clinical relevance of the detection of human leucocyte antigen (HLA) antibodies in sera of renal transplant recipients by highly sensitive methods such as Luminex alone is uncertain and a matter of debate. The choice of output thresholds affects antibody detection and thus organ allocation, yet there are no internationally agreed threshold levels. This study aims at evaluating our current practice of using an MFI threshold of 1000 in antibody detection. Methods We carried out a...

  6. Label-free electrochemical impedance biosensor to detect human interleukin-8 in serum with sub-pg/ml sensitivity

    OpenAIRE

    Sharma, R.; Deacon, S.E.; Nowak, D.; George, S.E.; Szymonik, M.P.; Tang, A.A.S.; Tomlinson, D.C.; Davies, A.G.; McPherson, M.J.; W?lti, C.

    2016-01-01

    Biosensors with high sensitivity and short time-to-result that are capable of detecting biomarkers in body fluids such as serum are an important prerequisite for early diagnostics in modern healthcare provision. Here, we report the development of an electrochemical impedance-based sensor for the detection in serum of human interleukin-8 (IL-8), a pro-angiogenic chemokine implicated in a wide range of inflammatory diseases. The sensor employs a small and robust synthetic non-antibody capture p...

  7. Endocrine sensitivity of the receptor-positive T61 human breast carcinoma serially grown in nude mice

    DEFF Research Database (Denmark)

    Brünner, N; Spang-Thomsen, M; Skovgaard Poulsen, H

    1985-01-01

    A study was made on the effect of ovariectomy, 17 beta-oestradiol, and tamoxifen on the oestrogen and progesterone receptor-positive T61 human breast carcinoma grown in nude mice. The effect of the treatment was evaluated by the specific growth delay calculated on the basis of Gompertz growth cur...... but is not a sufficiently clear marker to allow prediction of the endocrine sensitivity of individual breast tumours....

  8. Differentiated human midbrain-derived neural progenitor cells express excitatory strychnine-sensitive glycine receptors containing α2β subunits.

    Science.gov (United States)

    Wegner, Florian; Kraft, Robert; Busse, Kathy; Härtig, Wolfgang; Ahrens, Jörg; Leffler, Andreas; Dengler, Reinhard; Schwarz, Johannes

    2012-01-01

    Human fetal midbrain-derived neural progenitor cells (NPCs) may deliver a tissue source for drug screening and regenerative cell therapy to treat Parkinson's disease. While glutamate and GABA(A) receptors play an important role in neurogenesis, the involvement of glycine receptors during human neurogenesis and dopaminergic differentiation as well as their molecular and functional characteristics in NPCs are largely unknown. Here we investigated NPCs in respect to their glycine receptor function and subunit expression using electrophysiology, calcium imaging, immunocytochemistry, and quantitative real-time PCR. Whole-cell recordings demonstrate the ability of NPCs to express functional strychnine-sensitive glycine receptors after differentiation for 3 weeks in vitro. Pharmacological and molecular analyses indicate a predominance of glycine receptor heteromers containing α2β subunits. Intracellular calcium measurements of differentiated NPCs suggest that glycine evokes depolarisations mediated by strychnine-sensitive glycine receptors and not by D-serine-sensitive excitatory glycine receptors. Culturing NPCs with additional glycine, the glycine-receptor antagonist strychnine, or the Na(+)-K(+)-Cl(-) co-transporter 1 (NKCC1)-inhibitor bumetanide did not significantly influence cell proliferation and differentiation in vitro. These data indicate that NPCs derived from human fetal midbrain tissue acquire essential glycine receptor properties during neuronal maturation. However, glycine receptors seem to have a limited functional impact on neurogenesis and dopaminergic differentiation of NPCs in vitro.

  9. Differentiated human midbrain-derived neural progenitor cells express excitatory strychnine-sensitive glycine receptors containing α2β subunits.

    Directory of Open Access Journals (Sweden)

    Florian Wegner

    Full Text Available BACKGROUND: Human fetal midbrain-derived neural progenitor cells (NPCs may deliver a tissue source for drug screening and regenerative cell therapy to treat Parkinson's disease. While glutamate and GABA(A receptors play an important role in neurogenesis, the involvement of glycine receptors during human neurogenesis and dopaminergic differentiation as well as their molecular and functional characteristics in NPCs are largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we investigated NPCs in respect to their glycine receptor function and subunit expression using electrophysiology, calcium imaging, immunocytochemistry, and quantitative real-time PCR. Whole-cell recordings demonstrate the ability of NPCs to express functional strychnine-sensitive glycine receptors after differentiation for 3 weeks in vitro. Pharmacological and molecular analyses indicate a predominance of glycine receptor heteromers containing α2β subunits. Intracellular calcium measurements of differentiated NPCs suggest that glycine evokes depolarisations mediated by strychnine-sensitive glycine receptors and not by D-serine-sensitive excitatory glycine receptors. Culturing NPCs with additional glycine, the glycine-receptor antagonist strychnine, or the Na(+-K(+-Cl(- co-transporter 1 (NKCC1-inhibitor bumetanide did not significantly influence cell proliferation and differentiation in vitro. CONCLUSIONS/SIGNIFICANCE: These data indicate that NPCs derived from human fetal midbrain tissue acquire essential glycine receptor properties during neuronal maturation. However, glycine receptors seem to have a limited functional impact on neurogenesis and dopaminergic differentiation of NPCs in vitro.

  10. Sexual dimorphism in hepatic, adipose tissue and peripheral tissue insulin sensitivity in obese humans

    Directory of Open Access Journals (Sweden)

    Kasper W. ter Horst

    2015-11-01

    Full Text Available Glucose and lipid metabolism differ between men and women, and women tend to have better whole-body or muscle insulin sensitivity. This may be explained, in part, by differences in sex hormones and adipose tissue distribution. Few studies have investigated gender differences in hepatic, adipose tissue and whole-body insulin sensitivity between severely obese men and women. In this study, we aimed to determine the differences in glucose metabolism between severely obese men and women using tissue-specific measurements of insulin sensitivity. Insulin sensitivity was compared between age and body mass index (BMI-matched obese men and women by a two-step euglycemic hyperinsulinemic clamp with infusion of [6,6-2H2]glucose. Basal endogenous glucose production and insulin sensitivity of the liver, adipose tissue and peripheral tissues were assessed. Liver fat content was assessed by proton magnetic resonance spectroscopy in a subset of included subjects. We included 46 obese men and women (age, 48±2 vs 46±2 years, p=0.591; BMI, 41±1 vs 41±1 kg/m2, p=0.832. There was no difference in basal endogenous glucose production (14.4±1.0 vs 15.3±0.5 µmol•kg fat-free mass-1•min-1, p=0.410, adipose tissue insulin sensitivity (insulin-mediated suppression of free fatty acids, 71.6±3.6 vs 76.1±2.6%, p=0.314 or peripheral insulin sensitivity (insulin-stimulated rate of disappearance of glucose, 26.2±2.1 vs 22.7±1.7 µmol•kg-1•min-1, p=0.211. Obese men were characterized by lower hepatic insulin sensitivity (insulin-mediated suppression of endogenous glucose production, 61.7±4.1 vs 72.8±2.5% in men vs women, resp., p=0.028. Finally, these observations could not be explained by differences in liver fat content (men vs women, 16.5±3.1 vs 16.0±2.5%, p=0.913, n=27.We conclude that obese men have lower hepatic, but comparable adipose tissue and peripheral tissue, insulin sensitivity compared to similarly obese women. Hepatic insulin resistance may

  11. Omalizumab Increases the Intrinsic Sensitivity of Human Basophils to IgE-Mediated Stimulation

    Science.gov (United States)

    MacGlashan, Donald; Saini, Sarbjit S.

    2013-01-01

    Background Treatment of allergic patients with omalizumab results in a paradoxical increase in their basophil histamine release response, ex vivo, to crosslinking anti-IgE antibody. It is not known whether this change in response is associated with an increase in intrinsic cellular sensitivity, which would be a paradoxical response. Objective To determine if the increase in response to anti-IgE Ab is a reflection of an increased cellular sensitivity, expressed as molecules of antigen-specific IgE per basophil required to produce a 50% of maximal response. Methods Patients were treated with omalizumab or placebo agent for 12 weeks (NCT01003301 at ClinicalTrials.gov) and the metric of basophil sensitivity was assessed at 4 time points, baseline, 6–8 weeks, 12 weeks (after which treatment stopped) and 24 weeks (12 weeks after the end of treatment). Results As observed previously, treatment with omalizumab resulted in a marked increase in the maximal histamine release induced by crosslinking anti-IgE Ab. This change was accompanied by a marked shift in intrinsic basophil sensitivity, ranging from 2.5 to 125 fold, with an average of 6 fold at the midpoint of the treatment to 12 fold after 12 weeks. The magnitude of the increase in cellular sensitivity was inversely related to the starting sensitivity or the starting maximum histamine release. The increased cellular sensitivity also occurred when using LTC4 secretion as a metric of the basophil response. 12 weeks after the end of treatment, cellular sensitivity was found to shift towards the baseline level although the return to baseline was not yet complete at this time point. Conclusions Treatment with omalizumab results in a markedly increased sensitivity of basophils to IgE-mediated stimulation, in terms of the number of IgE molecules required to produce a given response. These results provide a better quantitative sense of the phenotypic change that occurs in basophils during omalizumab treatment which has

  12. Aging of non-visual spectral sensitivity to light in humans: compensatory mechanisms?

    Directory of Open Access Journals (Sweden)

    Raymond P Najjar

    Full Text Available The deterioration of sleep in the older population is a prevalent feature that contributes to a decrease in quality of life. Inappropriate entrainment of the circadian clock by light is considered to contribute to the alteration of sleep structure and circadian rhythms in the elderly. The present study investigates the effects of aging on non-visual spectral sensitivity to light and tests the hypothesis that circadian disturbances are related to a decreased light transmittance. In a within-subject design, eight aged and five young subjects were exposed at night to 60 minute monochromatic light stimulations at 9 different wavelengths (420-620 nm. Individual sensitivity spectra were derived from measures of melatonin suppression. Lens density was assessed using a validated psychophysical technique. Although lens transmittance was decreased for short wavelength light in the older participants, melatonin suppression was not reduced. Peak of non-visual sensitivity was, however, shifted to longer wavelengths in the aged participants (494 nm compared to young (484 nm. Our results indicate that increased lens filtering does not necessarily lead to a decreased non-visual sensitivity to light. The lack of age-related decrease in non-visual sensitivity to light may involve as yet undefined adaptive mechanisms.

  13. Central sensitization in spinal cord injured humans assessed by reflex receptive fields.

    Science.gov (United States)

    Biurrun Manresa, José Alberto; Finnerup, Nanna Susanne Brix; Johannesen, Inger Lauge; Biering-Sørensen, Fin; Jensen, Troels Staehelin; Arendt-Nielsen, Lars; Andersen, Ole Kæseler

    2014-02-01

    To investigate the effects of central sensitization, elicited by intramuscular injection of capsaicin, by comparing the reflex receptive fields (RRF) of spinally-intact volunteers and spinal cord injured volunteers that present presensitized spinal nociceptive mechanisms. Fifteen volunteers with complete spinal cord injury (SCI) and fourteen non-injured (NI) volunteers participated in the experiment. Repeated electrical stimulation was applied on eight sites on the foot sole to elicit the nociceptive withdrawal reflex (NWR). RRF were assessed before, 1min after and 60min after an intramuscular injection of capsaicin in the foot sole in order to induce central sensitization. Both groups presented RRF expansion and lowered NWR thresholds immediately after capsaicin injection, reflected by the enlargement of RRF sensitivity areas and RRF probability areas. Moreover, the topography of the RRF sensitivity and probability areas were significantly different in SCI volunteers compared to NI volunteers in terms of size and shape. SCI volunteers can develop central sensitization, despite adaptive/maladaptive changes in synaptic plasticity and lack of supraspinal control. Protective plastic mechanisms may still be functional in SCI volunteers. Copyright © 2013 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.

  14. Headache and mechanical sensitization of human pericranial muscles after repeated intake of monosodium glutamate (MSG)

    DEFF Research Database (Denmark)

    Shimada, Akiko; Cairns, B.E.; Vad, N.

    2013-01-01

    A single intake of monosodium glutamate (MSG) may cause headache and increased muscle sensitivity. We conducted a double-blinded, placebo-controlled, crossover study to examine the effect of repeated MSG intake on spontaneous pain, mechanical sensitivity of masticatory muscles, side effects...... pressure were evaluated before and 15, 30, and 50 min after MSG intake. Whole saliva samples were taken before and 30 min after MSG intake to assess glutamate concentrations. Headache occurred in 8/14 subjects during MSG and 2/14 during placebo (P = 0.041). Salivary glutamate concentrations on Day 5 were...

  15. A novel method of selective removal of human DNA improves PCR sensitivity for detection of Salmonella Typhi in blood samples.

    Science.gov (United States)

    Zhou, Liqing; Pollard, Andrew J

    2012-07-27

    Enteric fever is a major public health problem, causing an estimated 21million new cases and 216,000 or more deaths every year. Current diagnosis of the disease is inadequate. Blood culture only identifies 45 to 70% of the cases and is time-consuming. Serological tests have very low sensitivity and specificity. Clinical samples obtained for diagnosis of enteric fever in the field generally have blood, so that even PCR-based methods, widely used for detection of other infectious diseases, are not a straightforward option in typhoid diagnosis. We developed a novel method to enrich target bacterial DNA by selective removal of human DNA from blood samples, enhancing the sensitivity of PCR tests. This method offers the possibility of improving PCR assays directly using clinical specimens for diagnosis of this globally important infectious disease. Blood samples were mixed with ox bile for selective lysis of human blood cells and the released human DNA was then digested with addition of bile resistant micrococcal nuclease. The intact Salmonella Typhi bacteria were collected from the specimen by centrifugation and the DNA extracted with QIAamp DNA mini kit. The presence of Salmonella Typhi bacteria in blood samples was detected by PCR with the fliC-d gene of Salmonella Typhi as the target. Micrococcal nuclease retained activity against human blood DNA in the presence of up to 9% ox bile. Background human DNA was dramatically removed from blood samples through the use of ox bile lysis and micrococcal nuclease for removal of mammalian DNA. Consequently target Salmonella Typhi DNA was enriched in DNA preparations and the PCR sensitivity for detection of Salmonella Typhi in spiked blood samples was enhanced by 1,000 fold. Use of a combination of selective ox-bile blood cell lysis and removal of human DNA with micrococcal nuclease significantly improves PCR sensitivity and offers a better option for improved typhoid PCR assays directly using clinical specimens in diagnosis of

  16. In vivo imaging of human burn injuries with polarization-sensitive optical coherence tomography

    NARCIS (Netherlands)

    Kim, K.H.; Pierce, M. C.; Maguluri, G. N.; Park, B. H.; Yoon, S.J.; Lydan, M.; Sheridan, R.; de Boer, J.F.

    2012-01-01

    The accurate determination of burn depth is critical in the clinical management of burn wounds. Polarization- sensitive optical coherence tomography (PS-OCT) has been proposed as a potentially non-invasive method for determining burn depth by measuring thermally induced changes in the structure and

  17. Muscle triacylglycerol and hormone-sensitive lipase activity in untrained and trained human muscles

    DEFF Research Database (Denmark)

    Helge, Jørn Wulff; Biba, Taus O; Galbo, Henrik

    2006-01-01

    During exercise, triacylglycerol (TG) is recruited in skeletal muscles. We hypothesized that both muscle hormone-sensitive lipase (HSL) activity and TG recruitment would be higher in trained than in untrained subjects in response to prolonged exercise. Healthy male subjects (26 +/- 1 years, body ...

  18. Human skeletal muscle perilipin 2 and 3 expression varies with insulin sensitivity

    DEFF Research Database (Denmark)

    Vigelsø Hansen, Andreas; Prats Gavalda, Clara; Ploug, Thorkil

    2013-01-01

    was obtained from vastus lateralis, and a two-step sequential euglycaemic-hy- perinsulinaemic clamp was performed. Muscle sam- ples were analyzed by Western blot for expression of perilipin 2, 3, 5, adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), endothelial lipase (EL) and lipoprotein...

  19. Is Cytox 3522 (10% methylene-bis-thiocyanate) a human skin sensitizer?

    DEFF Research Database (Denmark)

    Andersen, Klaus Ejner; Hamann, K

    1983-01-01

    Methylene-bis-thiocyanate is an antimicrobial agent in Cytox 3522 (American Cyanamid Corporation) and Nalco 206 (Nalco Chemical Company). Both are wide-spectrum industrial biocides. Cytox 3522 showed a strong sensitization potential in guineau pigs using the Guinea Pig Maximization Test and the O...

  20. Lipid droplet size and location in human skeletal muscle fibers are associated with insulin sensitivity

    DEFF Research Database (Denmark)

    Nielsen, Joachim; Christensen, Anders E; Nellemann, Birgitte

    2017-01-01

    In skeletal muscle, an accumulation of lipid droplets (LDs) in the subsarcolemmal space is associated with insulin resistance, but the underlying mechanism is not clear. We aimed to investigate how the size, number and location of LDs are associated with insulin sensitivity and muscle fiber types...... are associated with insulin resistance in skeletal muscle....

  1. Resistance to cisplatin does not affect sensitivity of human ovarian cancer cell lines to mifepristone cytotoxicity

    Directory of Open Access Journals (Sweden)

    Seidel Erin E

    2009-02-01

    Full Text Available Abstract Background The prototypical antiprogestin mifepristone exhibits potent growth inhibition activity towards ovarian cancer cells in vitro and in vivo. The aim of this research was to establish whether mifepristone is capable of inhibiting cell proliferation and inducing apoptotic cell death regardless of the degree of sensitivity ovarian cancer cells exhibit to cisplatin. Methods OV2008, OV2008/C13, A2780, A2780/CP70, Caov-3, and SK-OV-3 cell lines exhibiting a range of sensitivities to cisplatin were used. Growth inhibition, cell viability, and sub-diploid DNA content in response to treatment with escalating doses of either mifepristone or cisplatin were assessed by microcapillary cytometry. Apoptotic cell death was evaluated by measuring genomic DNA fragmentation and cleavage of caspase-3 and poly (ADP ribose polymerase (PARP. Results The sensitivities to cisplatin manifested by the cell lines were OV2008 > A2780 > Caov-3 > SK-OV-3 > OV2008/C13 > A2780/CP70. Mifepristone inhibited the growth of all six cell lines in a dose-related manner with IC50s ranging from ~6–12 μM and without significant correlation with the relative sensitivities the cells displayed for cisplatin. Moreover, at the highest concentration studied, mifepristone triggered apoptotic death in all six cell lines as evidenced by the increase in sub-diploid fragmented DNA content and cleavage of caspase-3 and of its downstream substrate PARP. Conclusion Mifepristone is cytotoxic towards ovarian cancer cells independent of the sensitivity exhibited by the cells to cisplatin, displaying cytostatic effects at lower concentrations and lethal effects at higher concentrations. Mifepristone monotherapy emerges as a valuable therapeutic alternative for platinum-resistant ovarian cancers.

  2. A novel biomarker for beryllium sensitization in humans. 1998 annual progress report

    Energy Technology Data Exchange (ETDEWEB)

    Albertini, R.J.

    1998-06-01

    'Beryllium reactive T-lymphocytes can be used as an indicator of sensitization. Traditionally, their presence is detected by an in vitro proliferation assay. However, this test is capricious (results varying from day to day in the same laboratory) and insensitive (rarely positive before clinical symptoms ). The objective of this project is to obtain and characterize beryllium reactive T-cells from peripheral blood using the hprt T-cell mutation assay. T-cells are selected on the basis of their mutation of the hprt gene which renders them insensitive to 6-thioguanine in culture. Such mutant populations are expected to be enriched for cells which are proliferating in vivo as a result of the sensitizing process. This hypothesis has been verified in a number of studies. The seven specific aims of this study will: (i) identify the in vivo proliferating T-cell clones in sensitized individuals by selecting for hprt mutants, (ii) determine T-cell receptor (TCR) gene usages and commonalities among these clones, (iii) demonstrate reactivity to beryllium of these clones, (iv) generate beryllium sensitized T-cells in vitro from peripheral blood of the same individual, (v) determine TCR gene usages and commonalities for these in vitro derived cells, (vi) compare TCR gene patterns between the in vivo and in vitro derived clones, and (vii) develop a quantitative PCR (qPCR) method for amplifying the common (and therefore relevant) TCR genes directly from peripheral blood. The last of these is the novel biomarker of early beryllium sensitization. This report summarizes studies of the first 20 months of this project.'

  3. Elucidation of Pertussis Toxin-Sensitive Migration Signaling in Human Breast Cancer Cells

    National Research Council Canada - National Science Library

    Rust, William

    2001-01-01

    The long range goal of this laboratory is to identify integrin-associated signaling events that contribute to the constitutive migration of human breast cancer cells on the laminin extracellular matrix proteins...

  4. Elucidation of Pertussis Toxin-Sensitive Migration Signaling in Human Breast Cancer Cells

    National Research Council Canada - National Science Library

    Rust, William

    2002-01-01

    The long range goal of this laboratory is to identify integrin-associated signaling events that contribute to the constitutive migration of human breast cancer cells on the laminin extracellular matrix proteins...

  5. The Tactile Modality: A Review of Tactile Sensitivity and Human Tactile Interfaces

    National Research Council Canada - National Science Library

    Myles, Kimberly; Binseel, Mary S

    2007-01-01

    ... the most important. Hearing also is viewed as necessary for interpreting environmental stimuli. In contrast, touch, smell, and taste are largely ignored as being essential to humans' interaction with the environment...

  6. The Tactile Modality: A Review of Tactile Sensitivity and Human Tactile Interfaces

    National Research Council Canada - National Science Library

    Myles, Kimberly; Binseel, Mary S

    2007-01-01

    .... Because humans have a limited capacity to receive, hold in working memory, and cognitively process information taken from the environment, the use of one sensory modality to convey information within...

  7. Sensitive and rapid detection of Paragonimus westermani infection in humans and animals by loop-mediated isothermal amplification (LAMP).

    Science.gov (United States)

    Chen, M X; Ai, L; Zhang, R L; Xia, J J; Wang, K; Chen, S H; Zhang, Y N; Xu, M J; Li, X; Zhu, X Q; Chen, J X

    2011-05-01

    In the present study, a loop-mediated isothermal amplification (LAMP) assay was developed and validated for the detection of Paragonimus westermani adults, metacercariae, and eggs in human and animal samples. The LAMP amplification can be finished in 45 min under isothermal condition at 60°C by employing a set of four species-specific primer mixtures and the results can be checked by naked-eye visualization. No amplification products were detected with deoxyribunucleic acid (DNA) of related trematode species including Fasciola hepatica, Fasciola gigantica, Clonorchis sinensis, Opisthorchis viverrini, Schistosoma mansoni, and Schistosoma japonicum. The method was further validated by examining P. westermani DNA in intermediate hosts including freshwater crabs and crayfish, as well as in sputum and pleural fluid samples from patients of paragonimiasis. These results indicated that the LAMP assay was highly specific, sensitive, and rapid, and it was approximately 100 times more sensitive than conventional specific PCR. The LAMP assay established in this study provides a rapid and sensitive tool for the detection of P. westermani DNA in freshwater crabs, crayfish, sputum, and pleural fluid samples, which has important implications for effective control of human paragonimiasis.

  8. Two consensus primer systems and nested polymerase chain reaction for human papillomavirus detection in cervical biopsies: A study of sensitivity.

    Science.gov (United States)

    Zehbe, I; Wilander, E

    1996-08-01

    Polymerase chain reaction (PCR) is being increasingly used in clinical laboratories for the diagnosis of human papillomavirus. From the L1 region, there are two commonly used consensus primer systems designated CP5+/G6+ and MY09/MY11. Both detect a wide variety of human papillomaviruses (HPVs). In this investigation, the authors compared the sensitivity of these approaches with the modification of hot-start PCR on 148 neutral-buffered formaldehyde-fixed cervical biopsies classified as cervical intraepithelial neoplasia (CIN) I to III. The authors chose hot-start PCR because in a previous study it proved more sensitive than cold-start PCR. Furthermore, the authors combined GP5+/GP6+ and MY09/MY11 in a two-step amplification (nested PCR) to analyze further those cases that proved negative with either GP5+/GP6+ or MY09/MY11. The authors found that the two consensus primer systems were equally sensitive with a correlation of 98%. By using GP5+/GP6+, the authors achieved an HPV positivity rate of 95% and with MY09/MY11 94%. Nested PCR did not improve HPV positivity in the CINs included in this study.

  9. FENOFIBRATE ADMINISTRATION DOES NOT AFFECT MUSCLE TRIGLYCERIDE CONCENTRATION OR INSULIN SENSITIVITY IN HUMANS

    Science.gov (United States)

    Perreault, Leigh; Bergman, Bryan C.; Hunerdosse, Devon M.; Howard, David J.; Eckel, Robert H.

    2010-01-01

    Objective Animal data suggest that males, in particular, rely on PPAR-α activity to maintain normal muscle triglyceride metabolism. We sought to examine whether this was also true in men vs. women and its relationship to insulin sensitivity. Materials/Methods Normolipidemic obese men (n=9) and women (n=9) underwent an assessment of insulin sensitivity (IVGTT) and intramuscular triglyceride metabolism (GC/MS and GC/C/IRMS from plasma and muscle biopsies taken after infusion of [U-13C]palmitate) before and after 12 weeks of fenofibrate treatment. Results Women were more insulin sensitive (Si; 5.2(0.7 vs. 2.4(0.4 ×10−4/uU/ml, W vs. M, ptriglyceride (IMTG) concentration (41.9(15.5 vs. 30.8(5.1 ug/mg dry weight, W vs. M, p=0.43), and IMTG fractional synthesis rate (FSR; 0.27(0.07 vs. 0.35(0.06/hr, W vs. M, p=0.41) as men. Fenofibrate enhanced FSR in men (0.35(0.06 to 0.54(0.06, p=0.05), with no such change seen in women (0.27(0.07 to 0.32(0.13, p=0.73), and no change in IMTG concentration in either group (23.0(3.9 in M, p=0.26 vs. baseline; 36.3(12.0 in W, p=0.79 vs. baseline). Insulin sensitivity was unaffected by fenofibrate (p>0.68). Lower percent saturation of IMTG in women vs. men before (29.1(2.3 vs. 35.2(1.7%, p=0.06) and after (27.3(2.8 vs. 35.1(1.9%, p=0.04) fenofibrate most closely related to their greater insulin sensitivity (R2=0.34, p=0.10), and was largely unchanged by the drug. Conclusions PPAR-α agonist therapy had little effect on IMTG metabolism in men or women. IMTG saturation, rather than IMTG concentration or FSR, most closely (but not significantly) related to insulin sensitivity and was unchanged by fenofibrate administration. PMID:21306746

  10. Demethylation restores SN38 sensitivity in cells with acquired resistance to SN38 derived from human cervical squamous cancer cells

    Science.gov (United States)

    TANAKA, TETSUJI; BAI, TAO; TOUJIMA, SAORI; UTSUNOMIYA, TOMOKO; MATSUOKA, TOSHIHIDE; KOBAYASHI, AYA; YAMAMOTO, MADOKA; SASAKI, NORIYUKI; TANIZAKI, YUKO; UTSUNOMIYA, HIROTOSHI; TANAKA, JUNKO; YUKAWA, KAZUNORI

    2012-01-01

    Using seven monoclonal SN38-resistant subclones established from ME180 human cervical squamous cell carcinoma cells, we examined the demethylation effects of 5-aza-2′-deoxycytidine (5-aza-CdR) on the SN38-sensitivity of the cells as well as the expression of death-associated protein kinase (DAPK) in the SN38-resistant cells. The DAPK expression levels were evaluated among parent ME180 cells, SN38-resistant ME180 cells and cisplatin-resistant ME180 cells by methylation-specific DAPK-PCR, quantitative RT-PCR and western blot analysis. The SN38-resistant cells co-treated with SN38 and 5-aza-CdR strongly exhibited enhanced SN38-sensitivities resembling those found in the parent cells. In the SN38-resistant subclones, no relationships were found between the restored SN38 sensitivity and hypermethylation of the DAPK promoter, DAPK mRNA expression, DAPK protein expression and induction of DAPK protein after 5-aza-CdR treatment, unlike the strong suppression of 5-aza-CdR-induced DAPK protein expression in the cisplatin-resistant subclones. These findings indicate that reversibly methylated molecules, but not DAPK, may regulate SN38 resistance, and that demethylating agents can be strong sensitizing anticancer chemotherapeutic drugs for SN38-resistant cancers. PMID:22246465

  11. Traditional Chinese Medicine Curcumin Sensitizes Human Colon Cancer to Radiation by Altering the Expression of DNA Repair-related Genes.

    Science.gov (United States)

    Yang, Guangen; Qiu, Jianming; Wang, Dong; Tao, Yong; Song, Yihuan; Wang, Hongtao; Tang, Juping; Wang, Xing; Sun, Y U; Yang, Zhijian; Hoffman, Robert M

    2018-01-01

    The aim of the present study was to investigate the radio-sensitizing efficacy of curcumin, a traditional Chinese medicine (TCM) on colon cancer cells in vitro and in vivo. Human colon cancer HT-29 cells were treated with curcumin (2.5 μM), irradiation (10 Gy) and the combination of irradiation and curcumin. Cell proliferation was assessed using the MTT assay. Apoptotic cells were detected by Annexin V-PE/7-AAD analysis. PCR was performed to determine differential-expression profiling of 95 DNA-repair genes in irradiated cells and cells treated with both irradiation and curcumin. Differentially-expressed genes were confirmed by Western blotting. In vivo radio-sensitizing efficacy of curcumin was assessed in a xenograft mouse model of HT-29 colon cancer. Curcumin was administrated daily by intraperitoneal injection at 20 mg/kg/dose. Mice received irradiation (10 Gy) twice weekly. Apoptosis of the cancer cells following treatment was determined by TUNEL staining. Irradiation induced proliferation inhibition and apoptosis of HT-29 cells in vitro. Concurrent curcumin treatment sensitized the HT-29 tumor to irradiation (phuman colon cancer in vitro and in vivo to radiation. Downregulation of LIG4 and PNKP and upregulation of XRCC5 and CCNH DNA-repair-related genes were involved in the radio-sensitizing efficacy of curcumin in colon cancer. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  12. Exercise increases human skeletal muscle insulin sensitivity via coordinated increases in microvascular perfusion and molecular signaling

    DEFF Research Database (Denmark)

    Sjøberg, Kim Anker; Frøsig, Christian; Kjøbsted, Rasmus

    2017-01-01

    and increased similarly in both legs during the clamp and L-NMMA had no effect on these insulin-stimulated signaling pathways. Therefore, acute exercise increases insulin sensitivity of muscle by a coordinated increase in insulin-stimulated microvascular perfusion and molecular signaling at the level of TBC1D4...... and glycogen synthase in muscle. This secures improved glucose delivery on the one hand and increased ability to take up and dispose of the delivered glucose on the other hand....

  13. High sensitivity LC-MS/MS method for direct quantification of human parathyroid 1-34 (teriparatide) in human plasma.

    Science.gov (United States)

    Chambers, Erin E; Lame, Mary E; Bardsley, Jon; Hannam, Sally; Legido-Quigley, Cristina; Smith, Norman; Fountain, Kenneth J; Collins, Eileen; Thomas, Elizabeth

    2013-11-01

    Teriparatide, the 1-34 fragment of human parathyroid hormone, is used to treat osteoporosis patients with a high risk of fracture by stimulating new bone formation. Routinely teriparatide is quantified using radioimmunoassay however the LC-MS/MS described here has the potential to achieve greater accuracy and precision, higher specificity, and is readily implemented in routine bioanalytical laboratories. Hence a complete method combining effective sample prep with appropriate LC separation and selected reaction monitoring (SRM) MS detection was developed to selectively separate teriparatide from closely related endogenous peptides and to reduce interferences. Samples were concentrated without evaporation, minimizing the risk of adsorptive losses. Chromatography was performed on a sub 2μm particle charged surface hybrid column, which provided significantly higher peak capacity than a traditional C18 column when formic acid was used as the mobile phase modifier. Total LC cycle time was 6min. An LOD of 15pg/mL (3.6fmol/mL) from 200μL of human plasma was readily achieved and standard curves were accurate and precise from 15pg/mL to 500pg/mL. Mean QC accuracies ranged from 90% to 106%. Mean QC precision was better than 7%. The CV of matrix factors across 6 sources of human plasma was 5%. The assay presented here is the first LC-MS method which reaches clinically relevant detection limits for teriparatide. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Headache and mechanical sensitization of human pericranial muscles after repeated intake of monosodium glutamate (MSG).

    Science.gov (United States)

    Shimada, Akiko; Cairns, Brian E; Vad, Nynne; Ulriksen, Kathrine; Pedersen, Anne Marie Lynge; Svensson, Peter; Baad-Hansen, Lene

    2013-01-24

    A single intake of monosodium glutamate (MSG) may cause headache and increased muscle sensitivity. We conducted a double-blinded, placebo-controlled, crossover study to examine the effect of repeated MSG intake on spontaneous pain, mechanical sensitivity of masticatory muscles, side effects, and blood pressure. Fourteen healthy subjects participated in 5 daily sessions for one week of MSG intake (150 mg/kg) or placebo (24 mg/kg NaCl) (randomized, double-blinded). Spontaneous pain, pressure pain thresholds and tolerance levels for the masseter and temporalis muscles, side effects, and blood pressure were evaluated before and 15, 30, and 50 min after MSG intake. Whole saliva samples were taken before and 30 min after MSG intake to assess glutamate concentrations. Headache occurred in 8/14 subjects during MSG and 2/14 during placebo (P = 0.041). Salivary glutamate concentrations on Day 5 were elevated significantly (P < 0.05). Pressure pain thresholds in masseter muscle were reduced by MSG on Day 2 and 5 (P < 0.05). Blood pressure was significantly elevated after MSG (P < 0.040). In conclusion, MSG induced mechanical sensitization in masseter muscle and adverse effects such as headache and short-lasting blood pressure elevation for which tolerance did not develop over 5 days of MSG intake.

  15. Human adipocytes are highly sensitive to intermittent hypoxia induced NF-kappaB activity and subsequent inflammatory gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Taylor, Cormac T. [School of Medicine and Medical Science, The Conway Institute, University College Dublin (Ireland); Kent, Brian D.; Crinion, Sophie J.; McNicholas, Walter T. [School of Medicine and Medical Science, The Conway Institute, University College Dublin (Ireland); Pulmonary and Sleep Disorders Unit, St. Vincent’s University Hospital, Dublin (Ireland); Ryan, Silke, E-mail: silke.ryan@ucd.ie [School of Medicine and Medical Science, The Conway Institute, University College Dublin (Ireland); Pulmonary and Sleep Disorders Unit, St. Vincent’s University Hospital, Dublin (Ireland)

    2014-05-16

    Highlights: • Intermittent hypoxia (IH) leads to NF-κB activation in human primary adipocytes. • Adipocytes bear higher pro-inflammatory potential than other human primary cells. • IH leads to upregulation of multiple pro-inflammatory genes in human adipocytes. - Abstract: Introduction: Intermittent hypoxia (IH)-induced activation of pro-inflammatory pathways is a major contributing factor to the cardiovascular pathophysiology associated with obstructive sleep apnea (OSA). Obesity is commonly associated with OSA although it remains unknown whether adipose tissue is a major source of inflammatory mediators in response to IH. The aim of this study was to test the hypothesis that IH leads to augmented inflammatory responses in human adipocytes when compared to cells of non-adipocyte lineages. Methods and results: Human primary subcutaneous and visceral adipocytes, human primary microvascular pulmonary endothelial cells (HUMEC-L) and human primary small airway epithelial cells (SAEC) were exposed to 0, 6 or 12 cycles of IH or stimulated with tumor necrosis factor (TNF)-α. IH led to a robust increase in NF-κB DNA-binding activity in adipocytes compared with normoxic controls regardless of whether the source of adipocytes was visceral or subcutaneous. Notably, the NF-κB response of adipocytes to both IH and TNF-α was significantly greater than that in HUMEC-L and SAEC. Western blotting confirmed enhanced nuclear translocation of p65 in adipocytes in response to IH, accompanied by phosphorylation of I-κB. Parallel to p65 activation, we observed a significant increase in secretion of the adipokines interleukin (IL)-8, IL-6 and TNF-α with IH in adipocytes accompanied by significant upregulation of mRNA expression. PCR-array suggested profound influence of IH on pro-inflammatory gene expression in adipocytes. Conclusion: Human adipocytes demonstrate strong sensitivity to inflammatory gene expression in response to acute IH and hence, adipose tissue may be a key

  16. Human adipocytes are highly sensitive to intermittent hypoxia induced NF-kappaB activity and subsequent inflammatory gene expression

    International Nuclear Information System (INIS)

    Taylor, Cormac T.; Kent, Brian D.; Crinion, Sophie J.; McNicholas, Walter T.; Ryan, Silke

    2014-01-01

    Highlights: • Intermittent hypoxia (IH) leads to NF-κB activation in human primary adipocytes. • Adipocytes bear higher pro-inflammatory potential than other human primary cells. • IH leads to upregulation of multiple pro-inflammatory genes in human adipocytes. - Abstract: Introduction: Intermittent hypoxia (IH)-induced activation of pro-inflammatory pathways is a major contributing factor to the cardiovascular pathophysiology associated with obstructive sleep apnea (OSA). Obesity is commonly associated with OSA although it remains unknown whether adipose tissue is a major source of inflammatory mediators in response to IH. The aim of this study was to test the hypothesis that IH leads to augmented inflammatory responses in human adipocytes when compared to cells of non-adipocyte lineages. Methods and results: Human primary subcutaneous and visceral adipocytes, human primary microvascular pulmonary endothelial cells (HUMEC-L) and human primary small airway epithelial cells (SAEC) were exposed to 0, 6 or 12 cycles of IH or stimulated with tumor necrosis factor (TNF)-α. IH led to a robust increase in NF-κB DNA-binding activity in adipocytes compared with normoxic controls regardless of whether the source of adipocytes was visceral or subcutaneous. Notably, the NF-κB response of adipocytes to both IH and TNF-α was significantly greater than that in HUMEC-L and SAEC. Western blotting confirmed enhanced nuclear translocation of p65 in adipocytes in response to IH, accompanied by phosphorylation of I-κB. Parallel to p65 activation, we observed a significant increase in secretion of the adipokines interleukin (IL)-8, IL-6 and TNF-α with IH in adipocytes accompanied by significant upregulation of mRNA expression. PCR-array suggested profound influence of IH on pro-inflammatory gene expression in adipocytes. Conclusion: Human adipocytes demonstrate strong sensitivity to inflammatory gene expression in response to acute IH and hence, adipose tissue may be a key

  17. Histone Deacetylase Inhibitor Induced Radiation Sensitization Effects on Human Cancer Cells after Photon and Hadron Radiation Exposure

    Directory of Open Access Journals (Sweden)

    Ariungerel Gerelchuluun

    2018-02-01

    Full Text Available Suberoylanilide hydroxamic acid (SAHA is a histone deacetylase inhibitor, which has been widely utilized throughout the cancer research field. SAHA-induced radiosensitization in normal human fibroblasts AG1522 and lung carcinoma cells A549 were evaluated with a combination of γ-rays, proton, and carbon ion exposure. Growth delay was observed in both cell lines during SAHA treatment; 2 μM SAHA treatment decreased clonogenicity and induced cell cycle block in G1 phase but 0.2 μM SAHA treatment did not show either of them. Low LET (Linear Energy Transfer irradiated A549 cells showed radiosensitization effects on cell killing in cycling and G1 phase with 0.2 or 2 μM SAHA pretreatment. In contrast, minimal sensitization was observed in normal human cells after low and high LET radiation exposure. The potentially lethal damage repair was not affected by SAHA treatment. SAHA treatment reduced the rate of γ-H2AX foci disappearance and suppressed RAD51 and RPA (Replication Protein A focus formation. Suppression of DNA double strand break repair by SAHA did not result in the differences of SAHA-induced radiosensitization between human cancer cells and normal cells. In conclusion, our results suggest SAHA treatment will sensitize cancer cells to low and high LET radiation with minimum effects to normal cells.

  18. Hyper-radiation sensitivity of murine scid mutation and mapping of the human homologue HYRC1 gene

    International Nuclear Information System (INIS)

    Komatsu, Kenshi; Ohta, Tohru; Niikawa, Norio; Okumura, Yutaka; Kubota, Nobuo.

    1994-01-01

    The murine severe combined immunodeficient mutation (scid) is characterized by a lack of both B and T cells, due to a defect in lymphoid variable-(diversity)-joining(V(D)J) rearrangement. Scid cells are highly sensitive to both radiation-induced killing and chromosomal aberrations. Present experiments also demonstrated the high sensitivity of scid cells to killing, because of a deficient repair of double strand breaks(DSB). Scid cells can repair only 60% of radiation-induced DSB for 3 hours, while normal cells repair 85% of the DSB. Significantly reduced Do and n values were obtained from survival curves of scid cells and were similar to ataxia-telangiectasia(AT) cells (a unique human disease conferring whole body radiosensitivity). However, the kinetics of DNA synthesis after irradiation were different between the two cell types. In contrast with the radioresistant DNA synthesis of AT cells, DNA synthesis of scid cells was markedly inhibited after irradiation. The existence of different mutations was also supported by evidence of complementation in somatic cell hybrids between scid cells and AT cells. Using these hybrid cells, fragments of human chromosome 8 were introduced into scid cells HPRT mutant via X-irradiation and somatic cell fusion. The resulting hybrid clones contained human DNA fragment(s) which complemented the hyper-radiosensitivity of the scid cells. Alu-PCR products from these hybrids were used for chromosome painting using the technique of chromosome in situ suppression hybridization, allowing assignment of the human HYRC1 (hyper-radiosensitivity of murine scid mutation, complementing 1) gene, a candidate for a V(D)J recombinant gene, to human chromosome 8q11. (author)

  19. Suppression of NRF2–ARE activity sensitizes chemotherapeutic agent-induced cytotoxicity in human acute monocytic leukemia cells

    International Nuclear Information System (INIS)

    Peng, Hui; Wang, Huihui; Xue, Peng; Hou, Yongyong; Dong, Jian; Zhou, Tong; Qu, Weidong; Peng, Shuangqing; Li, Jin; Carmichael, Paul L.; Nelson, Bud; Clewell, Rebecca; Zhang, Qiang; Andersen, Melvin E.; Pi, Jingbo

    2016-01-01

    Nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of the antioxidant response element (ARE)-dependent transcription, plays a pivotal role in chemical detoxification in normal and tumor cells. Consistent with previous findings that NRF2–ARE contributes to chemotherapeutic resistance of cancer cells, we found that stable knockdown of NRF2 by lentiviral shRNA in human acute monocytic leukemia (AML) THP-1 cells enhanced the cytotoxicity of several chemotherapeutic agents, including arsenic trioxide (As 2 O 3 ), etoposide and doxorubicin. Using an ARE-luciferase reporter expressed in several human and mouse cells, we identified a set of compounds, including isonicotinic acid amides, isoniazid and ethionamide, that inhibited NRF2–ARE activity. Treatment of THP-1 cells with ethionamide, for instance, significantly reduced mRNA expression of multiple ARE-driven genes under either basal or As 2 O 3 -challenged conditions. As determined by cell viability and cell cycle, suppression of NRF2–ARE by ethionamide also significantly enhanced susceptibility of THP-1 and U937 cells to As 2 O 3 -induced cytotoxicity. In THP-1 cells, the sensitizing effect of ethionamide on As 2 O 3 -induced cytotoxicity was highly dependent on NRF2. To our knowledge, the present study is the first to demonstrate that ethionamide suppresses NRF2–ARE signaling and disrupts the transcriptional network of the antioxidant response in AML cells, leading to sensitization to chemotherapeutic agents. - Highlights: • Identification of novel inhibitors of ARE-dependent transcription • Suppression of NRF2–ARE sensitizes THP-1 cells to chemotherapy. • Ethionamide suppresses ARE-dependent transcriptional activity. • Ethionamide and isoniazid increase the cytotoxicity of As 2 O 3 in AML cells. • Sensitization of THP-1 cells to As 2 O 3 toxicity by ethionamide is NRF2-dependent.

  20. Characterization of lipid metabolism in insulin-sensitive adipocytes differentiated from immortalized human mesenchymal stem cells

    DEFF Research Database (Denmark)

    Prawitt, Janne; Niemeier, Andreas; Kassem, Moustapha

    2008-01-01

    There is a great demand for cell models to study human adipocyte function. Here we describe the adipogenic differentiation of a telomerase-immortalized human mesenchymal stem cell line (hMSC-Tert) that maintains numerous features of terminally differentiated adipocytes even after prolonged...... receptor (VLDLR, LRP1) and apolipoprotein E expression during differentiation. Despite this increased expression, the receptor-mediated endocytosis of lipoproteins was decreased in differentiated adipocytes, suggesting that these proteins may have an additional function in adipose tissue beyond lipoprotein...

  1. Polarization sensitive changes in the human macula associated with normal aging and age-related macular degeneration

    Science.gov (United States)

    VanNasdale, Dean Allan, Jr.

    2011-12-01

    The human macula occupies a relatively small, but crucial retinal area, as it is the location responsible for our most acute spatial vision and best color discrimination. Localizing important landmarks in the retina is difficult even in normal eyes where morphological inter-individual variability is high. This becomes even more challenging in the presence of sight-threatening pathology. With respect to the human macula, there remains a significant gap in the understanding of normal structure and function. Even less is known about the pathological mechanisms that occur in sight-threatening diseases including age-related macular degeneration. Because relatively little is known about normal aging changes, it is also difficult to differentiate those changes from changes associated with retinal disease. To better understand normal and pathological changes in the macula, imaging techniques using specific optical signatures are required. Structural features in the macula can be distinguished based on their intrinsic properties using specific light/tissue interactions. Because of the high degree of structural regularity in the macula, polarization sensitive imaging is potentially a useful tool for evaluating the morphology and integrity of the cellular architecture for both normal individuals and those affected by disease. In our investigations, we used polarization sensitive imaging to determining normal landmarks that are important clinically and for research investigations. We found that precision and accuracy in localizing the central macula was greatly improved through the use of polarization sensitive imaging. We also found that specific polarization alterations can be used to demonstrate systematic changes as a function of age, disproportionately affecting the central macular region. When evaluating patients with age-related macular degeneration, we found that precision and accuracy of localizing the central macula was also improved, even when significant pathology

  2. Integrating human and environmental health in antibiotic risk assessment: A critical analysis of protection goals, species sensitivity and antimicrobial resistance.

    Science.gov (United States)

    Le Page, Gareth; Gunnarsson, Lina; Snape, Jason; Tyler, Charles R

    2017-12-01

    Antibiotics are vital in the treatment of bacterial infectious diseases but when released into the environment they may impact non-target organisms that perform vital ecosystem services and enhance antimicrobial resistance development with significant consequences for human health. We evaluate whether the current environmental risk assessment regulatory guidance is protective of antibiotic impacts on the environment, protective of antimicrobial resistance, and propose science-based protection goals for antibiotic manufacturing discharges. A review and meta-analysis was conducted of aquatic ecotoxicity data for antibiotics and for minimum selective concentration data derived from clinically relevant bacteria. Relative species sensitivity was investigated applying general linear models, and predicted no effect concentrations were generated for toxicity to aquatic organisms and compared with predicted no effect concentrations for resistance development. Prokaryotes were most sensitive to antibiotics but the range of sensitivities spanned up to several orders of magnitude. We show reliance on one species of (cyano)bacteria and the 'activated sludge respiration inhibition test' is not sufficient to set protection levels for the environment. Individually, neither traditional aquatic predicted no effect concentrations nor predicted no effect concentrations suggested to safeguard for antimicrobial resistance, protect against environmental or human health effects (via antimicrobial resistance development). Including data from clinically relevant bacteria and also more species of environmentally relevant bacteria in the regulatory framework would help in defining safe discharge concentrations for antibiotics for patient use and manufacturing that would protect environmental and human health. It would also support ending unnecessary testing on metazoan species. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  3. Antibody-Mediated Rejection in Sensitized Nonhuman Primates: Modeling Human Biology.

    Science.gov (United States)

    Burghuber, C K; Kwun, J; Page, E J; Manook, M; Gibby, A C; Leopardi, F V; Song, M; Farris, A B; Hong, J J; Villinger, F; Adams, A B; Iwakoshi, N N; Knechtle, S J

    2016-06-01

    We have established a model of sensitization in nonhuman primates and tested two immunosuppressive regimens. Animals underwent fully mismatched skin transplantation, and donor-specific antibody (DSA) response was monitored by flow cross-match. Sensitized animals subsequently underwent kidney transplantation from their skin donor. Immunosuppression included tacrolimus, mycophenolate, and methylprednisolone. Three animals received basiliximab induction; compared with nonsensitized animals, they showed a shorter mean survival time (4.7 ± 3.1 vs. 187 ± 88 days). Six animals were treated with T cell depletion (anti-CD4/CD8 mAbs), which prolonged survival (mean survival time 21.6 ± 19.0 days). All presensitized animals showed antibody-mediated rejection (AMR). In two of three basiliximab-injected animals, cellular rejection (ACR) was prominent. After T cell depletion, three of six monkeys experienced early acute rejection within 8 days with histological evidence of thrombotic microangiopathy and AMR. The remaining three monkeys survived 27-44 days, with mixed AMR and ACR. Most T cell-depleted animals experienced a rebound of DSA that correlated with deteriorating kidney function. We also found an increase in proliferating memory B cells (CD20(+) CD27(+) IgD(-) Ki67(+) ), lymph node follicular helper T cells (ICOS(+) PD-1(hi) CXCR5(+) CD4(+) ), and germinal center (GC) response. Depletion controlled cell-mediated rejection in sensitized nonhuman primates better than basiliximab, yet grafts were rejected with concomitant DSA rise. This model provides an opportunity to test novel desensitization strategies. © Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.

  4. mTOR inhibition sensitizes human hepatocellular carcinoma cells to resminostat

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Xingang, E-mail: pengxinggang26@sina.com [Department of Emergency General Surgery, The Affiliated Hospital of Qingdao University, Qingdao (China); Zhang, Donghui, E-mail: zhangdonghuiyx@sina.com [Department of Infectious Disease, Linyi People’s Hospital, Linyi (China); Li, Zhengling, E-mail: lizhenglingzz@sina.com [Department of Nursing, Tengzhou Central People’s Hospital, Tengzhou (China); Fu, Meili, E-mail: fumeilidrlinyi@tom.com [Department of Infectious Disease, Linyi People’s Hospital, Linyi (China); Liu, Haiyan, E-mail: liuhaiyanlinyi5@sina.com [Department of Nursing, Linyi People’s Hospital, Linyi (China)

    2016-09-02

    Histone deacetylases (HDACs) hyper-activity in hepatocellular carcinoma (HCC) is often associated with patients’ poor prognosis. Our previous study has shown that resminostat, a novel HDAC inhibitor (HDACi), activated mitochondrial permeability transition pore (mPTP)-dependent apoptosis pathway in HCC cells. Here we explored the potential resminostat resistance factor by focusing on mammalian target of rapamycin (mTOR). We showed that AZD-2014, a novel mTOR kinase inhibitor, potentiated resminostat-induced cytotoxicity and proliferation inhibition in HCC cells. Molecularly, AZD-2014 enhanced resminostat-induced mPTP apoptosis pathway activation in HCC cells. Inhibition of this apoptosis pathway, by the caspase-9 specific inhibitor Ac-LEHD-CHO, the mPTP blockers (sanglifehrin A/cyclosporine A), or by shRNA-mediated knockdown of mPTP component cyclophilin-D (Cyp-D), significantly attenuated resminostat plus AZD-2014-induced cytotoxicity and apoptosis in HCC cells. Significantly, mTOR shRNA knockdown or kinase-dead mutation (Asp-2338-Ala) also sensitized HCC cells to resminostat, causing profound cytotoxicity and apoptosis induction. Together, these results suggest that mTOR could be a primary resistance factor of resminostat. Targeted inhibition of mTOR may thus significantly sensitize HCC cells to resminostat. - Highlights: • AZD-2014 potentiates resminostat’s cytotoxicity against HCC cells. • AZD-2014 facilitates resminostat-induced HCC cell apoptosis. • AZD-2014 augments resminostat-induced mitochondrial apoptosis pathway activation. • mTOR shRNA or kinase-dead mutation significantly sensitizes HCC cells to resminostat.

  5. In Situ Capture RT-qPCR: A New Simple and Sensitive Method to Detect Human Norovirus in Oysters

    OpenAIRE

    Zhou, Zhenhuan; Tian, Zhengan; Li, Qianqian; Tian, Peng; Wu, Qingping; Wang, Dapeng; Shi, Xianming

    2017-01-01

    Human noroviruses (HuNoVs) are the major cause worldwide for non-bacterial acute gastroenteritis. In this study, we applied a novel viral receptor mediated in situ capture RT-qPCR (ISC-RT-qPCR) to detect HuNoVs in oysters and compared with the traditional RT-qPCR method. Ten HuNoVs RT-PCR positive and 5 negative clinical samples from gastroenteritis patients were used to compare specificity and sensitivity of ISC-RT-qPCR against that of the RT-qPCR assay. ISC-RT-qPCR had at a one-log and a tw...

  6. Imaging of human breast tissue using polarization sensitive optical coherence tomography

    Science.gov (United States)

    Verma, Y.; Gautam, M.; Divakar Rao, K.; Swami, M. K.; Gupta, P. K.

    2011-12-01

    We report a study on the use of polarization sensitive optical coherence tomography (PSOCT) for discriminating malignant (invasive ductal carcinoma), benign (fibroadenoma) and normal (adipocytes) breast tissue sites. The results show that while conventional OCT, that utilizes only the intensity of light back-scattered from tissue microstructures, is able to discriminate breast tissues as normal (adipocytes) and abnormal (malignant and benign) tissues, PS-OCT helps in discriminating between malignant and benign tissue sites also. The estimated values of birefringence obtained from the PSOCT imaging show that benign breast tissue samples have significantly higher birefringence as compared to the malignant tissue samples.

  7. Human skeletal muscle ceramide content is not a major factor in muscle insulin sensitivity

    DEFF Research Database (Denmark)

    Skovbro, M; Baranowski, M; Skov-Jensen, C

    2008-01-01

    : The middle-aged male participants (n=33) were matched for lean body mass and divided into four groups: type 2 diabetes (T2D, n=8), impaired glucose tolerance (IGT, n=9), healthy controls (CON, n=8) and endurance-trained (TR, n=8). A two step (28 and 80 mU m(-2) min(-1)) sequential euglycaemic......AIMS/HYPOTHESIS: In skeletal muscle, ceramides may be involved in the pathogenesis of insulin resistance through an attenuation of insulin signalling. This study investigated total skeletal muscle ceramide fatty acid content in participants exhibiting a wide range of insulin sensitivities. METHODS...

  8. Brown adipose tissue improves whole-body glucose homeostasis and insulin sensitivity in humans

    Science.gov (United States)

    Brown adipose tissue (BAT) has attracted scientific interest as an antidiabetic tissue owing to its ability to dissipate energy as heat. Despite a plethora of data concerning the role of BAT in glucose metabolism in rodents, the role of BAT (if any) in glucose metabolism in humans remains unclear. T...

  9. Accuracy of Diagnostic Methods and Surveillance Sensitivity for Human Enterovirus, South Korea, 1999–2011

    Science.gov (United States)

    Hyeon, Ji-Yeon; Hwang, Seoyeon; Kim, Hyejin; Song, Jaehyoung; Ahn, Jeongbae; Kang, Byunghak; Kim, Kisoon; Choi, Wooyoung; Chung, Jae Keun; Kim, Cheon-Hyun; Cho, Kyungsoon; Jee, Youngmee; Kim, Jonghyun; Kim, Kisang; Kim, Sun-Hee; Kim, Min-Ji

    2013-01-01

    The epidemiology of enteroviral infection in South Korea during 1999–2011 chronicles nationwide outbreaks and changing detection and subtyping methods used over the 13-year period. Of 14,657 patients whose samples were tested, 4,762 (32.5%) samples were positive for human enterovirus (human EV); as diagnostic methods improved, the rate of positive results increased. A seasonal trend of outbreaks was documented. Genotypes enterovirus 71, echovirus 30, coxsackievirus B5, enterovirus 6, and coxsackievirus B2 were the most common genotypes identified. Accurate test results correlated clinical syndromes to enterovirus genotypes: aseptic meningitis to echovirus 30, enterovirus 6, and coxsackievirus B5; hand, foot and mouth disease to coxsackievirus A16; and hand, foot and mouth disease with neurologic complications to enterovirus 71. There are currently no treatments specific to human EV infections; surveillance of enterovirus infections such as this study provides may assist with evaluating the need to research and develop treatments for infections caused by virulent human EV genotypes. PMID:23876671

  10. Human-Related Factors Regulate the Spatial Ecology of Domestic Cats in Sensitive Areas for Conservation

    Science.gov (United States)

    Ferreira, Joaquim P.; Leitão, Inês; Santos-Reis, Margarida; Revilla, Eloy

    2011-01-01

    Background Domestic cats ranging freely in natural areas are a conservation concern due to competition, predation, disease transmission or hybridization with wildcats. In order to improve our ability to design effective control policies, we investigate the factors affecting their numbers and space use in natural areas of continental Europe. Methodology/Principal Findings We describe the patterns of cat presence, abundance and space use and analyse the associated environmental and human constraints in a well-preserved Mediterranean natural area with small scattered local farms. We failed in detecting cats in areas away from human settlements (trapping effort above 4000 trap-nights), while we captured 30 individuals near inhabited farms. We identified 130 cats, all of them in farms still in use by people (30% of 128 farms). All cats were free-ranging and very wary of people. The main factor explaining the presence of cats was the presence of people, while the number of cats per farm was mostly affected by the occasional food provisioning with human refuse and the presence of people. The home ranges of eight radio tagged cats were centred at inhabited farms. Males went furthest away from the farms during the mating season (3.8 km on average, maximum 6.3 km), using inhabited farms as stepping-stones in their mating displacements (2.2 km of maximum inter-farm distance moved). In their daily movements, cats notably avoided entering in areas with high fox density. Conclusions The presence, abundance and space use of cats were heavily dependent on human settlements. Any strategy aiming at reducing their impact in areas of conservation concern should aim at the presence of settlements and their spatial spread and avoid any access to human refuse. The movements of domestic cats would be limited in areas with large patches of natural vegetation providing good conditions for other carnivore mammals such as red foxes. PMID:22043298

  11. Human-related factors regulate the spatial ecology of domestic cats in sensitive areas for conservation.

    Science.gov (United States)

    Ferreira, Joaquim P; Leitão, Inês; Santos-Reis, Margarida; Revilla, Eloy

    2011-01-01

    Domestic cats ranging freely in natural areas are a conservation concern due to competition, predation, disease transmission or hybridization with wildcats. In order to improve our ability to design effective control policies, we investigate the factors affecting their numbers and space use in natural areas of continental Europe. We describe the patterns of cat presence, abundance and space use and analyse the associated environmental and human constraints in a well-preserved Mediterranean natural area with small scattered local farms. We failed in detecting cats in areas away from human settlements (trapping effort above 4000 trap-nights), while we captured 30 individuals near inhabited farms. We identified 130 cats, all of them in farms still in use by people (30% of 128 farms). All cats were free-ranging and very wary of people. The main factor explaining the presence of cats was the presence of people, while the number of cats per farm was mostly affected by the occasional food provisioning with human refuse and the presence of people. The home ranges of eight radio tagged cats were centred at inhabited farms. Males went furthest away from the farms during the mating season (3.8 km on average, maximum 6.3 km), using inhabited farms as stepping-stones in their mating displacements (2.2 km of maximum inter-farm distance moved). In their daily movements, cats notably avoided entering in areas with high fox density. The presence, abundance and space use of cats were heavily dependent on human settlements. Any strategy aiming at reducing their impact in areas of conservation concern should aim at the presence of settlements and their spatial spread and avoid any access to human refuse. The movements of domestic cats would be limited in areas with large patches of natural vegetation providing good conditions for other carnivore mammals such as red foxes.

  12. Human-related factors regulate the spatial ecology of domestic cats in sensitive areas for conservation.

    Directory of Open Access Journals (Sweden)

    Joaquim P Ferreira

    Full Text Available BACKGROUND: Domestic cats ranging freely in natural areas are a conservation concern due to competition, predation, disease transmission or hybridization with wildcats. In order to improve our ability to design effective control policies, we investigate the factors affecting their numbers and space use in natural areas of continental Europe. METHODOLOGY/PRINCIPAL FINDINGS: We describe the patterns of cat presence, abundance and space use and analyse the associated environmental and human constraints in a well-preserved Mediterranean natural area with small scattered local farms. We failed in detecting cats in areas away from human settlements (trapping effort above 4000 trap-nights, while we captured 30 individuals near inhabited farms. We identified 130 cats, all of them in farms still in use by people (30% of 128 farms. All cats were free-ranging and very wary of people. The main factor explaining the presence of cats was the presence of people, while the number of cats per farm was mostly affected by the occasional food provisioning with human refuse and the presence of people. The home ranges of eight radio tagged cats were centred at inhabited farms. Males went furthest away from the farms during the mating season (3.8 km on average, maximum 6.3 km, using inhabited farms as stepping-stones in their mating displacements (2.2 km of maximum inter-farm distance moved. In their daily movements, cats notably avoided entering in areas with high fox density. CONCLUSIONS: The presence, abundance and space use of cats were heavily dependent on human settlements. Any strategy aiming at reducing their impact in areas of conservation concern should aim at the presence of settlements and their spatial spread and avoid any access to human refuse. The movements of domestic cats would be limited in areas with large patches of natural vegetation providing good conditions for other carnivore mammals such as red foxes.

  13. Effect of recombinant adenovirus encoding human p53 tumor suppressor gene (rAd-p53) on the growth and radiotherapeutic sensitivity of human lymphoma cell lines

    International Nuclear Information System (INIS)

    Yu Zeyang; Fan Wo; Li Dongqing; Zhu Ran; Wang Yongqing; Wu Jinchang

    2008-01-01

    Objective: To explore the inhibitory effect and radiation sensitization of recombinant adenovirus encoding human p53 tumor suppressor gene (rAd-p53) on human lymphoma cell lines. Methods: Human lymphoma cell lines Raji and Daudi were treated with rAd-p53, radiation therapy and combined treatment, respectively. The cell growth inhibition was assessed by MTT. The p53 protein expression was detected by Western blotting, and p53 mRNA was detected by BT-PCB. Results: The MTT results showed that the inhibitory effect and radiosensitivity enhancement of rAd-p53 on human lymphoma cell lines were not obvious [Raji: (27.5±4.1)%; Daudi: (28.1±1.6)%]. The results of Western blotting and BT-PCB showed that extrinsic p53 protein and p53 mRNA were expressed to some degree, but not at high-level. In addition, the results didn't demonstrate obvious radiosensitivity enhancement. Conclusions: The role of inhibition and radiosensitivity enhancement of rAd-p53 was not significant on human lymphoma cell lines. (authors)

  14. Fasting boosts sensitivity of human skin melanoma to cisplatin-induced cell death.

    Science.gov (United States)

    Antunes, Fernanda; Corazzari, Marco; Pereira, Gustavo; Fimia, Gian Maria; Piacentini, Mauro; Smaili, Soraya

    2017-03-25

    Melanoma is one of leading cause of tumor death worldwide. Anti-cancer strategy includes combination of different chemo-therapeutic agents as well as radiation; however these treatments have limited efficacy and induce significant toxic effects on healthy cells. One of most promising novel therapeutic approach to cancer therapy is the combination of anti-cancer drugs with calorie restriction. Here we investigated the effect Cisplatin (CDDP), one of the most potent chemotherapeutic agent used to treat tumors, in association with fasting in wild type and mutated BRAF V600E melanoma cell lines. Here we show that nutrient deprivation can consistently enhance the sensitivity of tumor cells to cell death induction by CDDP, also of those malignancies particularly resistant to any treatment, such as oncogenic BRAF melanomas. Mechanistic studies revealed that the combined therapy induced cell death is characterized by ROS accumulation and ATF4 in the absence of ER-stress. In addition, we show that autophagy is not involved in the enhanced sensitivity of melanoma cells to combined CDDP/EBSS-induced apoptosis. While, the exposure to 2-DG further enhanced the apoptotic rate observed in SK Mel 28 cells upon treatment with both CDDP and EBSS. Copyright © 2016. Published by Elsevier Inc.

  15. Berberine modulates cisplatin sensitivity of human gastric cancer cells by upregulation of miR-203.

    Science.gov (United States)

    You, He-Yi; Xie, Xue-Meng; Zhang, Wei-Jian; Zhu, Heng-Liang; Jiang, Fei-Zhao

    2016-09-01

    Chemotherapeutic resistance is the main reason of the failure in clinical treatment of gastric cancer. Berberine (BER) is the active compound of traditional Chinese medicine Huang Lian. The aim of this present study is to evaluate the effect of BER on cisplatin resistance in gastric cancer cells and to investigate its possible mechanism. Gastric cancer cell lines SGC-7901 and BGC-823 and their respective cisplatin-resistant variants SGC-7901/DDP and BGC-823/DDP were used in this study. We found that BER treatment significantly reversed cisplatin sensitivity and induced caspase-dependent apoptosis in SGC-7901/DDP and BGC-823/DDP cells; BER treatment induced miR-203 expression, and overexpression of miR-203 mimicked the cisplatin-sensitizing effect of BER. Importantly, we showed that miR-203 was able to target the 3'UTR of Bcl-w. Therefore, we conclude that BER treatment reduces cisplatin resistance of gastric cancer cells by modulating the miR-203/Bcl-w apoptotic axis. BER may be a novel agent to enhance chemotherapeutic responses in cisplatin-resistant gastric cancer patients.

  16. Identification of human papillomavirus type 156, the prototype of a new human gammapapillomavirus species, by a generic and highly sensitive PCR strategy for long DNA fragments.

    Science.gov (United States)

    Chouhy, Diego; Bolatti, Elisa M; Piccirilli, Gustavo; Sánchez, Adriana; Fernandez Bussy, Ramón; Giri, Adriana A

    2013-03-01

    This study developed a hanging-droplet long PCR, a generic and highly sensitive strategy to facilitate the identification of new human papillomavirus (HPV) genomes. This novel procedure used for the first time the hanging-droplet PCR technique for the amplification of long DNA fragments with generic primers targeting the L1 and E1 regions. It was first applied to the amplification of types belonging to the highly divergent genus Gammapapillovirus (γ-PV). The hanging-droplet long PCR was 100-fold more sensitive than a simple long PCR procedure, detecting as few as ten copies of HPV-4. Nineteen skin samples, potentially containing putative HPV types from the γ-PV genus, were also screened. The method identified four γ-PV genomic halves from new and previously described putative types, and made the full characterization of HPV-156 possible. This novel virus meets the criteria for a new species within the γ-PV genus, with nucleotide identities in the L1 ORF ranging from 58.3 to 67.3 % compared with representative types of the current γ-PV species. HPV-156 showed the highest identity to HPV-60 (67.3 %) from species γ-4, and was consistently closely related to it in both late- and early-gene-derived phylogenies. In conclusion, this report provides a versatile and highly sensitive approach that allowed identification of the prototype of a new species within the γ-PV genus. Its application with primers targeting the different genera in which both human and non-human PVs are distributed may facilitate characterization of the missing members of the family Papillomaviridae.

  17. Rapid and sensitive liquid chromatography-tandem mass spectrometry method for the quantitation of levodropropizine in human plasma.

    Science.gov (United States)

    Tang, Yunbiao; Zhao, Limei; Wang, Yingwu; Fawcett, J Paul; Gu, Jingkai

    2005-05-05

    A rapid and sensitive LC-MS-MS method for quantifying levodropropizine in human plasma after oral administration of a single-dose (60 mg/day) was developed and validated. The sample preparation used liquid-liquid extraction with a mixture of dichloromethane-diethyl ether (2:3, v/v) in a basic environment. The retention time of levodropropizne and zolmitriptan (used as internal standard) was 1.6 and 1.4 min, respectively. The assay was linear over the range 0.25-500 ng/mL with a LOQ of 0.25 ng/mL. The intra- and inter-day precision were levodropropizine concentration profile in human plasma was determined.

  18. A Modified LC/MS/MS Method with Enhanced Sensitivity for the Determination of Scopolamine in Human Plasma

    Science.gov (United States)

    Wang, Zuwei; Vaksman, Zalman; Putcha, Lakshmi

    2008-01-01

    Intranasal scopolamine is a choice drug for the treatment of motion sickness during space flight because of its quick onset of action, short half-life and favorable sideeffects profile. The dose administered usually ranges between 0.1 and 0.4 mg. Such small doses make it difficult to detect concentrations of scopolamine in biological fluids using existing sensitive LC/MS/MS method, especially when the biological sample volumes are limited. To measure scopolamine in human plasma to facilitate pharmacokinetic evaluation of the drug, we developed a sensitive LC/MS/MS method using 96 well micro elution plates for solid phase extraction (SPE) of scopolamine in human plasma. Human plasma (100-250 micro L) were loaded onto Waters Oasis HLB 96 well micro elution plate and eluted with 50 L of organic solvent without evaporation and reconstitution. HPLC separation of the eluted sample was performed using an Agilent Zorbax SB-CN column (50 x 2.1 mm) at a flow rate of 0.2 mL/min for 3 minutes. The mobile phase for separation was 80:20 (v/v) methanol: ammonium acetate (30 mM) in water. Concentrations of scopolamine were determined using a Micromass Quattro Micro(TM) mass spectrometer with electrospray ionization (ESI). ESI mass spectra were acquired in positive ion mode with multiple reaction monitoring for the determination of scopolamine m/z = 304.2 right arrow 138.1 and internal standard hyoscyamine m/z = 290.2 right arrow 124.1. The method is rapid, reproducible, specific and has the following parameters: scopolamine and the IS are eluted at about 1.1 and 1.7 min respectively. The linear range is 25-10000 pg/mL for scopolamine in human plasma with correlation coefficients greater than 0.99 and CV less than 0.5%. The intra-day and inter-day CVs are less than 15% for quality control samples with concentrations of 75,300, and 750 pg/mL of scopolamine in human plasma. SPE using 96 well micro elution plates allows rapid sample preparation and enhanced sensitivity for the LC

  19. PKCδ regulates hepatic insulin sensitivity and hepatosteatosis in mice and humans

    DEFF Research Database (Denmark)

    Bezy, Olivier; Tran, Thien T; Pihlajamäki, Jussi

    2011-01-01

    C57BL/6J and 129S6/Sv (B6 and 129) mice differ dramatically in their susceptibility to developing diabetes in response to diet- or genetically induced insulin resistance. A major locus contributing to this difference has been mapped to a region on mouse chromosome 14 that contains the gene encodi...... of PKCδ between strains of mice and in obese humans play an important role in the genetic risk of hepatic insulin resistance, glucose intolerance, and hepatosteatosis; and thus PKCδ may be a potential target in the treatment of metabolic syndrome....... PKCδ. Here, we found that PKCδ expression in liver was 2-fold higher in B6 versus 129 mice from birth and was further increased in B6 but not 129 mice in response to a high-fat diet. PRKCD gene expression was also elevated in obese humans and was positively correlated with fasting glucose...

  20. Sensitivity labels and invisible identification markings in human-readable output

    Science.gov (United States)

    Busch, Christoph; Wolthusen, Stephen D.

    2002-04-01

    This paper presents a mechanism for embedding both immediately readable and steganographically hidden information in human-readable output, particularly in hard copy format. The mechanism is embedded within a domain inaccessible to unprivileged users in the operating system's Trusted Computing Base. A realization is presented which permits the embedding of such markings in arbitrary printing systems under the Microsoft Windows NT family of operating systems.

  1. Response of sensitive human ataxia and resistant T-1 cell lines to accelerated heavy ions

    Energy Technology Data Exchange (ETDEWEB)

    Tobias, C.A.; Blakely, E.A.; Chang, P.Y.; Lommel, L.; Roots, R.

    1983-07-01

    The radiation dose responses of fibroblast from a patient with Ataxia telangiectasis (AT-2SF) and an established line of human T-1 cells were studied. Nearly monoenergetic accelerated neon and argon ions were used at the Berkeley Bevalac with various residual range values. The LET of the particles varied from 30 keV/..mu..m to over 1000 keV/..mu..m. All Ataxia survival curves were exponential functions of the dose. Their radiosensitivity reached peak values at 100 to 200 keV/..mu..m. Human T-1 cells have effective sublethal damage repair as has been evidenced by split dose experiments, and they are much more resistant to low LET than to high LET radiation. The repair-misrepair model has been used to interpret these results. We have obtained mathematical expressions that describe the cross sections and inactivation coefficients for both human cell lines as a function of the LET and the type of particle used. The results suggest either that high-LET particles induce a greater number of radiolesions per track or that heavy-ions at high LET induce lesions that kill cells more effectively and that are different from those produced at low LET. We assume that the lesions induced in T-1 and Ataxia cells are qualitatively similar and that each cell line attempts to repair these lesions. The result in most irradiated Ataxia cells, however, is either lethal misrepair or incomplete repair leading to cell death. 63 references, 10 figures, 1 table.

  2. Identification of proteins sensitive to thermal stress in human neuroblastoma and glioma cell lines.

    Directory of Open Access Journals (Sweden)

    Guilian Xu

    Full Text Available Heat-shock is an acute insult to the mammalian proteome. The sudden elevation in temperature has far-reaching effects on protein metabolism, leads to a rapid inhibition of most protein synthesis, and the induction of protein chaperones. Using heat-shock in cells of neuronal (SH-SY5Y and glial (CCF-STTG1 lineage, in conjunction with detergent extraction and sedimentation followed by LC-MS/MS proteomic approaches, we sought to identify human proteins that lose solubility upon heat-shock. The two cell lines showed largely overlapping profiles of proteins detected by LC-MS/MS. We identified 58 proteins in detergent insoluble fractions as losing solubility in after heat shock; 10 were common between the 2 cell lines. A subset of the proteins identified by LC-MS/MS was validated by immunoblotting of similarly prepared fractions. Ultimately, we were able to definitively identify 3 proteins as putatively metastable neural proteins; FEN1, CDK1, and TDP-43. We also determined that after heat-shock these cells accumulate insoluble polyubiquitin chains largely linked via lysine 48 (K-48 residues. Collectively, this study identifies human neural proteins that lose solubility upon heat-shock. These proteins may represent components of the human proteome that are vulnerable to misfolding in settings of proteostasis stress.

  3. Gap-junction coupling and ATP-sensitive potassium channels in human β -cell clusters: Effects on emergent dynamics

    Science.gov (United States)

    Loppini, A.; Pedersen, M. G.; Braun, M.; Filippi, S.

    2017-09-01

    The importance of gap-junction coupling between β cells in pancreatic islets is well established in mouse. Such ultrastructural connections synchronize cellular activity, confine biological heterogeneity, and enhance insulin pulsatility. Dysfunction of coupling has been associated with diabetes and altered β -cell function. However, the role of gap junctions between human β cells is still largely unexplored. By using patch-clamp recordings of β cells from human donors, we previously estimated electrical properties of these channels by mathematical modeling of pairs of human β cells. In this work we revise our estimate by modeling triplet configurations and larger heterogeneous clusters. We find that a coupling conductance in the range 0.005 -0.020 nS/pF can reproduce experiments in almost all the simulated arrangements. We finally explore the consequence of gap-junction coupling of this magnitude between β cells with mutant variants of the ATP-sensitive potassium channels involved in some metabolic disorders and diabetic conditions, translating studies performed on rodents to the human case. Our results are finally discussed from the perspective of therapeutic strategies. In summary, modeling of more realistic clusters with more than two β cells slightly lowers our previous estimate of gap-junction conductance and gives rise to patterns that more closely resemble experimental traces.

  4. The timing and duration of a sensitive period in human flavor learning: a randomized trial.

    Science.gov (United States)

    Mennella, Julie A; Lukasewycz, Laura D; Castor, Sara M; Beauchamp, Gary K

    2011-05-01

    By using the response to protein hydrolysate formula (PHF) as a model system, we discovered the existence of a sensitive period, before 4 mo, when exposure determines the hedonic tone to flavors. We aimed to characterize the timing and duration of this sensitive period. Healthy infants, whose parents had chosen formula feeding, were randomly assigned into 1 of 6 groups at age 0.5 mo: 2 control groups, one fed cow milk-based formula (CMF) and the other fed PHF for 7 mo; 2 groups fed PHF for either 1 or 3 mo beginning at 1.5 mo and CMF otherwise; and 2 groups fed PHF for 1 mo beginning at either 2.5 or 3.5 mo and CMF otherwise. Brief access taste tests were conducted monthly, and complete "meals" of both formulas occurred at the end of the study. Three months of PHF exposure led to acceptance similar to that at 1 mo of exposure. Although these infants were more accepting than were infants with no exposure, they were less accepting than were infants with 7 mo of exposure, which suggests a dosing effect. The time when flavor experiences began was also significant. Among infants exposed to PHF for 1 mo, those who were first fed PHF at 3.5 mo rejected PHF relative to CMF more than did infants exposed at younger ages. The general principles observed are likely of broader significance, indicating a fundamental feature of mammalian development and reflecting the importance of familiarizing infants with flavors that their mothers consume and transmit to breast milk. This trial was registered at clinicaltrials.gov as NCT00994747.

  5. A photonic sintering derived Ag flake/nanoparticle-based highly sensitive stretchable strain sensor for human motion monitoring.

    Science.gov (United States)

    Kim, Inhyuk; Woo, Kyoohee; Zhong, Zhaoyang; Ko, Pyungsam; Jang, Yunseok; Jung, Minhun; Jo, Jeongdai; Kwon, Sin; Lee, Seung-Hyun; Lee, Sungwon; Youn, Hongseok; Moon, Jooho

    2018-03-21

    Recently, the demand for stretchable strain sensors used for detecting human motion is rapidly increasing. This paper proposes high-performance strain sensors based on Ag flake/Ag nanocrystal (NC) hybrid materials incorporated into a polydimethylsiloxane (PDMS) elastomer. The addition of Ag NCs into an Ag flake network enhances the electrical conductivity and sensitivity of the strain sensors. The intense localized heating of Ag flakes/NCs is induced by intense pulsed light (IPL) irradiation, to achieve efficient sintering of the Ag NCs within a second, without damaging the PDMS matrix. This leads to significant improvement in the sensor sensitivity. Our strain sensors are highly stretchable (maximum strain = 80%) and sensitive (gauge factor = 7.1) with high mechanical stability over 10 000 stretching cycles under 50% strain. For practical demonstration, the fabrication of a smart glove for detecting the motions of fingers and a sports band for measuring the applied arm strength is also presented. This study provides an effective method for fabricating elastomer-based high-performance stretchable electronics.

  6. TERRA Expression Levels Do Not Correlate With Telomere Length and Radiation Sensitivity in Human Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Alexandra eSmirnova

    2013-05-01

    Full Text Available Mammalian telomeres are transcribed into long non-coding telomeric RNA molecules (TERRA that seem to play a role in the maintenance of telomere stability. In human cells, CpG island promoters drive TERRA transcription and are regulated by methylation. It was suggested that the amount of TERRA may be related to telomere length. To test this hypothesis we measured telomere length and TERRA levels in single clones isolated from five human cell lines: HeLa (cervical carcinoma, BRC-230 (breast cancer, AKG and GK2 (gastric cancers and GM847 (SV40 immortalized skin fibroblasts. We observed great clonal heterogeneity both in TRF (Terminal Restriction Fragment length and in TERRA levels. However, these two parameters did not correlate with each other. Moreover, cell survival to γ-rays did not show a significant variation among the clones, suggesting that, in this cellular system, the intra-population variability in telomere length and TERRA levels does not influence sensitivity to ionizing radiation. This conclusion was supported by the observation that in a cell line in which telomeres were greatly elongated by the ectopic expression of telomerase, TERRA expression levels and radiation sensitivity were similar to the parental HeLa cell line.

  7. Elevated cyclin A associated kinase activity promotes sensitivity of metastatic human cancer cells to DNA antimetabolite drug.

    Science.gov (United States)

    Wang, Jin; Yin, Hailin; Panandikar, Ashwini; Gandhi, Varsha; Sen, Subrata

    2015-08-01

    Drug resistance is a major obstacle in successful systemic therapy of metastatic cancer. We analyzed the involvement of cell cycle regulatory proteins in eliciting response to N (phosphonoacetyl)-L-aspartate (PALA), an inhibitor of de novo pyrimidine synthesis, in two metastatic variants of human cancer cell line MDA-MB-435 isolated from lung (L-2) and brain (Br-1) in nude mouse, respectively. L-2 and Br-l cells markedly differed in their sensitivity to PALA. While both cell types displayed an initial S phase delay/arrest, Br-l cells proliferated but most L-2 cells underwent apoptosis. There was distinct elevation in cyclin A, and phosphorylated Rb proteins concomitant with decreased expression of bcl-2 protein in the PALA treated L-2 cells undergoing apoptosis. Markedly elevated cyclin A associated and cdk2 kinase activities together with increased E2F1-DNA binding were detected in these L-2 cells. Induced ectopic cyclin A expression sensitized Br-l cells to PALA by activating an apoptotic pathway. Our findings demonstrate that elevated expression of cyclin A and associated kinase can activate an apoptotic pathway in cells exposed to DNA antimetabolites. Abrogation of this pathway can lead to resistance against these drugs in metastatic variants of human carcinoma cells.

  8. Label-free electrochemical impedance biosensor to detect human interleukin-8 in serum with sub-pg/ml sensitivity.

    Science.gov (United States)

    Sharma, R; Deacon, S E; Nowak, D; George, S E; Szymonik, M P; Tang, A A S; Tomlinson, D C; Davies, A G; McPherson, M J; Wälti, C

    2016-06-15

    Biosensors with high sensitivity and short time-to-result that are capable of detecting biomarkers in body fluids such as serum are an important prerequisite for early diagnostics in modern healthcare provision. Here, we report the development of an electrochemical impedance-based sensor for the detection in serum of human interleukin-8 (IL-8), a pro-angiogenic chemokine implicated in a wide range of inflammatory diseases. The sensor employs a small and robust synthetic non-antibody capture protein based on a cystatin scaffold that displays high affinity for human IL-8 with a KD of 35 ± 10 nM and excellent ligand specificity. The change in the phase of the electrochemical impedance from the serum baseline, ∆θ(ƒ), measured at 0.1 Hz, was used as the measure for quantifying IL-8 concentration in the fluid. Optimal sensor signal was observed after 15 min incubation, and the sensor exhibited a linear response versus logarithm of IL-8 concentration from 900 fg/ml to 900 ng/ml. A detection limit of around 90 fg/ml, which is significantly lower than the basal clinical levels of 5-10 pg/ml, was observed. Our results are significant for the development of point-of-care and early diagnostics where high sensitivity and short time-to-results are essential. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  9. TERRA Expression Levels Do Not Correlate with Telomere Length and Radiation Sensitivity in Human Cancer Cell Lines.

    Science.gov (United States)

    Smirnova, Alexandra; Gamba, Riccardo; Khoriauli, Lela; Vitelli, Valerio; Nergadze, Solomon G; Giulotto, Elena

    2013-01-01

    Mammalian telomeres are transcribed into long non-coding telomeric repeat-containing RNA (TERRA) molecules that seem to play a role in the maintenance of telomere stability. In human cells, CpG-island promoters drive TERRA transcription and are regulated by methylation. It was suggested that the amount of TERRA may be related to telomere length. To test this hypothesis we measured telomere length and TERRA levels in single clones isolated from five human cell lines: HeLa (cervical carcinoma), BRC-230 (breast cancer), AKG and GK2 (gastric cancers), and GM847 (SV40 immortalized skin fibroblasts). However, these two parameters did not correlate with each other. Moreover, cell survival to γ-rays did not show a significant variation among the clones, suggesting that, in this cellular system, the intra-population variability in telomere length and TERRA levels does not influence sensitivity to ionizing radiation. This conclusion was supported by the observation that in a cell line in which telomeres were greatly elongated by the ectopic expression of telomerase, TERRA expression levels and radiation sensitivity were similar to the parental HeLa cell line.

  10. Peripheral erythrocytes decrease upon specific respiratory challenge with grass pollen allergen in sensitized mice and in human subjects.

    Directory of Open Access Journals (Sweden)

    Galateja Jordakieva

    Full Text Available BACKGROUND AND AIMS: Specific hyper-responsiveness towards an allergen and non-specific airway hyperreactivity both impair quality of life in patients with respiratory allergic diseases. We aimed to investigate cellular responses following specific and non-specific airway challenges locally and systemically in i sensitized BALB/c mice challenged with grass pollen allergen Phl p 5, and in ii grass pollen sensitized allergic rhinitis subjects undergoing specific airway challenge in the Vienna Challenge Chamber (VCC. METHODS AND RESULTS: BALB/c mice (n = 20 were intraperitoneally immunized with grass pollen allergen Phl p 5 and afterwards aerosol challenged with either the specific allergen Phl p 5 (n = 10 or the non-specific antigen ovalbumin (OVA (n = 10. A protocol for inducing allergic asthma as well as allergic rhinitis, according to the united airway concept, was used. Both groups of exposed mice showed significantly reduced physical activity after airway challenge. Specific airway challenge further resulted in goblet cell hyperplasia, enhanced mucous secretion, intrapulmonary leukocyte infiltration and lymphoid follicle formation, associated with significant expression of IL-4, IL-5 and IL-13 in splenocytes and also partially in lung tissue. Concerning circulating blood cell dynamics, we observed a significant drop of erythrocyte counts, hemoglobin and hematocrit levels in both mouse groups, challenged with allergen or OVA. A significant decrease in circulating erythrocytes and hematocrit levels after airway challenges with grass pollen allergen was also found in grass pollen sensitized human rhinitis subjects (n = 42 at the VCC. The effects on peripheral leukocyte counts in mice and humans however were opposed, possibly due to the different primary inflammation sites. CONCLUSION: Our data revealed that, besides significant leukocyte dynamics, particularly erythrocytes are involved in acute hypersensitivity reactions to respiratory allergens

  11. Effects of extracellular plaque components on the chlorhexidine sensitivity of strains of Streptococcus mutans and human dental plaque

    International Nuclear Information System (INIS)

    Wolinsky, L.E.; Hume, W.R.

    1985-01-01

    An in vitro study was undertaken to determine the effects of sucrose-derived extracellular plaque components on the sensitivity of selected oral bacteria to chlorhexidine (CX). Cultures of Streptococcus mutans HS-6, OMZ-176, Ingbritt C, 6715-wt13, and pooled human plaque were grown in trypticase soy media with or without 1% sucrose. The sensitivity to CX of bacteria grown in each medium was determined by fixed-time exposure to CX and subsequent measurement of 3 H-thymidine uptake. One-hour exposure to CX at concentrations of 10(-4) M (0.01% w/v) or greater substantially inhibited subsequent cellular division among all the S. mutans strains and human plaque samples tested. An IC50 (the CX concentration which depressed 3 H-thymidine incorporation to 50% of control level) of close to 10(-4) M was noted for S. mutans strains HS-6, OMZ-176, and 6715-wt13 when grown in the presence of sucrose. The same strains grown in cultures without added sucrose showed about a ten-fold greater sensitivity to CX (IC50 close to 10(-5) M). A three-fold difference was noted for S. mutans Ingbritt C. Only a slight increase in the IC50 was noted for the plaque samples cultured in sucrose-containing media, but their threshold for depression of 3 H-thymidine uptake by CX was lower than that for the sucrose-free plaque samples. The study showed that extracellular products confer some protection against CX to the bacteria examined, and provided an explanation for the disparity between clinically-recommended concentrations for plaque suppression and data on in vitro susceptibility

  12. Processing of natural sounds in human auditory cortex: tonotopy, spectral tuning, and relation to voice sensitivity.

    Science.gov (United States)

    Moerel, Michelle; De Martino, Federico; Formisano, Elia

    2012-10-10

    Auditory cortical processing of complex meaningful sounds entails the transformation of sensory (tonotopic) representations of incoming acoustic waveforms into higher-level sound representations (e.g., their category). However, the precise neural mechanisms enabling such transformations remain largely unknown. In the present study, we use functional magnetic resonance imaging (fMRI) and natural sounds stimulation to examine these two levels of sound representation (and their relation) in the human auditory cortex. In a first experiment, we derive cortical maps of frequency preference (tonotopy) and selectivity (tuning width) by mathematical modeling of fMRI responses to natural sounds. The tuning width maps highlight a region of narrow tuning that follows the main axis of Heschl's gyrus and is flanked by regions of broader tuning. The narrowly tuned portion on Heschl's gyrus contains two mirror-symmetric frequency gradients, presumably defining two distinct primary auditory areas. In addition, our analysis indicates that spectral preference and selectivity (and their topographical organization) extend well beyond the primary regions and also cover higher-order and category-selective auditory regions. In particular, regions with preferential responses to human voice and speech occupy the low-frequency portions of the tonotopic map. We confirm this observation in a second experiment, where we find that speech/voice selective regions exhibit a response bias toward the low frequencies characteristic of human voice and speech, even when responding to simple tones. We propose that this frequency bias reflects the selective amplification of relevant and category-characteristic spectral bands, a useful processing step for transforming a sensory (tonotopic) sound image into higher level neural representations.

  13. A Sensitive Sensor Cell Line for the Detection of Oxidative Stress Responses in Cultured Human Keratinocytes

    Directory of Open Access Journals (Sweden)

    Ute Hofmann

    2014-06-01

    Full Text Available In the progress of allergic and irritant contact dermatitis, chemicals that cause the generation of reactive oxygen species trigger a heat shock response in keratinocytes. In this study, an optical sensor cell line based on cultured human keratinocytes (HaCaT cells expressing green fluorescent protein (GFP under the control of the stress-inducible HSP70B’ promoter were constructed. Exposure of HaCaT sensor cells to 25 µM cadmium, a model substance for oxidative stress induction, provoked a 1.7-fold increase in total glutathione and a ~300-fold induction of transcript level of the gene coding for heat shock protein HSP70B’. An extract of Arnica montana flowers resulted in a strong induction of the HSP70B’ gene and a pronounced decrease of total glutathione in keratinocytes. The HSP70B’ promoter-based sensor cells conveniently detected cadmium-induced stress using GFP fluorescence as read-out with a limit of detection of 6 µM cadmium. In addition the sensor cells responded to exposure of cells to A. montana extract with induction of GFP fluorescence. Thus, the HaCaT sensor cells provide a means for the automated detection of the compromised redox status of keratinocytes as an early indicator of the development of human skin disorders and could be applied for the prediction of skin irritation in more complex in vitro 3D human skin models and in the development of micro-total analysis systems (µTAS that may be utilized in dermatology, toxicology, pharmacology and drug screenings.

  14. Investigation of touch-sensitive responses by hyphae of the human pathogenic fungus Candida albicans.

    Science.gov (United States)

    Gow, N A; Perera, T H; Sherwood-Higham, J; Gooday, G W; Gregory, D W; Marshall, D

    1994-01-01

    Candida albicans is a fungus that commonly infects the mucosal surface of humans. The hyphal growth form of this fungus may initiate the primary invasion of the host. Here we show that hyphae respond thigmotropically and morphologically to cues such as the presence of a surface, pores, grooves and ridges. Growth on some firm surfaces elicits a helical growth response. Hyphae follow grooves and ridges of inert substrates and penetrate pores of filtration membranes. Our in vitro experiments suggest that thigmotropism may enhance the ability of a hypha to invade epithelia of a host at sites of weakened integrity.

  15. A sensitive radioimmunoassay of parathyroid hormone in human serum using a specific extraction procedure

    International Nuclear Information System (INIS)

    Christensen, M.S.

    1976-01-01

    A radioimmunoassay of human parathyroid hormone (PTH) is described. PTH was extracted from serum by adsorption to and elution from microfine silica, causing a 3.2-fold greater hormone concentration in extract than in serum. The extraction procedure eliminated from the assay unspecific interference caused by serum factors. The detection limit was 10 pg bovine PTH. The concentration of PTH was measurable in serum from 95 % of normal subjects (mean, 68 pg/ml; S.D., 18), was undetectable in sera from hypoparathyroid patients, and elevated in 96 % of sera from patients with primary hyperthyroidism. The data presented suggest that the assay mainly measures PTH(1-84). (Auth.)

  16. Differential contributory roles of nucleotide excision and homologous recombination repair for enhancing cisplatin sensitivity in human ovarian cancer cells

    Science.gov (United States)

    2011-01-01

    Background While platinum-based chemotherapeutic agents are widely used to treat various solid tumors, the acquired platinum resistance is a major impediment in their successful treatment. Since enhanced DNA repair capacity is a major factor in conferring cisplatin resistance, targeting of DNA repair pathways is an effective stratagem for overcoming cisplatin resistance. This study was designed to delineate the role of nucleotide excision repair (NER), the principal mechanism for the removal of cisplatin-induced DNA intrastrand crosslinks, in cisplatin resistance and reveal the impact of DNA repair interference on cisplatin sensitivity in human ovarian cancer cells. Results We assessed the inherent NER efficiency of multiple matched pairs of cisplatin-sensitive and -resistant ovarian cancer cell lines and their expression of NER-related factors at mRNA and protein levels. Our results showed that only the cisplatin-resistant ovarian cancer cell line PEO4 possessed an increased NER capacity compared to its inherently NER-inefficient parental line PEO1. Several other cisplatin-resistant cell lines, including CP70, CDDP and 2008C13, exhibited a normal and parental cell-comparable NER capacity for removing cisplatin-induced DNA intrastrand cross-links (Pt-GG). Concomitant gene expression analysis revealed discordance in mRNA and protein levels of NER factors in various ovarian cancer cell lines and NER proteins level were unrelated to the cisplatin sensitivity of these cell lines. Although knockdown of NER factors was able to compromise the NER efficiency, it only caused a minimal effect on cisplatin sensitivity. On the contrary, downregulation of BRCA2, a critical protein for homologous recombination repair (HRR), significantly enhanced the efficacy of cisplatin in killing ovarian cancer cell line PEO4. Conclusion Our studies indicate that the level of NER factors in ovarian cancer cell lines is neither a determinant of their NER capacity nor of the sensitivity to

  17. Polyamine Metabolism Is Sensitive to Glycolysis Inhibition in Human Neuroblastoma Cells*

    Science.gov (United States)

    Ruiz-Pérez, M. Victoria; Medina, Miguel Ángel; Urdiales, José Luis; Keinänen, Tuomo A.; Sánchez-Jiménez, Francisca

    2015-01-01

    Polyamines are essential for cell proliferation, and their levels are elevated in many human tumors. The oncogene n-myc is known to potentiate polyamine metabolism. Neuroblastoma, the most frequent extracranial solid tumor in children, harbors the amplification of n-myc oncogene in 25% of the cases, and it is associated with treatment failure and poor prognosis. We evaluated several metabolic features of the human neuroblastoma cell lines Kelly, IMR-32, and SK-N-SH. We further investigated the effects of glycolysis impairment in polyamine metabolism in these cell lines. A previously unknown linkage between glycolysis impairment and polyamine reduction is unveiled. We show that glycolysis inhibition is able to trigger signaling events leading to the reduction of N-Myc protein levels and a subsequent decrease of both ornithine decarboxylase expression and polyamine levels, accompanied by cell cycle blockade preceding cell death. New anti-tumor strategies could take advantage of the direct relationship between glucose deprivation and polyamine metabolism impairment, leading to cell death, and its apparent dependence on n-myc. Combined therapies targeting glucose metabolism and polyamine synthesis could be effective in the treatment of n-myc-expressing tumors. PMID:25593318

  18. Polyamine metabolism is sensitive to glycolysis inhibition in human neuroblastoma cells.

    Science.gov (United States)

    Ruiz-Pérez, M Victoria; Medina, Miguel Ángel; Urdiales, José Luis; Keinänen, Tuomo A; Sánchez-Jiménez, Francisca

    2015-03-06

    Polyamines are essential for cell proliferation, and their levels are elevated in many human tumors. The oncogene n-myc is known to potentiate polyamine metabolism. Neuroblastoma, the most frequent extracranial solid tumor in children, harbors the amplification of n-myc oncogene in 25% of the cases, and it is associated with treatment failure and poor prognosis. We evaluated several metabolic features of the human neuroblastoma cell lines Kelly, IMR-32, and SK-N-SH. We further investigated the effects of glycolysis impairment in polyamine metabolism in these cell lines. A previously unknown linkage between glycolysis impairment and polyamine reduction is unveiled. We show that glycolysis inhibition is able to trigger signaling events leading to the reduction of N-Myc protein levels and a subsequent decrease of both ornithine decarboxylase expression and polyamine levels, accompanied by cell cycle blockade preceding cell death. New anti-tumor strategies could take advantage of the direct relationship between glucose deprivation and polyamine metabolism impairment, leading to cell death, and its apparent dependence on n-myc. Combined therapies targeting glucose metabolism and polyamine synthesis could be effective in the treatment of n-myc-expressing tumors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Optimization of PIXE-sensitivity for detection of Ti in thin human skin sections

    Science.gov (United States)

    Pallon, Jan; Garmer, Mats; Auzelyte, Vaida; Elfman, Mikael; Kristiansson, Per; Malmqvist, Klas; Nilsson, Christer; Shariff, Asad; Wegdén, Marie

    2005-04-01

    Modern sunscreens contain particles like TiO2 having sizes of 25-70 nm and acting as a reflecting substance. For cosmetic reasons the particle size is minimized. Questions have been raised to what degree these nano particles penetrate the skin barrier, and how they do affect the human. The EU funded project "Quality of skin as a barrier to ultra-fine particles" - NANODERM has started with the purpose to evaluate the possible risks of TiO2 penetration into vital skin layers. The purpose of the work presented here was to find the optimal conditions for micro-PIXE analysis of Ti in thin skin sections. In the skin region where Ti is expected to be found, the naturally occurring major elements phosphorus, chlorine, sulphur and potassium have steep gradients and thus influence the X-ray background in a non-predictable manner. Based on experimental studies of Ti-exposed human skin sections using proton energies ranging from 1.8-2.55 MeV, the corresponding PIXE detection limits for Ti were calculated. The energy that was found to be the most favourable, 1.9 MeV, was then selected for future studies.

  20. Implied motion because of instability in Hokusai Manga activates the human motion-sensitive extrastriate visual cortex: an fMRI study of the impact of visual art.

    Science.gov (United States)

    Osaka, Naoyuki; Matsuyoshi, Daisuke; Ikeda, Takashi; Osaka, Mariko

    2010-03-10

    The recent development of cognitive neuroscience has invited inference about the neurosensory events underlying the experience of visual arts involving implied motion. We report functional magnetic resonance imaging study demonstrating activation of the human extrastriate motion-sensitive cortex by static images showing implied motion because of instability. We used static line-drawing cartoons of humans by Hokusai Katsushika (called 'Hokusai Manga'), an outstanding Japanese cartoonist as well as famous Ukiyoe artist. We found 'Hokusai Manga' with implied motion by depicting human bodies that are engaged in challenging tonic posture significantly activated the motion-sensitive visual cortex including MT+ in the human extrastriate cortex, while an illustration that does not imply motion, for either humans or objects, did not activate these areas under the same tasks. We conclude that motion-sensitive extrastriate cortex would be a critical region for perception of implied motion in instability.

  1. Substrate Metabolism and Insulin Sensitivity During Fasting in Obese Human Subjects: Impact of GH Blockade.

    Science.gov (United States)

    Pedersen, Morten Høgild; Svart, Mads Vandsted; Lebeck, Janne; Bidlingmaier, Martin; Stødkilde-Jørgensen, Hans; Pedersen, Steen Bønløkke; Møller, Niels; Jessen, Niels; Jørgensen, Jens O L

    2017-04-01

    Insulin resistance and metabolic inflexibility are features of obesity and are amplified by fasting. Growth hormone (GH) secretion increases during fasting and GH causes insulin resistance. To study the metabolic effects of GH blockade during fasting in obese subjects. Nine obese males were studied thrice in a randomized design: (1) after an overnight fast (control), (2) after 72 hour fasting (fasting), and (3) after 72 hour fasting with GH blockade (pegvisomant) [fasting plus GH antagonist (GHA)]. Each study day consisted of a 4-hour basal period followed by a 2-hour hyperinsulinemic, euglycemic clamp combined with indirect calorimetry, assessment of glucose and palmitate turnover, and muscle and fat biopsies. GH levels increased with fasting (P fasting-induced reduction of serum insulin-like growth factor I was enhanced by GHA (P Fasting increased lipolysis and lipid oxidation independent of GHA, but fasting plus GHA caused a more pronounced suppression of lipid intermediates in response to hyperinsulinemic, euglycemic clamp. Fasting-induced insulin resistance was abrogated by GHA (P Fasting plus GHA also caused elevated glycerol levels and reduced levels of counterregulatory hormones. Fasting significantly reduced the expression of antilipolytic signals in adipose tissue independent of GHA. Suppression of GH activity during fasting in obese subjects reverses insulin resistance and amplifies insulin-stimulated suppression of lipid intermediates, indicating that GH is an important regulator of substrate metabolism, insulin sensitivity, and metabolic flexibility also in obese subjects. Copyright © 2017 by the Endocrine Society

  2. In vitro phagocytosis of methicillin resistant and methicillin sensitive staphylococcus aureus by human polymorphonuclear leucocytes

    International Nuclear Information System (INIS)

    Javed, N.; Tahir, R.; Abbas, A.

    2009-01-01

    Staphylococcus aureus is a gram positive bacterium that causes a number of diseases such as abscesses, infective endocarditis, septic arthritis, etc. It is acquiring resistance against many antibiotics like methicillin; therefore its control is becoming increasingly difficult. Peripheral blood phagocytes particularly polymorphonuclear leucocytes play an important role in the protective mechanisms against these organisms. Phagocytes interact with bacteria and phagocytose these microorganisms to kill them. Phenotypically different isolates of Staphylococcus aureus including methicillin resistant Staphylococcus aureus (MRSA) and methicillin sensitive Staphylococcus aureus (MSSA) were collected from various hospitals of Lahore, Pakistan. Fresh polymorphonuclaer leucocytes were obtained from healthy individuals by centrifugation using Ficol-Hypaque gradient combined with dextran sedimentation. Microbiological method was used for the determination of phagocytic index of phenotypic variants of Staphylococcus aureus. A significant difference was observed between the phagocytic index of both bacterial groups. MSSA group showed the Mean+-SD of 79.46%+-3.9 while MRSA group showed 72.35%+-2.5. Significant difference in phagocytic index indicates that it can be one of the mechanisms of MRSA to evade host immune system as compare to MSSA. (author)

  3. Simultaneous and sensitive LC-MS/MS determination of tetrahydrocannabinol and metabolites in human plasma.

    Science.gov (United States)

    Ferreirós, N; Labocha, S; Walter, C; Lötsch, J; Geisslinger, G

    2013-02-01

    Cannabis is not only a widely used illicit drug but also a substance which can be used in pharmacological therapy because of its analgesic, antiemetic, and antispasmodic properties. A very rapid and sensitive method for determination of ∆(9)-tetrahydrocannabinol (THC), the principal active component of cannabis, and two of its phase I metabolites in plasma has been developed and validated. After solid-phase extraction of plasma (0.2 mL), the clean extracts were analyzed by tandem mass spectrometry after a 5-min liquid chromatographic separation. The linear calibration ranges were from 0.05 to 30 ng mL(-1) for THC and 11-nor-∆(9)-carboxy-tetrahydrocannabinol (THC-COOH) and from 0.2 to 30 ng mL(-1) for ∆(9)-(11-OH)-tetrahydrocannabinol (11-OH-THC). Imprecision and inaccuracy were always below 7 and 12 % (expressed as relative standard deviation and relative error), respectively. The method has been successfully applied to determination of the three analytes in plasma obtained from healthy volunteers after oral administration of 20 mg dronabinol.

  4. Nebivolol, but not metoprolol, lowers blood pressure in nitric oxide-sensitive human hypertension.

    Science.gov (United States)

    Okamoto, Luis E; Gamboa, Alfredo; Shibao, Cyndya A; Arnold, Amy C; Choi, Leena; Black, Bonnie K; Raj, Satish R; Robertson, David; Biaggioni, Italo

    2014-12-01

    Nebivolol, unlike other selective β1-receptor blockers, induces vasodilation attributable to increased NO bioavailability. The relative contribution of this mechanism to the blood pressure (BP)-lowering effects of nebivolol is unclear because it is normally masked by baroreflex buffering. Autonomic failure provides a unique model of hypertension devoid of autonomic modulation but sensitive to the hypotensive effects of NO potentiation. We tested the hypothesis that nebivolol would decrease BP in these patients through a mechanism independent of β-blockade. We randomized 20 autonomic failure patients with supine hypertension (14 men; 69±2 years) to receive a single oral dose of placebo, nebivolol 5 mg, metoprolol 50 mg (negative control), and sildenafil 25 mg (positive control) on separate nights in a double-blind, crossover study. Supine BP was monitored every 2 hours from 8:00 pm to 8:00 am. Compared with placebo, sildenafil and nebivolol decreased systolic BP during the night (P20 mm Hg at 4:00 am) and nonresponders. Nebivolol significantly lowered systolic BP in sildenafil responders (-44±13 mm Hg) but not in nonresponders (1±11 mm Hg). Despite lowering nighttime BP, nebivolol did not worsen morning orthostatic tolerance compared with placebo. In conclusion, nebivolol effectively lowered supine hypertension in autonomic failure, independent of β1-blockade. These results are consistent with the hypothesis that NO potentiation contributes significantly to the antihypertensive effect of nebivolol. © 2014 American Heart Association, Inc.

  5. The effects of instructions on the sensitivity of negatively reinforced human behavior to extinction.

    Science.gov (United States)

    Alessandri, Jérôme; Cançado, Carlos R X

    2017-03-01

    The effects of instructions on the sensitivity of negatively reinforced (escape) behavior to extinction were studied. Initially, responding produced timeouts from pressing a force cell on a variable-ratio (VR) schedule, which was then discontinued (extinction). Based on extinction data, participants were distributed into two groups. Participants in the Persistence Group (for which response rates were low in extinction) were instructed that the experimenter expected them to continue responding in extinction after a second exposure to the VR schedule. Participants in the Extinction group (for which response rates were high in extinction) were instructed that the experimenter expected them to stop responding in extinction. Relative to the condition in which instructions were absent, extinction-response rates increased and decreased, respectively, for participants in the Persistence and Extinction groups. These results replicate and extend to negatively reinforced responding previous findings that showed behavioral control by instructions formulated as explicit experimenter demands or expectations. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Inhibition of mTOR promotes hyperthermia sensitivity in SMMC-7721 human hepatocellular carcinoma cell line

    Science.gov (United States)

    WANG, QING-LIANG; LIU, BO; LI, XIAO-JIE; HU, KUN-PENG; ZHAO, KUN; YE, XIAO-MING

    2016-01-01

    The mammalian target of rapamycin (mTOR) is a critical mediator of the phosphoinositide 3-kinase/protein kinase B/mTOR signaling pathway, and mTOR activity is induced following heat shock. Thermotherapy is used to treat hepatocellular carcinoma (HCC). However, the role of mTOR in modulating thermosensitivity in HCC has yet to be elucidated. In the present study, the antisense plasmid pEGFP-C1-mTOR was transfected into SMMC-7721 cells, and the expression levels of mTOR were analyzed by reverse transcription-polymerase chain reaction and western blot analysis. The thermal responses of the transfected cells were also examined. The results revealed that SMMC-7721 cells were sensitive to heat treatment, and cell viability was significantly inhibited following hyperthermia treatment (P<0.01). The mRNA and protein expression levels of mTOR decreased post-transfection. Cell proliferation, colony-forming ability and motility were all significantly decreased following hyperthermia treatment in the transfected cells. Flow cytometry analysis demonstrated that apoptosis was significantly increased following treatment (P<0.01). The number of cells in S phase was increased, and the cell cycle was arrested in S phase. In conclusion, inhibition of mTOR increased the thermosensitivity of SMMC-7721 cells by increasing cellular apoptosis and inducing S phase arrest. PMID:26998020

  7. Capacitive DNA sensor for rapid and sensitive detection of whole genome human herpesvirus-1 dsDNA in serum.

    Science.gov (United States)

    Cheng, Cheng; Oueslati, Rania; Wu, Jayne; Chen, Jiangang; Eda, Shigetoshi

    2017-06-01

    This work presents a rapid, highly sensitive, low-cost, and specific capacitive DNA sensor for detection of whole genome human herpesvirus-1 DNA. This sensor is capable of direct DNA detection with a response time of 30 s, and it can be used to test standard buffer or serum samples. The sensing approach for DNA detection is based on alternating current (AC) electrokinetics. By applying an inhomogeneous AC electric field on sensor electrodes, positive dielectrophoresis is induced to accelerate DNA hybridization. The same applied AC signal also directly measures the hybridization of target with the probe on the sensor surface. Experiments are conducted to optimize the AC signal, as well as the buffers for probe immobilization and target DNA hybridization. The assay is highly sensitive and specific, with no response to human herpesvirus-2 DNA at 5 ng/mL and a LOD of 1.0 pg/mL (6.5 copies/μL or 10.7 aM) in standard buffer. When testing the double stranded (ds) DNA spiked in human serum samples, the sensor yields a LOD of 20.0 pg/mL (129.5 copies/μL or 0.21 femtomolar (fM)) in neat serum. In this work, the target is whole genome dsDNA, consequently the test can be performed without the use of enzyme or amplification, which considerably simplifies the sensor operation and is highly suitable for point of care disease diagnosis. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Sensitization of human breast cancer cells to gemcitabine by the protein kinase C modulator bryostatin 1.

    Science.gov (United States)

    Ali, Shadan; Aranha, Olivia; Li, Yiwei; Pettit, George R; Sarkar, Fazlul H; Philip, Philip Agop

    2003-09-01

    Protein kinase C (PKC) plays an important role in cell proliferation, differentiation, and apoptosis. The interaction between the PKC modulator bryostatin 1 (BRYO), and gemcitabine in human breast cancer MCF-7 and MDA-MB-231 cells and in the non-transformed MCF-10A human breast epithelial cells was investigated. Immunoblotting was used to determine the expression of PKC isoenzymes and proteins involved in the cell cycle and apoptosis. MTT, ELISA and flow cytometry assays were used to determine cell survival. Treatment of cells with BRYO 200 n M resulted in a significant downregulation of cytoplasmic PKC in all three cell lines. However, the expression of membranous PKC was differentially affected in these cells. BRYO (1-200 n M) had no significant effects on cell viability in any of the cell lines. Nevertheless, BRYO significantly enhanced the antiproliferative and apoptotic effects of gemcitabine in the MCF-7 and MDA-MB-231 cells, but not in the MCF-10A cells. This was associated with significant reduction in the bcl-2/bax ratio. There was a significant upregulation of p53, p21(waf1), and p27 in MCF7 and MCF-10A cells treated with the combination of gemcitabine and BRYO compared to gemcitabine-treated cells. The potentiation of the effect of gemcitabine by BRYO was demonstrated in MCF-7 and MDA-MB-231 cells and was associated with a specific pattern of PKC modulation. Further investigation of the role of specific isoforms of PKC in the downstream molecular events of gemcitabine-induced cytotoxicity is warranted.

  9. SIRT1 inhibition restores apoptotic sensitivity in p53-mutated human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Herbert, Katharine J.; Cook, Anthony L., E-mail: Anthony.Cook@utas.edu.au; Snow, Elizabeth T., E-mail: elizabeth.snow@utas.edu.au

    2014-06-15

    Mutations to the p53 gene are common in UV-exposed keratinocytes and contribute to apoptotic resistance in skin cancer. P53-dependent activity is modulated, in part, by a complex, self-limiting feedback loop imposed by miR-34a-mediated regulation of the lysine deacetylase, SIRT1. Expression of numerous microRNAs is dysregulated in squamous and basal cell carcinomas; however the contribution of specific microRNAs to the pathogenesis of skin cancer remains untested. Through use of RNAi, miRNA target site blocking oligonucleotides and small molecule inhibitors, this study explored the influence of p53 mutational status, SIRT1 activity and miR-34a levels on apoptotic sensitivity in primary (NHEK) and p53-mutated (HaCaT) keratinocyte cell lines. SIRT1 and p53 are overexpressed in p53-mutated keratinocytes, whilst miR-34a levels are 90% less in HaCaT cells. HaCaTs have impaired responses to p53/SIRT1/miR-34a axis manipulation which enhanced survival during exposure to the chemotherapeutic agent, camptothecin. Inhibition of SIRT1 activity in this cell line increased p53 acetylation and doubled camptothecin-induced cell death. Our results demonstrate that p53 mutations increase apoptotic resistance in keratinocytes by interfering with miR-34a-mediated regulation of SIRT1 expression. Thus, SIRT1 inhibitors may have a therapeutic potential for overcoming apoptotic resistance during skin cancer treatment. - Highlights: • Impaired microRNA biogenesis promotes apoptotic resistance in HaCaT keratinocytes. • TP53 mutations suppress miR-34a-mediated regulation of SIRT1 expression. • SIRT1 inhibition increases p53 acetylation in HaCaTs, restoring apoptosis.

  10. Reduction of Orc6 expression sensitizes human colon cancer cells to 5-fluorouracil and cisplatin.

    Directory of Open Access Journals (Sweden)

    Elaine J Gavin

    Full Text Available Previous studies from our group have shown that the expression levels of Orc6 were highly elevated in colorectal cancer patient specimens and the induction of Orc6 was associated with 5-fluorouracil (5-FU treatment. The goal of this study was to investigate the molecular and cellular impact of Orc6 in colon cancer. In this study, we use HCT116 (wt-p53 and HCT116 (null-p53 colon cancer cell lines as a model system to investigate the impact of Orc6 on cell proliferation, chemosensitivity and pathways involved with Orc6. We demonstrated that the down regulation of Orc6 sensitizes colon cancer cells to both 5-FU and cisplatin (cis-pt treatment. Decreased Orc6 expression in HCT-116 (wt-p53 cells by RNA interference triggered cell cycle arrest at G1 phase. Prolonged inhibition of Orc6 expression resulted in multinucleated cells in HCT-116 (wt-p53 cell line. Western immunoblot analysis showed that down regulation of Orc6 induced p21 expression in HCT-116 (wt-p53 cells. The induction of p21 was mediated by increased level of phosphorylated p53 at ser-15. By contrast, there is no elevated expression of p21 in HCT-116 (null-p53 cells. Orc6 down regulation also increased the expression of DNA damaging repair protein GADD45beta and reduced the expression level of JNK1. Orc6 may be a potential novel target for future anti cancer therapeutic development in colon cancer.

  11. Assignment of the human amiloride-sensitive Na{sup +} channel {delta} isoform to chromosome 1p36.3-p36.2

    Energy Technology Data Exchange (ETDEWEB)

    Waldmann, R.; Bassilana, F.; Voilley, N. [Institut de Pharmacologie Moleculaire et Cellulaire, Valbonne (France)] [and others

    1996-06-01

    This report describes the localization of the human amiloride-sensitive Na{sup +} channel {delta} isoform to human chromosome 1p36.3-p36.2 using in situ hybridization. Mutations in this group of ion channels have been implicated in various hereditary diseases. 18 refs., 1 fig.

  12. Immunoregulatory adherent cells in human tuberculosis: radiation-sensitive antigen-specific suppression by monocytes

    Energy Technology Data Exchange (ETDEWEB)

    Kleinhenz, M.E.; Ellner, J.J.

    1985-07-01

    In human tuberculosis, adherent mononuclear cells (AMC) selectively depress in vitro responses to the mycobacterial antigen tuberculin purified protein derivative (PPD). The phenotype of this antigen-specific adherent suppressor cell was characterized by examining the functional activity of adherent cells after selective depletion of sheep erythrocyte-rosetting T cells or OKM1-reactive monocytes. Adherent cell suppression was studied in the (/sup 3/H)thymidine-incorporation microculture assay by using T cells rigorously depleted of T cells with surface receptors for the Fc portion of IgG (T gamma cells) as antigen-responsive cells. PPD-induced (/sup 3/H)thymidine incorporation by these non gamma T cells was uniformly reduced (mean, 42% +/- 10% (SD)) when autologous AMC were added to non gamma T cells at a ratio of 1:2. Antigen-specific suppression by AMC was not altered by depletion of sheep erythrocyte-rosetting T cells or treatment with indomethacin. However, AMC treated with OKM1 and complement or gamma irradiation (1,500 rads) no longer suppressed tuberculin responses in vitro. These studies identify the antigen-specific adherent suppressor cell in tuberculosis as an OKM1-reactive, non-erythrocyte-rosetting monocyte. The radiosensitivity of this monocyte immunoregulatory function may facilitate its further definition.

  13. Immunoregulatory adherent cells in human tuberculosis: radiation-sensitive antigen-specific suppression by monocytes

    International Nuclear Information System (INIS)

    Kleinhenz, M.E.; Ellner, J.J.

    1985-01-01

    In human tuberculosis, adherent mononuclear cells (AMC) selectively depress in vitro responses to the mycobacterial antigen tuberculin purified protein derivative (PPD). The phenotype of this antigen-specific adherent suppressor cell was characterized by examining the functional activity of adherent cells after selective depletion of sheep erythrocyte-rosetting T cells or OKM1-reactive monocytes. Adherent cell suppression was studied in the [ 3 H]thymidine-incorporation microculture assay by using T cells rigorously depleted of T cells with surface receptors for the Fc portion of IgG (T gamma cells) as antigen-responsive cells. PPD-induced [ 3 H]thymidine incorporation by these non gamma T cells was uniformly reduced (mean, 42% +/- 10% [SD]) when autologous AMC were added to non gamma T cells at a ratio of 1:2. Antigen-specific suppression by AMC was not altered by depletion of sheep erythrocyte-rosetting T cells or treatment with indomethacin. However, AMC treated with OKM1 and complement or gamma irradiation (1,500 rads) no longer suppressed tuberculin responses in vitro. These studies identify the antigen-specific adherent suppressor cell in tuberculosis as an OKM1-reactive, non-erythrocyte-rosetting monocyte. The radiosensitivity of this monocyte immunoregulatory function may facilitate its further definition

  14. Human Sarcoma growth is sensitive to small-molecule mediated AXIN stabilization.

    Directory of Open Access Journals (Sweden)

    Alessandra De Robertis

    Full Text Available Sarcomas are mesenchymal tumors showing high molecular heterogeneity, reflected at the histological level by the existence of more than fifty different subtypes. Genetic and epigenetic evidences link aberrant activation of the Wnt signaling to growth and progression of human sarcomas. This phenomenon, mainly accomplished by autocrine loop activity, is sustained by gene amplification, over-expression of Wnt ligands and co-receptors or epigenetic silencing of endogenous Wnt antagonists. We previously showed that pharmacological inhibition of Wnt signaling mediated by Axin stabilization produced in vitro and in vivo antitumor activity in glioblastoma tumors. Here, we report that targeting different sarcoma cell lines with the Wnt inhibitor/Axin stabilizer SEN461 produces a less transformed phenotype, as supported by modulation of anchorage-independent growth in vitro. At the molecular level, SEN461 treatment enhanced the stability of the scaffold protein Axin1, a key negative regulator of the Wnt signaling with tumor suppressor function, resulting in downstream effects coherent with inhibition of canonical Wnt signaling. Genetic phenocopy of small molecule Axin stabilization, through Axin1 over-expression, coherently resulted in strong impairment of soft-agar growth. Importantly, sarcoma growth inhibition through pharmacological Axin stabilization was also observed in a xenograft model in vivo in female CD-1 nude mice. Our findings suggest the usefulness of Wnt inhibitors with Axin stabilization activity as a potentialyl clinical relevant strategy for certain types of sarcomas.

  15. Human Sarcoma growth is sensitive to small-molecule mediated AXIN stabilization.

    Science.gov (United States)

    De Robertis, Alessandra; Mennillo, Federica; Rossi, Marco; Valensin, Silvia; Tunici, Patrizia; Mori, Elisa; Caradonna, Nicola; Varrone, Maurizio; Salerno, Massimiliano

    2014-01-01

    Sarcomas are mesenchymal tumors showing high molecular heterogeneity, reflected at the histological level by the existence of more than fifty different subtypes. Genetic and epigenetic evidences link aberrant activation of the Wnt signaling to growth and progression of human sarcomas. This phenomenon, mainly accomplished by autocrine loop activity, is sustained by gene amplification, over-expression of Wnt ligands and co-receptors or epigenetic silencing of endogenous Wnt antagonists. We previously showed that pharmacological inhibition of Wnt signaling mediated by Axin stabilization produced in vitro and in vivo antitumor activity in glioblastoma tumors. Here, we report that targeting different sarcoma cell lines with the Wnt inhibitor/Axin stabilizer SEN461 produces a less transformed phenotype, as supported by modulation of anchorage-independent growth in vitro. At the molecular level, SEN461 treatment enhanced the stability of the scaffold protein Axin1, a key negative regulator of the Wnt signaling with tumor suppressor function, resulting in downstream effects coherent with inhibition of canonical Wnt signaling. Genetic phenocopy of small molecule Axin stabilization, through Axin1 over-expression, coherently resulted in strong impairment of soft-agar growth. Importantly, sarcoma growth inhibition through pharmacological Axin stabilization was also observed in a xenograft model in vivo in female CD-1 nude mice. Our findings suggest the usefulness of Wnt inhibitors with Axin stabilization activity as a potentialyl clinical relevant strategy for certain types of sarcomas.

  16. HepG2 human hepatocarcinomas cells sensitization by endogenous porphyrins

    Science.gov (United States)

    Vonarx-Coinsmann, Veronique; Foultier, Marie-Therese; de Brito, Leonor X.; Morlet, Laurent; Patrice, Thierry

    1995-03-01

    We assessed the ability of the human hepatocarcinoma cell line HepG2 to synthesize PpIX in vitro from exogenous ALA and analyzed ALA-induced toxicity and phototoxicity on this cell line. ALA induced a slight dose-dependent dark toxicity, with 79 and 66% cell survival respectively for ALA 50 and 100 mg/ml after 3-h incubation. Whereas the same treatment followed by laser irradiation (l equals 632 nm, 25 J/sq cm) induced dose-dependent phototoxicity, with 54 and 19% cell survival 24 h after PDT. Whatever the incubation time with ALA, a 3-h delay before light exposure was found optimal to reach a maximal phototoxicity. Photoproducts induced by porphyrin light irradiation absorbed light in the red spectral region at longer wavelengths than did the original porphyrins. The possible enhancement of PDT effects after ALA HepG2 cell incubation was investigated by irradiating cells successively with red light (l equals 632 nm) and light (l equals 650 nm). Total fluence was kept constant at 25 J/sq cm. Phototoxicity was lower when cells were irradiated for increased periods of l equals 650 nm light than with l equals 632 nm light alone. Any photoproducts involved had either a short life or were poorly photoreactive. HepG2 cells, synthesizing enzymes and precursors of endogenous porphyrin synthesis, represent a good in vitro model for experiments using ALA-PpIX-PDT.

  17. Could periodic patterns in human mortality be sensitive to solar activity?

    Directory of Open Access Journals (Sweden)

    R. Díaz-Sandoval

    2011-06-01

    Full Text Available Seasonal behaviour of human diseases have been observed and reported in the literature for years. Although the Sun plays an essential role in the origin and evolution of life on Earth, it is barely taken into account in biological processes for the development of a specific disease. Higher mortality rates occur during the winter season in the Northern Hemisphere for several diseases, particularly diseases of the cardiovascular and respiratory systems. This increment has been associated with seasonal and social causes. However, is there more behind these correlations, in particular in terms of solar variability? In this paper we attempt to make a first step towards answering this question. A detailed wavelet analysis of periodicities for diseases from England and Wales seem to reveal that mortality periodicities (3 days to half a year could be due to the Earth's position around the Sun. Moreover, crosswavelet and wavelet coherence analysis show common features between medical diseases and solar proxies around solar maximum activity suggesting that this relation, if any, has to be searched in times of high solar activity.

  18. A Rapid and Sensitive Method to Measure the Functional Activity of Shiga Toxins in Human Serum

    Science.gov (United States)

    Arfilli, Valentina; Carnicelli, Domenica; Ardissino, Gianluigi; Torresani, Erminio; Scavia, Gaia; Brigotti, Maurizio

    2015-01-01

    Shiga toxins (Stx) have a definite role in the development of hemolytic uremic syndrome in children with hemorrhagic colitis caused by pathogenic Stx-producing Escherichia coli (STEC) strains. The dramatic effects of these toxins on the microvasculature of different organs, particularly of the kidney, are well known, whereas there is no consensus on the mechanism by which Stx reach the endothelia of target organs and/or indirectly injure these body sites. We hereby describe a quick (4 h), radioactive, Raji cell-based method designed for the detection of Stx in human sera. The assay monitors the translation impairment induced by these powerful inhibitors of protein synthesis, which are identified properly by neutralizing their activity with specific monoclonal antibodies. By this method, we detected for the first time the functional activity of Stx in sera of STEC-infected patients during hemorrhagic colitis. Recent research has pointed to a dynamic process of Stx-induced renal intoxication in which concurrent and interactive steps are involved. Our rapid and specific method could be useful for studying the kinetics of Stx during the natural course of STEC infection and the interplay between Stx activity in serum and Stx presence in different blood fractions (neutrophils, monocytes, platelets, leukocyte-platelet aggregates, microvesicles, lipoproteins). PMID:26556372

  19. Long non-coding RNA LINC00261 sensitizes human colon cancer cells to cisplatin therapy.

    Science.gov (United States)

    Wang, Z K; Yang, L; Wu, L L; Mao, H; Zhou, Y H; Zhang, P F; Dai, G H

    2017-12-11

    Colon cancer is one of the most common digestive tumors. The present study aimed to explore the functional role, as well as the underlying mechanism of long non-coding RNA LINC00261 in colon cancer. Expression of LINC00261 was analyzed in colon cancer cell lines and human normal cell lines. Acquired resistance cell lines were then built and the acquired resistance efficiency was detected by evaluating cell viability. Thereafter, the effects of LINC00261 overexpression on cisplatin-resistant colon cancer cells were measured, as well as cell apoptosis, viability, migration, and invasion. Subsequently, we investigated the interaction of LINC00261 and β-catenin. The results showed that the LINC00261 gene was down-regulated in colon cancer cell lines and tissues, and in cisplatin-resistant cells. LINC00261 overexpression might relieve cisplatin resistance of colon cancer cells via promoting cell apoptosis, and inhibiting cell viability, migration, and invasion. Moreover, LINC00261 might down-regulate nuclear β-catenin through restraining β-catenin from cytoplasm into nuclei or it could also promote β-catenin degradation and inhibit activation of Wnt pathway. Finally, LINC00261 reduced cisplatin resistance of colon cancer in vivo and enhanced the anti-colon cancer effect of cisplatin through reducing tumor volume and weight.

  20. Sensitive quantification of diphemanil methyl sulphate in human plasma by liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Do, B; Goulay-Dufaÿ, S; Le Hoang, M D; Adoui, N; Graffard, H; Guyon, F; Pradeau, D

    2007-01-01

    A simple detection system with a high-performance liquid chromatography (HPLC) with positive ionisation-tandem mass spectrometry (ESI-MS/MS) for determining diphemanil methylsulphate (DMS) levels in human plasma using 4-diphemanylmethylene,1-methylpiperidine as an internal standard (I.S.), is proposed. The acquisition was performed with the multiple reactional monitoring (MRM) mode, by monitoring the transitions: m/z 278>262 for DMS and m/z 263>247 for the I.S. The method involved a simple single-step deproteinisation with acetonitrile. The analyte was chromatographed on a Zorbax C18 reversed-phase chromatographic column by isocratic elution with 10(-3)M ammonium acetate and 10(-3)M hexafluorobutyric acid, adjusted to pH 7.0 with ammoniac/acetonitrile (40/60, v/v). The results were linear over the studied range (0.5-50.0 ng mL(-1)) and the total analysis time for each run was 10 min. The mean extraction apparent recoveries expressed at the 95% intervals of confidence were 94-104% for DMS and 92-106% for the I.S. The intra- and inter-assay precisions were 4.6-8.4% and 2.9-10.6%, respectively. The limit of quantification was 0.15 ng mL(-1). The devised assay was successfully applied to the residual concentrations monitoring in infant.

  1. Radiation induced bystander effects in modification of cellular radio-sensitivity in human cancer cells

    International Nuclear Information System (INIS)

    Pandey, B.N.

    2012-01-01

    Radiation-induced Bystander Effect is manifestation of radiation effects in non-irradiated cells in the population. The phenomenon may have significant implication in risk of radiation induced cancer incidence and outcome of cancer radiotherapy. To understand the bystander interaction in tumor cells, we have studied secretion of diffusible factors from control and irradiated tumor cells of different origin. Our results showed a good correlation between magnitude of secretion of diffusible factors and survival of tumor cells. These diffusible factors are shown to affect proliferation and survival of tumor cells involving regulation of kinases and genes/proteins involved in apoptotic machinery. Our experiments using pharmacological inhibitors showed involvement of activating transcription factor 2 (ATF-2) signaling in survival of tumor cells after treatment with diffusible factors. These factors seem to be involved in exerting radio-resistance in tumor cells. Furthermore, in proton microbeam irradiation studies showed induction of double strand break measured as gH2AX foci in human lung carcinoma cells, which was found to propagate to bystander tumor cells during post-irradiation incubation. Implication of these observations in outcome of cancer radiotherapy scenario would be discussed. (author)

  2. A Role for REM Sleep in Recalibrating the Sensitivity of the Human Brain to Specific Emotions

    Science.gov (United States)

    Gujar, Ninad; McDonald, Steven Andrew; Nishida, Masaki

    2011-01-01

    Although the impact of sleep on cognitive function is increasingly well established, the role of sleep in modulating affective brain processes remains largely uncharacterized. Using a face recognition task, here we demonstrate an amplified reactivity to anger and fear emotions across the day, without sleep. However, an intervening nap blocked and even reversed this negative emotional reactivity to anger and fear while conversely enhancing ratings of positive (happy) expressions. Most interestingly, only those subjects who obtained rapid eye movement (REM) sleep displayed this remodulation of affective reactivity for the latter 2 emotion categories. Together, these results suggest that the evaluation of specific human emotions is not static across a daytime waking interval, showing a progressive reactivity toward threat-related negative expressions. However, an episode of sleep can reverse this predisposition, with REM sleep depotentiating negative reactivity toward fearful expressions while concomitantly facilitating recognition and ratings of reward-relevant positive expressions. These findings support the view that sleep, and specifically REM neurophysiology, may represent an important factor governing the optimal homeostasis of emotional brain regulation. PMID:20421251

  3. Weather and place-based human behavior: recreational preferences and sensitivity.

    Science.gov (United States)

    de Freitas, C R

    2015-01-01

    This study examines the links between biometeorological variables and the behavior of beach recreationists along with their rating of overall weather conditions. To identify and describe significance of on-site atmospheric conditions, two separate forms of response are examined. The first is sensory perception of the immediate atmospheric surround expressed verbally, which was the subject of earlier work. In the research reported here, on-site observations of behavior that reflect the effects of weather and climate are examined. By employing, independently, separate indicators of on-site experience, the reliability of each is examined and interpreted and apparent threshold conditions verified. The study site is King's Beach located on the coast of Queensland, Australia. On-site observations of atmospheric variables and beach user behavior are made for the daylight hours of 45 days spread over a 12-month period. The results show that behavioral data provide reliable and meaningful indications of the significance of the atmospheric environment for leisure. Atmospheric conditions within the zone of acceptability are those that the beach users can readily cope with or modify by a range of minor behavioral adjustments. Optimal weather conditions appear to be those requiring no specific behavioral adjustment. Attendance levels reflect only the outer limits of acceptability of the meteorological environment, while duration of visit enables calibration of levels of approval in so far as it reflects rating of on-site weather within a broad zone of tolerance. In a broad theoretical sense, the results add to an understanding of the relationship between weather and human behavior. This information is potentially useful in effective tourism management and planning.

  4. Weather and place-based human behavior: recreational preferences and sensitivity

    Science.gov (United States)

    de Freitas, C. R.

    2015-01-01

    This study examines the links between biometeorological variables and the behavior of beach recreationists along with their rating of overall weather conditions. To identify and describe significance of on-site atmospheric conditions, two separate forms of response are examined. The first is sensory perception of the immediate atmospheric surround expressed verbally, which was the subject of earlier work. In the research reported here, on-site observations of behavior that reflect the effects of weather and climate are examined. By employing, independently, separate indicators of on-site experience, the reliability of each is examined and interpreted and apparent threshold conditions verified. The study site is King's Beach located on the coast of Queensland, Australia. On-site observations of atmospheric variables and beach user behavior are made for the daylight hours of 45 days spread over a 12-month period. The results show that behavioral data provide reliable and meaningful indications of the significance of the atmospheric environment for leisure. Atmospheric conditions within the zone of acceptability are those that the beach users can readily cope with or modify by a range of minor behavioral adjustments. Optimal weather conditions appear to be those requiring no specific behavioral adjustment. Attendance levels reflect only the outer limits of acceptability of the meteorological environment, while duration of visit enables calibration of levels of approval in so far as it reflects rating of on-site weather within a broad zone of tolerance. In a broad theoretical sense, the results add to an understanding of the relationship between weather and human behavior. This information is potentially useful in effective tourism management and planning.

  5. Variation in sensitizing effect of caffeine in human tumour cell lines after γ-irradiation

    International Nuclear Information System (INIS)

    Valenzuela, M.T.; Almodovar, M.R. de; Mateos, S.; McMillan, T.J.

    2000-01-01

    We have investigated whether the protective role of the G2 checkpoint has increasing importance when the p53-dependent G1 checkpoint is inactivated. We have studied the differential effect of caffeine by clonogenic assays and flow cytometry in three human tumour cell lines with different functionality of p53 protein. The radiosensitizing effect of caffeine (2 mM) expressed itself as a significant decrease in surviving fraction at 2 Gy and a significant increase in α-values in RT112 and TE671, both with non-functional p53. However, no radiosensitizing effect was seen in cells with a normal p53 function (MCF-7 BUS). Two millimoles of caffeine also caused important changes in the cell cycle progression after irradiation. MCF-7 BUS showed a G1 arrest after irradiation and an early G2 arrest but those cells that reached the second G2 did not arrest significantly. In contrast, TE671 exhibited radiosensitization by caffeine, no G1 arrest, a G2 arrest in those cells irradiated in G2, no significant accumulation in the second G2 but an overall delay in release from the first cell cycle, which could be abrogated by caffeine. RT112 was similar to TE671 except that the emphasis in a G2 arrest was shifted from the block in cells irradiated in G2 to those irradiated at other cell cycle phases. The data presented confirm that p53 status can be a significant determinant of the efficacy of caffeine as radiosensitizer in these tumour cell lines, and document the importance of the G2 checkpoint in this effect. (author)

  6. Changes in glucose tolerance and insulin sensitivity following 2 weeks of daily cinnamon ingestion in healthy humans

    DEFF Research Database (Denmark)

    Solomon, Thomas; Blannin, Andrew K

    2009-01-01

    Cinnamon can improve fasting glucose in humans yet data on insulin sensitivity are limited and controversial. Eight male volunteers (aged 25 +/- 1 years, body mass 76.5 +/- 3.0 kg, BMI 24.0 +/- 0.7 kg m(-2); mean +/- SEM) underwent two 14-day interventions involving cinnamon or placebo...... supplementation (3 g day(-1)). Placebo supplementation was continued for 5 days following this 14 day period. Oral glucose tolerance tests (OGTT) were performed on days 0, 1, 14, 16, 18, and 20. Cinnamon ingestion reduced the glucose response to OGTT on day 1 (-13.1 +/- 6.3% vs. day 0; P ....5 +/- 8.1% vs. day 0; P = 0.09). Cinnamon ingestion also reduced insulin responses to OGTT on day 14 (-27.1 +/- 6.2% vs. day 0; P cinnamon feeding. Cinnamon may improve glycaemic...

  7. Effects of melatonin on prepulse inhibition, habituation and sensitization of the human startle reflex in healthy volunteers

    DEFF Research Database (Denmark)

    Lehtinen, Emilia K; Ucar, Ebru; Glenthøj, Birte Y

    2014-01-01

    Prepulse inhibition of the startle reflex (PPI) is an operational measure of sensorimotor gating, which is demonstrated to be impaired in patients with schizophrenia. In addition, a disruption of the circadian rhythm together with blunted melatonin secretion is regularly found in patients...... with schizophrenia and it is theorized that these may contribute to their attentional deficits. The aim of this study was to assess the effects of acute melatonin on healthy human sensorimotor gating. Twenty-one healthy male volunteers were administered melatonin or placebo after which their levels of PPI were...... assessed. Melatonin significantly reduced startle magnitude and ratings of alertness, but did not influence PPI, nor sensitization and habituation. However, when taking baseline scores in consideration, melatonin significantly increased PPI in low scoring individuals while significantly decreasing...

  8. [The macrophage disappearance reaction in guinea pigs sensitized with bovine gamma globulin or human scrum albumin (author's transl)].

    Science.gov (United States)

    Schimke, R; Bernstein, B; Ambrosius, H

    1977-01-01

    The macrophage disappearance reaction (MDR) is a suitable test for detection of cell mediated immunity against bovine gamma globulin (BGG) and human serum albumin (HSA) in guinea pigs. The MDR is a technical simple, good manipulable, and quantifiable test. The optimal test conditions for the antigens BGC and HSA are the following: Peritoneal exudat cells (PEC) were stimulated with paraffin oil. On the 5th day after receiving oil the animals were injected with 80 microgram BGG or 30 microgram HSA i.p. 5 hours later the PEC were harvested and counted. With the MDR it is possible to detect differences with respect to degree of cell-mediated immunity. Supernatants of sensitized lymphocytes produces the MDR too.

  9. Sensitivity to cisplatin in primary cell lines derived from human glioma correlates with levels of EGR-1 expression

    Directory of Open Access Journals (Sweden)

    Ponti Donatella

    2011-03-01

    Full Text Available Abstract Background Less than 30% of malignant gliomas respond to adjuvant chemotherapy. Here, we have asked whether variations in the constitutive expression of early-growth response factor 1 (EGR-1 predicted acute cytotoxicity and clonogenic cell death in vitro, induced by six different chemotherapics. Materials and methods Cytotoxicity assays were performed on cells derived from fresh tumor explants of 18 human cases of malignant glioma. In addition to EGR-1, tumor cultures were investigated for genetic alterations and the expression of cancer regulating factors, related to the p53 pathway. Results We found that sensitivity to cisplatin correlates significantly with levels of EGR-1 expression in tumors with wild-type p53/INK4a/p16 status. Conclusion Increased knowledge of the mechanisms regulating EGR-1 expression in wild-type p53/INK4a/p16 cases of glioma may help in the design of new chemotherapeutic strategies for these tumors.

  10. Impact of sensitivity of human leucocyte antigen antibody detection by Luminex technology on graft loss at 1 year.

    Science.gov (United States)

    Szatmary, Peter; Jones, James; Hammad, Abdul; Middleton, Derek

    2013-06-01

    The clinical relevance of the detection of human leucocyte antigen (HLA) antibodies in sera of renal transplant recipients by highly sensitive methods such as Luminex alone is uncertain and a matter of debate. The choice of output thresholds affects antibody detection and thus organ allocation, yet there are no internationally agreed threshold levels. This study aims at evaluating our current practice of using an MFI threshold of 1000 in antibody detection. We carried out a case-control study by looking at 761 renal transplant recipients at one unit between 2000 and 2010. Of these, there were 93 cases of graft loss within 1 year and stored serum samples of 40 cases were available for testing. Controls were selected (graft function >2 years) and individually matched according to age, sex, number of transplants and date of transplant. All 40 cases and 40 controls had negative crossmatch by complement-dependent cytotoxicity (CDC) at the time of transplant, and pre-transplant sera were re-analysed for the presence of detectable HLA and donor-specific antibodies (DSAs) using Luminex screen and single-antigen beads and MFI threshold values of 1000, 2000 and 4000. In nearly 48% of cases with graft loss within a year, HLA antibodies were detectable by Luminex when using a 1000 MFI threshold. This was 25% greater than in controls (P = 0.017). There was also a 15% increase in detected DSAs; however, statistical significance depends on the inclusion or exclusion of one specific case. Using MFI thresholds of 2000 and 4000, no DSAs were found in any long-term surviving grafts. Selection of appropriate MFI cut-off values influences the detection of DSAs and, thus, organ allocation. Using a threshold of 1000 led to the detection of DSAs in 5% of long-term graft survivors in our population and should be considered too sensitive. Using a detection threshold of 2000 is sufficiently sensitive and leads to clinically relevant detection of DSA.

  11. SiRNA-mediated IGF-1R inhibition sensitizes human colon cancer SW480 cells to radiation

    International Nuclear Information System (INIS)

    Yavari, Kamal; Taghikhani, Mohammad; Mesbah-Namin, Seyed A.; Maragheh, Mohammad Ghannadi; Babaei, Mohammad Hosein; Arfaee, Ali Jabbary; Madani, Hossein; Mirzaei, Hamid Reza

    2010-01-01

    Purpose. Insulin like growth factor receptor 1 (IGF-1R) is well-documented to play a key role in radiation response and tumor radiosensitivity, thus offering an attractive clinic drug target to enhance tumor sensitivity to anti-cancer radiotherapy. Material and methods. Human colon carcinoma SW480 cells were transfected with the specific small interference RNA (siRNA) expression vector (pkD-shRNA-IGF-1R-V2) designed to target IGF-1R mRNA. The expression of IGF-1R mRNA and its protein among the transfected and untransfected cells were detected by semi-quantitative RT-PCR and ELISA assay. The changes in cell radiosensitivity were examined by MTT assay. Results. Transfection of mammalian expression vector pkD containing IGF-1R siRNA was shown to reduce IGF-1R mRNA levels by up to 95%. ELISA assay detected a similar inhibition of IGF-1R protein levels in cells transfected with IGF-1R siRNA. SW480 cells transfected with the expression vector for siRNA significantly rendered cells more sensitive to radiation and the highest radiation enhancement ratio was 2.02 ± 0.08. Conclusion. These data provide the first evidence that specific siRNA fragment (pkD-shRNA-IGF-1R-V2) targeting human IGF-1R mRNA is able to enhance colon cancer radiosensitivity. Also results indicated that, combining IGF-1R siRNA and radiation significantly enhances antitumor efficacy compared with either modality alone

  12. Large-scale assembly of highly sensitive Si-based flexible strain sensors for human motion monitoring

    Science.gov (United States)

    Zhang, Bing-Chang; Wang, Hui; Zhao, Yu; Li, Fan; Ou, Xue-Mei; Sun, Bao-Quan; Zhang, Xiao-Hong

    2016-01-01

    Silicon is the dominant semiconductor in modern society, but the rigid nature of most Si structures hinders its applications in flexible electronics. In this work, Si-based flexible strain sensors are fabricated with Si fabric consisting of long Si nanowires. The as-obtained sensors demonstrate a large strain range of 50% and a gauge factor of up to 350, which are sufficient to detect human motions with superior performance over traditional sensors. The results reveal that the assembling strategy may potentially be applied to large-scale fabrication of highly sensitive, flexible strain sensors for emerging applications such as healthcare and sports monitoring. Moreover, the Si fabric would also enable broad applications of Si materials in other flexible and wearable devices such as flexible optoelectronics and displays.Silicon is the dominant semiconductor in modern society, but the rigid nature of most Si structures hinders its applications in flexible electronics. In this work, Si-based flexible strain sensors are fabricated with Si fabric consisting of long Si nanowires. The as-obtained sensors demonstrate a large strain range of 50% and a gauge factor of up to 350, which are sufficient to detect human motions with superior performance over traditional sensors. The results reveal that the assembling strategy may potentially be applied to large-scale fabrication of highly sensitive, flexible strain sensors for emerging applications such as healthcare and sports monitoring. Moreover, the Si fabric would also enable broad applications of Si materials in other flexible and wearable devices such as flexible optoelectronics and displays. Electronic supplementary information (ESI) available: The morphological and structural characterization of the silicon nanowires, the plot of the relative resistance change versus cubic strain, and the relationship between the width of the gap and the exerted strain. See DOI: 10.1039/c5nr07546g

  13. Biotin uptake by mouse and human pancreatic beta cells/islets: a regulated, lipopolysaccharide-sensitive carrier-mediated process

    Science.gov (United States)

    Ghosal, Abhisek; Sekar, Thillai V.

    2014-01-01

    Biotin is essential for the normal function of pancreatic beta cells. These cells obtain biotin from their surroundings via transport across their cell membrane. Little is known about the uptake mechanism involved, how it is regulated, and how it is affected by internal and external factors. We addressed these issues using the mouse-derived pancreatic beta-TC-6 cells and freshly isolated mouse and human primary pancreatic beta cells as models. The results showed biotin uptake by pancreatic beta-TC-6 cells occurs via a Na+-dependent, carrier-mediated process, that is sensitive to desthiobiotin, as well as to pantothenic acid and lipoate; the process is also saturable as a function of concentration (apparent Km = 22.24 ± 5.5 μM). These cells express the sodium-dependent multivitamin transporter (SMVT), whose knockdown (with doxycycline-inducible shRNA) led to a sever inhibition in biotin uptake. Similarly, uptake of biotin by mouse and human primary pancreatic islets is Na+-dependent and carrier-mediated, and both cell types express SMVT. Biotin uptake by pancreatic beta-TC-6 cells is also adaptively regulated (via transcriptional mechanism) by extracellular substrate level. Chronic treatment of pancreatic beta-TC-6 cells with bacterial lipopolysaccharides (LPS) leads to inhibition in biotin uptake. This inhibition is mediated via a Toll-Like receptor 4-mediated process and involves a decrease in membrane expression of SMVT. These findings show, for the first time, that pancreatic beta cells/islets take up biotin via a specific and regulated carrier-mediated process, and that the process is sensitive to the effect of LPS. PMID:24904078

  14. Long-term exposure to abnormal glucose levels alters drug metabolism pathways and insulin sensitivity in primary human hepatocytes

    Science.gov (United States)

    Davidson, Matthew D.; Ballinger, Kimberly R.; Khetani, Salman R.

    2016-06-01

    Hyperglycemia in type 2 diabetes mellitus has been linked to non-alcoholic fatty liver disease, which can progress to inflammation, fibrosis/cirrhosis, and hepatocellular carcinoma. Understanding how chronic hyperglycemia affects primary human hepatocytes (PHHs) can facilitate the development of therapeutics for these diseases. Conversely, elucidating the effects of hypoglycemia on PHHs may provide insights into how the liver adapts to fasting, adverse diabetes drug reactions, and cancer. In contrast to declining PHH monocultures, micropatterned co-cultures (MPCCs) of PHHs and 3T3-J2 murine embryonic fibroblasts maintain insulin-sensitive glucose metabolism for several weeks. Here, we exposed MPCCs to hypo-, normo- and hyperglycemic culture media for ~3 weeks. While albumin and urea secretion were not affected by glucose level, hypoglycemic MPCCs upregulated CYP3A4 enzyme activity as compared to other glycemic states. In contrast, hyperglycemic MPCCs displayed significant hepatic lipid accumulation in the presence of insulin, while also showing decreased sensitivity to insulin-mediated inhibition of glucose output relative to a normoglycemic control. In conclusion, we show for the first time that PHHs exposed to hypo- and hyperglycemia can remain highly functional, but display increased CYP3A4 activity and selective insulin resistance, respectively. In the future, MPCCs under glycemic states can aid in novel drug discovery and mechanistic investigations.

  15. Hydrogen-bond network and pH sensitivity in transthyretin: Neutron crystal structure of human transthyretin.

    Science.gov (United States)

    Yokoyama, Takeshi; Mizuguchi, Mineyuki; Nabeshima, Yuko; Kusaka, Katsuhiro; Yamada, Taro; Hosoya, Takaaki; Ohhara, Takashi; Kurihara, Kazuo; Tomoyori, Katsuaki; Tanaka, Ichiro; Niimura, Nobuo

    2012-02-01

    Transthyretin (TTR) is a tetrameric protein associated with human amyloidosis. In vitro, the formation of amyloid fibrils by TTR is known to be promoted by low pH. Here we show the neutron structure of TTR, focusing on the hydrogen bonds, protonation states and pH sensitivities. A large crystal was prepared at pD 7.4 for neutron protein crystallography. Neutron diffraction studies were conducted using the IBARAKI Biological Crystal Diffractometer with the time-of-flight method. The neutron structure solved at 2.0Å resolution revealed the protonation states of His88 and the detailed hydrogen-bond network depending on the protonation states of His88. This hydrogen-bond network is composed of Thr75, Trp79, His88, Ser112, Pro113, Thr118-B and four water molecules, and is involved in both monomer-monomer and dimer-dimer interactions, suggesting that the double protonation of His88 by acidification breaks the hydrogen-bond network and causes the destabilization of the TTR tetramer. In addition, the comparison with X-ray structure at pH 4.0 indicated that the protonation occurred to Asp74, His88 and Glu89 at pH 4.0. Our neutron model provides insights into the molecular stability of TTR related to the hydrogen-bond network, the pH sensitivity and the CH···O weak hydrogen bond. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Age-Associated Impairments in Mitochondrial ADP Sensitivity Contribute to Redox Stress in Senescent Human Skeletal Muscle

    Directory of Open Access Journals (Sweden)

    Graham P. Holloway

    2018-03-01

    Full Text Available Summary: It remains unknown if mitochondrial bioenergetics are altered with aging in humans. We established an in vitro method to simultaneously determine mitochondrial respiration and H2O2 emission in skeletal muscle tissue across a range of biologically relevant ADP concentrations. Using this approach, we provide evidence that, although the capacity for mitochondrial H2O2 emission is not increased with aging, mitochondrial ADP sensitivity is impaired. This resulted in an increase in mitochondrial H2O2 and the fraction of electron leak to H2O2, in the presence of virtually all ADP concentrations examined. Moreover, although prolonged resistance training in older individuals increased muscle mass, strength, and maximal mitochondrial respiration, exercise training did not alter H2O2 emission rates in the presence of ADP, the fraction of electron leak to H2O2, or the redox state of the muscle. These data establish that a reduction in mitochondrial ADP sensitivity increases mitochondrial H2O2 emission and contributes to age-associated redox stress. : Holloway et al. show that an inability of ADP to decrease mitochondrial reactive oxygen species emission contributes to redox stress in skeletal muscle tissue of older individuals and that this process is not recovered following prolonged resistance-type exercise training, despite the general benefits of resistance training for muscle health. Keywords: mitochondria, aging, muscle, ROS, H2O2, ADP, respiration, bioenergetics, exercise, resistance training

  17. Use of γ-H2AX Foci Assay on Human Peripheral Blood Lymphocytes as Sensitive Biomarker of Exposure

    International Nuclear Information System (INIS)

    Gajski, G.; Garaj-Vrhovac, V.; Geric, M.; Filipic, M.; Nunic, J.; Straser, A.; Zegura, B.

    2013-01-01

    In modern medicine, it is impossible to imagine diagnostics and treatments without equipment that emit radiation (X-ray, CT, PET, etc.). At the same time there is a need to minimize the amount of radiation that the patient will gain during such medical examination. In that manner ALARA (As Low As Reasonably Achievable) principle and dosimetry are the bases of assuring patients safety. The induction of gamma phosphorylated H2AX histone is newly developed tool in biodosimetry, which is more sensitive for the detection of radiation caused DNA damage than currently used micronucleus and comet assay. Gamma phosphorylation of H2AX histone is a consequence of DNA double strand breaks and its role is to trigger the DNA repair mechanisms. In this study, we tested the effect of 2 and 4 Gy X-rays on human peripheral blood lymphocytes from two healthy volunteers using γ-H2AX foci assay. The FITC signal from labelled antibodies was monitored using flow cytometry and clearly demonstrated the difference in control samples and irradiated samples. There was also the difference between the exposed blood samples from the two volunteers. The results of present study reveal new sensitive method that is capable of detecting changes in DNA when exposed to different doses of radiation, and thus potentially optimizing the ALARA principle.(author)

  18. Development of ultra-sensitive soybean peroxidase-based CL-ELISA for the determination of human thyroglobulin.

    Science.gov (United States)

    Vdovenko, Marina M; Zubkov, Alexander V; Kuznetsova, Galina I; Ciana, Leopoldo Della; Kuzmina, Nina S; Sakharov, Ivan Yu

    2010-10-31

    Serum thyroglobulin (Tg) is a main marker of thyroid cancer relapses after total or near-total thyroidectomy of patients with differentiated thyroid carcinoma. In this study, we developed a chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for detecting Tg in human serum. Soybean peroxidase (SbP) in combination with 3-(10'-phenothiazinyl)propane-1-sulfonate (SPTZ) and 4-morpholinopyridine (MORPH) and horseradish peroxidase (HRP) with p-iodophenol (PIP) were used as detection systems in the sandwich CL-ELISA. Comparison of these two systems showed that a lower detection limit (LOD) of CL-ELISA with SbP/SPTZ/MORPH was 10 times lower than for the immunoassay with HRP/PIP. The LOD value for SbP-based CL-ELISA of 0.2 ng/mL was identical to LOD value typical of CL-ELISA Immulite kit produced with alkaline phosphatase. The sensitivity of Tg CL-ELISA using SbP/SPTZ/MORPH completely satisfies the requirements of modern endocrinology. Comparative study of clinical serum specimens assayed by the SbP-based CL-ELISA (x) and Immulite kit (y) for detecting Tg showed a good correlation between these two immunoassays (y=1.15 x -0.14, R=0.99). The obtained results open good perspectives for use of SbP/SPTZ/MORPH system in the development of ultra-sensitive immunoassays. Copyright © 2010 Elsevier B.V. All rights reserved.

  19. Small protease sensitive oligomers of PrPSc in distinct human prions determine conversion rate of PrP(C.

    Directory of Open Access Journals (Sweden)

    Chae Kim

    Full Text Available The mammalian prions replicate by converting cellular prion protein (PrP(C into pathogenic conformational isoform (PrP(Sc. Variations in prions, which cause different disease phenotypes, are referred to as strains. The mechanism of high-fidelity replication of prion strains in the absence of nucleic acid remains unsolved. We investigated the impact of different conformational characteristics of PrP(Sc on conversion of PrP(C in vitro using PrP(Sc seeds from the most frequent human prion disease worldwide, the Creutzfeldt-Jakob disease (sCJD. The conversion potency of a broad spectrum of distinct sCJD prions was governed by the level, conformation, and stability of small oligomers of the protease-sensitive (s PrP(Sc. The smallest most potent prions present in sCJD brains were composed only of∼20 monomers of PrP(Sc. The tight correlation between conversion potency of small oligomers of human sPrP(Sc observed in vitro and duration of the disease suggests that sPrP(Sc conformers are an important determinant of prion strain characteristics that control the progression rate of the disease.

  20. Antioxidants keep the potentially probiotic but highly oxygen-sensitive human gut bacterium Faecalibacterium prausnitzii alive at ambient air.

    Directory of Open Access Journals (Sweden)

    M Tanweer Khan

    Full Text Available The beneficial human gut microbe Faecalibacterium prausnitzii is a 'probiotic of the future' since it produces high amounts of butyrate and anti-inflammatory compounds. However, this bacterium is highly oxygen-senstive, making it notoriously difficult to cultivate and preserve. This has so far precluded its clinical application in the treatment of patients with inflammatory bowel diseases. The present studies were therefore aimed at developing a strategy to keep F. prausnitzii alive at ambient air. Our previous research showed that F. prausnitzii can survive in moderately oxygenized environments like the gut mucosa by transfer of electrons to oxygen. For this purpose, the bacterium exploits extracellular antioxidants, such as riboflavin and cysteine, that are abundantly present in the gut. We therefore tested to what extent these antioxidants can sustain the viability of F. prausnitzii at ambient air. The present results show that cysteine can facilitate the survival of F. prausnitzii upon exposure to air, and that this effect is significantly enhanced the by addition of riboflavin and the cryoprotectant inulin. The highly oxygen-sensitive gut bacterium F. prausnitzii can be kept alive at ambient air for 24 h when formulated with the antioxidants cysteine and riboflavin plus the cryoprotectant inulin. Improved formulations were obtained by addition of the bulking agents corn starch and wheat bran. Our present findings pave the way towards the biomedical exploitation of F. prausnitzii in redox-based therapeutics for treatment of dysbiosis-related inflammatory disorders of the human gut.

  1. Sensitive and selective liquid chromatography-tandem mass spectrometry method for the quantification of aniracetam in human plasma.

    Science.gov (United States)

    Zhang, Jingjing; Liang, Jiabi; Tian, Yuan; Zhang, Zunjian; Chen, Yun

    2007-10-15

    A rapid, sensitive and selective LC-MS/MS method was developed and validated for the quantification of aniracetam in human plasma using estazolam as internal standard (IS). Following liquid-liquid extraction, the analytes were separated using a mobile phase of methanol-water (60:40, v/v) on a reverse phase C18 column and analyzed by a triple-quadrupole mass spectrometer in the selected reaction monitoring (SRM) mode using the respective [M+H]+ ions, m/z 220-->135 for aniracetam and m/z 295-->205 for the IS. The assay exhibited a linear dynamic range of 0.2-100 ng/mL for aniracetam in human plasma. The lower limit of quantification (LLOQ) was 0.2 ng/mL with a relative standard deviation of less than 15%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The validated LC-MS/MS method has been successfully applied to study the pharmacokinetics of aniracetam in healthy male Chinese volunteers.

  2. Highly sensitive determination of estrone and estradiol in human serum by liquid chromatography-electrospray ionization tandem mass spectrometry.

    Science.gov (United States)

    Yamashita, Kouwa; Okuyama, Mitsunobu; Watanabe, Yoko; Honma, Seijiro; Kobayashi, Sayuri; Numazawa, Mitsuteru

    2007-10-01

    A highly sensitive and specific quantification method of estrone and estradiol in human serum was described based upon the use of picolinoyl derivatization and liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) in a positive mode. Estrogens were treated with picolinoyl chloride hydrochloride or picolinic acid and 2-methyl-6-nitrobenzoic anhydride followed by a solid-phase extraction with ODS cartridge. Picolinoyl derivatization proceeded quantitatively even in a microscale, and the picolinoyl esters provided simple positive ESI-mass spectra showing [M+H](+) as base peaks for these estrogens. The picolinoyl derivatives of these estrogens showed 100-fold higher detection response compared to underivatized intact molecules by LC-ESI-MS (selected reaction monitoring). Using this derivatization, estrogens spiked in the charcoal treated human serum samples were analyzed with limit of quantification (LOQ), intra-day accuracy and precision of 1.0pg/ml, 96.0% and 9.9% for estrone, and 0.5pg/ml, 84.4% and 12.8% for estradiol, respectively. Estrone and estradiol added to the crude serum samples were recovered with comparable LOQ and accuracy obtained for the charcoal treated serum samples as well.

  3. Small protease sensitive oligomers of PrPSc in distinct human prions determine conversion rate of PrP(C).

    Science.gov (United States)

    Kim, Chae; Haldiman, Tracy; Surewicz, Krystyna; Cohen, Yvonne; Chen, Wei; Blevins, Janis; Sy, Man-Sun; Cohen, Mark; Kong, Qingzhong; Telling, Glenn C; Surewicz, Witold K; Safar, Jiri G

    2012-01-01

    The mammalian prions replicate by converting cellular prion protein (PrP(C)) into pathogenic conformational isoform (PrP(Sc)). Variations in prions, which cause different disease phenotypes, are referred to as strains. The mechanism of high-fidelity replication of prion strains in the absence of nucleic acid remains unsolved. We investigated the impact of different conformational characteristics of PrP(Sc) on conversion of PrP(C) in vitro using PrP(Sc) seeds from the most frequent human prion disease worldwide, the Creutzfeldt-Jakob disease (sCJD). The conversion potency of a broad spectrum of distinct sCJD prions was governed by the level, conformation, and stability of small oligomers of the protease-sensitive (s) PrP(Sc). The smallest most potent prions present in sCJD brains were composed only of∼20 monomers of PrP(Sc). The tight correlation between conversion potency of small oligomers of human sPrP(Sc) observed in vitro and duration of the disease suggests that sPrP(Sc) conformers are an important determinant of prion strain characteristics that control the progression rate of the disease.

  4. Small Protease Sensitive Oligomers of PrPSc in Distinct Human Prions Determine Conversion Rate of PrPC

    Science.gov (United States)

    Kim, Chae; Haldiman, Tracy; Surewicz, Krystyna; Cohen, Yvonne; Chen, Wei; Blevins, Janis; Sy, Man-Sun; Cohen, Mark; Kong, Qingzhong; Telling, Glenn C.; Surewicz, Witold K.; Safar, Jiri G.

    2012-01-01

    The mammalian prions replicate by converting cellular prion protein (PrPC) into pathogenic conformational isoform (PrPSc). Variations in prions, which cause different disease phenotypes, are referred to as strains. The mechanism of high-fidelity replication of prion strains in the absence of nucleic acid remains unsolved. We investigated the impact of different conformational characteristics of PrPSc on conversion of PrPC in vitro using PrPSc seeds from the most frequent human prion disease worldwide, the Creutzfeldt-Jakob disease (sCJD). The conversion potency of a broad spectrum of distinct sCJD prions was governed by the level, conformation, and stability of small oligomers of the protease-sensitive (s) PrPSc. The smallest most potent prions present in sCJD brains were composed only of∼20 monomers of PrPSc. The tight correlation between conversion potency of small oligomers of human sPrPSc observed in vitro and duration of the disease suggests that sPrPSc conformers are an important determinant of prion strain characteristics that control the progression rate of the disease. PMID:22876179

  5. Rapid, simple and highly sensitive LC-ESI-MS/MS method for the quantification of tamsulosin in human plasma.

    Science.gov (United States)

    Ramakrishna, N V S; Vishwottam, K N; Manoj, S; Koteshwara, M; Wishu, S; Varma, D P

    2005-12-01

    A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of tamsulosin (I), a highly selective alpha1-adrenoceptor antagonist used for the treatment of patients with symptomatic benign prostatic hyperplasia. The analyte and internal standard, mosapride (II) were extracted by liquid-liquid extraction with diethyl ether-dichloromethane (70:30, v/v) using a Glas-Col Multi-Pulse Vortexer. The chromatographic separation was performed on a reverse phase Waters symmetry C18 column with a mobile phase of 0.03% formic acid-acetonitrile (30:70, v/v). The protonated analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions m/z 409.1 solidus in circle 228.1 and m/z 422.3 solidus in circle 198.3 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 0.1-50.0 ng/mL for tamsulosin in human plasma. The lower limit of quantitation was 100 pg/mL with a relative standard deviation of less than 10%. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A run time of 2.0 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies. (c) 2005 John Wiley & Sons, Ltd.

  6. Monocrotophos induces the expression and activity of xenobiotic metabolizing enzymes in pre-sensitized cultured human brain cells.

    Directory of Open Access Journals (Sweden)

    Vinay K Tripathi

    Full Text Available The expression and metabolic profile of cytochrome P450s (CYPs is largely missing in human brain due to non-availability of brain tissue. We attempted to address the issue by using human brain neuronal (SH-SY5Y and glial (U373-MG cells. The expression and activity of CYP1A1, 2B6 and 2E1 were carried out in the cells exposed to CYP inducers viz., 3-methylcholanthrene (3-MC, cyclophosphamide (CPA, ethanol and known neurotoxicant- monocrotophos (MCP, a widely used organophosphorous pesticide. Both the cells show significant induction in the expression and CYP-specific activity against classical inducers and MCP. The induction level of CYPs was comparatively lower in MCP exposed cells than cells exposed to classical inducers. Pre-exposure (12 h of cells to classical inducers significantly added the MCP induced CYPs expression and activity. The findings were concurrent with protein ligand docking studies, which show a significant modulatory capacity of MCP by strong interaction with CYP regulators-CAR, PXR and AHR. Similarly, the known CYP inducers- 3-MC, CPA and ethanol have also shown significantly high docking scores with all the three studied CYP regulators. The expression of CYPs in neuronal and glial cells has suggested their possible association with the endogenous physiology of the brain. The findings also suggest the xenobiotic metabolizing capabilities of these cells against MCP, if received a pre-sensitization to trigger the xenobiotic metabolizing machinery. MCP induced CYP-specific activity in neuronal cells could help in explaining its effect on neurotransmission, as these CYPs are known to involve in the synthesis/transport of the neurotransmitters. The induction of CYPs in glial cells is also of significance as these cells are thought to be involved in protecting the neurons from environmental insults and safeguard them from toxicity. The data provide better understanding of the metabolizing capability of the human brain cells against

  7. Radioecological sensitivity

    International Nuclear Information System (INIS)

    Howard, Brenda J.; Strand, Per; Assimakopoulos, Panayotis

    2003-01-01

    After the release of radionuclide into the environment it is important to be able to readily identify major routes of radiation exposure, the most highly exposed individuals or populations and the geographical areas of most concern. Radioecological sensitivity can be broadly defined as the extent to which an ecosystem contributes to an enhanced radiation exposure to Man and biota. Radioecological sensitivity analysis integrates current knowledge on pathways, spatially attributes the underlying processes determining transfer and thereby identifies the most radioecologically sensitive areas leading to high radiation exposure. This identifies where high exposure may occur and why. A framework for the estimation of radioecological sensitivity with respect to humans is proposed and the various indicators by which it can be considered have been identified. These are (1) aggregated transfer coefficients (Tag), (2) action (and critical) loads, (3) fluxes and (4) individual exposure of humans. The importance of spatial and temporal consideration of all these outputs is emphasized. Information on the extent of radionuclide transfer and exposure to humans at different spatial scales is needed to reflect the spatial differences which can occur. Single values for large areas, such as countries, can often mask large variation within the country. Similarly, the relative importance of different pathways can change with time and therefore assessments of radiological sensitivity are needed over different time periods after contamination. Radioecological sensitivity analysis can be used in radiation protection, nuclear safety and emergency preparedness when there is a need to identify areas that have the potential of being of particular concern from a risk perspective. Prior identification of radioecologically sensitive areas and exposed individuals improve the focus of emergency preparedness and planning, and contribute to environmental impact assessment for future facilities. The

  8. Targeting miR-21 enhances the sensitivity of human colon cancer HT-29 cells to chemoradiotherapy in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Deng, Jun; Lei, Wan; Fu, Jian-Chun; Zhang, Ling; Li, Jun-He; Xiong, Jian-Ping, E-mail: jpxiong@medmail.com.cn

    2014-01-17

    Highlight: •MiR-21 plays a significant role in 5-FU resistance. •This role might be attributed to targeting of hMSH2 as well as TP and DPD via miR-21 targeted hMSH2. •Indirectly targeted TP and DPD to influence 5-FU chemotherapy sensitivity. -- Abstract: 5-Fluorouracil (5-FU) is a classic chemotherapeutic drug that has been widely used for colorectal cancer treatment, but colorectal cancer cells are often resistant to primary or acquired 5-FU therapy. Several studies have shown that miR-21 is significantly elevated in colorectal cancer. This suggests that this miRNA might play a role in this resistance. In this study, we investigated this possibility and the possible mechanism underlying this role. We showed that forced expression of miR-21 significantly inhibited apoptosis, enhanced cell proliferation, invasion, and colony formation ability, promoted G1/S cell cycle transition and increased the resistance of tumor cells to 5-FU and X radiation in HT-29 colon cancer cells. Furthermore, knockdown of miR-21 reversed these effects on HT-29 cells and increased the sensitivity of HT-29/5-FU to 5-FU chemotherapy. Finally, we showed that miR-21 targeted the human mutS homolog2 (hMSH2), and indirectly regulated the expression of thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD). These results demonstrate that miR-21 may play an important role in the 5-FU resistance of colon cancer cells.

  9. Effects of a novel porphyrin-based photosensitizer on sensitive and multidrug-resistant human gastric cancer cell lines.

    Science.gov (United States)

    Chen, Jingjing; Mao, Lina; Liu, Shuping; Liang, Yanling; Wang, Sicheng; Wang, Yeyu; Zhao, Qiang; Zhang, Xiaojing; Che, Yanjun; Gao, Lijing; Liu, Tianjun

    2015-10-01

    Photodynamic therapy (PDT) has been considered to be a possible candidate approach in combating multidrug resistance (MDR) phenomenon during the treatment of cancer. To investigate the photocytotoxicity of a novel porphyrin-based photosensitizer, meso-5-[ρ-DTPA-aminophenyl]-10, 15, 20-triphenyl-porhyrin (DTP) (Fig. 1A), on MDR cells, the intracellular DTP uptake, phototoxicity and subcellular DTP localization were studied by using a human gastric cancer MGC803 cell line and its paclitaxel selected subline MGC803/PA expressing MDR phenotype. No significant difference was observed in intracellular DTP accumulation between sensitive and resistant cell lines after exposure to 1.56 μM concentration for 6h. DTP-PDT induced significant photocytotoxicity on both MGC803 and MGC803/PA cell lines and the photokilling was greater in MGC803 cell line in comparison to MGC803/PA. The fluence that caused 50% cell death was 4.42 and 6.29 J/cm(2) in MGC803 and MGC803/PA cell lines, respectively. The presence of Pgp inhibitors verapamil and cyclosporin A could not modify the intracellular DTP level in MGC803/PA cell line and the phototoxic effects. DTP was localized at lysosomes of MGC803 cell line but at lysosomes and mitochondria of MGC803/PA. Our results indicated that DTP-mediated PDT could eradicate gastric cancer cells whether or not they express MDR although the efficacy is slightly reduced in the MDR cells. The photokilling in MDR cells could not be altered by MDR inhibitor verapamil. The slightly different photocytotoxicity between sensitive and resistant cell lines could not explained by classical Pgp MDR and might be attributed to the differential intracellular DTP localization sites. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. ErbB-2 signaling plays a critical role in regulating androgen-sensitive and castration-resistant androgen receptor-positive prostate cancer cells.

    Science.gov (United States)

    Muniyan, Sakthivel; Chen, Siu-Ju; Lin, Fen-Fen; Wang, Zhengzhong; Mehta, Parmender P; Batra, Surinder K; Lin, Ming-Fong

    2015-11-01

    While androgen deprivation therapy (ADT) reduces tumor burden, autocrine growth factor loops such as human epidermal growth factor receptor 2 (HER2/ErbB-2/neu) have been proposed to contribute to prostate cancer (PCa) survival and relapse. However, the role of ErbB-2 in regulating androgen-sensitive (AS) and castration-resistant (CR) cell proliferation remains unclear. Here, we determined the role of ErbB-2 in PCa progression and survival under steroid-reduced conditions using two independent PCa cell progression models. In AR-positive androgen-independent (AI) PCa cells that exhibit the CR phenotype, ErbB-2 was constitutively activated, compared to corresponding AS PCa cells. In AS LNCaP C-33 cells, androgen-induced ErbB-2 activation through ERK1/2 mediates PCa cell proliferation. Further, the ErbB-2-specific but not EGFR-specific inhibitor suppresses basal and androgen-stimulated cell proliferation and also blocks ERK1/2 activation. ErbB-2 ectopic expression and cPAcP siRNA transfection of LNCaP C-33 cells each increases ErbB-2 tyrosine phosphorylation, correlating with increased AI PSA secretion and cell proliferation. Conversely, trapping ErbB-2 by transfected endoplasmic reticulum-targeting ScFv5R expression vector abolished DHT-induced LNCaP C-33 cell growth. Moreover, inhibition of ErbB-2 but not EGFR in AI LNCaP C-81 and MDA PCa2b-AI PCa cells significantly abolished AI cell growth. In contrast to androgens via ErbB-2/ERK1/2 signaling in AS PCa cells, the inhibition of ErbB-2 abrogated AI cell proliferation by inhibiting the cell survival protein Akt in those AI cells. These results suggest that ErbB-2 is a prominent player in mediating the ligand-dependent and -independent activation of AR in AS and AI/CR PCa cells respectively for PCa progression and survival. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. The implications of non-linear biological oscillations on human electrophysiology for electrohypersensitivity (EHS) and multiple chemical sensitivity (MCS).

    Science.gov (United States)

    Sage, Cindy

    2015-01-01

    maintenance; and resilience can be compromised. Electrohypersensitivity can be caused by successive assaults on human bioelectrochemical dynamics from exogenous electromagnetic fields (EMF) and RFR or a single acute exposure. Once sensitized, further exposures are widely reported to cause reactivity to lower and lower intensities of EMF/RFR, at which point thousand-fold lower levels can cause adverse health impacts to the electrosensitive person. Electrohypersensitivity (EHS) can be a precursor to, or linked with, multiple chemical sensitivity (MCS) based on reports of individuals who first develop one condition, then rapidly develop the other. Similarity of chemical biomarkers is seen in both conditions [histamines, markers of oxidative stress, auto-antibodies, heat shock protein (HSP), melatonin markers and leakage of the blood-brain barrier]. Low intensity pulsed microwave activation of voltage-gated calcium channels (VGCCs) is postulated as a mechanism of action for non-thermal health effects.

  12. Neuronal markers are expressed in human gliomas and NSE knockdown sensitizes glioblastoma cells to radiotherapy and temozolomide

    Directory of Open Access Journals (Sweden)

    Yan Tao

    2011-12-01

    Full Text Available Abstract Background Expression of neuronal elements has been identified in various glial tumors, and glioblastomas (GBMs with neuronal differentiation patterns have reportedly been associated with longer survival. However, the neuronal class III β-tubulin has been linked to increasing malignancy in astrocytomas. Thus, the significance of neuronal markers in gliomas is not established. Methods The expressions of class III β-tubulin, neurofilament protein (NFP, microtubule-associated protein 2 (MAP2 and neuron-specific enolase (NSE were investigated in five GBM cell lines and two GBM biopsies with immunocytochemistry and Western blot. Moreover, the expression levels were quantified by real-time qPCR under different culture conditions. Following NSE siRNA treatment we used Electric cell-substrate impedance sensing (ECIS to monitor cell growth and migration and MTS assays to study viability after irradiation and temozolomide treatment. Finally, we quantitated NSE expression in a series of human glioma biopsies with immunohistochemistry using a morphometry software, and collected survival data for the corresponding patients. The biopsies were then grouped according to expression in two halves which were compared by survival analysis. Results Immunocytochemistry and Western blotting showed that all markers except NFP were expressed both in GBM cell lines and biopsies. Notably, qPCR demonstrated that NSE was upregulated in cellular stress conditions, such as serum-starvation and hypoxia, while we found no uniform pattern for the other markers. NSE knockdown reduced the migration of glioma cells, sensitized them to hypoxia, radio- and chemotherapy. Furthermore, we found that GBM patients in the group with the highest NSE expression lived significantly shorter than patients in the low-expression group. Conclusions Neuronal markers are aberrantly expressed in human GBMs, and NSE is consistently upregulated in different cellular stress conditions

  13. miR-320 enhances the sensitivity of human colon cancer cells to chemoradiotherapy in vitro by targeting FOXM1

    Energy Technology Data Exchange (ETDEWEB)

    Wan, Lu-Ying; Deng, Jun; Xiang, Xiao-Jun; Zhang, Ling; Yu, Feng; Chen, Jun; Sun, Zhe; Feng, Miao; Xiong, Jian-Ping, E-mail: jpxiong@medmail.com.cn

    2015-02-06

    Highlights: • miR-320 plays a significant role in chemoresistance. • This role might be attribute to targeting FOXM1. • The Wnt/β-catenin pathway also involves in this chemotherapy sensitivity. - Abstract: miR-320 expression level is found to be down-regulated in human colon cancer. To date, however, its underlying mechanisms in the chemo-resistance remain largely unknown. In this study, we demonstrated that ectopic expression of miR-320 led to inhibit HCT-116 cell proliferation, invasion and hypersensitivity to 5-Fu and Oxaliplatin. Also, knockdown of miR-320 reversed these effects in HT-29 cells. Furthermore, we identified an oncogene, FOXM1, as a direct target of miR-320. In addition, miR-320 could inactive the activity of Wnt/β-catenin pathway. Finally, we found that miR-320 and FOXM1 protein had a negative correlation in colon cancer tissues and adjacent normal tissues. These findings implied that miR-320–FOXM1 axis may overcome chemo-resistance of colon cancer cells and provide a new therapeutic target for the treatment of colon cancer.

  14. Different oral sensitivities to and sensations of short-, medium-, and long-chain fatty acids in humans.

    Science.gov (United States)

    Running, Cordelia A; Mattes, Richard D

    2014-08-01

    Fatty acids that vary in chain length and degree of unsaturation have different effects on metabolism and human health. As evidence for a "taste" of nonesterified fatty acids (NEFA) accumulates, it may be hypothesized that fatty acid structures will also influence oral sensations. The present study examined oral sensitivity to caproic (C6), lauric (C12), and oleic (C18:1) acids over repeated visits. Analyses were also conducted on textural properties of NEFA emulsions and blank solutions. Oral thresholds for caproic acid were lower compared with oleic acid. Lauric acid thresholds were intermediate but not significantly different from either, likely due to lingering irritating sensations that prevented accurate discrimination. From particle size analysis, larger droplets were observed in blank solutions when mineral oil was used, leading to instability of the emulsion, which was not observed when emulsions contained NEFA or when mineral oil was removed from the blank. Rheological data showed no differences in viscosity among samples except for a slightly higher viscosity with oleic acid concentrations above 58 mM. Thus, texture was unlikely to be the property used to distinguish between the samples. Differences in oral detection and sensation of caproic, lauric, and oleic acids may be due to different properties of the fatty acid alkyl chains. Copyright © 2014 the American Physiological Society.

  15. Andrographolide sensitizes the cytotoxicity of human colorectal carcinoma cells toward cisplatin via enhancing apoptosis pathways in vitro and in vivo.

    Science.gov (United States)

    Lin, Hui-Hsuan; Shi, Ming-Der; Tseng, Hsien-Chun; Chen, Jing-Hsien

    2014-05-01

    Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, has been shown to suppress the growth and invasion of human colorectal carcinoma (CRC) Lovo cells, and trigger apoptosis in vitro. The potential of Andro as a chemotherapeutic agent in CRC was evaluated by investigating its cytotoxic effects as a single agent or in coadministration with cisplatin (CDDP). Andro potentiated the cytotoxic effect of CDDP in Lovo cells through apoptosis. The molecular mechanism for these favorable cellular response was further investigated by analyzing the apoptotic profiles, protein levels, and mRNA expression patterns of several key genes after treatments of Andro or/and CDDP. Molecular results indicated that the effect of Andro alone might be mediated via both intrinsic and extrinsic apoptotic pathways in Lovo cells. The addition of Andro to CDDP induced synergistic apoptosis, which could be corroborated to the changes in protein and mRNA levels of Bax and Bcl-2, and the increased Fas/FasL association in these cells, resulting in increased release of cytochrome c, and activation of caspases. Pretreatment of Nok-1 monoclonal antibody, a Fas signaling inhibitor, or Bax inhibitor peptide V5 repressed the Andro-induced cleavage of procaspase and the sensitization to CDDP-induced apoptosis. Finally, the combination therapy of Andro with CDDP was evidenced by its synergistic inhibition on the growth of Lovo cells in xenograft tumor studies. The results indicate that Andro, in combination with chemotherapeutics, is likely to represent a potential therapeutic strategy for CRC.

  16. Sensitive and Rapid UHPLC-MS/MS for the Analysis of Tomato Phenolics in Human Biological Samples

    Directory of Open Access Journals (Sweden)

    Miriam Martínez-Huélamo

    2015-11-01

    Full Text Available An UHPLC-MS/MS method for the quantification of tomato phenolic metabolites in human fluids was optimized and validated, and then applied in a pilot dietary intervention study with healthy volunteers. A 5-fold gain in speed (3.5 min of total run; 7-fold increase in MS sensitivity and 2-fold greater efficiency (50% peak width reduction were observed when comparing the proposed method with the reference-quality HPLC-MS/MS system, whose assay performance has been previously documented. The UHPLC-MS/MS method led to an overall improvement in the limits of detection (LOD and quantification (LOQ for all the phenolic compounds studied. The recoveries ranged between 68% and 100% in urine and 61% and 100% in plasma. The accuracy; intra- and interday precision; and stability met with the acceptance criteria of the AOAC International norms. Due to the improvements in the analytical method; the total phenolic metabolites detected in plasma and urine in the pilot intervention study were 3 times higher than those detected by HPLC-MS/MS. Comparing with traditional methods; which require longer time of analysis; the methodology described is suitable for the analysis of phenolic compounds in a large number of plasma and urine samples in a reduced time frame.

  17. An investigation of UV-induced chromosome changes in relation to lethality in normal and UV-sensitive human cells

    International Nuclear Information System (INIS)

    Scott, D.; Mashall, R.R.

    1978-01-01

    Cultured fibroblasts of excision-repair xeroderma pigmentosum (XP) cells sustain more conventional chromosome structural aberrations than normal human fibroblasts when exposed to UV. By measuring cell cycle times after UV-irradiation it is found that whereas in normal cells such aberrations are primarily confined to cells at their first post-irradiation mitosis, XP cells aberrations occur mainly in later cell cycles. The frequency of cells with chromosomal aberrations is totally inadequate to explain the degree of cell killing. Neither can an explanation be offered for the mechanism of cell killing in terms of abnormal segregation of chromosomes at mitosis (multipolarity) or failure to undergo first post-irradiation mitosis (interphase death), the frequency of the latter being small compared with the proportion of cells that die. Chromosomally normal cells pass through several cell cycles before dying. UV-sensitive cells from an individual (designated 11961), who is not an XP and has no detectable DNA repair defects, exhibit no chromosome aberrations or multipolar mitoses above control values even after UV doses that kill over 90% of cells. A small increase in sister chromatid exchange (SCE) frequencies is observed after these UV doses and the spontaneous frequency of SCEs in 11961 cells is a little higher than in normal cells studies in parallel. Cell death cannot be attributed to visible UV-induced chromosome changes. (author)

  18. QCM-D providing new horizon in the domain of sensitivity range and information for haemostasis of human plasma.

    Science.gov (United States)

    Hussain, Munawar; Northoff, Hinnak; Gehring, Frank K

    2015-04-15

    Monitoring of the haemostasis status is significant for proper therapeutic directions and decisions in surgery and innate coagulation disorders. In this regard, to gain a general overview of the plasmatic coagulation, prothrombin time (PT) tests are frequently combined with tests for activated partial thromboplastin time (aPTT). For aPTT we report for the first time that a QCM-D (Quartz Crystal Microbalances with Dissipation) based technique offers a better alternative to the standard coagulometer method in the perspective of range and information. We used heparin as anticoagulant to generate different coagulation times for human plasma. QCM-D astonishingly proved to be more sensitive and reliable than the standard coagulometer for aPTT range of upper limits of coagulation times. The established platform can monitor the fibrinogen concentration ranging from 1-6g/L (yielding R(2)=0.98 in calibration curves) along with aPTT from frequency and dissipation shifts together in a single set of measurements. Additionally the sensor layers have been tested for reusability, demonstrating no loss in sensor characteristics up to ten times measurements. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. An ultra-sensitive monoclonal antibody-based enzyme-linked immunosobent assay for dibutyl phthalate in human urinary

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Lifang [Institute of Pharmacology, Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical Science, Zhejiang University, Hangzhou 310058 (China); Lei, Yajing [Hangzhou EPIE Bio-detection Technology Limited, Hangzhou 310051 (China); Zhang, Dai; Ahmed, Shabbir [Institute of Pharmacology, Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical Science, Zhejiang University, Hangzhou 310058 (China); Chen, Shuqing, E-mail: chenshuqing@zju.edu.cn [Institute of Pharmacology, Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical Science, Zhejiang University, Hangzhou 310058 (China)

    2016-01-15

    Dibutyl phthalate (DBP) has been extensively used as a plasticizer in many daily products, which is highly toxic to human, notably affecting the reproductive and developmental function. As the previous method is expensive, time-consuming, low sensitivity and just focused on the environment. Present study was aimed to establish an ultra-sensitive and simple method based on good quality monoclonal antibody, applying to evaluate excretion level of DBP in urine samples of Chinese population directly. A monoclonal antibody was generated and characterized after fusion of myeloma cells with spleen cells isolated from BALB/c mouse. The mouse was previously immunized using a specially designed amino derivative of DBP conjugated with bovine serum albumin (BSA) as immunogen. Cross-reactivity values of the monoclonal antibody against DBP, di-isobutyl phthalate (DIBP) were observed 100% and 1.25%, while for dimethyl phthalate (DMP), butyl benzyl phthalate (BBP) and didecyl phthalate (DDP) the values were < 0.06%. The standard curve was constructed at 0–50 ng mL{sup −1} and good linearity (R{sup 2} = 0.994) was achieved. The observed IC{sub 50} (7.34 ng mL{sup −1}) and LOD (0.06 ng mL{sup −1}) values was improved 1000-fold to polyclonal antibody and 5-fold to other monoclonal antibodies. A total 1246 urine samples were analyzed and the detection frequency of DBP was observed 72.87% by ic-ELISA. The 95th percentile and mean concentration of DBP were 12.07 and 3.00 ng mL{sup −1}. Acceptable recovery rates of DBP were 97.8–114.3% and coefficients variation 5.93–11.09%. The concentrations of DBP in females were found significantly higher (p < 0.05) than males. Similarly, the DBP in middle aged and low educated individuals was found higher (p < 0.001) than the others. Considering the adverse health effects, DBP internal exposure in the Chinese population should be reduced. The ic-ELISA method has been proved as a cost effective, specific, and highly sensitive screening

  20. Comparative DNA microarray analysis of human monocyte derived dendritic cells and MUTZ-3 cells exposed to the moderate skin sensitizer cinnamaldehyde

    International Nuclear Information System (INIS)

    Python, Francois; Goebel, Carsten; Aeby, Pierre

    2009-01-01

    The number of studies involved in the development of in vitro skin sensitization tests has increased since the adoption of the EU 7th amendment to the cosmetics directive proposing to ban animal testing for cosmetic ingredients by 2013. Several studies have recently demonstrated that sensitizers induce a relevant up-regulation of activation markers such as CD86, CD54, IL-8 or IL-1β in human myeloid cell lines (e.g., U937, MUTZ-3, THP-1) or in human peripheral blood monocyte-derived dendritic cells (PBMDCs). The present study aimed at the identification of new dendritic cell activation markers in order to further improve the in vitro evaluation of the sensitizing potential of chemicals. We have compared the gene expression profiles of PBMDCs and the human cell line MUTZ-3 after a 24-h exposure to the moderate sensitizer cinnamaldehyde. A list of 80 genes modulated in both cell types was obtained and a set of candidate marker genes was selected for further analysis. Cells were exposed to selected sensitizers and non-sensitizers for 24 h and gene expression was analyzed by quantitative real-time reverse transcriptase-polymerase chain reaction. Results indicated that PIR, TRIM16 and two Nrf2-regulated genes, CES1 and NQO1, are modulated by most sensitizers. Up-regulation of these genes could also be observed in our recently published DC-activation test with U937 cells. Due to their role in DC activation, these new genes may help to further refine the in vitro approaches for the screening of the sensitizing properties of a chemical.

  1. Sulfite-induced protein radical formation in LPS aerosol-challenged mice: Implications for sulfite sensitivity in human lung disease

    Directory of Open Access Journals (Sweden)

    Ashutosh Kumar

    2018-05-01

    Full Text Available Exposure to (bisulfite (HSO3– and sulfite (SO32– has been shown to induce a wide range of adverse reactions in sensitive individuals. Studies have shown that peroxidase-catalyzed oxidation of (bisulfite leads to formation of several reactive free radicals, such as sulfur trioxide anion (.SO3–, peroxymonosulfate (–O3SOO., and especially the sulfate (SO4. – anion radicals. One such peroxidase in neutrophils is myeloperoxidase (MPO, which has been shown to form protein radicals. Although formation of (bisulfite-derived protein radicals is documented in isolated neutrophils, its involvement and role in in vivo inflammatory processes, has not been demonstrated. Therefore, we aimed to investigate (bisulfite-derived protein radical formation and its mechanism in LPS aerosol-challenged mice, a model of non-atopic asthma. Using immuno-spin trapping to detect protein radical formation, we show that, in the presence of (bisulfite, neutrophils present in bronchoalveolar lavage and in the lung parenchyma exhibit, MPO-catalyzed oxidation of MPO to a protein radical. The absence of radical formation in LPS-challenged MPO- or NADPH oxidase-knockout mice indicates that sulfite-derived radical formation is dependent on both MPO and NADPH oxidase activity. In addition to its oxidation by the MPO-catalyzed pathway, (bisulfite is efficiently detoxified to sulfate by the sulfite oxidase (SOX pathway, which forms sulfate in a two-electron oxidation reaction. Since SOX activity in rodents is much higher than in humans, to better model sulfite toxicity in humans, we induced SOX deficiency in mice by feeding them a low molybdenum diet with tungstate. We found that mice treated with the SOX deficiency diet prior to exposure to (bisulfite had much higher protein radical formation than mice with normal SOX activity. Altogether, these results demonstrate the role of MPO and NADPH oxidase in (bisulfite-derived protein radical formation and show the involvement of

  2. Sulfite-induced protein radical formation in LPS aerosol-challenged mice: Implications for sulfite sensitivity in human lung disease.

    Science.gov (United States)

    Kumar, Ashutosh; Triquigneaux, Mathilde; Madenspacher, Jennifer; Ranguelova, Kalina; Bang, John J; Fessler, Michael B; Mason, Ronald P

    2018-05-01

    Exposure to (bi)sulfite (HSO 3 - ) and sulfite (SO 3 2- ) has been shown to induce a wide range of adverse reactions in sensitive individuals. Studies have shown that peroxidase-catalyzed oxidation of (bi)sulfite leads to formation of several reactive free radicals, such as sulfur trioxide anion (.SO 3 - ), peroxymonosulfate ( - O 3 SOO.), and especially the sulfate (SO 4 . - ) anion radicals. One such peroxidase in neutrophils is myeloperoxidase (MPO), which has been shown to form protein radicals. Although formation of (bi)sulfite-derived protein radicals is documented in isolated neutrophils, its involvement and role in in vivo inflammatory processes, has not been demonstrated. Therefore, we aimed to investigate (bi)sulfite-derived protein radical formation and its mechanism in LPS aerosol-challenged mice, a model of non-atopic asthma. Using immuno-spin trapping to detect protein radical formation, we show that, in the presence of (bi)sulfite, neutrophils present in bronchoalveolar lavage and in the lung parenchyma exhibit, MPO-catalyzed oxidation of MPO to a protein radical. The absence of radical formation in LPS-challenged MPO- or NADPH oxidase-knockout mice indicates that sulfite-derived radical formation is dependent on both MPO and NADPH oxidase activity. In addition to its oxidation by the MPO-catalyzed pathway, (bi)sulfite is efficiently detoxified to sulfate by the sulfite oxidase (SOX) pathway, which forms sulfate in a two-electron oxidation reaction. Since SOX activity in rodents is much higher than in humans, to better model sulfite toxicity in humans, we induced SOX deficiency in mice by feeding them a low molybdenum diet with tungstate. We found that mice treated with the SOX deficiency diet prior to exposure to (bi)sulfite had much higher protein radical formation than mice with normal SOX activity. Altogether, these results demonstrate the role of MPO and NADPH oxidase in (bi)sulfite-derived protein radical formation and show the involvement of

  3. Detection of bony metastases of androgen-independent prostate cancer by PET-FDG

    International Nuclear Information System (INIS)

    Yeh, Samuel D. J.; Imbriaco, Massimo; Larson, Steven M.; Garza, Dahlia; Zhang Jiaju; Kalaigian, Hovanes; Finn, Ronald D.; Reddy, David; Horowitz, Steven M.; Goldsmith, Stanley J.; Scher, Howard I.

    1996-01-01

    Fourteen F-18 fluorodeoxyglucose (FDG) studies were carried out in 13 patients known to have bony metastases from carcinoma of the prostate. One patient was newly diagnosed. The remaining patients had various types of therapy and were considered hormonally resistant. The average age was 67. All patients had extensive bony metastases shown on the conventional Tc99m-MDP bone scans. Only about 18% of bony lesions apparent on the conventional bone scans showed corresponding increase of FDG uptake. Anatomical correlation was performed by using co-registered images of SPECT and PET in the same area. The positive FDG uptake was not related to the duration of illness, level of PSA, previous therapy, and magnitude of disease involvement. It appears that only a small percentage of bony metastases is associated with increased glycolysis. It is possible that other metabolic processes are more important than glycolysis for providing prostate cancer with a source of energy and nutrients

  4. Detection of bony metastases of androgen-independent prostate cancer by PET-FDG

    Energy Technology Data Exchange (ETDEWEB)

    Yeh, Samuel D. J.; Imbriaco, Massimo; Larson, Steven M.; Garza, Dahlia; Zhang Jiaju; Kalaigian, Hovanes; Finn, Ronald D.; Reddy, David; Horowitz, Steven M.; Goldsmith, Stanley J.; Scher, Howard I

    1996-08-01

    Fourteen F-18 fluorodeoxyglucose (FDG) studies were carried out in 13 patients known to have bony metastases from carcinoma of the prostate. One patient was newly diagnosed. The remaining patients had various types of therapy and were considered hormonally resistant. The average age was 67. All patients had extensive bony metastases shown on the conventional Tc99m-MDP bone scans. Only about 18% of bony lesions apparent on the conventional bone scans showed corresponding increase of FDG uptake. Anatomical correlation was performed by using co-registered images of SPECT and PET in the same area. The positive FDG uptake was not related to the duration of illness, level of PSA, previous therapy, and magnitude of disease involvement. It appears that only a small percentage of bony metastases is associated with increased glycolysis. It is possible that other metabolic processes are more important than glycolysis for providing prostate cancer with a source of energy and nutrients.

  5. A Novel Strategy to Inhibit Hedgehog Signaling and Control Growth of Androgen Independent Prostate Cancer Cells

    Science.gov (United States)

    2013-06-01

    activation) inhibition studies: Conduct western blotting and qRT-PCR for expression of Gli and Gli targets, including Ptch1, FoxL1, SNAIL , TWIST and...isolated for hybridization to Signal Transduction Pathway Finder oligo arrays (SA Biosciences) as described by the manufacturer. The resulting images were

  6. Evaluation of Roles of Interferon Gamma Regulated Genes in Inhibition of Androgen-Independent Prostate Cancer

    Science.gov (United States)

    2006-08-01

    glucose phosphate isomerase AI124792 2821 -1.8 RPN1 ribophorin I CD644128 6184 -1.8 AR SORD sorbitol dehydrogenase BC025295 6652 -1.6 AR GRHPR...identification of active principles. J Natl Cancer Inst 2002; 94: 1275-81. [151] Thomson JO, Dzubak P, Hajduch M. Prostate cancer and the food ...METABOLISM METABOLISM - CARBOHYDRATE UGDH UDP- glucose dehydrogenase BC022781 7358 -2.0 GALNT7 UDP-N-acetyl-alpha-D-galactosamine BM976847 51809 -1.8 GPI

  7. The Role of the Co-Chaperone, CHIP, in Androgen Independent Prostate Cancer

    National Research Council Canada - National Science Library

    Hassen, Waleed A

    2008-01-01

    Expression of Chip, a Co-Chaperone Which Interacts with the Androgen Receptor, Results in Loss of AR Expression and Growth Inhibition of Prostate Cancer Cells Waleed Hassen, Xiaoyoung Zheng, Antonio...

  8. An in vitro method for detecting chemical sensitization using human reconstructed skin models and its applicability to cosmetic, pharmaceutical, and medical device safety testing.

    Science.gov (United States)

    McKim, James M; Keller, Donald J; Gorski, Joel R

    2012-12-01

    Chemical sensitization is a serious condition caused by small reactive molecules and is characterized by a delayed type hypersensitivity known as allergic contact dermatitis (ACD). Contact with these molecules via dermal exposure represent a significant concern for chemical manufacturers. Recent legislation in the EU has created the need to develop non-animal alternative methods for many routine safety studies including sensitization. Although most of the alternative research has focused on pure chemicals that possess reasonable solubility properties, it is important for any successful in vitro method to have the ability to test compounds with low aqueous solubility. This is especially true for the medical device industry where device extracts must be prepared in both polar and non-polar vehicles in order to evaluate chemical sensitization. The aim of this research was to demonstrate the functionality and applicability of the human reconstituted skin models (MatTek Epiderm(®) and SkinEthic RHE) as a test system for the evaluation of chemical sensitization and its potential use for medical device testing. In addition, the development of the human 3D skin model should allow the in vitro sensitization assay to be used for finished product testing in the personal care, cosmetics, and pharmaceutical industries. This approach combines solubility, chemical reactivity, cytotoxicity, and activation of the Nrf2/ARE expression pathway to identify and categorize chemical sensitizers. Known chemical sensitizers representing extreme/strong-, moderate-, weak-, and non-sensitizing potency categories were first evaluated in the skin models at six exposure concentrations ranging from 0.1 to 2500 µM for 24 h. The expression of eight Nrf2/ARE, one AhR/XRE and two Nrf1/MRE controlled gene were measured by qRT-PCR. The fold-induction at each exposure concentration was combined with reactivity and cytotoxicity data to determine the sensitization potential. The results demonstrated that

  9. Phase-resolved optical coherence tomography and optical Doppler tomography for imaging blood flow in human skin with fast scanning speed and high velocity sensitivity

    NARCIS (Netherlands)

    Zhao, YH; Chen, ZP; Saxer, CE; Xiang, SH; de Boer, JF; Nelson, JS

    2000-01-01

    We have developed a novel phase-resolved optical coherence tomography (OCT) and optical Doppler tomography (ODT) system that uses phase information derived from a Hilbert transformation to image blood flow in human skin with fast scanning speed and high velocity sensitivity. Using the phase change

  10. Common genetic variation in the human FNDC5 locus, encoding the novel muscle-derived 'browning' factor irisin, determines insulin sensitivity.

    Directory of Open Access Journals (Sweden)

    Harald Staiger

    Full Text Available AIMS/HYPOTHESIS: Recently, the novel myokine irisin was described to drive adipose tissue 'browning', to increase energy expenditure, and to improve obesity and insulin resistance in high fat-fed mice. Here, we assessed whether common single nucleotide polymorphisms (SNPs in the FNDC5 locus, encoding the irisin precursor, contribute to human prediabetic phenotypes (overweight, glucose intolerance, insulin resistance, impaired insulin release. METHODS: A population of 1,976 individuals was characterized by oral glucose tolerance tests and genotyped for FNDC5 tagging SNPs. Subgroups underwent hyperinsulinaemic-euglycaemic clamps, magnetic resonance imaging/spectroscopy, and intravenous glucose tolerance tests. From 37 young and 14 elderly participants recruited in two different centres, muscle biopsies were obtained for the preparation of human myotube cultures. RESULTS: After appropriate adjustment and Bonferroni correction for the number of tested variants, SNPs rs16835198 and rs726344 were associated with in vivo measures of insulin sensitivity. Via interrogation of publicly available data from the Meta-Analyses of Glucose and Insulin-related traits Consortium, rs726344's effect on insulin sensitivity was replicated. Moreover, novel data from human myotubes revealed a negative association between FNDC5 expression and appropriately adjusted in vivo measures of insulin sensitivity in young donors. This finding was replicated in myotubes from elderly men. CONCLUSIONS/INTERPRETATION: This study provides evidence that the FNDC5 gene, encoding the novel myokine irisin, determines insulin sensitivity in humans. Our gene expression data point to an unexpected insulin-desensitizing effect of irisin.

  11. Common genetic variation in the human FNDC5 locus, encoding the novel muscle-derived 'browning' factor irisin, determines insulin sensitivity.

    Science.gov (United States)

    Staiger, Harald; Böhm, Anja; Scheler, Mika; Berti, Lucia; Machann, Jürgen; Schick, Fritz; Machicao, Fausto; Fritsche, Andreas; Stefan, Norbert; Weigert, Cora; Krook, Anna; Häring, Hans-Ulrich; de Angelis, Martin Hrabě

    2013-01-01

    Recently, the novel myokine irisin was described to drive adipose tissue 'browning', to increase energy expenditure, and to improve obesity and insulin resistance in high fat-fed mice. Here, we assessed whether common single nucleotide polymorphisms (SNPs) in the FNDC5 locus, encoding the irisin precursor, contribute to human prediabetic phenotypes (overweight, glucose intolerance, insulin resistance, impaired insulin release). A population of 1,976 individuals was characterized by oral glucose tolerance tests and genotyped for FNDC5 tagging SNPs. Subgroups underwent hyperinsulinaemic-euglycaemic clamps, magnetic resonance imaging/spectroscopy, and intravenous glucose tolerance tests. From 37 young and 14 elderly participants recruited in two different centres, muscle biopsies were obtained for the preparation of human myotube cultures. After appropriate adjustment and Bonferroni correction for the number of tested variants, SNPs rs16835198 and rs726344 were associated with in vivo measures of insulin sensitivity. Via interrogation of publicly available data from the Meta-Analyses of Glucose and Insulin-related traits Consortium, rs726344's effect on insulin sensitivity was replicated. Moreover, novel data from human myotubes revealed a negative association between FNDC5 expression and appropriately adjusted in vivo measures of insulin sensitivity in young donors. This finding was replicated in myotubes from elderly men. This study provides evidence that the FNDC5 gene, encoding the novel myokine irisin, determines insulin sensitivity in humans. Our gene expression data point to an unexpected insulin-desensitizing effect of irisin.

  12. High-sensitivity TMS/fMRI of the Human Motor Cortex Using a Dedicated Multichannel MR Coil.

    Science.gov (United States)

    Navarro de Lara, Lucia I; Tik, Martin; Woletz, Michael; Frass-Kriegl, Roberta; Moser, Ewald; Laistler, Elmar; Windischberger, Christian

    2017-04-15

    To validate a novel setup for concurrent TMS/fMRI in the human motor cortex based on a dedicated, ultra-thin, multichannel receive MR coil positioned between scalp and TMS system providing greatly enhanced sensitivity compared to the standard birdcage coil setting. A combined TMS/fMRI design was applied over the primary motor cortex based on 1Hz stimulation with stimulation levels of 80%, 90%, 100%, and 110% of the individual active motor threshold, respectively. Due to the use of a multichannel receive coil we were able to use multiband-accelerated (MB=2) EPI sequences for the acquisition of functional images. Data were analysed with SPM12 and BOLD-weighted signal intensity time courses were extracted in each subject from two local maxima (individual functional finger tapping localiser, fixed MNI coordinate of the hand knob) next to the hand area of the primary motor cortex (M1) and from the global maximum. We report excellent image quality without noticeable signal dropouts or image distortions. Parameter estimates in the three peak voxels showed monotonically ascending activation levels over increasing stimulation intensities. Across all subjects, mean BOLD signal changes for 80%, 90%, 100%, 110% of the individual active motor threshold were 0.43%, 0.63%, 1.01%, 2.01% next to the individual functional finger tapping maximum, 0.73%, 0.91%, 1.34%, 2.21% next to the MNI-defined hand knob and 0.88%, 1.09%, 1.65%, 2.77% for the global maximum, respectively. Our results show that the new setup for concurrent TMS/fMRI experiments using a dedicated MR coil array allows for high-sensitivity fMRI particularly at the site of stimulation. Contrary to the standard birdcage approach, the results also demonstrate that the new coil can be successfully used for multiband-accelerated EPI acquisition. The gain in flexibility due to the new coil can be easily combined with neuronavigation within the MR scanner to allow for accurate targeting in TMS/fMRI experiments. Copyright

  13. Adherence performances of pressure sensitive adhesives on a model viscoelastic synthetic film: a tool for the understanding of adhesion on the human skin.

    Science.gov (United States)

    Renvoise, Julien; Burlot, Delphine; Marin, Gérard; Derail, Christophe

    2009-02-23

    This work deals with the rheological behavior and adherence properties of pressure sensitive adhesive formulations dedicated to medical applications. We have developed a specific viscoelastic substrate which mimics adhesion on human skin to measure the adherence properties of PSAs when they are stuck on the human skin. By comparing peeling results of PSAs, dedicated to medical applications, stuck on human skin and on this viscoelastic substrate we show that this substrate, based on a blend of natural proteins, presents a better representation of the interactions occurring at the skin/adhesive interface than conventional substrates used for peel test (i.e. glass and steel).

  14. Semi-Metric Topology of the Human Connectome: Sensitivity and Specificity to Autism and Major Depressive Disorder.

    Directory of Open Access Journals (Sweden)

    Tiago Simas

    Full Text Available The human functional connectome is a graphical representation, consisting of nodes connected by edges, of the inter-relationships of blood oxygenation-level dependent (BOLD time-series measured by MRI from regions encompassing the cerebral cortices and, often, the cerebellum. Semi-metric analysis of the weighted, undirected connectome distinguishes an edge as either direct (metric, such that there is no alternative path that is accumulatively stronger, or indirect (semi-metric, where one or more alternative paths exist that have greater strength than the direct edge. The sensitivity and specificity of this method of analysis is illustrated by two case-control analyses with independent, matched groups of adolescents with autism spectrum conditions (ASC and major depressive disorder (MDD.Significance differences in the global percentage of semi-metric edges was observed in both groups, with increases in ASC and decreases in MDD relative to controls. Furthermore, MDD was associated with regional differences in left frontal and temporal lobes, the right limbic system and cerebellum. In contrast, ASC had a broadly increased percentage of semi-metric edges with a more generalised distribution of effects and some areas of reduction. In summary, MDD was characterised by localised, large reductions in the percentage of semi-metric edges, whilst ASC is characterised by more generalised, subtle increases. These differences were corroborated in greater detail by inspection of the semi-metric backbone for each group; that is, the sub-graph of semi-metric edges present in >90% of participants, and by nodal degree differences in the semi-metric connectome.These encouraging results, in what we believe is the first application of semi-metric analysis to neuroimaging data, raise confidence in the methodology as potentially capable of detection and characterisation of a range of neurodevelopmental and psychiatric disorders.

  15. Rapid and sensitive determination of strychnine and brucine in human urine by capillary electrophoresis with field-amplified sample stacking.

    Science.gov (United States)

    Li, Junmei; Jiang, Ye

    2010-02-01

    A simple, rapid, sensitive and low-cost method using capillary electrophoresis (CE) coupled with field-amplified sample stacking (FASS) has been developed and validated for the simultaneous determination of strychnine and brucine residues in human urine. Before sample loading, a water plug (3.5 kPa, 3 s) was injected to contain sample cations and to permit FASS. Electrokinetic injection at a voltage (20 kV, 25 s) was then used to introduce cations. Separation was performed using 20 mM acetate buffer (pH 3.8) with an applied voltage of 20 kV. The calibration curves were linear over a range of 8.00-2.56 infinity 10(2) ng/mL (r = 0.9995) for strychnine and 10.0-3.20 x 10(2) ng/mL (r = 0.9999) for brucine. Extraction recoveries in urine were greater than 79.6 and 82.8% for strychnine and brucine, respectively, with an RSD of less than 4.9%. The detection limits (signal-to-noise ratio 3) for strychnine and brucine were 2.00 and 2.50 ng/mL, respectively. A urine sample from one healthy female volunteer (26 years old, 50 kg) was pretreated and analyzed. Strychnine and brucine levels in urine could be detected 24 h after administration. On these grounds, this method was feasible for application to preliminary screening of trace levels of abused drugs for both doping control and forensic analysis. (c) 2009 John Wiley & Sons, Ltd.

  16. Simple and sensitive high performance liquid chromatographic method for the simultaneous quantitation of the phenylalanine in human plasma

    Directory of Open Access Journals (Sweden)

    Hossein Danafar

    2015-09-01

    Full Text Available Phenylalanine (Phe is the most reliable indicator for the diagnosis of phenylketonuria (PKU. The purpose of this study is to establish a reliable and quick method for the assignment of Phe in peripheral capillary blood from newborns and children by high performance liquid chromatography with ultraviolet detection (HPLC-UV. PKU is an inborn error of metabolism characterized by the inability of the body to use Phe. A rapid and sensitive high performance liquid chromatographic (HPLC method has been developed for determination of Phe in plasma. The method uses a protein precipitation step with sulfosalicilic acid for sample preparation by separation on a Nova-pack C18 column using sodium acetate buffer and acetonitrile (94: 6 v/v adjusted to pH 6.5 with glacial acetic acid. The eluted peaks detected by a UV detector was set at wavelength of 215 nm. The method was validated in the range of Phe concentrations from 0.1 to 20 µg/ml. The limits of detection (LOD and quantitation (LOQ of the method were 0.05 and 0.1 µg/ml, respectively. The average drug recovery from plasma was 88.60 percent throughout the linear concentration range., with the average within-run and between-run accuracy values of 103.3 and 115.350, respectively. The method is quick, easy, very steady and precise for the screen, assignment, and evaluation of Phe in human plasma by HPLC, which is particularly a useful way for screening and diagnosis of PKU and monitoring of a diet therapy.

  17. Small interfering RNA (siRNA) against Slug induces apoptosis and sensitizes human anaplastic thyroid carcinoma cells to doxorubicin.

    Science.gov (United States)

    Pan, Yinghua; Liu, Peiji; Chen, Deng; Dou, Linying

    2017-01-01

    Anaplastic thyroid carcinoma (ATC) is one of the most aggressive human cancers and often shows resistance to multimodal therapeutic approaches. It has been shown that the transcriptional repressor Slug inhibits the chemotherapeutic agent-induced apoptosis of cancer cells. We evaluated whether targeting of Slug could augment doxorubicin (DOX)-induced apoptosis of ATC cells. We also determined changes in PUMA (p53-upregulated modulator of apoptosis) expression levels to identify possible mechanisms of their combined actions. SW1736 cells were transfected with Slug siRNA or/and PUMA siRNA and then exposed to DOX (0.1, 1, and 5 μ M) for selected times. Scrambled siRNA was used as a control. The effects on cell viability were determined via MTT assay. Apoptosis was assessed using TUNEL assays and annexin V staining, and was confirmed by flow cytometry analyses. Slug and PUMA levels were determined using western blotting, RT-PCR and immunofluorescence analyses. We used a subcutaneous implanted tumor model of SW1736 cells in nude mice to assess the effects of Slug silencing in combination with DOX on tumor development. Apoptosis was assessed via TUNEL assay. Targeting of Slug using siRNA inhibits growth of SW1736 cells and sensitizes SW1736 cells to DOX in vitro and vivo. Targeting of Slug combined with DOX led to lower cell viability than treatment with DOX alone in SW1736 cells. TUNEL and flow cytometry analyses showed that targeting of Slug enhanced DOX-induced apoptosis of SW1736 cells. In addition, targeting of Slug increased PUMA expression, and targeting of PUMA restored the chemoresistance of SW1736/Slug siRNA cells to DOX. Knockdown of Slug enhanced the antitumor activity of DOX in SW1736 cells via induction of PUMA upregulation. Our results suggest that targeting of Slug has good potential for the development of new therapeutic strategies for ATC.

  18. ATP-sensitive potassium channel: a novel target for protection against UV-induced human skin cell damage.

    Science.gov (United States)

    Cao, Cong; Healey, Sarah; Amaral, Ashley; Lee-Couture, Avery; Wan, Shu; Kouttab, Nicola; Chu, Wenming; Wan, Yinsheng

    2007-07-01

    Ultraviolet radiation (UV) induces cell damages leading to skin photoaging and skin cancer. ATP-sensitive potassium (K(ATP)) channel openers (KCOs) have been shown to exert significant myocardial preservation and neuroprotection in vitro and in vivo, and yet the potential role of those KCOs in protection against UV-induced skin cell damage is unknown. We investigated the effects of pinacidil and diazoxide, two classical KCOs, on UV-induced cell death using cultured human keratinocytes (HaCat cells). Here, we demonstrated for the first time that Kir 6.1, Kir 6.2 and SUR2 subunits of K(ATP) channels are functionally expressed in HaCaT cells and both non-selective K(ATP) channel opener pinacidil and mitoK(ATP) (mitochondrial K(ATP)) channel opener diazoxide attenuated UV-induced keratinocytes cell death. The protective effects were abolished by both non-selective K(ATP) channel blocker glibenclamide and selective mitoK(ATP) channel blocker 5-hydroxydecanoate (5-HD). Also, activation of K(ATP) channel with pinacidil or diazoxide resulted in suppressive effects on UV-induced MAPK activation and reactive oxygen species (ROS) production. Unexpectedly, we found that the level of intracellular ROS was slightly elevated in HaCaT cells when treated with pinacidil or diazoxide alone. Furthermore, UV-induced mitochondrial membrane potential loss, cytochrome c release and ultimately apoptotic cell death were also inhibited by preconditioning with pinacidil and diazoxide, and their effects were reversed by glibenclamide and 5-HD. Taken together, we contend that mitoK(ATP) is likely to contribute the protection against UV-induced keratinocytes cell damage. Our findings suggest that K(ATP) openers such as pinacidil and diazoxide may be utilized to prevent from UV-induced skin aging.

  19. Sensitive Detection of Measles Virus Infection in the Blood and Tissues of Humanized Mouse by One-step Quantitative RT-PCR

    Directory of Open Access Journals (Sweden)

    Shota eIkeno

    2013-10-01

    Full Text Available Live attenuated measles virus (MV has long been recognized as a safe and effective vaccine, and it has served as the basis for development of various MV-based vaccines. However, because MV is a human-tropic virus, the evaluation of MV-based vaccines has been hampered by the lack of a small-animal model. The humanized mouse, a recently developed system in which an immunodeficient mouse is transplanted with human fetal tissues or hematopoietic stem cells, may represent a suitable model. Here, we developed a sensitive one-step quantitative reverse transcription (qRT PCR that simultaneously measures nucleocapsid (N and human RNase P mRNA levels. The results can be used to monitor MV infection in a humanized mouse model. Using this method, we elucidated the replication kinetics of MV expressing EGFP both in vitro and in humanized mice in parallel with flow-cytometric analysis. Because our qRT-PCR system was sensitive enough to detect MV expression using RNA extracted from a small number of cells, it can be used to monitor MV infection in humanized mice by sequential blood sampling.

  20. Suppression of gastric acid increases the risk of developing immunoglobulin E-mediated drug hypersensitivity: human diclofenac sensitization and a murine sensitization model.

    Science.gov (United States)

    Riemer, A B; Gruber, S; Pali-Schöll, I; Kinaciyan, T; Untersmayr, E; Jensen-Jarolim, E

    2010-03-01

    Hypersensitivity reactions towards non-steroidal anti-inflammatory drugs (NSAID) are common, although true allergies are detectable only in a subgroup of patients. The current study was prompted by a case observation, where a patient experienced generalized urticaria following his second course of diclofenac and proton pump inhibitor medication, and was found to have diclofenac-specific IgE. During recent years, our group has been investigating the importance of gastric digestion in the development of food allergies, demonstrating anti-acid medication as a risk factor for sensitization against food proteins. Here, we aimed to investigate whether the mechanism of food allergy induction described can also be causative in NSAID allergy, using diclofenac as a paradigm. We subjected BALB/c mice to several oral immunization regimens modelled after the patient's medication intake. Diclofenac was applied with or without gastric acid suppression, in various doses, alone or covalently coupled to albumin, a protein abundant in gastric juices. Immune responses were assessed on the antibody level, and functionally examined by in vitro and in vivo crosslinking assays. Only mice receiving albumin-coupled diclofenac under gastric acid suppression developed anti-diclofenac IgG1 and IgE, whereas no immune responses were induced by the drug alone or without gastric acid suppression. Antibody induction was dose dependent with the group receiving the higher dose of the drug showing sustained anti-diclofenac titres. The antibodies induced triggered basophil degranulation in vitro and positive skin tests in vivo. Gastric acid suppression was found to be a causative mechanism in the induction of IgE-mediated diclofenac allergy.

  1. Antibodies Elicited by Multiple Envelope Glycoprotein Immunogens in Primates Neutralize Primary Human Immunodeficiency Viruses (HIV-1) Sensitized by CD4-Mimetic Compounds.

    Science.gov (United States)

    Madani, Navid; Princiotto, Amy M; Easterhoff, David; Bradley, Todd; Luo, Kan; Williams, Wilton B; Liao, Hua-Xin; Moody, M Anthony; Phad, Ganesh E; Vázquez Bernat, Néstor; Melillo, Bruno; Santra, Sampa; Smith, Amos B; Karlsson Hedestam, Gunilla B; Haynes, Barton; Sodroski, Joseph

    2016-05-15

    The human immunodeficiency virus (HIV-1) envelope glycoproteins (Env) mediate virus entry through a series of complex conformational changes triggered by binding to the receptors CD4 and CCR5/CXCR4. Broadly neutralizing antibodies that recognize conserved Env epitopes are thought to be an important component of a protective immune response. However, to date, HIV-1 Env immunogens that elicit broadly neutralizing antibodies have not been identified, creating hurdles for vaccine development. Small-molecule CD4-mimetic compounds engage the CD4-binding pocket on the gp120 exterior Env and induce Env conformations that are highly sensitive to neutralization by antibodies, including antibodies directed against the conserved Env region that interacts with CCR5/CXCR4. Here, we show that CD4-mimetic compounds sensitize primary HIV-1 to neutralization by antibodies that can be elicited in monkeys and humans within 6 months by several Env vaccine candidates, including gp120 monomers. Monoclonal antibodies directed against the gp120 V2 and V3 variable regions were isolated from the immunized monkeys and humans; these monoclonal antibodies neutralized a primary HIV-1 only when the virus was sensitized by a CD4-mimetic compound. Thus, in addition to their direct antiviral effect, CD4-mimetic compounds dramatically enhance the HIV-1-neutralizing activity of antibodies that can be elicited with currently available immunogens. Used as components of microbicides, the CD4-mimetic compounds might increase the protective efficacy of HIV-1 vaccines. Preventing HIV-1 transmission is a high priority for global health. Eliciting antibodies that can neutralize transmitted strains of HIV-1 is difficult, creating problems for the development of an effective vaccine. We found that small-molecule CD4-mimetic compounds sensitize HIV-1 to antibodies that can be elicited in vaccinated humans and monkeys. These results suggest an approach to prevent HIV-1 sexual transmission in which a virus-sensitizing

  2. Antibodies Elicited by Multiple Envelope Glycoprotein Immunogens in Primates Neutralize Primary Human Immunodeficiency Viruses (HIV-1) Sensitized by CD4-Mimetic Compounds

    Science.gov (United States)

    Madani, Navid; Princiotto, Amy M.; Easterhoff, David; Bradley, Todd; Luo, Kan; Williams, Wilton B.; Liao, Hua-Xin; Moody, M. Anthony; Phad, Ganesh E.; Vázquez Bernat, Néstor; Melillo, Bruno; Santra, Sampa; Smith, Amos B.; Karlsson Hedestam, Gunilla B.; Haynes, Barton

    2016-01-01

    ABSTRACT The human immunodeficiency virus (HIV-1) envelope glycoproteins (Env) mediate virus entry through a series of complex conformational changes triggered by binding to the receptors CD4 and CCR5/CXCR4. Broadly neutralizing antibodies that recognize conserved Env epitopes are thought to be an important component of a protective immune response. However, to date, HIV-1 Env immunogens that elicit broadly neutralizing antibodies have not been identified, creating hurdles for vaccine development. Small-molecule CD4-mimetic compounds engage the CD4-binding pocket on the gp120 exterior Env and induce Env conformations that are highly sensitive to neutralization by antibodies, including antibodies directed against the conserved Env region that interacts with CCR5/CXCR4. Here, we show that CD4-mimetic compounds sensitize primary HIV-1 to neutralization by antibodies that can be elicited in monkeys and humans within 6 months by several Env vaccine candidates, including gp120 monomers. Monoclonal antibodies directed against the gp120 V2 and V3 variable regions were isolated from the immunized monkeys and humans; these monoclonal antibodies neutralized a primary HIV-1 only when the virus was sensitized by a CD4-mimetic compound. Thus, in addition to their direct antiviral effect, CD4-mimetic compounds dramatically enhance the HIV-1-neutralizing activity of antibodies that can be elicited with currently available immunogens. Used as components of microbicides, the CD4-mimetic compounds might increase the protective efficacy of HIV-1 vaccines. IMPORTANCE Preventing HIV-1 transmission is a high priority for global health. Eliciting antibodies that can neutralize transmitted strains of HIV-1 is difficult, creating problems for the development of an effective vaccine. We found that small-molecule CD4-mimetic compounds sensitize HIV-1 to antibodies that can be elicited in vaccinated humans and monkeys. These results suggest an approach to prevent HIV-1 sexual transmission in

  3. A sensitive ELISA for human tissue-type plasminogen activator applicable to the study of acute release from cultured human endothelial cells

    NARCIS (Netherlands)

    Schrauwen, Y.; Emeis, J.J.; Kooistra, T.

    1994-01-01

    The development of a highly sensitive, enzyme-linked immunosorbent assay (ELISA) for tissue-type plasminogen activator (tPA) is described. The use of a biotin-avidin system resulted in a detection limit of 10 pg of tPA per ml, which is 50 to 150 times more sensitive than commercially-available

  4. Ionizing radiation and nitric oxide donor sensitize Fas-induced apoptosis via up-regulation of Fas in human cervical cancer cells

    International Nuclear Information System (INIS)

    Park, In Chul; Woo, Sang Hyeok; Park, Myung Jin; Lee, Hyung Chahn; Lee Su Jae; Hong, Young Joon; Lee, Seung Hoon; Hong, Seok II; Rhee, Chang Hun

    2004-01-01

    Fas/CD95/Apo1 is a transmembrane receptor known to trigger apoptotic cell death in several cell types. In the present study, we showed that ionizing radiation (IR) and NO donor, S-nitroso-N-acetyl-penicillamine (SNAP), sensitized Fas-induced apoptotic cell death of HeLa human cervical cancers. Suboptimal dose of IR and SNAP up-regulated cell-surface Fas antigen, detected by FACScan using FITC-anti-Fas antibody. When combined with IR or SNAP, agonistic anti-Fas antibody CH-11 resulted in marked enhancement of apoptosis. This sensitization was completely abrogated by anti-Fas neutralizing antibody ZB4. During the IR and SNAP sensitized Fas-induced apoptosis, mitochondria permeabilization, cytochrome c release, and DNA fragmentation were detected. Furthermore, combined treatment of IR and SNAP additively up-regulated the surface Fas protein expression and sensitized Fas-induced apoptosis. Our finding demonstrate that sensitization of HeLa cervical cells to Fas-mediated apoptosis by IR and NO donor is most likely due to the up-regulation of Fas expression and also provides a means with which to sensitize tumors to the killing effects of cancer therapy via the Fas receptor

  5. Improved physiologically based pharmacokinetic model for oral exposures to chromium in mice, rats, and humans to address temporal variation and sensitive populations.

    Science.gov (United States)

    Kirman, C R; Suh, M; Proctor, D M; Hays, S M

    2017-06-15

    A physiologically based pharmacokinetic (PBPK) model for hexavalent chromium [Cr(VI)] in mice, rats, and humans developed previously (Kirman et al., 2012, 2013), was updated to reflect an improved understanding of the toxicokinetics of the gastrointestinal tract following oral exposures. Improvements were made to: (1) the reduction model, which describes the pH-dependent reduction of Cr(VI) to Cr(III) in the gastrointestinal tract under both fasted and fed states; (2) drinking water pattern simulations, to better describe dosimetry in rodents under the conditions of the NTP cancer bioassay; and (3) parameterize the model to characterize potentially sensitive human populations. Important species differences, sources of non-linear toxicokinetics, and human variation are identified and discussed within the context of human health risk assessment. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Human serum levels of fetal antigen 1 (FA1/Dlk1) increase with obesity, are negatively associated with insulin sensitivity and modulate inflammation in vitro

    DEFF Research Database (Denmark)

    Chacón, M R; Miranda, M; Jensen, C H

    2008-01-01

    in the context of obesity and insulin sensitivity (S(i)); and n=61 severely obese women before and after bariatric surgery. The response in vitro to FA1 protein on human cell lines of monocytes, preadipocytes and mature adipocytes was studied. MEASUREMENTS: Anthropometrical parameters: body mass index, waist...... levels. In severe obesity, serum levels of FA1 decreased 1.4-fold 6 months after bariatric surgery. In vitro assays with FA1 protein on human monocytes and adipocytes cell lines modified the expression of pro-inflammatory cytokines and adipokines (tumor necrosis factor-alpha (TNFalpha), monocyte...

  7. Interferon-gamma and tumor necrosis factor-alpha sensitize primarily resistant human endometrial stromal cells to Fas-mediated apoptosis

    DEFF Research Database (Denmark)

    Fluhr, Herbert; Krenzer, Stefanie; Stein, Gerburg M

    2007-01-01

    of Fas by an agonistic anti-Fas antibody. Incubation of ESCs with the early embryonic signal human chorionic gonadotropin (hCG, CGB) does not influence their reaction to Fas stimulation. The sensitizing effect of IFN-gamma and TNF-alpha was accompanied by a significant upregulation of Fas and FLICE......The subtle interaction between the implanting embryo and the maternal endometrium plays a pivotal role during the process of implantation. Human endometrial stromal cells (ESCs) express Fas and the implanting trophoblast cells secrete Fas ligand (FASLG, FasL), suggesting a possible role for Fas...

  8. A Sensitivity Study of Human Errors in Optimizing Surveillance Test Interval (STI) and Allowed Outage Time (AOT) of Standby Safety System

    International Nuclear Information System (INIS)

    Chung, Dae Wook; Shin, Won Ky; You, Young Woo; Yang, Hui Chang

    1998-01-01

    In most cases, the surveillance test intervals (STIs), allowed outage times (AOTS) and testing strategies of safety components in nuclear power plant are prescribed in plant technical specifications. And, in general, it is required that standby safety system shall be redundant (i.e., composed of multiple components) and these components are tested by either staggered test strategy or sequential test strategy. In this study, a linear model is presented to incorporate the effects of human errors associated with test into the evaluation of unavailability. The average unavailabilities of 1/4, 2/4 redundant systems are computed considering human error and testing strategy. The adverse effects of test on system unavailability, such as component wear and test-induced transient have been modelled. The final outcome of this study would be the optimized human error domain from 3-D human error sensitivity analysis by selecting finely classified segment. The results of sensitivity analysis show that the STI and AOT can be optimized provided human error probability is maintained within allowable range. (authors)

  9. Inhibition of WNT signaling reduces differentiation and induces sensitivity to doxorubicin in human malignant neuroblastoma SH-SY5Y cells.

    Science.gov (United States)

    Suebsoonthron, Junjira; Jaroonwitchawan, Thiranut; Yamabhai, Montarop; Noisa, Parinya

    2017-06-01

    Neuroblastoma is one of the most common cancers in infancy, arising from the neuroblasts during embryonic development. This cancer is difficult to treat and resistance to chemotherapy is often found; therefore, clinical trials of novel therapeutic approaches, such as targeted-cancer signaling, could be an alternative for a better treatment. WNT signaling plays significant roles in the survival, proliferation, and differentiation of human neuroblastoma. In this report, WNT signaling of a malignant human neuroblastoma cell line, SH-SY5Y cells, was inhibited by XAV939, a specific inhibitor of the Tankyrase enzyme. XAV939 treatment led to the reduction of β-catenin within the cells, confirming its inhibitory effect of WNT. The inhibition of WNT signaling by XAV939 did not affect cell morphology, survival, and proliferation; however, the differentiation and sensitivity to anticancer drugs of human neuroblastoma cells were altered. The treatment of XAV939 resulted in the downregulation of mature neuronal markers, including β-tubulin III, PHOX2A, and PHOX2B, whereas neural progenitor markers (PAX6, TFAP2α, and SLUG) were upregulated. In addition, the combination of XAV939 significantly enhanced the sensitivity of SH-SY5Y and IMR-32 cells to doxorubicin in both 2D and 3D culture systems. Microarray gene expression profiling suggested numbers of candidate target genes of WNT inhibition by XAV939, in particular, p21, p53, ubiquitin C, ZBED8, MDM2, CASP3, and FZD1, and this explained the enhanced sensitivity of SH-SY5Y cells to doxorubicin. Altogether, these results proposed that the altered differentiation of human malignant neuroblastoma cells by inhibiting WNT signaling sensitized the cells to anticancer drugs. This approach could thus serve as an effective treatment option for aggressive brain malignancy.

  10. Common Genetic Variation in the Human FNDC5 Locus, Encoding the Novel Muscle-Derived ‘Browning’ Factor Irisin, Determines Insulin Sensitivity

    Science.gov (United States)

    Staiger, Harald; Böhm, Anja; Scheler, Mika; Berti, Lucia; Machann, Jürgen; Schick, Fritz; Machicao, Fausto; Fritsche, Andreas; Stefan, Norbert; Weigert, Cora; Krook, Anna; Häring, Hans-Ulrich; de Angelis, Martin Hrabě

    2013-01-01

    Aims/hypothesis Recently, the novel myokine irisin was described to drive adipose tissue ‘browning’, to increase energy expenditure, and to improve obesity and insulin resistance in high fat-fed mice. Here, we assessed whether common single nucleotide polymorphisms (SNPs) in the FNDC5 locus, encoding the irisin precursor, contribute to human prediabetic phenotypes (overweight, glucose intolerance, insulin resistance, impaired insulin release). Methods A population of 1,976 individuals was characterized by oral glucose tolerance tests and genotyped for FNDC5 tagging SNPs. Subgroups underwent hyperinsulinaemic-euglycaemic clamps, magnetic resonance imaging/spectroscopy, and intravenous glucose tolerance tests. From 37 young and 14 elderly participants recruited in two different centres, muscle biopsies were obtained for the preparation of human myotube cultures. Results After appropriate adjustment and Bonferroni correction for the number of tested variants, SNPs rs16835198 and rs726344 were associated with in vivo measures of insulin sensitivity. Via interrogation of publicly available data from the Meta-Analyses of Glucose and Insulin-related traits Consortium, rs726344’s effect on insulin sensitivity was replicated. Moreover, novel data from human myotubes revealed a negative association between FNDC5 expression and appropriately adjusted in vivo measures of insulin sensitivity in young donors. This finding was replicated in myotubes from elderly men. Conclusions/interpretation This study provides evidence that the FNDC5 gene, encoding the novel myokine irisin, determines insulin sensitivity in humans. Our gene expression data point to an unexpected insulin-desensitizing effect of irisin. PMID:23637927

  11. How sensitizing is chlorocresol?

    DEFF Research Database (Denmark)

    Andersen, Klaus Ejner; Hamann, K

    1984-01-01

    Chlorocresol is a biocide with widespread use in industry and pharmaceutical products. It is an occasional human contact sensitizer. The sensitizing potential of chlorocresol was judged strong using the guinea pig maximization test (GPMT) and doubtful in the less sensitive open epicutaneous test ...

  12. A rapid, sensitive and validated method for the determination of ondansetron in human plasma by reversed-phase high-pressure liquid chromatography.

    Science.gov (United States)

    Chandrasekar, Durairaj; Ramakrishna, Sistla; Diwan, Prakash V

    2004-01-01

    A simple and sensitive method for the determination of ondansetron (CAS 116002-70-1) in human plasma was developed using high-pressure liquid chromatography (HPLC). The procedure involves extraction of human plasma with tertiary butyl methyl ether containing 2 mol/l sodium hydroxide, followed by reversed-phase HPLC using a LiChrospher 100 RP-18e 5 microm column and UV detection at 305 nm. The retention times of ondansetron and internal standard (propranolol hydrochloride, CAS 318-98-9) were 9.38 and 13.40 min, respectively. The calibration curves were linear over the range of 10 ng/ml (lower limit of quantitation, LOQ) and 380 ng/ml for ondansetron. The intra- and inter-assay coefficients of variation for all the criteria of validation were less than 15% over the linearity range. Ondansetron was stable upon storage in human plasma. The sensitivity and precision of the method were within the accepted limits (< 15 %) throughout the validation period. The present method is useful for determination of plasma concentrations of ondansetron during human pharmacokinetic studies.

  13. Drug and radiation sensitivity measurements of successful primary monolayer culturing of human tumor cells using cell-adhesive matrix and supplemented medium

    International Nuclear Information System (INIS)

    Baker, F.L.; Spitzer, G.; Ajani, J.A.

    1986-01-01

    The limitations of the agar suspension culture method for primary culturing of human tumor cells prompted development of a monolayer system optimized for cell adhesion and growth. This method grew 83% of fresh human tumor cell biopsy specimens, cultured and not contaminated, from a heterogeneous group of 396 tumors including lung cancer (93 of 114, 82%); melanoma (54 of 72, 75%); sarcoma (46 of 59, 78%); breast cancer (35 of 39, 90%); ovarian cancer (16 of 21, 76%); and a miscellaneous group consisting of gastrointestinal, genitourinary, mesothelioma, and unknown primaries (78 of 91, 86%). Cell growth was characterized morphologically with Papanicolaoustained coverslip cultures and cytogenetically with Giemsastained metaphase spreads. Morphological features such as nuclear pleomorphism, chromatin condensation, basophilic cytoplasm, and melanin pigmentation were routinely seen. Aneuploid metaphases were seen in 90% of evaluable cultures, with 15 of 28 showing 70% or more aneuploid metaphases. Colony-forming efficiency ranged between 0.01 and 1% of viable tumor cells, with a median efficiency of 0.2%. This culture system uses a low inoculum of 25,000 viable cells per well which permitted chemosensitivity testing of nine drugs at four doses in duplicate from 2.2 X 10(6) viable tumor cells and radiation sensitivity testing at five doses in quadruplicate from 0.6 X 10(6) cells. Cultures were analyzed for survival by computerized image analysis of crystal violet-stained cells. Drug sensitivity studies showed variability in sensitivity and in survival curve shape with exponential cell killing for cisplatin, Adriamycin, and etoposide, and shouldered survival curves for 5-fluorouracil frequently seen. Radiation sensitivity studies also showed variability in both sensitivity and survival curve shape. Many cultures showed exponential cell killing, although others had shouldered survival curves

  14. Role of IgG4 in histamine release from human basophil leucocytes. I. Sensitization of cells from normal donors

    DEFF Research Database (Denmark)

    Poulsen, L K; Stahl Skov, P; Mosbech, H

    1988-01-01

    by a phorbol ester TPA, although not up to the level of anti-IgE. The cells from the high responding donor and a monoclonal anti-IgG4 were selected for further studies. Serum pools from patients allergic to house dust mite (Dermatophagoides pteronyssinus) were used for passive sensitization. The pools...

  15. Role of IgG4 in histamine release from human basophil leucocytes. I. Sensitization of cells from normal donors

    DEFF Research Database (Denmark)

    Poulsen, L K; Stahl Skov, P; Mosbech, H

    1988-01-01

    Several conflicting reports on the ability of IgG4 to mediate type I allergic reactions have appeared lately. We have developed a model system for testing this possibility, using passive sensitization of basophil leucocytes from normal individuals. At first, the system was optimized with regard...

  16. Simple and sensitive method for identification of human DNA by allele-specific polymerase chain reaction of FOXP2.

    Science.gov (United States)

    Hiroshige, Kenichi; Soejima, Mikiko; Nishioka, Tomoki; Kamimura, Shigeo; Koda, Yoshiro

    2009-07-01

    The forkhead box P2 (FOXP2) gene is specifically involved in speech and language development in humans. The sequence is well conserved among many vertebrate species but has accumulated amino acid changes in the human lineage. The aim of this study was to develop a simple method to discriminate between human and nonhuman vertebrate DNA in forensic specimens by amplification of a human-specific genomic region. In the present study, we designed an allele-specific polymerase chain reaction (PCR) using primers to amplify smaller than 70-bp regions of FOXP2 to identify DNA as being of human or nonhuman, including ape, origin. PCR amplification was also successfully performed using fluorescence-labeled primers, and this method allows a single PCR reaction with a genomic DNA sample as small as 0.01 ng. This system also identified the presence of human DNA in two blood stains stored for 20 and 38 years. The results suggested the potential usefulness of FOXP2 as an identifier of human DNA in forensic samples.

  17. Need for a gender-sensitive human security framework: results of a quantitative study of human security and sexual violence in Djohong District, Cameroon.

    Science.gov (United States)

    Parmar, Parveen Kaur; Agrawal, Pooja; Goyal, Ravi; Scott, Jennifer; Greenough, P Gregg

    2014-01-01

    Human security shifts traditional concepts of security from interstate conflict and the absence of war to the security of the individual. Broad definitions of human security include livelihoods and food security, health, psychosocial well-being, enjoyment of civil and political rights and freedom from oppression, and personal safety, in addition to absence of conflict. In March 2010, we undertook a population-based health and livelihood study of female refugees from conflict-affected Central African Republic living in Djohong District, Cameroon and their female counterparts within the Cameroonian host community. Embedded within the survey instrument were indicators of human security derived from the Leaning-Arie model that defined three domains of psychosocial stability suggesting individuals and communities are most stable when their core attachments to home, community and the future are intact. While the female refugee human security outcomes describe a population successfully assimilated and thriving in their new environments based on these three domains, the ability of human security indicators to predict the presence or absence of lifetime and six-month sexual violence was inadequate. Using receiver operating characteristic (ROC) analysis, the study demonstrates that common human security indicators do not uncover either lifetime or recent prevalence of sexual violence. These data suggest that current gender-blind approaches of describing human security are missing serious threats to the safety of one half of the population and that efforts to develop robust human security indicators should include those that specifically measure violence against women.

  18. Modelling ecological and human exposure to POPs in Venice lagoon - Part II: Quantitative uncertainty and sensitivity analysis in coupled exposure models.

    Science.gov (United States)

    Radomyski, Artur; Giubilato, Elisa; Ciffroy, Philippe; Critto, Andrea; Brochot, Céline; Marcomini, Antonio

    2016-11-01

    The study is focused on applying uncertainty and sensitivity analysis to support the application and evaluation of large exposure models where a significant number of parameters and complex exposure scenarios might be involved. The recently developed MERLIN-Expo exposure modelling tool was applied to probabilistically assess the ecological and human exposure to PCB 126 and 2,3,7,8-TCDD in the Venice lagoon (Italy). The 'Phytoplankton', 'Aquatic Invertebrate', 'Fish', 'Human intake' and PBPK models available in MERLIN-Expo library were integrated to create a specific food web to dynamically simulate bioaccumulation in various aquatic species and in the human body over individual lifetimes from 1932 until 1998. MERLIN-Expo is a high tier exposure modelling tool allowing propagation of uncertainty on the model predictions through Monte Carlo simulation. Uncertainty in model output can be further apportioned between parameters by applying built-in sensitivity analysis tools. In this study, uncertainty has been extensively addressed in the distribution functions to describe the data input and the effect on model results by applying sensitivity analysis techniques (screening Morris method, regression analysis, and variance-based method EFAST). In the exposure scenario developed for the Lagoon of Venice, the concentrations of 2,3,7,8-TCDD and PCB 126 in human blood turned out to be mainly influenced by a combination of parameters (half-lives of the chemicals, body weight variability, lipid fraction, food assimilation efficiency), physiological processes (uptake/elimination rates), environmental exposure concentrations (sediment, water, food) and eating behaviours (amount of food eaten). In conclusion, this case study demonstrated feasibility of MERLIN-Expo to be successfully employed in integrated, high tier exposure assessment. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Vulnerability or Sensitivity to the Environment? Methodological Issues, Trends, and Recommendations in Gene?Environment Interactions Research in Human Behavior

    OpenAIRE

    Leighton, Caroline; Botto, Alberto; Silva, Jaime R.; Jim?nez, Juan Pablo; Luyten, Patrick

    2017-01-01

    Research on the potential role of gene–environment interactions (GxE) in explaining vulnerability to psychopathology in humans has witnessed a shift from a diathesis-stress perspective to differential susceptibility approaches. This paper critically reviews methodological issues and trends in this body of research. Databases were screened for studies of GxE in the prediction of personality traits, behavior, and mental health disorders in humans published between January 2002 and January 2015....

  20. The human cyclin B1 protein modulates sensitivity of DNA mismatch repair deficient prostate cancer cell lines to alkylating agents.

    Science.gov (United States)

    Rasmussen, L J; Rasmussen, M; Lützen, A; Bisgaard, H C; Singh, K K

    2000-05-25

    DNA damage caused by alkylating agents results in a G2 checkpoint arrest. DNA mismatch repair (MMR) deficient cells are resistant to killing by alkylating agents and are unable to arrest the cell cycle in G2 phase after alkylation damage. We investigated the response of two MMR-deficient prostate cancer cell lines DU145 and LNCaP to the alkylating agent MNNG. Our studies reveal that DU145 cancer cells are more sensitive to killing by MNNG than LNCaP. Investigation of the underlying reasons for lower resistance revealed that the DU145 cells contain low endogenous levels of cyclin B1. We provide direct evidence that the endogenous level of cyclin B1 modulates the sensitivity of MMR-deficient prostate cancer cells to alkylating agents.

  1. Macrophages and Adipocytes in Human Obesity Adipose Tissue Gene Expression and Insulin Sensitivity During Calorie Restriction and Weight Stabilization

    DEFF Research Database (Denmark)

    Capel, F.; Klimcakova, E.; Viguerie, N.

    2009-01-01

    OBJECTIVE-We investigated the regulation of adipose tissue gene expression during different phases of a dietary weight loss program and its relation with insulin sensitivity. RESEARCH DESIGN AND METHODS-Twenty-two obese women followed a dietary intervention program composed of an energy restriction...... macrophages and adipocytes show distinct patterns of gene regulation and association with insulin sensitivity during the various phases of a dietary weight loss program. Diabetes 58:1558-1567, 2009...... phase with a 4-week very-low-calorie diet and a weight stabilization period composed of a 2-month low-calorie diet followed by 3-4 months of a weight maintenance diet. At each time point, a euglycemic-hyperinsulinemic clamp and subcutaneous adipose tissue biopsies were performed. Adipose tissue gene...

  2. Applying antibody-sensitive hypervariable region 1-deleted hepatitis C virus to the study of escape pathways of neutralizing human monoclonal antibody AR5A

    DEFF Research Database (Denmark)

    Velazquez-Moctezuma, Rodrigo; Law, Mansun; Bukh, Jens

    2017-01-01

    isolates with high antibody resistance, or antibodies with moderate potency, it remains challenging to induce escape mutations in vitro. Here, as proof-of-concept, we used antibody-sensitive HVR1-deleted (ΔHVR1) viruses to generate escape mutants for a human monoclonal antibody, AR5A, targeting a rare...... effect but sensitized the virus to AR5A. Of note, H77/JFH1L665S was non-viable. The resistance mutations did not affect cell-to-cell spread or E1/E2 interactions. Finally, introducing L665W, identified in genotype 1, into genotypes 2–6 parental and HVR1-deleted variants (not available for genotype 4a) we...... observed diverse effects on viral fitness and a universally pronounced reduction in AR5A sensitivity. Thus, we were able to take advantage of the neutralization-sensitive HVR1-deleted viruses to rapidly generate escape viruses aiding our understanding of the divergent escape pathways used by HCV to evade...

  3. Subject-specific finite element modelling of the human foot complex during walking: sensitivity analysis of material properties, boundary and loading conditions.

    Science.gov (United States)

    Akrami, Mohammad; Qian, Zhihui; Zou, Zhemin; Howard, David; Nester, Chris J; Ren, Lei

    2018-04-01

    The objective of this study was to develop and validate a subject-specific framework for modelling the human foot. This was achieved by integrating medical image-based finite element modelling, individualised multi-body musculoskeletal modelling and 3D gait measurements. A 3D ankle-foot finite element model comprising all major foot structures was constructed based on MRI of one individual. A multi-body musculoskeletal model and 3D gait measurements for the same subject were used to define loading and boundary conditions. Sensitivity analyses were used to investigate the effects of key modelling parameters on model predictions. Prediction errors of average and peak plantar pressures were below 10% in all ten plantar regions at five key gait events with only one exception (lateral heel, in early stance, error of 14.44%). The sensitivity analyses results suggest that predictions of peak plantar pressures are moderately sensitive to material properties, ground reaction forces and muscle forces, and significantly sensitive to foot orientation. The maximum region-specific percentage change ratios (peak stress percentage change over parameter percentage change) were 1.935-2.258 for ground reaction forces, 1.528-2.727 for plantar flexor muscles and 4.84-11.37 for foot orientations. This strongly suggests that loading and boundary conditions need to be very carefully defined based on personalised measurement data.

  4. Factors affecting the sensitivity of human-derived esophageal carcinoma cell lines to 5-fluorouracil and cisplatin

    Science.gov (United States)

    MINEGAKI, TETSUYA; TAKARA, KOHJI; HAMAGUCHI, RYOHEI; TSUJIMOTO, MASAYUKI; NISHIGUCHI, KOHSHI

    2013-01-01

    Effective chemotherapy against esophageal carcinoma is considered achievable with a combination of 5-fluorouracil (5-FU) and cisplatin (CDDP). However, chemo-therapy remains ineffective in certain patients. The aim of this study was to clarify the factors which affect sensitivity to 5-FU and CDDP. The effects of factors known to influence sensitivity to 5-FU and CDDP, namely transporters, DNA repair enzymes and metabolic enzymes, were examined. mRNA levels of four transporters, SLC22A2, SLC23A2, ABCB1 and ABCC2, two DNA repair-related enzymes, Rad51 and MSH2, and one metabolic enzyme, dihydropyrimidine dehydrogenase (DPYD), showed a strong correlation (|r|>0.7) with IC50 values for 5-FU. In addition, the mRNA levels of ABCC2, MSH2 and DPYD showed a strong correlation (|r|>0.7) with the IC50 values for CDDP. Gimeracil, a DPYD inhibitor, enhanced the sensitivity of some cells to 5-FU but decreased the sensitivity of all the cells to CDDP. The inhibitory effects of ABCC2 with MK571 did not correspond to those observed in the correlation analysis. In conclusion, mRNA levels of SLC22A2, SLC23A2, ABCB1, ABCC2, Rad51, MSH2 and DPYD were confirmed to be strongly correlated with IC50 values for 5-FU, and mRNA levels of ABCC2, MSH2 and DPYD were confirmed to be strongly correlated with IC50 values for CDDP. In addition, the inhibition of DPYD appeared to affect the cytotoxicity of CDDP. PMID:23420099

  5. Uterine and ovarian carcinosarcomas overexpressing Trop-2 are sensitive to hRS7, a humanized anti-Trop-2 antibody

    Directory of Open Access Journals (Sweden)

    Raji Rhoda

    2011-11-01

    Full Text Available Abstract Background We evaluated the expression of human trophoblastic cell-surface marker (Trop-2 and the potential of hRS7 - a humanized monoclonal anti-Trop-2 antibody - as a therapeutic strategy against treatment-refractory human uterine (UMMT and ovarian (OMMT carcinosarcoma cell lines. Materials and methods Trop-2 expression was evaluated by immunohistochemistry (IHC in paraffin-embedded tumor tissues, by real-time polymerase-chain-reaction (RT-PCR and flow-cytometry in cell lines. Sensitivity to hRS7 antibody-dependent cellular cytotoxicity (ADCC and complement-dependent cytotoxicity was tested using 5-hour chromium-release assays against UMMT and OMMT cells. Results Trop-2 expression was elevated in 9 of 26 (35% UMMT and 8 of 14 (57% OMMT tissues tested by IHC. Positivity for Trop-2 mRNA by RT-PCR and surface expression by flow cytometry were detected in 2 of 4 cell lines, with high positivity noted in OMMT-ARK-2. OMMT-ARK-2 was highly sensitive to hRS7 ADCC (range: 34.7-41.0%; P Conclusion Trop-2 is overexpressed in a proportion of UMMT and OMMT, and hRS7 may represent a novel, potentially highly effective treatment option for patients with treatment-refractory carcinosarcomas overexpressing Trop-2.

  6. Auxetic Foam-Based Contact-Mode Triboelectric Nanogenerator with Highly Sensitive Self-Powered Strain Sensing Capabilities to Monitor Human Body Movement

    KAUST Repository

    Zhang, Steven L.

    2017-05-15

    The first contact-mode triboelectric self-powered strain sensor using an auxetic polyurethane foam, conductive fabric, and polytetrafluroethylene (PTFE) is fabricated. Utilizing the auxetic properties of the polyurethane foam, the auxetic polyurethane foam would expand into the PTFE when the foam is stretched, causing contact electrification. Due to a larger contact area between the PTFE and the foam as the foam is stretched, this device can serve effectively as a strain sensor. The sensitivity of this method is explored, and this sensor has the highest sensitivity in all triboelectric nanogenerator devices that are used previously as a strain sensor. Different applications of this strain sensor are shown, and this sensor can be used as a human body monitoring system, self-powered scale to measure weight, and a seat belt to measure body movements inside a car seat.

  7. Study on sensitivity of southern blotting hybridization using a 32P-labeled probe of PCR products in detecting human cytomegalovirus

    International Nuclear Information System (INIS)

    Bu Hengfu; Chen Juan; Shen Rongsen; Ma Liren; Xu Yongqiang

    1996-01-01

    Southern blotting hybridization (SBH) using a 32 P-labeled probe is one of the most practical methods for genetic diagnosis of pathogen. On the basis of establishing PCR and nested PCR for detecting human cytomegalovirus (HCMV), a 32 P-labeled probe was prepared with the amplified products of 613 bp PCR outer primers and hybridized with 300 bp inner primer amplified product, resulting in increase in detecting sensitivity from 17 ng (in 1.2% agarose electrophoresis) before SBH to 500 pg (autoradiographed), in other words, increasing the sensitivity of detecting HCMV by 10 2 dilutions after using SBH. The method of PCR and SBH using a 32 P-labeled probe could detect less than 1 gene copy of HCMV, therefore, it is a rapid and reliable diagnosis method for detecting HCMV latent infection

  8. Rapid and sensitive LC-MS-MS determination of 2-mercaptobenzothiazole, a rubber additive, in human urine.

    Science.gov (United States)

    Gries, Wolfgang; Küpper, Katja; Leng, Gabriele

    2015-05-01

    2-Mercaptobenzothiazole (MBT) is one of the most important vulcanization accelerators in the industrial production of rubber, especially car tires. Given its wide use in household articles and industrial rubber products it has a high potential to migrate into the environment. Humans can be exposed by dermal, oral, or inhalative routes. Incorporated MBT is excreted in urine, mainly as conjugates to glucuronide, sulfate, and mercapturic acid. On the basis of these facts MBT has been selected as a substance of high interest in the large scale 10-year German project on human biomonitoring (HBM); a cooperation between the German Federal Ministry for the Environment (BMUB) and the German Chemical Industry Association (VCI) with the objective of developing new analytical methods for relevant chemicals. The presented method was developed to determine MBT in human urine to reliably investigate the internal human MBT dose. Total MBT is measured after enzymatic hydrolysis followed by application of high-pressure liquid chromatography tandem mass spectrometry (HPLC-MS-MS) in positive-electrospray-ionization mode (ESI+) using isotope-dilution quantification. High sample throughput could be obtained by use of the column-switching technique. Optimization yielded an analytical method with a low and reproducible limit of detection (LOD) of 0.4 μg L(-1) and a limit of quantification (LOQ) of 1 μg L(-1), and low relative standard deviations in the range 1.6-5.8 %. A small biomonitoring study covering unexposed humans and occupationally exposed workers was performed to establish the feasibility and reliability of the method. MBT was found in only one urine sample from the unexposed humans, at a value of 10.8 μg MBT per liter, whereas it was found in all samples from the tested workers at values of up to 6210 μg MBT per liter.

  9. Using detergent to enhance detection sensitivity of African trypanosomes in human CSF and blood by loop-mediated isothermal amplification (LAMP.

    Directory of Open Access Journals (Sweden)

    Dennis J Grab

    2011-08-01

    Full Text Available The loop-mediated isothermal amplification (LAMP assay, with its advantages of simplicity, rapidity and cost effectiveness, has evolved as one of the most sensitive and specific methods for the detection of a broad range of pathogenic microorganisms including African trypanosomes. While many LAMP-based assays are sufficiently sensitive to detect DNA well below the amount present in a single parasite, the detection limit of the assay is restricted by the number of parasites present in the volume of sample assayed; i.e. 1 per µL or 10(3 per mL. We hypothesized that clinical sensitivities that mimic analytical limits based on parasite DNA could be approached or even obtained by simply adding detergent to the samples prior to LAMP assay.For proof of principle we used two different LAMP assays capable of detecting 0.1 fg genomic DNA (0.001 parasite. The assay was tested on dilution series of intact bloodstream form Trypanosoma brucei rhodesiense in human cerebrospinal fluid (CSF or blood with or without the addition of the detergent Triton X-100 and 60 min incubation at ambient temperature. With human CSF and in the absence of detergent, the LAMP detection limit for live intact parasites using 1 µL of CSF as the source of template was at best 10(3 parasites/mL. Remarkably, detergent enhanced LAMP assay reaches sensitivity about 100 to 1000-fold lower; i.e. 10 to 1 parasite/mL. Similar detergent-mediated increases in LAMP assay analytical sensitivity were also found using DNA extracted from filter paper cards containing blood pretreated with detergent before card spotting or blood samples spotted on detergent pretreated cards.This simple procedure for the enhanced detection of live African trypanosomes in biological fluids by LAMP paves the way for the adaptation of LAMP for the economical and sensitive diagnosis of other protozoan parasites and microorganisms that cause diseases that plague the developing world.

  10. Using Detergent to Enhance Detection Sensitivity of African Trypanosomes in Human CSF and Blood by Loop-Mediated Isothermal Amplification (LAMP)

    Science.gov (United States)

    Grab, Dennis J.; Nikolskaia, Olga V.; Inoue, Noboru; Thekisoe, Oriel M. M.; Morrison, Liam J.; Gibson, Wendy; Dumler, J. Stephen

    2011-01-01

    Background The loop-mediated isothermal amplification (LAMP) assay, with its advantages of simplicity, rapidity and cost effectiveness, has evolved as one of the most sensitive and specific methods for the detection of a broad range of pathogenic microorganisms including African trypanosomes. While many LAMP-based assays are sufficiently sensitive to detect DNA well below the amount present in a single parasite, the detection limit of the assay is restricted by the number of parasites present in the volume of sample assayed; i.e. 1 per µL or 103 per mL. We hypothesized that clinical sensitivities that mimic analytical limits based on parasite DNA could be approached or even obtained by simply adding detergent to the samples prior to LAMP assay. Methodology/Principal Findings For proof of principle we used two different LAMP assays capable of detecting 0.1 fg genomic DNA (0.001 parasite). The assay was tested on dilution series of intact bloodstream form Trypanosoma brucei rhodesiense in human cerebrospinal fluid (CSF) or blood with or without the addition of the detergent Triton X-100 and 60 min incubation at ambient temperature. With human CSF and in the absence of detergent, the LAMP detection limit for live intact parasites using 1 µL of CSF as the source of template was at best 103 parasites/mL. Remarkably, detergent enhanced LAMP assay reaches sensitivity about 100 to 1000-fold lower; i.e. 10 to 1 parasite/mL. Similar detergent-mediated increases in LAMP assay analytical sensitivity were also found using DNA extracted from filter paper cards containing blood pretreated with detergent before card spotting or blood samples spotted on detergent pretreated cards. Conclusions/Significance This simple procedure for the enhanced detection of live African trypanosomes in biological fluids by LAMP paves the way for the adaptation of LAMP for the economical and sensitive diagnosis of other protozoan parasites and microorganisms that cause diseases that plague the

  11. Sensitive analysis of blonanserin, a novel antipsychotic agent, in human plasma by ultra-performance liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Ogawa, Tadashi; Hattori, Hideki; Kaneko, Rina; Ito, Kenjiro; Iwai, Masayo; Mizutani, Yoko; Arinobu, Tetsuya; Ishii, Akira; Suzuki, Osamu; Seno, Hiroshi

    2010-01-01

    A rapid and sensitive method for analysis of blonanserin in human plasma by ultra-performance liquid chromatography-tandem mass spectrometry is presented. After pretreatment of a plasma sample by solid-phase extraction, blonanserin was analyzed by the system with a C(18) column. This method gave satisfactory recovery rates, reproducibility, and good linearity of calibration curve in the range of 0.01-10.0 ng/mL for quality control samples spiked with blonanserin. The detection limit was as low as 1 pg/mL. This method seems very useful in forensic and clinical toxicology and pharmacokinetic studies.

  12. Simple and Sensitive Analysis of Blonanserin and Blonanserin C in Human Plasma by Liquid Chromatography Tandem Mass Spectrometry and Its Application

    OpenAIRE

    Zheng, Yunliang; Hu, Xingjiang; Liu, Jian; Wu, Guolan; Zhou, Huili; Zhu, Meixiang; Zhai, You; Wu, Lihua; ShenTu, Jianzhong

    2014-01-01

    A highly sensitive, simple, and rapid liquid chromatography tandem mass spectrometry method to simultaneously determine blonanserin and blonanserin C in human plasma with AD-5332 as internal standard (IS) was established. A simple direct protein precipitation method was used for the sample pretreatment, and chromatographic separation was performed on a Waters XBridge C8 (4.6 × 150 mm, 3.5  μ m) column. The mobile phase consists of a mixture of 10 mM ammonium formate and 0.1% formic acid in wa...

  13. Molecular cloning and expression of a transformation-sensitive human protein containing the TPR motif and sharing identity to the stress-inducible yeast protein STI1

    DEFF Research Database (Denmark)

    Honoré, B; Leffers, H; Madsen, Peder

    1992-01-01

    A transformation-sensitive human protein (IEF SSP 3521) that is 2-fold up-regulated in SV40-transformed MRC-5 fibroblasts has been purified by two-dimensional gel electrophoresis, microsequenced, and cDNA cloned using oligodeoxyribonucleotides. The 2.1-kilobase cDNA encodes a 543-amino acid protein...... transformed cells. Immunofluorescence studies using a polyclonal antibody raised against the purified protein revealed that the antigen is present mainly in the nucleus of SV40 transformed MRC-5 fibroblasts, while it localizes to the Golgi apparatus and small vesicles in their normal counterparts...

  14. Dose-response assessments of Kathon biocide (I). Diagnostic use and diagnostic threshold patch testing with sensitized humans.

    Science.gov (United States)

    Weaver, J E; Cardin, C W; Maibach, H I

    1985-03-01

    Nearly all effective, commercially available preservatives possess skin sensitization potential. This manuscript describes a program of diagnostic patch and practical use testing of consumer products that contained Kathon CG, a relatively new biocide. A series of threshold diagnostic patch tests demonstrated that the minimal elicitation concentration in occluded patch testing of allergic subjects considerably exceeded the concentrations of the biocide typically present in normal diluted use of the test products. Use testing further confirmed a threshold exposure for eliciting allergic reactions. It showed that even subjects who have delayed contact hypersensitivity to Kathon CG used rinse-off personal care products preserved with this agent without experiencing elicitation of these allergies.

  15. Sulfite-induced protein radical formation in LPS aerosol-challenged mice: Implications for sulfite sensitivity in human lung disease

    OpenAIRE

    Kumar, Ashutosh; Triquigneaux, Mathilde; Madenspacher, Jennifer; Ranguelova, Kalina; Bang, John J.; Fessler, Michael B.; Mason, Ronald P.

    2017-01-01

    Exposure to (bi)sulfite (HSO3 –) and sulfite (SO3 2–) has been shown to induce a wide range of adverse reactions in sensitive individuals. Studies have shown that peroxidase-catalyzed oxidation of (bi)sulfite leads to formation of several reactive free radicals, such as sulfur trioxide anion (.SO3 –), peroxymonosulfate (–O3SOO.), and especially the sulfate (SO4 . –) anion radicals. One such peroxidase in neutrophils is myeloperoxidase (MPO), which has been shown to form protein radicals. Alth...

  16. The immune response during the luteal phase of the ovarian cycle : increasing sensitivity of human monocytes to endotoxin

    NARCIS (Netherlands)

    Bouman, Annechien; Moes, H; Heineman, MJ; de Leij, LFMH; Faas, MM

    Objective: To test the hypothesis that during the luteal phase of the human ovarian cycle, as compared with the follicular phase, the percentage of cytokines producing peripheral monocytes after in vitro stimulation with endotoxin is increased. Design: Prospective study. Setting: Academic research

  17. A Household-Based Distribution-Sensitive Human Development Index: An Empirical Application to Mexico, Nicaragua and Peru

    Science.gov (United States)

    Lopez-Calva, Luis F.; Ortiz-Juarez, Eduardo

    2012-01-01

    In measuring human development, one of the main concerns relates to the inclusion of a measure that penalizes inequalities in the distribution of achievements across the population. Using indicators from nationally representative household surveys and census data, this paper proposes a straightforward methodology to estimate a household-based…

  18. Sensitive genotyping of foodborne-associated human noroviruses and hepatitis A virus using an array-based platform

    Science.gov (United States)

    The viral pathogens, human norovirus (NoV) and hepatitis A virus (HAV), are significant contributors of foodborne associated outbreaks. To develop a typing tool for foodborne viruses, a focused, low-density DNA microarray was developed in conjunction with a rapid and high-throughput fluorescent meth...

  19. Sensitization of multidrug-resistant human cancer cells to Hsp90 inhibitors by down-regulation of SIRT1

    Science.gov (United States)

    Kim, Hak-Bong; Lee, Su-Hoon; Um, Jee-Hyun; Oh, Won Keun; Kim, Dong-Wan; Kang, Chi-Dug; Kim, Sun-Hee

    2015-01-01

    The effectiveness of Hsp90 inhibitors as anticancer agents was limited in multidrug-resistant (MDR) human cancer cells due to induction of heat shock proteins (Hsps) such as Hsp70/Hsp27 and P-glycoprotein (P-gp)-mediated efflux. In the present study, we showed that resistance to Hsp90 inhibitors of MDR human cancer cells could be overcome with SIRT1 inhibition. SIRT1 knock-down or SIRT1 inhibitors (amurensin G and EX527) effectively suppressed the resistance to Hsp90 inhibitors (17-AAG and AUY922) in several MDR variants of human lymphoblastic leukemia and human breast cancer cell lines. SIRT1 inhibition down-regulated the expression of heat shock factor 1 (HSF1) and subsequently Hsps and facilitated Hsp90 multichaperone complex disruption via hyperacetylation of Hsp90/Hsp70. These findings were followed by acceleration of ubiquitin ligase CHIP-mediated mutant p53 (mut p53) degradation and subsequent down-regulation of P-gp in 17-AAG-treated MDR cancer cells expressing P-gp and mut p53 after inhibition of SIRT1. Therefore, combined treatment with Hsp90 inhibitor and SIRT1 inhibitor could be a more effective therapeutic approach for Hsp90 inhibitor-resistant MDR cells via down-regulation of HSF1/Hsps, mut p53 and P-gp. PMID:26416354

  20. Increased chromosome exchange frequencies in iodo-deoxyuridine-sensitized human SW-1573 cells after gamma-irradiation

    NARCIS (Netherlands)

    Franken, N. A.; van Bree, C.; Veltmaat, M. A.; Ludwików, G.; Kipp, J. B.; Barendsen, G. W.

    1999-01-01

    The induction of chromosome exchanges was investigated in SW-1573 human lung tumour cells radiosensitized with iododeoxyuridine (IdUrd) and irradiated with gamma-rays. Following treatment chromosome 2 and X were analyzed using fluorescence in situ hybridization (FISH) with chromosome-specific DNA

  1. Antioxidants keep the potentially probiotic but highly oxygen-sensitive human gut bacterium Faecalibacterium prausnitzii alive at ambient air

    NARCIS (Netherlands)

    Khan, M. Tanweer; van Dijl, Jan Maarten; Harmsen, Hermie J M

    2014-01-01

    The beneficial human gut microbe Faecalibacterium prausnitzii is a 'probiotic of the future' since it produces high amounts of butyrate and anti-inflammatory compounds. However, this bacterium is highly oxygen-senstive, making it notoriously difficult to cultivate and preserve. This has so far

  2. Endocrine sensitivity of the receptor-positive T61 human breast carcinoma serially grown in nude mice

    DEFF Research Database (Denmark)

    Brünner, N; Spang-Thomsen, M; Skovgaard Poulsen, H

    1985-01-01

    A study was made on the effect of ovariectomy, 17 beta-oestradiol, and tamoxifen on the oestrogen and progesterone receptor-positive T61 human breast carcinoma grown in nude mice. The effect of the treatment was evaluated by the specific growth delay calculated on the basis of Gompertz growth...

  3. In Situ CaptureRT-qPCR: A new simple and sensitive method to detect human norovirus in oysters

    Science.gov (United States)

    Human noroviruses (HuNoVs) are the major cause for the non-bacterial acute gastroenteritis worldwide. RT-qPCR is a widely used method to detect HuNoVs. However, the method is unable to discriminate between infectious and non-infectious viruses. Previously, we reported that the receptor mediated in s...

  4. Single-cell analysis of targeted transcriptome predicts drug sensitivity of single cells within human myeloma tumors.

    Science.gov (United States)

    Mitra, A K; Mukherjee, U K; Harding, T; Jang, J S; Stessman, H; Li, Y; Abyzov, A; Jen, J; Kumar, S; Rajkumar, V; Van Ness, B

    2016-05-01

    Multiple myeloma (MM) is characterized by significant genetic diversity at subclonal levels that have a defining role in the heterogeneity of tumor progression, clinical aggressiveness and drug sensitivity. Although genome profiling studies have demonstrated heterogeneity in subclonal architecture that may ultimately lead to relapse, a gene expression-based prediction program that can identify, distinguish and quantify drug response in sub-populations within a bulk population of myeloma cells is lacking. In this study, we performed targeted transcriptome analysis on 528 pre-treatment single cells from 11 myeloma cell lines and 418 single cells from 8 drug-naïve MM patients, followed by intensive bioinformatics and statistical analysis for prediction of proteasome inhibitor sensitivity in individual cells. Using our previously reported drug response gene expression profile signature at the single-cell level, we developed an R Statistical analysis package available at https://github.com/bvnlabSCATTome, SCATTome (single-cell analysis of targeted transcriptome), that restructures the data obtained from Fluidigm single-cell quantitative real-time-PCR analysis run, filters missing data, performs scaling of filtered data, builds classification models and predicts drug response of individual cells based on targeted transcriptome using an assortment of machine learning methods. Application of SCATT should contribute to clinically relevant analysis of intratumor heterogeneity, and better inform drug choices based on subclonal cellular responses.

  5. RETICULOCYE ENRICHMENT AND IMPROVED SENSITIVITY OF ZINC PROTOPORPHYRIN/HEME RATIOS IN HUMAN CORD BLOOD AND SUCKLING RATS

    Science.gov (United States)

    Blohowiak, Sharon E.; Chen, Melinda E.; Repyak, Kristin S.; Carlton, David P.; Georgieff, Michael K.; Crenshaw, Thomas D.; Kling, Pamela J.

    2010-01-01

    In infants and children, whole blood ZnPP/H ratios measure iron-deficient erythropoiesis. Because immature erythrocytes are less dense than mature erythrocytes, we hypothesized that the sensitivity of ZnPP/H is improved if measured in the least dense cells. Blood was collected from control suckling, mild and severe iron-deficient (ID) suckling rats. Cord blood was collected after uncomplicated (control), diabetic (severe ID) and intermediate iron status pregnancies (mild ID). ZnPP/H was measured before and after density centrifugation. ZnPP/H in the lightest cells, the top fraction, was reproducible. The difference between whole and top fraction was defined as Δ ZnPP/H. In rats, although the whole blood or top ZnPP/H differed by postnatal age, PZnPP/H was greatest after the interval with least body iron accrual, PZnPP/H was similar to, but Δ ZnPP/H was greater than controls, PZnPP/H was similar to, but Δ ZnPP/H was relatively greater than controls, PZnPP/H is more sensitive than whole blood ZnPP/H in identifying conditions associated with impaired erythrocyte iron delivery and may become a useful tool in measuring erythrocyte iron incorporation in early development. PMID:18360311

  6. Andrographolide sensitizes cisplatin-induced apoptosis via suppression of autophagosome-lysosome fusion in human cancer cells.

    Science.gov (United States)

    Zhou, Jing; Hu, Shuai-Er; Tan, Shi-Hao; Cao, Ruoxi; Chen, Yiyang; Xia, Dajing; Zhu, Xinqiang; Yang, Xing-Fen; Ong, Choon-Nam; Shen, Han-Ming

    2012-03-01

    Suppression of autophagy has been increasingly recognized as a novel cancer therapeutic approach. Andrographolide (Andro), a diterpenoid lactone isolated from an herbal plant Andrographis paniculata, is known to possess anti-inflammatory and anticancer activity. In this study, we sought to examine the effect of Andro on autophagy, and to evaluate whether such effect is relevant to the sensitization effect of Andro on apoptosis induced by DNA damage agents in cancer cells. First, we found that Andro is able to significantly enhance autophagic markers in various cancer cell lines, including GFP-LC3 puncta and LC3-II level. Interestingly, Andro treatment also led to marked increase of p62 protein level and addition of chloroquine (CQ) failed to further enhance either LC3-II or p62 level, indicating that Andro is likely to suppress autophagic flux at the maturation and degradation stage. Next, we provided evidence that Andro inhibits autophagosome maturation not by affecting the lysosomal function, but by impairing autophagosome-lysosome fusion. Lastly, we demonstrated that treatment with cisplatin, a DNA damage agent, induces autophagy in cancer cells. Importantly, Andro is capable of sensitizing cisplatin-induced cell killing determined with both short-term apoptosis assays and long-term clonogenic test, via suppression of autophagy, a process independent of p53. In summary, these observations collectively suggest that Andro could be a promising anti-cancer agent in combination therapy via its potent inhibitory effect on autophagy by disrupting autophagosome-lysosome fusion.

  7. Gold nanoparticle labeling of cells is a sensitive method to investigate cell distribution and migration in animal models of human disease.

    Science.gov (United States)

    Menk, Ralf Hendrik; Schültke, Elisabeth; Hall, Christopher; Arfelli, Fulvia; Astolfo, Alberto; Rigon, Luigi; Round, Adam; Ataelmannan, Khalid; MacDonald, Sarah Rigley; Juurlink, Bernhard H J

    2011-10-01

    The ability to track cells in small-animal models of human disease is important because it gives the potential to improve our understanding of the processes of disease progression as well as our understanding of the therapeutic effects of interventions. In this study gold nanoparticles have been used as a permanent marker of implanted normal and malignant cell grafts in combination with a suitable x-ray apparatus. Using x-ray computed tomography the micrometric three-dimensional distribution of these marked cells could be displayed with penetration depth, high cell sensitivity and high spatial resolution in rodent models of human diseases. In principle the method allows quantification of cell numbers at any anatomical location over time in small animals. Copyright © 2011 Elsevier Inc. All rights reserved.

  8. IgE mediates killing of intracellular Toxoplasma gondii by human macrophages through CD23-dependent, interleukin-10 sensitive pathway.

    Directory of Open Access Journals (Sweden)

    Ioannis Vouldoukis

    Full Text Available BACKGROUND: In addition to helminthic infections, elevated serum IgE levels were observed in many protozoal infections, while their contribution during immune response to these pathogens remained unclear. As IgE/antigen immune complexes (IgE-IC bind to human cells through FcεRI or FcεRII/CD23 surface molecules, the present study aimed to identify which functional receptor may be involved in IgE-IC interaction with human macrophages, the major effector cell during parasite infection. METHODOLOGY/PRINCIPAL FINDINGS: Human monocyte-derived macrophages were infected with Toxoplasma gondii before being incubated with IgE-IC. IgE receptors were then identified using appropriate blocking antibodies. The activation of cells and parasiticidal activity were evaluated by mediator quantification and direct counting of infected macrophages. RNAs were extracted and cell supernatants were also collected for their content in tumor necrosis factor (TNF-α, interleukin-10 (IL-10 and nitrites. Sera from symptomatic infected patients were also tested for their content of IgE, IL-10 and nitrites, and compared to values found in healthy donors. Results showed that IgE-IC induced intracellular elimination of parasites by human macrophages. IgE-mediated effect was FcεRI-independent, but required cross-linking of surface FcεRII/CD23, cell activation and the generation of nitric oxide (NO. Although TNF-α was shown to be produced during cell activation, this cytokine had minor contribution in this phenomenon while endogenous and exogenous IL-10 down-regulated parasite killing. Inverse relationship was found between IL-10 and NO expression by infected human macrophages at both mRNA and mediator levels. The relationship between these in vitro data and in vivo levels of various factors in T. gondii infected patients supports the involvement of CD23 antigen and IL-10 expression in disease control. CONCLUSION: Thus, IgE may be considered as immune mediator during

  9. TAS-116, a novel Hsp90 inhibitor, selectively enhances radio-sensitivity of human cancer cells to X-rays and carbon ion radiation

    Science.gov (United States)

    Lee, Younghyun; Sunada, Shigeaki; Hirakawa, Hirokazu; Fujimori, Akira; Nickoloff, Jac A.; Okayasu, Ryuichi

    2016-01-01

    Hsp90 inhibitors have been investigated as cancer therapeutics in mono-therapy and to augment radiotherapy, however serious adverse effects of early generation Hsp90 inhibitors limited their development. TAS-116 is a novel Hsp90 inhibitor with lower adverse effects than other Hsp90 inhibitors, and here we investigated the radio-sensitizing effects of TAS-116 in low LET X-ray, and high LET carbon ion irradiated human cancer cells and mouse tumor xenografts. TAS-116 decreased cell survival of both X-ray and carbon ion-irradiated human cancer cell lines (HeLa and H1299 cells), and similar to other Hsp90 inhibitors, it did not affect radiosensitivity of non-cancerous human fibroblasts. TAS-116 increased the number of radiation-induced γ-H2AX foci, and delayed the repair of DNA double-strand breaks (DSBs). TAS-116 reduced the expression of proteins that mediate repair of DSBs by homologous recombination (RAD51) and non-homologous end joining (Ku, DNA-PKcs), and suppressed formation of RAD51 foci and phosphorylation/activation of DNA-PKcs. TAS-116 also decreased expression of the cdc25 cell cycle progression marker, markedly increasing G2/M arrest. Combined treatment of mouse tumor xenografts with carbon ions and TAS-116 showed promising delay in tumor growth compared to either individual treatment. These results demonstrate that TAS-116 radio-sensitizes human cancer cells to both X rays and carbon ions by inhibiting the two major DSB repair pathways, and these effects were accompanied by marked cell cycle arrest. The promising results of combination TAS-116 + carbon ion radiation therapy of tumor xenografts justify further exploration of TAS-116 as an adjunct to radiotherapy using low or high LET radiation. PMID:28062703

  10. A Sensitive Medium-Throughput Method to Predict Intestinal Absorption in Humans Using Rat Intestinal Tissue Segments.

    Science.gov (United States)

    Da Silva, Laís Cristina; Da Silva, Taynara Lourenço; Antunes, Alisson Henrique; Rezende, Kênnia Rocha

    2015-09-01

    A range of in vitro, ex vivo, and in vivo approaches are currently used for drug development. Highly predictive human intestinal absorption models remain lagging behind the times because of numerous variables concerning permeability through gastrointestinal tract in humans. However, there is a clear need for a drug permeability model early in the drug development process that can balance the requirements for high throughput and effective predictive potential. The present study developed a medium throughput screening Snapwell (MTS-Snapwell) ex vivo model to provide an alternative method to classify drug permeability. Rat small intestine tissue segments were mounted in commercial Snapwell™ inserts. Unidirectional drug transport (A-B) was measured by collecting samples at different time points. Viability of intestinal tissue segments was measured by examining transepithelial electric resistance (TEER) and phenol red and caffeine transport. As a result, the apparent permeability (Papp; ×10(-6) cm/s) was determined for atenolol (10.7 ± 1.2), caffeine (17.6 ± 3.1), cimetidine (6.9 ± 0.1), metoprolol (12.6 ± 0.7), theophylline (15.3 ± 1.6) and, ranitidine (3.8 ± 0.4). All drugs were classified in high/low permeability according to Biopharmaceutics Classification System showing high correlation with human data (r = 0.89). These findings showed a high correlation with human data (r = 0.89), suggesting that this model has potential predictive capacity for paracellular and transcellular passively absorbed molecules. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  11. Sensitivity of PCR and real-time PCR for the diagnosis of human visceral leishmaniasis using peripheral blood

    OpenAIRE

    da Costa Lima, Manoel Sebastião; Zorzenon, Denielly Christina Rodrigues; Dorval, Maria Elizabeth Cavalheiros; Pontes, Elenir Rose Jardim Cury; Oshiro, Elisa Teruya; Cunha, Rodrigo; Andreotti, Renato; Matos, Maria de Fatima Cepa

    2013-01-01

    Objective: To evaluate the effectiveness of PCR and real-time PCR for the diagnosis of human visceral leishmaniasis using peripheral blood samples. Methods: DNA extraction was performed using Promega Wizard襅 Genomic kits. PCR employing RV1/RV2 primers yielded 1 45-bp amplicons. Real-time PCR was performed with the same primers and SYBR Green ROX Plus mix. These techniques were used to analyze 100 peripheral blood samples from patients with clinical signs of the disease. Results...

  12. [125I]RTI-55 binding to cocaine-sensitive dopaminergic and serotonergic uptake sites in the human brain.

    Science.gov (United States)

    Little, K Y; Kirkman, J A; Carroll, F I; Breese, G R; Duncan, G E

    1993-12-01

    [125I]RTI-55 is a newly synthesized cocaine congener that may offer advantages over other ligands previously used to examine cocaine binding sites. However, the in vitro pharmacological and anatomical characterization of [125I]RTI-55 binding sites has not been previously performed in human brain. To determine the specificity, stability, and feasibility of [125I]RTI-55 for use in radioligand binding assays in postmortem human tissue, a series of experiments were performed characterizing [125I]RTI-55 binding sites in human brain using homogenized membrane preparations and quantitative autoradiography. Analysis of the association, dissociation, and saturation data favored two-phase processes. A curve-fitting analysis of the data derived in saturation experiments found a high-affinity site with KD = 66 +/- 35 pM and Bmax = 13.2 +/- 10.1 pmol/g of tissue and a low-affinity site with KD = 1.52 +/- 0.55 nM and Bmax of 47.5 +/- 11.2 pmol/g of tissue. Competition by ligands known to bind to the dopamine transporter showed a rank order of RTI-55 > GBR-12909 > maxindol > WIN 35428 > = methylphenidate > (-)-cocaine > buproprion > (+)-amphetamine. Binding to serotonergic sites was evaluated in the midbrain. Results of the saturation experiment performed autoradiographically in the midbrain showed a single site with KD = 370 +/- 84 pM. It appears that [125I]RTI-55 should be useful in further studies of the regulation of cocaine binding sites using postmortem human specimens.

  13. MSH3 mismatch repair protein regulates sensitivity to cytotoxic drugs and a histone deacetylase inhibitor in human colon carcinoma cells.

    Directory of Open Access Journals (Sweden)

    Jae Myung Park

    Full Text Available MSH3 is a DNA mismatch repair (MMR gene that undergoes frequent somatic mutation in colorectal cancers (CRCs with MMR deficiency. MSH3, together with MSH2, forms the MutSβ heteroduplex that interacts with interstrand cross-links induced by drugs such as cisplatin. To date, the impact of MSH3 on chemosensitivity is unknown.We utilized isogenic HCT116 (MLH1-/MSH3- cells where MLH1 is restored by transfer of chromosome 3 (HCT116+ch3 and also MSH3 by chromosome 5 (HCT116+3+5. We generated HCT116+3+5, SW480 (MLH1+/MSH3+ and SW48 (MLH1-/MSH3+ cells with shRNA knockdown of MSH3. Cells were treated with 5-fluorouracil (5-FU, SN-38, oxaliplatin, or the histone deacetylase (HDAC inhibitor PCI-24781 and cell viability, clonogenic survival, DNA damage and apoptosis were analyzed.MSH3-deficient vs proficient CRC cells showed increased sensitivity to the irinotecan metabolite SN-38 and to oxaliplatin, but not 5-FU, as shown in assays for apoptosis and clonogenic survival. In contrast, suppression of MLH1 attenuated the cytotoxic effect of 5-FU, but did not alter sensitivity to SN-38 or oxaliplatin. The impact of MSH3 knockdown on chemosensitivity to SN-38 and oxaliplatin was maintained independent of MLH1 status. In MSH3-deficient vs proficient cells, SN-38 and oxaliplatin induced higher levels of phosphorylated histone H2AX and Chk2, and similar results were found in MLH1-proficient SW480 cells. MSH3-deficient vs proficient cells showed increased 53BP1 nuclear foci after irradiation, suggesting that MSH3 can regulate DNA double strand break (DSB repair. We then utilized PCI-24781 that interferes with homologous recombination (HR indicated by a reduction in Rad51 expression. The addition of PCI-24781 to oxaliplatin enhanced cytotoxicity to a greater extent compared to either drug alone.MSH3 status can regulate the DNA damage response and extent of apoptosis induced by chemotherapy. The ability of MSH3 to regulate chemosensitivity was independent of MLH1

  14. Effects of supplemented isoenergetic diets differing in cereal fiber and protein content on insulin sensitivity in overweight humans.

    Science.gov (United States)

    Weickert, Martin O; Roden, Michael; Isken, Frank; Hoffmann, Daniela; Nowotny, Peter; Osterhoff, Martin; Blaut, Michael; Alpert, Carl; Gögebakan, Ozlem; Bumke-Vogt, Christiane; Mueller, Friederike; Machann, Jürgen; Barber, Tom M; Petzke, Klaus J; Hierholzer, Johannes; Hornemann, Silke; Kruse, Michael; Illner, Anne-Kathrin; Kohl, Angela; Loeffelholz, Christian V; Arafat, Ayman M; Möhlig, Matthias; Pfeiffer, Andreas F H

    2011-08-01

    Despite their beneficial effects on weight loss and blood lipids, high-protein (HP) diets have been shown to increase insulin resistance and diabetes risk, whereas high-cereal-fiber (HCF) diets have shown the opposite effects on these outcomes. We compared the effects of isoenergetic HP and HCF diets and a diet with moderate increases in both cereal fibers and dietary protein (Mix diet) on insulin sensitivity, as measured by using euglycemic-hyperinsulinemic clamps with infusion of [6,6-(2)H(2)]glucose. We randomly assigned 111 overweight adults with features of the metabolic syndrome to 1 of 4 two-phased, 18-wk isoenergetic diets by group-matching. Per 3-d food protocols, the percentages of energy derived from protein and carbohydrates and the intake of cereal fiber per day, respectively, were as follows-after 6 wk: 17%, 52%, and 14 g (control); 17%, 52%, and 43 g (HCF); 28%, 43%, and 13 g (HP); 23%, 44%, and 26 g (Mix); after 18 wk: 17%, 51%, and 15 g (control); 17%, 51%, and 41 g (HCF); 26%, 45%, and 14 g (HP); and 22%, 46%, and 26 g (Mix). Eighty-four participants completed the study successfully and were included in the final analyses. Adherence was supported by the provision of tailored dietary supplements twice daily in all groups. Insulin sensitivity expressed as an M value was 25% higher after 6 wk of the HCF diet than after 6 wk of the HP diet (subgroup analysis: 4.61 ± 0.38 compared with 3.71 ± 0.36 mg · kg(-1) · min(-1), P = 0.008; treatment × time interaction: P = 0.005). Effects were attenuated after 18 wk (treatment × time interaction: P = 0.054), which was likely explained by lower adherence to the HP diet. HP intake was associated with a tendency to increased protein expression in adipose tissue of the translation initiation factor serine-kinase-6-1, which is known to mediate amino acid-induced insulin resistance. Biomarkers of protein intake indicated interference of cereal fibers with dietary protein absorption. Greater changes in insulin

  15. Sensiprobe—a miniature thermal device incorporating Peltier technology as a diagnostic tool for studying human oesophageal sensitivity

    International Nuclear Information System (INIS)

    Reeves, J W; Birch, M J; Al-Zinaty, M; Woodland, P; Sifrim, D; Aziz, Q

    2014-01-01

    Heightened perception of gastrointestinal sensation is termed visceral hypersensitivity (VH) and is commonly observed in patients with gastrointestinal disorders. VH is thought to be a major contributory factor in oesophageal disease, particularly gastro-oesophageal reflux disease that does not respond to standard (proton pump inhibitor) treatment, and in functional heartburn. Clinical tools that can help phenotype according to the mechanism of chronic pain and thus allow targeted drug treatment (e.g. with pain modulator therapy) would be very desirable. A technique that produces repeatable and controllable thermal stimuli within the oesophagus could meet this need. The aims of this study were to develop a method for linear control of the heat stimulation in the oesophagus, to assess the reproducibility of this method, and obtain normal thermal sensitivity values in the distal and proximal oesophagus. The 7 mm diameter Peltier-based thermal device was investigated on 27 healthy subjects using a heating ramp of 0.2 °C s −1 . The pain detection threshold (PDT) temperature was recorded. To assess the reproducibility of the device, each subject underwent the procedure twice, with a minimum of two weeks between each procedure. The mean PDT temperature measured in the distal oesophagus, was 53.8 ± 2.9 °C and 53.6 ± 2.6 °C, for visits 1 and 2 respectively. The mean PDT temperature measured in the proximal oesophagus was 54.1 ± 2.4 °C and 54.0 ± 2.8 °C, for visits 1 and 2 respectively. The reproducibility of the PDT temperature in the distal and proximal oesophagus, was good (intra-class correlation >0.6). Future studies should be aimed to determine whether oesophageal thermal sensitivity can act as a biomarker of transient receptor potential vallanoid 1 upregulation. (paper)

  16. Increased sensitivity for detecting malaria parasites in human umbilical cord blood using scaled-up DNA preparation.

    Science.gov (United States)

    Polley, Spencer D; Sutherland, Colin J; Regan, Fiona; Hassan, Maha; Chiodini, Peter L

    2012-03-05

    All mothers donating umbilical cord blood units to the NHS cord blood bank undergo an assessment for the likelihood of prior exposure to malaria infection. Those deemed at risk due to a history of travel to, or residence in, malaria endemic regions are screened serologically to detect anti-malaria antibodies. A positive result excludes the use of the cord blood for transplant therapy unless a risk assessment can ensure that malaria transmission is extremely unlikely. This paper details the screening of cord blood units from malaria serology positive mothers to detect malaria parasite DNA using a highly sensitive nested PCR. Uninfected blood from a healthy volunteer was spiked with known quantities of malaria parasites and 5 millilitre and 200 microlitre aliquots were subjected to DNA extraction using QIAamp DNA maxi and DNA mini kits respectively. Nested PCR, to detect malarial SSU rRNA sequences, was performed on the purified DNA samples to determine the limit of detection for this assay with both extraction methodologies. Following assay validation, 54 cord blood units donated by mothers who were positive for anti-malaria antibodies were screened by this approach. When DNA was purified from 5 millilitres of blood it was possible to routinely detect as few as 50 malaria parasites per millilitre using nested PCR. This equates to a significant increase in the sensitivity of the current gold standard nucleic acid amplification technique used to detect malaria parasites (routinely performed from > 200 microlitre volumes of blood). None of the 54 donated cord blood units from serology positive mothers tested positive for malaria parasites using this scaled up DNA preparation method. Serological testing for malaria parasites may be overly conservative, leading to unnecessary rejection of cord blood donations that lack malaria parasites and which are, therefore, safe for use in stem cell therapy.

  17. Apoptosis-related molecular differences for response to tyrosin kinase inhibitors in drug-sensitive and drug-resistant human bladder cancer cells

    Directory of Open Access Journals (Sweden)

    Jixia Li

    2013-01-01

    Full Text Available Context: The epidermal growth factor receptor (EGFR family is reportedly overexpressed in bladder cancer, and tyrosine kinaseinhibitors (TKIs have been suggested as treatment. Gefitinib is a selective inhibitor of the EGFR and lapatinib is a dual inhibitor of both the EGFR and HER2 (human EGFR type 2 receptor. Both compounds compete with the binding of adenosine triphosphate (ATP to the tyrosine kinase domain of the respective receptors to inhibit receptor autophosphorylation causing suppression of signal transduction. Unfortunately, resistance to these inhibitors is a major clinical problem. Aims: To compare the apoptosis signaling pathway(s induced by gefitinib and lapatinib, in UM-UC-5 (drug-sensitive and UM-UC-14 (drug-resistant bladder cancer cells and to identify molecular differences that might be useful predictors of their efficacy. Materials and Methods: Cell proliferation, cell cycle and apoptosis assay were used to detect the effect of TKIs on UM-UC-5 and UM-UC-14 cells. Molecular differences for response to TKIs were examined by protein array. Results: TKIs strongly inhibited cell proliferation and induced cell cycle G1 arrest and apoptosis in UM-UC-5 cells. Most notable apoptosis molecular differences included decreased claspin, trail, and survivin by TKIs in the sensitive cells. In contrast, TKIs had no effect on resistant cells. Conclusions: Claspin, trail, and survivin might be used to determine the sensitivity of bladder cancers to TKIs.

  18. Regulating Chondrogenesis of Human Mesenchymal Stromal Cells with a Retinoic Acid Receptor-Beta Inhibitor: Differential Sensitivity of Chondral Versus Osteochondral Development

    Directory of Open Access Journals (Sweden)

    Solvig Diederichs

    2014-05-01

    Full Text Available Aim: Main objective was to investigate whether the synthetic retinoic acid receptor (RAR-β antagonist LE135 is able to drive in vitro chondrogenesis of human mesenchymal stromal cells (MSCs or improve differentiation by suppressing hypertrophic chondrocyte development. Methods: Chondrogenesis of human bone marrow and adipose tissue-derived MSCs was induced in micromass pellet culture for six weeks. Effects of LE135 alone and in combinatorial treatment with TGF-β on deposition of cartilaginous matrix including collagen type II and glycosaminoglycans, on deposition of non-hyaline cartilage collagens type I and X, and on hypertrophy markers including alkaline phosphatase (ALP, indian hedghehog (IHH and matrix metalloproteinase (MMP-13 were assessed. Results: LE135 was no inducer of chondrogenesis and failed to stimulate deposition of collagen type II and glycosaminoglycans. Moreover, addition of LE135 to TGF-β-treated pellets inhibited cartilaginous matrix deposition and gene expression of COL2A1. In contrast, non-hyaline cartilage collagens were less sensitive to LE135 and hypertrophy markers remained unaffected. Conclusion: This demonstrates a differential sensitivity of chondral versus endochondral differentiation pathways to RARβ signaling; however, opposite to the desired direction. The relevance of trans-activating versus trans-repressing RAR signaling, including effects on activator protein (AP-1 is discussed and implications for overcoming current limits of hMSC chondrogenesis are considered.

  19. Regulating chondrogenesis of human mesenchymal stromal cells with a retinoic Acid receptor-Beta inhibitor: differential sensitivity of chondral versus osteochondral development.

    Science.gov (United States)

    Diederichs, Solvig; Zachert, Kerstin; Raiss, Patric; Richter, Wiltrud

    2014-01-01

    Main objective was to investigate whether the synthetic retinoic acid receptor (RAR)-β antagonist LE135 is able to drive in vitro chondrogenesis of human mesenchymal stromal cells (MSCs) or improve differentiation by suppressing hypertrophic chondrocyte development. Chondrogenesis of human bone marrow and adipose tissue-derived MSCs was induced in micromass pellet culture for six weeks. Effects of LE135 alone and in combinatorial treatment with TGF-β on deposition of cartilaginous matrix including collagen type II and glycosaminoglycans, on deposition of non-hyaline cartilage collagens type I and X, and on hypertrophy markers including alkaline phosphatase (ALP), indian hedghehog (IHH) and matrix metalloproteinase (MMP)-13 were assessed. LE135 was no inducer of chondrogenesis and failed to stimulate deposition of collagen type II and glycosaminoglycans. Moreover, addition of LE135 to TGF-β-treated pellets inhibited cartilaginous matrix deposition and gene expression of COL2A1. In contrast, non-hyaline cartilage collagens were less sensitive to LE135 and hypertrophy markers remained unaffected. This demonstrates a differential sensitivity of chondral versus endochondral differentiation pathways to RARβ signaling; however, opposite to the desired direction. The relevance of trans-activating versus trans-repressing RAR signaling, including effects on activator protein (AP)-1 is discussed and implications for overcoming current limits of hMSC chondrogenesis are considered. © 2014 S. Karger AG, Basel.

  20. Current research trends in early life stress and depression: review of human studies on sensitive periods, gene-environment interactions, and epigenetics.

    Science.gov (United States)

    Heim, Christine; Binder, Elisabeth B

    2012-01-01

    Early life stress, such as childhood abuse, neglect and loss, is a well established major risk factor for developing depressive disorders later in life. We here summarize and discuss current developments in human research regarding the link between early life stress and depression. Specifically, we review the evidence for the existence of sensitive periods for the adverse effects of early life stress in humans. We further review the current state of knowledge regarding gene×environment (G×E) interactions in the effects of early life stress. While multiple genes operate in multiple environments to induce risk for depression after early life stress, these same genes also seem to enhance the beneficial effects of a positive early environment. Also, we discuss the epigenetic mechanisms that might underlie these G×E interactions. Finally, we discuss the potential importance of identifying sensitive time periods of opportunity, as well as G×E interactions and epigenetic mechanisms, for early interventions that might prevent or reverse the detrimental outcomes of early life stress and its transmission across generations. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. Stability-indicating spectrofluorimetric method with enhanced sensitivity for determination of vancomycin hydrochloride in pharmaceuticals and spiked human plasma: Application to degradation kinetics

    Directory of Open Access Journals (Sweden)

    Mohie Khaled Sharaf El-Din

    2018-04-01

    Full Text Available Based on investigating the relative fluorescence intensity of vancomycin hydrochloride (VCM in methanol, a simple, highly sensitive, time-saving and specific spectrofluorimetric method was developed and validated. VCM fluorescence was measured at 335 nm when excited at 268 nm. Excellent linearity is obeyed in the concentration range 1–100 ng/mL with a detection limit of 5.94 pg/mL, a quantitation limit of 18.03 pg/mL and a very good correlation coefficient (r = 0.9999. Our method was applied to analyze VCM in pharmaceuticals as well as spiked human plasma. Moreover, VCM stability was studied when exposed to various degradation conditions such as oxidative, alkaline as well as acidic stress. Acidic and alkaline degradation kinetics of VCM was studied for the first time. The degradation follows pseudo-first-order kinetics. The apparent rate constants and half-life times were calculated. The Arrhenius equation was assessed and the activation energies of the degradation were also calculated. The developed method can be easily applied in quality control laboratories due to its sensitivity, specificity, simplicity and low cost. Keywords: Vancomycin hydrochloride, Spectrofluorimetry, Dosage form, Human plasma, Stability-indicating

  2. Selective and sensitive quantification of the cytochrome P450 3A4 protein in human liver homogenates through multiple reaction monitoring mass spectrometry.

    Science.gov (United States)

    Cieślak, Anna; Kelly, Isabelle; Trottier, Jocelyn; Verreault, Mélanie; Wunsch, Ewa; Milkiewicz, Piotr; Poirier, Guy; Droit, Arnaud; Barbier, Olivier

    2016-11-01

    This study aimed at establishing a sensitive multiple reaction monitoring-mass spectrometry (MRM-MS) method for the quantification of the drug metabolizing cytochrome P450 (CYP)3A4 enzyme in human liver homogenates. Liver samples were subjected to trypsin digestion. MRM-MS analyses were performed using three transitions optimized on one purified synthetic peptide unique to CYP3A4 and the standardizing protein, calnexin. Coefficient of variations for the precision and reproducibility of the MRM-MS measurement were also determined. The method was applied to liver samples from ten non-cholestatic donors and 34 cholestatic patients with primary biliary cholangitis (n = 12; PBC), primary sclerosing cholangitis (n = 10; PSC) or alcoholic liver disease (n = 12; ALD). The established method presented high sensitivity with limit of detection lower than 5 fmol, and was successfully applied for the absolute and relative quantification of CYP3A4 in both whole liver homogenate and microsomal fractions. When all groups were analyzed together, a significant correlation was observed for the MRM-based CYP3A4 protein quantification in homogenates and microsomes (r = 0.49, p < 0.001). No statistically significant difference was detected between CYP3A4 levels in PSC, PBC, ALD and control samples. Finally, the MRM-MS quantification of CYP3A4 in homogenates also correlated (r = 0.44; p < 0.05) with the level of enzyme activity in the same samples, as determined by measuring the chenodeoxycholic to hyocholic acid conversion. The established method provides a sensitive tool to evaluate the CYP3A4 protein in human liver homogenates from patients with normal or chronic/severe hepatic injury. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Development and optimization of a sensitive TaqMan® real-time PCR with synthetic homologous extrinsic control for quantitation of Human cytomegalovirus viral load.

    Science.gov (United States)

    Slavov, Svetoslav Nanev; Otaguiri, Katia Kaori; de Figueiredo, Glauciane Garcia; Yamamoto, Aparecida Yulie; Mussi-Pinhata, Marisa Marcia; Kashima, Simone; Covas, Dimas Tadeu

    2016-09-01

    Human cytomegalovirus (Human herpesvirus 5, HCMV) causes frequent asymptomatic infections in the general population. However, in immunosuppressed patients or congenitally infected infants, HCMV is related to high morbidity and mortality. In such cases, a rapid viral detection is crucial for monitoring the clinical outcome and the antiviral treatment. In this study, we optimized a sensitive biplex TaqMan® real-time PCR for the simultaneous detection and differentiation of a partial HCMV UL97 sequence and homologous extrinsic control (HEC) in the same tube. HEC was represented by a plasmid containing a modified HCMV sequence retaining the original primer binding sites, while the probe sequence was substituted by a phylogenetically divergent one (chloroplast CF0 subunit plant gene). It was estimated that the optimal HEC concentration, which did not influence the HCMV amplification is 1,000 copies/reaction. The optimized TaqMan® PCR demonstrated high analytical sensitivity (6.97 copies/reaction, CI = 95%) and specificity (100%). Moreover, the reaction showed adequate precision (repeatability, CV = 0.03; reproducibility, CV = 0.0027) and robustness (no carry-over or cross-contamination). The diagnostic sensitivity (100%) and specificity (97.8%) were adequate for the clinical application of the molecular platform. The optimized TaqMan® real-time PCR is suitable for HCMV detection and quantitation in predisposed patients and monitoring of the applied antiviral therapy. J. Med. Virol. 88:1604-1612, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  4. Sorafenib and its derivative SC-49 sensitize hepatocellular carcinoma cells to CS-1008, a humanized anti-TNFRSF10B (DR5) antibody.

    Science.gov (United States)

    Chen, Kuen-Feng; Chen, Hui-Ling; Shiau, Chung-Wai; Liu, Chun-Yu; Chu, Pei-Yi; Tai, Wei-Tien; Ichikawa, Kimihisa; Chen, Pei-Jer; Cheng, Ann-Lii

    2013-02-01

    Previously, we have shown that sorafenib sensitizes hepatocellular carcinoma (HCC) to apoptosis induced by TNF-related apoptosis-inducing ligand (TNFSF10; TRAIL). Here, we report that sorafenib and SC-49 sensitize HCC cells to CS-1008, a novel anti-human death receptor 5 (TNFRSF10B) antibody. HCC cell lines (PLC5, Huh-7, and Hep3B) were treated with CS-1008 and/or sorafenib and analysed in terms of apoptosis and signal transductions. SC-49 is a sorafenib derivative, which is devoid of kinase inhibitory activity. Both sorafenib and SC-49 down-regulated the phosphorylation of STAT3 at Tyr(705) and subsequently reduced the levels of STAT3-regulated proteins, Mcl-1, survivin and cylcin D1, in CS-1008-treated HCC cells. Knockdown of STAT3 by RNA interference overcame apoptotic resistance to CS-1008 in HCC cells, and ectopic expression of STAT3 in HCC cells abolished the sensitizing effects of sorafenib and SC-49 on CS-1008-induced apoptosis, indicating that inhibition of STAT3 mediates the enhancing effects of these compounds when combined with CS-1008. Importantly, inhibition of SHP-1 by adding a specific SHP-1 inhibitor reduced the effects of SC-49 and CS-1008 on p-STAT3 and apoptosis, whereas co-treatment of CS-1008 with SC-49 increased the activity of SHP-1. These data indicate that the combined effects of CS-1008 and SC-49 on HCC are mediated by SHP-1. Moreover, the combination of CS-1008 and SC-49 inhibited HCC xenograft tumour growth in vivo. Sorafenib and its derivative SC-49 sensitize HCC cells to the antitumour effects of CS-1008 through SHP-1-dependent inactivation of STAT3. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

  5. Rational design of multifunctional micelles against doxorubicin-sensitive and doxorubicin-resistant MCF-7 human breast cancer cells

    Science.gov (United States)

    Hong, Wei; Shi, Hong; Qiao, Mingxi; Gao, Xiang; Yang, Jie; Tian, Chunlian; Zhang, Dexian; Niu, Shengli; Liu, Mingchun

    2017-01-01

    Even though a tremendous number of multifunctional nanocarriers have been developed to tackle heterogeneous cancer cells, little attention has been paid to elucidate how to rationally design a multifunctional nanocarrier. In this study, three individual functions (active targeting, stimuli-triggered release and endo-lysosomal escape) were evaluated in doxorubicin (DOX)-sensitive MCF-7 cells and DOX-resistant MCF-7/ADR cells by constructing four kinds of micelles with active-targeting (AT-M), passive targeting, pH-triggered release (pHT-M) and endo-lysosomal escape (endoE-M) function, respectively. AT-M demonstrated the strongest cytotoxicity against MCF-7 cells and the highest cellular uptake of DOX due to the folate-mediated endocytosis. However, AT-M failed to exhibit the best efficacy against MCF-7/ADR cells, while endoE-M exhibited the strongest cytotoxicity against MCF-7/ADR cells and the highest cellular uptake of DOX due to the lowest elimination of DOX from the cells. This was attributed to the carrier-facilitated endo-lysosomal escape of DOX, which avoided exocytosis by lysosome secretion, resulting in an effective accumulation of DOX in the cytoplasm. The enhanced elimination of DOX from the MCF-7/ADR cells also accounted for the remarkable decrease in cytotoxicity against the cells of AT-M. Three micelles were further evaluated with MCF-7 cells and MCF-7/ADR-resistant cells xenografted mice model. In accordance with the in vitro results, AT-M and endoE-M demonstrated the strongest inhibition on the MCF-7 and MCF-7/ADR xenografted tumor, respectively. Active targeting and active targeting in combination with endo-lysosomal escape have been demonstrated to be the primary function for a nanocarrier against doxorubicin-sensitive and doxorubicin-resistant MCF-7 cells, respectively. These results indicate that the rational design of multifunctional nanocarriers for cancer therapy needs to consider the heterogeneous cancer cells and the primary function needs

  6. Distinct Mechanism of Cysteine Oxidation-Dependent Activation and Cold Sensitization of Human Transient Receptor Potential Ankyrin 1 Channel by High and Low Oxaliplatin

    Directory of Open Access Journals (Sweden)

    Takahito Miyake

    2017-11-01

    Full Text Available Oxaliplatin, a third-generation platinum-based chemotherapeutic agent, displays unique acute peripheral neuropathy triggered or enhanced by cold, and accumulating evidence suggests that transient receptor potential ankyrin 1 (TRPA1 is responsible. TRPA1 is activated by oxaliplatin via a glutathione-sensitive mechanism. However, oxaliplatin interrupts hydroxylation of a proline residue located in the N-terminal region of TRPA1 via inhibition of prolyl hydroxylase (PHD, which causes sensitization of TRPA1 to reactive oxygen species (ROS. Furthermore, PHD inhibition endows cold-insensitive human TRPA1 (hTRPA1 with ROS-dependent cold sensitivity. Since cysteine oxidation and proline hydroxylation regulate its activity, their association with oxaliplatin-induced TRPA1 activation and acquirement of cold sensitivity were investigated in the present study. A high concentration of oxaliplatin (1 mM induced outward-rectifier whole-cell currents and increased the intracellular Ca2+ concentration in hTRPA1-expressing HEK293 cells, but did not increase the probability of hTRPA1 channel opening in the inside-out configuration. Oxaliplatin also induced the rapid generation of hydrogen peroxide, and the resultant Ca2+ influx was prevented in the presence of glutathione and in cysteine-mutated hTRPA1 (Cys641Ser-expressing cells, whereas proline-mutated hTRPA1 (Pro394Ala-expressing cells showed similar whole-cell currents and Ca2+ influx. By contrast, a lower concentration of oxaliplatin (100 μM did not increase the intracellular Ca2+ concentration but did confer cold sensitivity on hTRPA1-expressing cells, and this was inhibited by PHD2 co-overexpression. Cold sensitivity was abolished by the mitochondria-targeting ROS scavenger mitoTEMPO and was minimal in cysteine-mutated hTRPA1 (Cys641Ser or Cys665Ser-expressing cells. Thus, high oxaliplatin evokes ROS-mediated cysteine oxidation-dependent hTRPA1 activation independent of PHD activity, while a lower

  7. Cucurbitacin B Causes Increased Radiation Sensitivity of Human Breast Cancer Cells via G2/M Cell Cycle Arrest

    Directory of Open Access Journals (Sweden)

    Suwit Duangmano

    2012-01-01

    Full Text Available Purpose. To explore the effects of cucurbitacin B on the radiation survival of human breast cancer cells and to elucidate the cellular mechanism of radiosensitization if any. Materials and Methods. Human breast carcinoma cell lines were treated with cucurbitacin B before irradiation with 0–10 Gy of C137s gamma rays. The effect of cucurbitacin B on cell-survival following irradiation was evaluated by colony-forming assay. Cell cycle distributions were investigated using flow cytometry. Real-time PCR and western blots were performed to investigate the expression of cell cycle checkpoints. Results. Cucurbitacin B inhibited breast cancer cell proliferation in a dose-dependent manner. Only MDA-MB-231 and MCF7:5C cells but not SKBR-3 cells were radiosensitized by cucurbitacin B. Flow cytometric analysis for DNA content indicated that cucurbitacin B resulted in G2/M arrest in MDA-MB-231 and MCF7:5C but not SKBR-3 cells. Moreover, Real-time PCR and western blot analysis demonstrated upregulated p21 expression before irradiation, a likely cause of the cell cycle arrest. Conclusion. Taken together, these findings suggest that cucurbitacin B causes radiosensitization of some breast cancer cells, and that cucurbitacin B induced G2/M arrest is an important mechanism. Therefore, combinations of cucurbitacin B with radiotherapy may be appropriate for experimental breast cancer treatment.

  8. Etoposide (VP-16) sensitizes p53-deficient human non-small cell lung cancer cells to caspase-7-mediated apoptosis.

    Science.gov (United States)

    Chiu, C-C; Lin, C-H M Y; Fang, K

    2005-05-01

    Human non-small-cell-lung-cancer (NSCLC) cells of (p)53-null genotype were exposed to low-dosage topoisomearse II inhibitor etoposide (VP-16). The cellular proliferation rate could be effectively inhibited by VP-16 in dose-dependent manner. The effective drug concentration for growth inhibition could be as low as 0.5 microM and the apoptotic phenotype became evident 48 h later. In H1299 cells, VP-16-induced cytotoxic effect was demonstrated associated with apoptosis that disappeared when restored with wild-type p53. Cell cycle analysis revealed that, upon VP-16 induction, cell death began with growth arrest by accumulating cells at the G(2)-M phase. The cells at sub-G(1) phase increased at the expense of those at G(2)-M transition state. To assess the regulation of cell cycle modulators, western blot analysis of H1299 cell lysates showed the release of apoptosis initiator, cytochrome c and apaf-1 hours following drug induction. The cleavage of downstream effectors, procaspase-9 and procaspase-7, but not procaspase-3, was accompanied with proteolysis of poly-(ADP-ribose) polymerase (PARP). VP-16-activated procaspase-7 cleavage was abrogated in cells with ectopically expressed p53. On the other hand, the inhibited procaspase-7 fragmentation by caspase-specific inhibitor reversed apoptotic phenotype caused by drug induction. Thus, VP-16-induced apoptotic cell death was contributed by caspase-7 activation in(p)53-deficient human NSCLC cells.

  9. Amphetamine-induced loss of human dopamine transporter activity: An internalization-dependent and cocaine-sensitive mechanism

    Science.gov (United States)

    Saunders, Christine; Ferrer, Jasmine V.; Shi, Lei; Chen, Jiayun; Merrill, Gerald; Lamb, Maria E.; Leeb-Lundberg, L. M. Fredrik; Carvelli, Lucia; Javitch, Jonathan A.; Galli, Aurelio

    2000-01-01

    The dopamine transporter (DAT) is a target of amphetamine (AMPH) and cocaine. These psychostimulants attenuate DAT clearance efficiency, thereby increasing synaptic dopamine (DA) levels. Re-uptake rate is determined by the number of functional transporters at the cell surface as well as by their turnover rate. Here, we present evidence that DAT substrates, including AMPH and DA, cause internalization of human DAT, thereby reducing transport capacity. Acute treatment with AMPH reduced the maximal rate of [3H]DA uptake, decreased AMPH-induced currents, and significantly redistributed the immunofluorescence of an epitope-tagged DAT from the plasma membrane to the cytosol in human embryonic kidney 293 cells. Conversely, DAT inhibitors, such as cocaine, mazindol, and nomifensine, when administered with AMPH, blocked the reduction in [3H]DA uptake and the redistribution of DAT immunofluorescence to the cytosol. The reductions of [3H]DA uptake and AMPH-induced DAT internalization also were inhibited by coexpression of a dominant negative mutant of dynamin I (K44A), indicating that endocytosis modulates transport capacity, likely through a clathrin-mediated pathway. With this mechanism of regulation, acute application of AMPH would reduce DA uptake not only by direct competition for uptake, but also by reducing the available cell-surface DAT. Moreover, AMPH-induced internalization might diminish the amount of DAT available for DA efflux, thereby modulating the cytotoxic effects of elevated extracellular DA. PMID:10823899

  10. The sensitivity of human tissues to changes in dose fractionation: deductions from the RCR survey among UK radiotherapists.

    Science.gov (United States)

    Hendry, J H; Roberts, S A

    1991-01-01

    The dosage prescriptions reported in the Royal College of Radiologists' fractionation survey among radiotherapists have been further analysed using model equations in order to deduce estimates of fractionation-sensitivity parameters for each of the six cases under consideration. For example, using the linear-quadratic model, including a (significant) time factor, with radical treatments of a T2 breast carcinoma or a T1N0 squamous carcinoma of the vocal cord, the (alpha/beta) ratios were 26 +/- 20 Gy and 37 +/- 46 Gy, respectively. The values of the time factor, expressed as the maximum extra dose required per day to counteract the decrease in effect with increasing overall time (gamma/alpha), were 0.60 and 0.45 Gy/day respectively. Using the Ellis formula, which provided a significantly better fit to the dosage prescription (P = 0.003), the exponents of N were calculated to be 0.24 +/- 0.05 and 0.27 +/- 0.07, respectively. The corresponding values of the T exponent were 0.16 +/- 0.06 and 0.014 +/- 0.075. About 20% of radiotherapists prescribed doses greater than +/- 10% from the mean fitted values for the breast treatment, and about 6% of them in the case of the vocal cord.

  11. Estimated Aortic Stiffness is Independently Associated with Cardiac Baroreflex Sensitivity in Humans: Role of Aging and Habitual Endurance Exercise

    Science.gov (United States)

    Pierce, Gary L.; Harris, Stephen A.; Seals, Douglas R.; Casey, Darren P.; Barlow, Patrick B.; Stauss, Harald M.

    2016-01-01

    We hypothesized that differences in cardiac baroreflex sensitivity (BRS) would be independently associated with aortic stiffness and augmentation index (AI), clinical biomarkers of cardiovascular disease (CVD) risk, among young sedentary and middle-aged/older sedentary and endurance-trained adults. A total of 36 healthy middle-aged/older (age 55-76 years, n=22 sedentary; n=14 endurance-trained) and 5 young sedentary (age 18-31 years) adults were included in a cross-sectional study. A subset of the middle-aged/older sedentary adults (n=12) completed an 8-week aerobic exercise intervention. Invasive brachial artery blood pressure waveforms were used to compute spontaneous cardiac BRS (via sequence technique) and estimated aortic pulse wave velocity (PWV) and AI (AI, via brachial-aortic transfer function and wave separation analysis). In the cross-sectional study, cardiac BRS was 71% lower in older compared with young sedentary adults (Pbaroreflex and aortic stiffness with age and exercise. PMID:26911535

  12. PAR-2 activation enhances weak acid-induced ATP release through TRPV1 and ASIC sensitization in human esophageal epithelial cells.

    Science.gov (United States)

    Wu, Liping; Oshima, Tadayuki; Shan, Jing; Sei, Hiroo; Tomita, Toshihiko; Ohda, Yoshio; Fukui, Hirokazu; Watari, Jiro; Miwa, Hiroto

    2015-10-15

    Esophageal visceral hypersensitivity has been proposed to be the pathogenesis of heartburn sensation in nonerosive reflux disease. Protease-activated receptor-2 (PAR-2) is expressed in human esophageal epithelial cells and is believed to play a role in inflammation and sensation. PAR-2 activation may modulate these responses through adenosine triphosphate (ATP) release, which is involved in transduction of sensation and pain. The transient receptor potential vanilloid receptor 1 (TRPV1) and acid-sensing ion channels (ASICs) are both acid-sensitive nociceptors. However, the interaction among these molecules and the mechanisms of heartburn sensation are still not clear. We therefore examined whether ATP release in human esophageal epithelial cells in response to acid is modulated by TRPV1 and ASICs and whether PAR-2 activation influences the sensitivity of TRPV1 and ASICs. Weak acid (pH 5) stimulated the release of ATP from primary human esophageal epithelial cells (HEECs). This effect was significantly reduced after pretreatment with 5-iodoresiniferatoxin (IRTX), a TRPV1-specific antagonist, or with amiloride, a nonselective ASIC blocker. TRPV1 and ASIC3 small interfering RNA (siRNA) transfection also decreased weak acid-induced ATP release. Pretreatment of HEECs with trypsin, tryptase, or a PAR-2 agonist enhanced weak acid-induced ATP release. Trypsin treatment led to the phosphorylation of TRPV1. Acid-induced ATP release enhancement by trypsin was partially blocked by IRTX, amiloride, or a PAR-2 antagonist. Conversely, acid-induced ATP release was augmented by PAR-2 activation through TRPV1 and ASICs. These findings suggested that the pathophysiology of heartburn sensation or esophageal hypersensitivity may be associated with the activation of PAR-2, TRPV1, and ASICs. Copyright © 2015 the American Physiological Society.

  13. Sensitivity of populations of bats (Mammalia: Chiroptera in relation to human development in northern Paraná, southern Brazil

    Directory of Open Access Journals (Sweden)

    NR. Reis

    Full Text Available Most natural forests have been converted for human use, restricting biological life to small forest fragments. Many animals, including some species of bats are disappearing and the list of these species grows every day. It seems that the destruction of the habitat is one of its major causes. This study aimed to analyze how this community of bats was made up in environments with different sizes and quality of habitat. Data from studies conducted in the region of Londrina, Parana, Brazil, from 1982 to 2000 were used. Originally, this area was covered by a semi deciduous forest, especially Aspidosperma polyneuron (Apocynaceae, Ficus insipida (Moraceae, Euterpe edulis (Arecaceae, Croton floribundus (Euforbiaceae, and currently, only small remnants of the original vegetation still exist. The results showed a decline in the number of species caught in smaller areas compared to the largest remnant. In about 18 years of sampling, 42 species of bats were found in the region, representing 67% of the species that occur in Paraná and 24.4% in Brazil. There were two species of Noctilionidae; 21 of Phyllostoma; 11 Vespertilionidae and eight Molossidae. Eight of these were captured only in the largest fragment, Mata dos Godoy State Park (680 ha. Ten species had a low capture rate in the smaller areas with less than three individuals. Of the total sampled, 14 species were found in human buildings, and were able to tolerate modified environments, foraging and even using them as shelter. As the size of the forest area increases, there is a greater variety of ecological opportunities and their physical conditions become more stable, i.e., conditions favorable for growth and survival of a greater number of species. Forest fragmentation limits and creates subpopulations, preserving only long-lived K-strategist animals for some time, where the supporting capacity of the environment is a limiting factor. The reduction of habitats, species and genetic diversity

  14. Evolutionary Dynamics of Pandemic Methicillin-Sensitive Staphylococcus aureus ST398 and Its International Spread via Routes of Human Migration.

    Science.gov (United States)

    Uhlemann, Anne-Catrin; McAdam, Paul R; Sullivan, Sean B; Knox, Justin R; Khiabanian, Hossein; Rabadan, Raul; Davies, Peter R; Fitzgerald, J Ross; Lowy, Franklin D

    2017-01-17

    Methicillin-susceptible Staphylococcus aureus (MSSA) accounts for the majority of S. aureus infections globally, and yet surprisingly little is known about its clonal evolution. We applied comparative whole-genome sequencing (WGS) analyses to epidemiologically and geographically diverse ST398-MSSA, a pandemic lineage affecting both humans and livestock. Bayesian phylogenetic analysis predicted divergence of human-associated ST398-MSSA ~40 years ago. Isolates from Midwestern pigs and veterinarians differed substantially from those in New York City (NYC). Pig ST398 strains contained a large region of recombination representing imports from multiple sequence types (STs). Phylogeographic analyses supported the spread of ST398-MSSA along local cultural and migratory links between parts of the Caribbean, North America, and France, respectively. Applying pairwise single-nucleotide polymorphism (SNP) distances as a measure of genetic relatedness between isolates, we observed that ST398 not only clustered in households but also frequently extended across local social networks. Isolates collected from environmental surfaces reflected the full diversity of colonizing individuals, highlighting their potentially critical role as reservoirs for transmission and diversification. Strikingly, we observed high within-host SNP variability compared to our previous studies on the dominant methicillin-resistant Staphylococcus aureus (MRSA) clone USA300. Our data indicate that the dynamics of colonization, persistence, and transmission differ substantially between USA300-MRSA and ST398-MSSA. Taken together, our study reveals local and international routes of transmission for a major MSSA clone, indicating key impacts of recombination and mutation on genetic diversification and highlighting important ecological differences from epidemic USA300. Our study demonstrates extensive local and international routes of transmission for a major MSSA clone despite the lack of substantial

  15. Sensitivity of populations of bats (Mammalia: Chiroptera) in relation to human development in northern Paraná, southern Brazil.

    Science.gov (United States)

    Reis, N R; Gallo, P H; Peracchi, A L; Lima, L P; Fregonezi, M N

    2012-08-01

    Most natural forests have been converted for human use, restricting biological life to small forest fragments. Many animals, including some species of bats are disappearing and the list of these species grows every day. It seems that the destruction of the habitat is one of its major causes. This study aimed to analyze how this community of bats was made up in environments with different sizes and quality of habitat. Data from studies conducted in the region of Londrina, Parana, Brazil, from 1982 to 2000 were used. Originally, this area was covered by a semi deciduous forest, especially Aspidosperma polyneuron (Apocynaceae), Ficus insipida (Moraceae), Euterpe edulis (Arecaceae), Croton floribundus (Euforbiaceae), and currently, only small remnants of the original vegetation still exist. The results showed a decline in the number of species caught in smaller areas compared to the largest remnant. In about 18 years of sampling, 42 species of bats were found in the region, representing 67% of the species that occur in Paraná and 24.4% in Brazil. There were two species of Noctilionidae; 21 of Phyllostoma; 11 Vespertilionidae and eight Molossidae. Eight of these were captured only in the largest fragment, Mata dos Godoy State Park (680 ha). Ten species had a low capture rate in the smaller areas with less than three individuals. Of the total sampled, 14 species were found in human buildings, and were able to tolerate modified environments, foraging and even using them as shelter. As the size of the forest area increases, there is a greater variety of ecological opportunities and their physical conditions become more stable, i.e., conditions favorable for growth and survival of a greater number of species. Forest fragmentation limits and creates subpopulations, preserving only long-lived K-strategist animals for some time, where the supporting capacity of the environment is a limiting factor. The reduction of habitats, species and genetic diversity resulting from human

  16. Human mast cell line-1 (HMC-1) cells transfected with FcεRIα are sensitive to IgE/antigen-mediated stimulation demonstrating selectivity towards cytokine production.

    Science.gov (United States)

    Xia, YuXiu C; Sun, ShanShan; Kuek, Li Eon; Lopata, Andreas L; Hulett, Mark D; Mackay, Graham A

    2011-08-01

    Mast cells play important roles in allergic and inflammatory diseases. Efforts to better understand human mast cell activation and develop novel inhibitory agents have been hampered by the lack of suitable human mast cell lines. The HMC-1 mast cell line has been extensively used, but lacks native expression of the human high-affinity IgE receptor FcεRI limiting its applications. We have stably transfected HMC-1 cells with the IgE-binding α-subunit of FcεRI to generate HMCα cells that are antigen-responsive. We have used flow cytometry, cell signaling assays, pharmacological pathway inhibitors and cell functional assays to characterize the properties of HMCα cells. IgE/antigen responses were compared with those of the adenosine receptor agonist NECA. Surface expression of FcεRI in HMCα cells was demonstrated and was enhanced by prior sensitization with IgE. Activation of HMCα cells with IgE/antigen did not produce degranulation, but did lead to release of numerous cytokines. Whilst there was no measurable increase of intracellular Ca(2+) or marked general changes in protein tyrosine phosphorylation, IgE/antigen stimulation of HMCα cells enhanced phosphorylation of p38(MAPK) and Erk. Inhibitors of these pathways, as well as the src kinase inhibitor PP2, attenuated IgE/antigen-induced cytokine release. In summary, we have generated and characterized HMCα cells and show that they are a useful and relevant human mast cell model to examine FcεRI stabilization, signaling and mediator release. We envisage that HMCα cells will have utility in understanding the importance of mast cells in human allergic disease and in assessing the activity of novel anti-allergic compounds. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. Vulnerability or Sensitivity to the Environment? Methodological Issues, Trends, and Recommendations in Gene–Environment Interactions Research in Human Behavior

    Science.gov (United States)

    Leighton, Caroline; Botto, Alberto; Silva, Jaime R.; Jiménez, Juan Pablo; Luyten, Patrick

    2017-01-01

    Research on the potential role of gene–environment interactions (GxE) in explaining vulnerability to psychopathology in humans has witnessed a shift from a diathesis-stress perspective to differential susceptibility approaches. This paper critically reviews methodological issues and trends in this body of research. Databases were screened for studies of GxE in the prediction of personality traits, behavior, and mental health disorders in humans published between January 2002 and January 2015. In total, 315 papers were included. Results showed that 34 candidate genes have been included in GxE studies. Independent of the type of environment studied (early or recent life events, positive or negative environments), about 67–83% of studies have reported significant GxE interactions, which is consistent with a social susceptibility model. The percentage of positive results does not seem to differ depending on the gene studied, although publication bias might be involved. However, the number of positive findings differs depending on the population studied (i.e., young adults vs. older adults). Methodological considerations limit the ability to draw strong conclusions, particularly as almost 90% (n = 283/315) of published papers are based on samples from North America and Europe, and about 70% of published studies (219/315) are based on samples that were also used in other reports. At the same time, there are clear indications of methodological improvements over time, as is shown by a significant increase in longitudinal and experimental studies as well as in improved minimum genotyping. Recommendations for future research, such as minimum quality assessment of genes and environmental factors, specifying theoretical models guiding the study, and taking into account of cultural, ethnic, and lifetime perspectives, are formulated. PMID:28674505

  18. Impact of anemia prevention by recombinant human erythropoietin on the sensitivity of xenografted glioblastomas to fractionated irradiation

    International Nuclear Information System (INIS)

    Stueben, G.; Poettgen, C.; Knuehmann, K.; Sack, H.; Stuschke, M.; Thews, O.; Vaupel, P.

    2003-01-01

    Background: Pronounced oxygen deficiency in tumors which might be caused by a diminished oxygen transport capacity of the blood (e.g., in anemia) reduces the efficacy of ionizing radiation. The aim of this study was to analyze whether anemia prevention by recombinant human erythropoietin (rHuEPO) affects the radiosensitivity of human glioblastoma xenografts during fractionated irradiation. Material and Methods: Anemia was induced by total body irradiation (TBI, 2 x 4 Gy) of mice prior to tumor implantation into the subcutis of the hind leg. In one experimental group, the development of anemia was prevented by rHuEPO (750 U/kg s.c.) given three times weekly starting 10 days prior to TBI. 13 days after tumor implantation (tumor volume approx. 40 mm 3 ), fractionated irradiation (4 x 7 Gy, one daily fraction) of the glioblastomas was performed resulting in a growth delay with subsequent regrowth of the tumors. Results: Compared to nonanemic control animals (hemoglobin concentration cHb = 14.7 g/dl), the growth delay in anemic mice (cHb = 9.9 g/dl) was significantly shorter (49 ± 5 days vs. 79 ± 4 days to reach four times the initial tumor volume) upon fractionated radiation. The prevention of anemia by rHuEPO treatment (cHb = 13.3 g/dl) resulted in a significantly prolonged growth delay (61 ± 5 days) compared to the anemia group, even though the growth inhibition found in control animals was not completely achieved. Conclusions: These data indicate that moderate anemia significantly reduces the efficacy of radiotherapy. Prevention of anemia with rHuEPO partially restores the radiosensitivity of xenografted glioblastomas to fractionated irradiation. (orig.)

  19. Vulnerability or Sensitivity to the Environment? Methodological Issues, Trends, and Recommendations in Gene-Environment Interactions Research in Human Behavior.

    Science.gov (United States)

    Leighton, Caroline; Botto, Alberto; Silva, Jaime R; Jiménez, Juan Pablo; Luyten, Patrick

    2017-01-01

    Research on the potential role of gene-environment interactions (GxE) in explaining vulnerability to psychopathology in humans has witnessed a shift from a diathesis-stress perspective to differential susceptibility approaches. This paper critically reviews methodological issues and trends in this body of research. Databases were screened for studies of GxE in the prediction of personality traits, behavior, and mental health disorders in humans published between January 2002 and January 2015. In total, 315 papers were included. Results showed that 34 candidate genes have been included in GxE studies. Independent of the type of environment studied (early or recent life events, positive or negative environments), about 67-83% of studies have reported significant GxE interactions, which is consistent with a social susceptibility model. The percentage of positive results does not seem to differ depending on the gene studied, although publication bias might be involved. However, the number of positive findings differs depending on the population studied (i.e., young adults vs. older adults). Methodological considerations limit the ability to draw strong conclusions, particularly as almost 90% ( n  = 283/315) of published papers are based on samples from North America and Europe, and about 70% of published studies (219/315) are based on samples that were also used in other reports. At the same time, there are clear indications of methodological improvements over time, as is shown by a significant increase in longitudinal and experimental studies as well as in improved minimum genotyping. Recommendations for future research, such as minimum quality assessment of genes and environmental factors, specifying theoretical models guiding the study, and taking into account of cultural, ethnic, and lifetime perspectives, are formulated.

  20. Vulnerability or Sensitivity to the Environment? Methodological Issues, Trends, and Recommendations in Gene–Environment Interactions Research in Human Behavior

    Directory of Open Access Journals (Sweden)

    Caroline Leighton

    2017-06-01

    Full Text Available Research on the potential role of gene–environment interactions (GxE in explaining vulnerability to psychopathology in humans has witnessed a shift from a diathesis-stress perspective to differential susceptibility approaches. This paper critically reviews methodological issues and trends in this body of research. Databases were screened for studies of GxE in the prediction of personality traits, behavior, and mental health disorders in humans published between January 2002 and January 2015. In total, 315 papers were included. Results showed that 34 candidate genes have been included in GxE studies. Independent of the type of environment studied (early or recent life events, positive or negative environments, about 67–83% of studies have reported significant GxE interactions, which is consistent with a social susceptibility model. The percentage of positive results does not seem to differ depending on the gene studied, although publication bias might be involved. However, the number of positive findings differs depending on the population studied (i.e., young adults vs. older adults. Methodological considerations limit the ability to draw strong conclusions, particularly as almost 90% (n = 283/315 of published papers are based on samples from North America and Europe, and about 70% of published studies (219/315 are based on samples that were also used in other reports. At the same time, there are clear indications of methodological improvements over time, as is shown by a significant increase in longitudinal and experimental studies as well as in improved minimum genotyping. Recommendations for future research, such as minimum quality assessment of genes and environmental factors, specifying theoretical models guiding the study, and taking into account of cultural, ethnic, and lifetime perspectives, are formulated.

  1. A quantum dot-based lateral flow immunoassay for the sensitive detection of human heart fatty acid binding protein (hFABP) in human serum.

    Science.gov (United States)

    Savin, Mihaela; Mihailescu, Carmen-Marinela; Matei, Iulia; Stan, Dana; Moldovan, Carmen Aura; Ion, Marian; Baciu, Ion

    2018-02-01

    We describe the preparation and validation of a novel lateral flow immunoassay test for the detection of human heart fatty acid binding protein (hFABP). Water-soluble CdTe quantum dots (QDs) were selected as the fluorescent label and were linked covalently to anti-hFABP antibodies. Upon conjugation, the secondary structure of the anti-hFABP was preserved and the fluorescence quantum yield of the CdTe QDs increased. The labelled antibodies were transferred to the immunoassay test strip and the antigen-antibody reaction was successfully performed. This evidenced the preserved antibody activity of QD-labelled anti-hFABP towards hFABP, and provided a rapid means for the quantitation of hFABP in human serum within the range of 0-160ng ∙ ml -1 , with a much lower detection limit of 221pg.∙ ml -1 compared with other rapid tests based on lateral flow immunoassays. This new immunoassay test has been successfully used for the early detection of acute myocardial infarction. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Oncolytic viruses sensitize human tumor cells for NY-ESO-1 tumor antigen recognition by CD4+ effector T cells.

    Science.gov (United States)

    Delaunay, Tiphaine; Violland, Mathilde; Boisgerault, Nicolas; Dutoit, Soizic; Vignard, Virginie; Münz, Christian; Gannage, Monique; Dréno, Brigitte; Vaivode, Kristine; Pjanova, Dace; Labarrière, Nathalie; Wang, Yaohe; Chiocca, E Antonio; Boeuf, Fabrice Le; Bell, John C; Erbs, Philippe; Tangy, Frédéric; Grégoire, Marc; Fonteneau, Jean-François

    2018-01-01

    Oncolytic immunotherapy using oncolytic viruses (OV) has been shown to stimulate the antitumor immune response by inducing the release of tumor-associated antigens (TAA) and danger signals from the dying infected tumor cells. In this study, we sought to determine if the lysis of tumor cells induced by different OV: measles virus, vaccinia virus, vesicular stomatitis virus, herpes simplex type I virus, adenovirus or enterovirus, has consequences on the capacity of tumor cells to present TAA, such as NY-ESO-1. We show that the co-culture of NY-ESO-1 neg /HLA-DP4 pos melanoma cells with NY-ESO-1 pos /HLA-DP4 neg melanoma cells infected and killed by different OV induces an intercellular transfer of NY-ESO-1 that allows the recognition of NY-ESO-1 neg /HLA-DP4 pos tumor cells by an HLA-DP4/NY-ESO-1 (157-170) -specific CD4+ cytotoxic T cell clone, NY67. We then confirmed this result in a second model with an HLA-DP4+ melanoma cell line that expresses a low amount of NY-ESO-1. Recognition of this cell line by the NY67 clone is largely increased in the presence of OV productive infection. Altogether, our results show for the first time another mechanism of stimulation of the anti-tumor immune response by OV, via the loading of tumor cells with TAA that sensitizes them for direct recognition by specific effector CD4+ T cells, supporting the use of OV for cancer immunotherapy.

  3. Highly Sensitive Micellar Enhanced Spectrofluorimetric Method for Determination of Mirtazapine in Tablets and Human Urine: Application to In Vitro Drug Release and Content Uniformity Test

    Directory of Open Access Journals (Sweden)

    Hany W. Darwish

    2016-01-01

    Full Text Available A highly sensitive and simple micelle enhanced spectrofluorimetric method was developed for assaying mirtazapine (MRZ in REMERON® tablets and spiked human urine directly without the need of derivatizing agent. The basis of the current procedure is the examination of the relative fluorescence intensity (RFI of MRZ in sodium lauryl sulphate (SLS micellar medium. The RFI of MRZ in water was enhanced markedly on addition of SLS. The RFI was measured at 403 nm after excitation at 320 nm. The fluorescence-concentration relationship was linear over the range 1–500 ng/mL, with lower detection limit of 0.399 ng/mL. The proposed method was successfully applied to the determination of MRZ in dosage form and spiked human urine. Recovery percentages of MRZ utilizing the current method were 99.05±1.83, 98.37±1.96, and 100.41±2.61% for pure powder, pharmaceutical dosage form, and spiked human urine, respectively. The application of the proposed method was extended to test content uniformity and the in vitro drug release of REMERON tablets, according to USP guidelines.

  4. Ghrelin stimulation of growth hormone isoforms: parallel secretion of total and 20-kDa growth hormone and relation to insulin sensitivity in healthy humans.

    Science.gov (United States)

    Tong, Jenny; D'Alessio, David; Ramisch, Juliane; Davis, Harold W; Stambrook, Elizabeth; Tschöp, Matthias H; Bidlingmaier, Martin

    2012-09-01

    The 20-kDa human GH (hGH) is produced in the pituitary by alternative splicing of the hGH-N gene. The 20-kDa hGH promotes growth similarly to 22-kDa or total hGH, the predominant form in circulation, but the relative effects of these isoforms on glucose metabolism have been debated. To investigate the effect of ghrelin on 20-kDa and total hGH secretion in healthy, nonobese subjects. We also studied associations between basal GH concentration and fasting glucose and insulin as well as between dynamic GH secretion and insulin sensitivity. Synthetic human acyl ghrelin (0.2 or 0.6 nmol/kg · h) or saline was infused in random order in 14 healthy subjects (six males, eight females; age 27.7 ± 6.3 yr; body mass index 22.0 ± 2.7 kg/m(2), mean ± SEM) on 3 separate days. Ghrelin was infused for 45 min to achieve steady-state levels and continued through a 3-h frequently sampled i.v. glucose tolerance test. Insulin sensitivity index was quantified using the minimal model of glucose kinetics. Basal 20-kDa and total GH concentrations were 0.4 ± 0.1 and 2.2 ± 0.4 ng/ml, respectively, with a 20-kDa to total GH ratio of 0.13 ± 0.02. Females had significantly higher baseline GH levels. Ghrelin administration increased 20-kDa and total GH levels in a parallel and dose-dependent fashion, with no significant change in the ratio of the isoforms. Basal 20-kDa and total GH levels were negatively correlated with fasting glucose, insulin, and homeostasis model assessment of insulin resistance. During the frequently sampled iv glucose tolerance test, GH secretion was positively correlated with insulin sensitivity index with saline infusion. Ghrelin dose-dependently increases endogenous 20-kDa and total GH secretion in a parallel fashion in healthy subjects. Both basal and stimulated levels of the different GH isoforms were positively associated with insulin sensitivity in this cohort of healthy men and women.

  5. Genetic reconstitution of the human Adenovirus type 2 temperature-sensitive 1 mutant defective in endosomal escape

    Directory of Open Access Journals (Sweden)

    Gastaldelli Michele

    2009-10-01

    Full Text Available Abstract Human Adenoviruses infect the upper and lower respiratory tracts, the urinary and digestive tracts, lymphoid systems and heart, and give rise to epidemic conjunctivitis. More than 51 human serotypes have been identified to-date, and classified into 6 species A-F. The species C Adenoviruses Ad2 and Ad5 (Ad2/5 cause upper and lower respiratory disease, but how viral structure relates to the selection of particular infectious uptake pathways is not known. An adenovirus mutant, Ad2-ts1 had been isolated upon chemical mutagenesis in the past, and shown to have unprocessed capsid proteins. Ad2-ts1 fails to package the viral protease L3/p23, and Ad2-ts1 virions do not efficiently escape from endosomes. It had been suggested that the C22187T point mutation leading to the substitution of the conserved proline 137 to leucine (P137L in the L3/p23 protease was at least in part responsible for this phenotype. To clarify if the C22187T mutation is necessary and sufficient for the Ad2-ts1 phenotype, we sequenced the genes encoding the structural proteins of Ad2-ts1, and confirmed that the Ad2-ts1 DNA carries the point mutation C22187T. Introduction of C22187T to the wild-type Ad2 genome in a bacterial artificial chromosome (Ad2-BAC gave Ad2-BAC46 virions with the full Ad2-ts1 phenotype. Reversion of Ad2-BAC46 gave wild-type Ad2 particles indicating that P137L is necessary and sufficient for the Ad2-ts1 phenotype. The kinetics of Ad2-ts1 uptake into cells were comparable to Ad2 suggesting similar endocytic uptake mechanisms. Surprisingly, infectious Ad2 or Ad5 but not Ad2-ts1 uptake required CALM (clathrin assembly lymphoid myeloid protein, which controls clathrin-mediated endocytosis and membrane transport between endosomes and the trans-Golgi-network. The data show that no other mutations than P137L in the viral protease are necessary to give rise to particles that are defective in capsid processing and endosomal escape. This provides a basis for

  6. LeftyA sensitive cytosolic pH regulation and glycolytic flux in Ishikawa human endometrial cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Salker, Madhuri S.; Zhou, Yuetao; Singh, Yogesh [Department of Physiology, University of Tuebingen, 72076 Tuebingen (Germany); Brosens, Jan [Division of Reproductive Health, Warwick Medical School, Clinical Sciences Research Laboratories, University Hospital, Coventry CV2 2DX (United Kingdom); Lang, Florian, E-mail: florian.lang@uni-tuebingen.de [Department of Physiology, University of Tuebingen, 72076 Tuebingen (Germany)

    2015-05-08

    Objective: LeftyA, a powerful regulator of stemness, embryonic differentiation, and reprogramming of cancer cells, counteracts cell proliferation and tumor growth. Key properties of tumor cells include enhanced glycolytic flux, which is highly sensitive to cytosolic pH and thus requires export of H{sup +} and lactate. H{sup +} extrusion is in part accomplished by Na{sup +}/H{sup +} exchangers, such as NHE1. An effect of LeftyA on transport processes has, however, never been reported. The present study thus explored whether LeftyA modifies regulation of cytosolic pH (pHi) in Ishikawa cells, a well differentiated endometrial carcinoma cell model. Methods: NHE1 transcript levels were determined by qRT-PCR, NHE1 protein abundance quantified by Western blotting, pH{sub i} estimated utilizing (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein [BCECF] fluorescence, Na{sup +}/H{sup +} exchanger activity from Na{sup +} dependent realkalinization after an ammonium pulse, and lactate concentration in the supernatant utilizing an enzymatic assay and subsequent colorimetry. Results: A 2 h treatment with LeftyA (8 ng/ml) significantly decreased NHE1 transcript levels (by 99.6%), NHE1 protein abundance (by 71%), Na{sup +}/H{sup +} exchanger activity (by 55%), pHi (from 7.22 ± 0.02 to 7.05 ± 0.02), and lactate release (by 41%). Conclusions: LeftyA markedly down-regulates NHE1 expression, Na{sup +}/H{sup +} exchanger activity, pHi, and lactate release in Ishikawa cells. Those effects presumably contribute to cellular reprogramming and growth inhibition. - Highlights: • LeftyA, an inhibitor of tumor growth, reduces Na{sup +}/H{sup +}-exchanger activity by 55%. • LeftyA decreases NHE1 transcripts by 99.6% and NHE1 protein by 71%. • LeftyA decreases cytosolic pH from 7.22 ± 0.02 to 7.05 ± 0.02. • Cytosolic acidification by Lefty A decreases glycolysis by 41%. • Cytosolic acidification by Lefty A compromises energy production of tumor cells.

  7. The hepatitis B virus ribonuclease H is sensitive to inhibitors of the human immunodeficiency virus ribonuclease H and integrase enzymes.

    Directory of Open Access Journals (Sweden)

    John E Tavis

    2013-01-01

    Full Text Available Nucleos(tide analog therapy blocks DNA synthesis by the hepatitis B virus (HBV reverse transcriptase and can control the infection, but treatment is life-long and has high costs and unpredictable long-term side effects. The profound suppression of HBV by the nucleos(tide analogs and their ability to cure some patients indicates that they can push HBV to the brink of extinction. Consequently, more patients could be cured by suppressing HBV replication further using a new drug in combination with the nucleos(tide analogs. The HBV ribonuclease H (RNAseH is a logical drug target because it is the second of only two viral enzymes that are essential for viral replication, but it has not been exploited, primarily because it is very difficult to produce active enzyme. To address this difficulty, we expressed HBV genotype D and H RNAseHs in E. coli and enriched the enzymes by nickel-affinity chromatography. HBV RNAseH activity in the enriched lysates was characterized in preparation for drug screening. Twenty-one candidate HBV RNAseH inhibitors were identified using chemical structure-activity analyses based on inhibitors of the HIV RNAseH and integrase. Twelve anti-RNAseH and anti-integrase compounds inhibited the HBV RNAseH at 10 µM, the best compounds had low micromolar IC(50 values against the RNAseH, and one compound inhibited HBV replication in tissue culture at 10 µM. Recombinant HBV genotype D RNAseH was more sensitive to inhibition than genotype H. This study demonstrates that recombinant HBV RNAseH suitable for low-throughput antiviral drug screening has been produced. The high percentage of compounds developed against the HIV RNAseH and integrase that were active against the HBV RNAseH indicates that the extensive drug design efforts against these HIV enzymes can guide anti-HBV RNAseH drug discovery. Finally, differential inhibition of HBV genotype D and H RNAseHs indicates that viral genetic variability will be a factor during drug

  8. A facile and sensitive peptide-modulating graphene oxide nanoribbon catalytic nanoplasmon analytical platform for human chorionic gonadotropin.

    Science.gov (United States)

    Liang, Aihui; Li, Chongning; Li, Dan; Luo, Yanghe; Wen, Guiqing; Jiang, Zhiliang

    2017-01-01

    The nanogold reaction between HAuCl 4 and citrate is very slow, and the catalyst graphene oxide nanoribbon (GONR) enhanced the nanoreaction greatly to produce gold nanoparticles (AuNPs) that exhibited strong surface plasmon resonance (SPR) absorption (Abs) at 550 nm and resonance Rayleigh scattering (RRS) at 550 nm. Upon addition of the peptide of human chorionic gonadotropin (hCG), the peptide could adsorb on the GONR surface, which inhibited the catalysis. When hCG was added, peptides were separated from the GONR surface due to the formation of stable peptide-hCG complex, which led to the activation of GONR catalytic effect. With the increase in hCG concentration, the RRS and Abs signal enhanced linearly. The enhanced RRS value showed a good linear relationship with hCG concentration in the range of 0.2-20 ng/mL, with a detection limit of 70 pg/mL. Accordingly, two new GONR catalytic RRS/Abs methods were established for detecting hCG in serum samples.

  9. A sensitive and reproducible in vivo imaging mouse model for evaluation of drugs against late-stage human African trypanosomiasis.

    Science.gov (United States)

    Burrell-Saward, Hollie; Rodgers, Jean; Bradley, Barbara; Croft, Simon L; Ward, Theresa H

    2015-02-01

    To optimize the Trypanosoma brucei brucei GVR35 VSL-2 bioluminescent strain as an innovative drug evaluation model for late-stage human African trypanosomiasis. An IVIS® Lumina II imaging system was used to detect bioluminescent T. b. brucei GVR35 parasites in mice to evaluate parasite localization and disease progression. Drug treatment was assessed using qualitative bioluminescence imaging and real-time quantitative PCR (qPCR). We have shown that drug dose-response can be evaluated using bioluminescence imaging and confirmed quantification of tissue parasite load using qPCR. The model was also able to detect drug relapse earlier than the traditional blood film detection and even in the absence of any detectable peripheral parasites. We have developed and optimized a new, efficient method to evaluate novel anti-trypanosomal drugs in vivo and reduce the current 180 day drug relapse experiment to a 90 day model. The non-invasive in vivo imaging model reduces the time required to assess preclinical efficacy of new anti-trypanosomal drugs. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  10. Identity Rights and Sensitive Ethical Questions: The European Convention on Human Rights and the Regulation of Surrogacy Arrangements.

    Science.gov (United States)

    Mulligan, Andrea

    2018-02-05

    Attitudes to surrogacy vary widely across Europe, leading to great variation in the domestic legal regimes of the Member States of the Council of Europe. Confronted with such diverse approaches, the European Court of Human Rights (ECtHR) faces a difficult task in seeking to apply Convention rights in the surrogacy context, which it has tackled in the recent cases of Mennesson v France and Paradiso and Campanelli v Italy. The primary purpose of this article is to propose an argument as to what the Convention requires of the Member States in the field of surrogacy. It is argued that while tensions exist between the leading cases, they may be reconciled by appreciating the importance of the right to identity, a facet of the right to respect for private life. Properly understood, the case law imposes obligations on the Member States as regards the legal status of surrogate-born children in both cross-border and domestic surrogacy. The secondary purpose of this article is to argue that, in the surrogacy context, the concept of identity should be given a richer interpretation which encompasses the child's relationship with genetic, gestational, and intended parents, and therefore that a narrow margin of appreciation must apply to all State interventions concerning the legal status of surrogate-born children. © The Author(s) 2018. Published by Oxford University Press; All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  11. Enhancing the Sensitivity of DNA Microarray Using Dye-Doped Silica Nanoparticles: Detection of Human Papilloma Virus

    Science.gov (United States)

    Enrichi, F.; Riccò, R.; Meneghello, A.; Pierobon, R.; Canton, G.; Cretaio, E.

    2010-10-01

    DNA microarray is a high-throughput technology used for detection and quantification of nucleic acid molecules and others of biological interest. The analysis is based on the specific hybridization between probe sequences deposited in array and a target ss-DNA amplified by PCR and functionalized by a fluorescent dye. Organic labels have well known disadvantages like photobleaching and low signal intensities, which put a limitation to the lower amount of DNA material that can be detected. Therefore for trace analysis the development of more efficient biomarkers is required. With this aim we present in this paper the synthesis and application of alternative hybrid nanosystems obtained by incorporating standard fluorescent molecules into monodisperse silica nanoparticles. Efficient application to the detection of Human Papilloma Virus is demonstrated. This virus is associated to the formation of cervical cancer, a leading cause of death by cancer for women worldwide. It is shown that the use of the novel biomarkers increases the optical signal of about one order of magnitude with respect to the free dyes or quantum dots in conventional instruments. This is due to the high number of molecules that can be accommodated into each nanoparticle, to the reduced photobleaching and to the improved environmental protection of the dyes when encapsulated in the silica matrix. The cheap and easy synthesis of these luminescent particles, the stability in water, the surface functionalizability and bio-compatibility make them very promising for present and future bio-labeling and bio-imaging applications.

  12. Ultra-high sensitivity of the non-immunological affinity of graphene oxide-peptide-based surface plasmon resonance biosensors to detect human chorionic gonadotropin.

    Science.gov (United States)

    Chiu, Nan-Fu; Kuo, Chia-Tzu; Lin, Ting-Li; Chang, Chia-Chen; Chen, Chen-Yu

    2017-08-15

    Specific peptide aptamers can be used in place of expensive antibody proteins, and they are gaining increasing importance as sensing probes due to their potential in the development of non-immunological assays with high sensitivity, affinity and specificity for human chorionic gonadotropin (hCG) protein. We combined graphene oxide (GO) sheets with a specific peptide aptamer to create a novel, simple and label-free tool to detect abnormalities at an early stage of pregnancy, a GO-peptide-based surface plasmon resonance (SPR) biosensor. This is the first binding interface experiment to successfully demonstrate binding specificity in kinetic analysis biomechanics in peptide aptamers and GO sheets. In addition to the improved affinity offered by the high compatibility with the target hCG protein, the major advantage of GO-peptide-based SPR sensors was their reduced nonspecific adsorption and enhanced sensitivity. The calculation of total electric field intensity (ΔE) in the GO-based sensing interfaces was significantly enhanced by up to 1.2 times that of a conventional SPR chip. The GO-peptide-based chip (1mM) had a high affinity (K A ) of 6.37×10 12 M -1 , limit of detection of 0.065nM and ultra-high sensitivity of 16 times that of a conventional SPR chip. The sensitivity of the slope ratio of the low concentration hCG protein assay in linear regression analysis was GO-peptide (1mM): GO-peptide (0.1mM): conventional chip (8-mercaptooctanoic acid)-peptide (0.1mM)=8.6: 3.3: 1. In summary, the excellent binding affinity, low detection limit, high sensitivity, good stability and specificity suggest the potential of this GO-peptide-based SPR chip detection method in clinical application. The development of real-time whole blood analytic and diagnostic tools to detect abnormalities at an early stage of pregnancy is a promising technique for future clinical application. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Cranberry Proanthocyanidins are Cytotoxic to Human Cancer Cells and Sensitize Platinum-Resistant Ovarian Cancer Cells to Paraplatin

    Science.gov (United States)

    Singh, Ajay P.; Singh, Rakesh K.; Kim, Kyu Kwang; Satyan, K. S.; Nussbaum, Roger; Torres, Monica; Brard, Laurent; Vorsa, Nicholi

    2010-01-01

    Polyphenolic extracts of the principal flavonoid classes present in cranberry were screened in vitro for cytotoxicity against solid tumor cells lines, identifying two fractions composed principally of proanthocyanidins (PACs) with potential anticancer activity. Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF-MS) analysis of the proanthocyanidins (PACs) fractions indicated the presence of A-type PACs with 1–4 linkages containing between 2–8 epicatechin units with a maximum of 1 epigallocatechin unit. PACs exhibited in vitro cytotoxicity against platinum-resistant human ovarian, neuroblastoma and prostate cancer cell lines (IC50 = 79–479 μg/mL) but were non-cytotoxic to lung fibroblast cells (IC50 > 1000 μg/ml). SKOV-3 ovarian cancer cells treated with PACs exhibited classic apoptotic changes. PACs acted synergistically with paraplatin in SKOV-3 cells. Pretreatment of SKOV-3 cells with PACs (106 μg/ ml) resulted in a significant reduction of the paraplatin IC50 value. Similarly, in a BrdU incorporation assay, co-treatment of SKOV-3 cells with PACs and paraplatin revealed reduced cell proliferation at lower concentrations than with either individually. In SKOV-3 cell cultures co-treated with PAC-1 and paraplatin, an HPLC analysis indicated differential quantitative presence of various PAC oligomers such as DP-8, -9, -11 and -14 indicating either selective binding or uptake. Cranberry proanthocyanidins exhibit cell-line specific cytotoxicity, induce apoptotic markers and augment cytotoxicity of paraplatin in platinum-resistant SKOV-3 ovarian cancer cells. PMID:19172579

  14. Single-channel properties of a stretch-sensitive chloride channel in the human mast cell line HMC-1.

    Science.gov (United States)

    Wang, Lina; Ding, Guanghong; Gu, Quanbao; Schwarz, Wolfgang

    2010-04-01

    A stretch-activated (SA) Cl(-) channel in the plasma membrane of the human mast cell line HMC-1 was identified in outside-out patch-clamp experiments. SA currents, induced by pressure applied to the pipette, exhibited voltage dependence with strong outward rectification (55.1 pS at +100 mV and an about tenfold lower conductance at -100 mV). The probability of the SA channel being open (P (o)) also showed steep outward rectification and pressure dependence. The open-time distribution was fitted with three components with time constants of tau(1o) = 755.1 ms, tau(2o) = 166.4 ms, and tau(3o) = 16.5 ms at +60 mV. The closed-time distribution also required three components with time constants of tau(1c) = 661.6 ms, tau(2c) = 253.2 ms, and tau(3c) = 5.6 ms at +60 mV. Lowering extracellular Cl(-) concentration reduced the conductance, shifted the reversal potential toward chloride reversal potential, and decreased the P (o) at positive potentials. The SA Cl(-) currents were reversibly blocked by the chloride channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) but not by (Z)-1-(p-dimethylaminoethoxyphenyl)-1,2-diphenyl-1-butene (tamoxifen). Furthermore, in HMC-1 cells swelling due to osmotic stress, DIDS could inhibit the increase in intracellular [Ca(2+)] and degranulation. We conclude that in the HMC-1 cell line, the SA outward currents are mediated by Cl(-) influx. The SA Cl(-) channel might contribute to mast cell degranulation caused by mechanical stimuli or accelerate membrane fusion during the degranulation process.

  15. High-sensitivity gamma spectroscopy for extended sources. Application to activity measurements on the human body, on glass, and on soil

    International Nuclear Information System (INIS)

    Jouve, B.

    1962-01-01

    The measurement and location by gamma spectroscopy of human body internal contaminations at maximum permissible levels, and, in certain cases, at lower activities such as that due to 40 K was investigated. The characteristics of the high-sensitivity apparatus used are given, and several assemblies using large-volume NaI(Tl) scintillators are described. The relatively light shielding required for natural radioactivity permitted construction of mobile assembly. Conditions of use are described, and the results are given. All gamma emitting elements were measured in 15 min at levels lower than the tolerance dose. Gamma spectroscopy was also used to determine fission products in the earth and to study radioactive elements in the presence of other emitters. (author) [fr

  16. Differential expression of microRNA expression in tamoxifen-sensitive MCF-7 versus tamoxifen-resistant LY2 human breast cancer cells

    Science.gov (United States)

    Manavalan, Tissa T.; Teng, Yun; Appana, Savitri N.; Datta, Susmita; Kalbfleisch, Theodore S.; Li, Yong; Klinge, Carolyn M.

    2011-01-01

    Microarrays identified miRNAs differentially expressed and 4-hydroxytamoxifen (4-OHT) regulated in MCF-7 endocrine- sensitive versus resistant LY2 human breast cancer cells. 97 miRNAs were differentially expressed in MCF-7 versus LY2 cells. Opposite expression of miRs- 10a, 21, 22, 29a, 93, 125b, 181, 200a, 200b, 200c, 205, and 222 was confirmed. Bioinformatic analyses to impute the biological significance of these miRNAs identified 36 predicted gene targets from those regulated by 4-OHT in MCF-7 cells. Agreement in the direction of anticipated regulation was detected for 12 putative targets. These miRNAs with opposite expression between the two cell lines may be involved in endocrine resistance. PMID:21955614

  17. Rapid, sensitive, type specific PCR detection of the E7 region of human papillomavirus type 16 and 18 from paraffin embedded sections of cervical carcinoma

    DEFF Research Database (Denmark)

    Lesnikova, Iana; Lidang, Marianne; Hamilton-Dutoit, Steven

    2010-01-01

    ABSTRACT: Human papillomavirus (HPV) infection, and in particularly infection with HPVs 16 and 18, is a central carcinogenic factor in the uterine cervix. We established and optimized a PCR assay for the detection and discrimination of HPV types 16 and 18 in archival formaldehyde fixed and paraffin...... embedded (FFPE) sections of cervical cancer.Tissue blocks from 35 cases of in situ or invasive cervical squamous cell carcinoma and surrogate FFPE sections containing the cell lines HeLa and SiHa were tested for HPV 16 and HPV18 by conventional PCR using type specific primers, and for the housekeeping gene...... beta-actin. Using HPV 16 E7 primers, PCR products with the expected length were detected in 18 of 35 of FFPE sections (51%). HPV 18 E7 specific sequences were detected in 3 of 35 FFPE sections (9%).In our experience, the PCR technique is a robust, simple and sensitive way of type specific detection...

  18. Schistosoma mansoni Infection of Mice, Rats and Humans Elicits a Strong Antibody Response to a Limited Number of Reduction-Sensitive Epitopes on Five Major Tegumental Membrane Proteins.

    Directory of Open Access Journals (Sweden)

    Greice Krautz-Peterson

    2017-01-01

    Full Text Available Schistosomiasis is a major disease of the developing world for which no vaccine has been successfully commercialized. While numerous Schistosoma mansoni worm antigens have been identified that elicit antibody responses during natural infections, little is known as to the identities of the schistosome antigens that are most prominently recognized by antibodies generated through natural infection. Non-reducing western blots probed with serum from schistosome-infected mice, rats and humans on total extracts of larval or adult schistosomes revealed that a small number of antigen bands predominate in all cases. Recognition of each of these major bands was lost when the blots were run under reducing condition. We expressed a rationally selected group of schistosome tegumental membrane antigens in insect host cells, and used the membrane extracts of these cells to unambiguously identify the major antigens recognized by S. mansoni infected mouse, rat and human serum. These results revealed that a limited number of dominant, reduction-sensitive conformational epitopes on five major tegumental surface membrane proteins: SmTsp2, Sm23, Sm29, SmLy6B and SmLy6F, are primary targets of mouse, rat and human S. mansoni infection sera antibodies. We conclude that, Schistosoma mansoni infection of both permissive (mouse and non-permissive (rat rodent models, as well as humans, elicit a dominant antibody response recognizing a limited number of conformational epitopes on the same five tegumental membrane proteins. Thus it appears that neither infecting schistosomula nor mature adult schistosomes are substantively impacted by the robust circulating anti-tegumental antibody response they elicit to these antigens. Importantly, our data suggest a need to re-evaluate host immune responses to many schistosome antigens and has important implications regarding schistosome immune evasion mechanisms and schistosomiasis vaccine development.

  19. Cytotoxicity and cellular mechanisms of toxicity of CuO NPs in mussel cells in vitro and comparative sensitivity with human cells.

    Science.gov (United States)

    Katsumiti, Alberto; Thorley, Andrew J; Arostegui, Inmaculada; Reip, Paul; Valsami-Jones, Eugenia; Tetley, Teresa D; Cajaraville, Miren P

    2018-04-01

    There is a need to assess human and ecosystem health effects of copper oxide nanoparticles (CuO NPs), extensively used in many industrial products. Here, we aimed to determine the cytotoxicity and cellular mechanisms involved in the toxicity of CuO NPs in mussel cells (hemocytes and gill cells) in parallel with exposures to ionic Cu and bulk CuO, and to compare the sensitivity of mussel primary cells with a well-established human cell line (pulmonary TT1 cells). At similar doses, CuO NPs promoted dose-dependent cytotoxicity and increased reactive oxygen species (ROS) production in mussel and human cells. In mussel cells, ionic Cu was more toxic than CuO NPs and the latter more than bulk CuO. Ionic Cu and CuO NPs increased catalase and acid phosphatase activities in both mussel cells and decreased gill cells Na-K-ATPase activity. All Cu forms produced DNA damage in hemocytes, whereas in gill cells only ionic Cu and CuO NPs were genotoxic. Induction of the MXR transport activity was found in gill cells exposed to all forms of Cu and in hemocytes exposed to ionic Cu and CuO NPs. Phagocytosis increased only in hemocytes exposed to CuO NPs, indicating a nanoparticle-specific immunostimulatory effect. In conclusion, toxicity of CuO NPs is driven by ROS in human and mussel cells. Mussel cells respond to CuO NP exposure by triggering an array of defensive mechanisms. Copyright © 2018 Elsevier Ltd. All rights reserved.

  20. Sensitive monitoring of monoterpene metabolites in human urine using two-step derivatisation and positive chemical ionisation-tandem mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Schmidt, Lukas [Institute and Outpatient Clinic of Occupational, Social and Environmental Medicine, University of Erlangen-Nuremberg, Schillerstrasse 25/29, 91054 Erlangen (Germany); Belov, Vladimir N. [Max Planck Institute for Biophysical Chemistry, Facility for Synthetic Chemistry, Am Fassberg 11, 37077 Göttingen (Germany); Göen, Thomas, E-mail: Thomas.Goeen@ipasum.med.uni-erlangen.de [Institute and Outpatient Clinic of Occupational, Social and Environmental Medicine, University of Erlangen-Nuremberg, Schillerstrasse 25/29, 91054 Erlangen (Germany)

    2013-09-02

    Highlights: •Sensitive monitoring of 10 metabolites of (R)-limonene, α-pinene, and Δ{sup 3}-carene in human urine samples. •Fast and simple sample preparation and derivatisation procedure using two-step silylation for unreactive tertiary hydroxyl groups. •Synthesis of reference substances and isotopically labelled internal standards of (R)-limonene, α-pinene, and Δ{sup 3}-carene metabolites. •Study on (R)-limonene, α-pinene, and Δ{sup 3}-carene metabolite background exposure of 36 occupationally unexposed volunteers. -- Abstract: A gas chromatographic–positive chemical ionisation-tandem mass spectrometric (GC–PCI-MS/MS) method for the simultaneous determination of 10 oxidative metabolites of the monoterpenoid hydrocarbons α-pinene, (R)-limonene, and Δ{sup 3}-carene ((+)-3-carene) in human urine was developed and tested for the monoterpene biomonitoring of the general population (n = 36). The method involves enzymatic cleavage of the glucuronides followed by solid-supported liquid–liquid extraction and derivatisation using a two-step reaction with N,O-bis(trimethylsilyl)-trifluoroacetamide and N-(trimethylsilyl)imidazole. The method proved to be both sensitive and reliable with detection limits ranging from 0.1 to 0.3 μg L{sup −1}. In contrast to the frequent and distinct quantities of (1S,2S,4R)-limonene-1,2-diol, the (1R,2R,4R)-stereoisomer could not be detected. The expected metabolite of (+)-3-carene, 3-caren-10-ol was not detected in any of the samples. All other metabolites were detected in almost all urine samples. The procedure enables for the first time the analysis of trace levels of a broad spectrum of mono- and bicyclic monoterpenoid metabolites (alcohols, diols, and carboxylic acids) in human urine. This analytical procedure is a powerful tool for population studies as well as for the discovery of human metabolism and toxicokinetics of monoterpenes.

  1. Radiation survival parameters of antineoplastic drug-sensitive and -resistant human ovarian cancer cell lines and their modification by buthionine sulfoximine

    International Nuclear Information System (INIS)

    Louie, K.G.; Behrens, B.C.; Kinsella, T.J.; Hamilton, T.C.; Grotzinger, K.R.; McKoy, W.M.; Winker, M.A.; Ozols, R.F.

    1985-01-01

    The optimum integration of chemotherapy and irradiation is of potential clinical significance in the treatment of ovarian cancer. A series of human ovarian cancer cell lines have been developed in which dose-response relationships to standard anticancer drugs have been determined, and the patterns of cross-resistance between these drugs and irradiation have been established. By stepwise incubation with drugs, sublines of A2780, a drug-sensitive cell line, have been made 100-fold, 10-fold, and 10-fold more resistant to Adriamycin (2780AD), melphalan (2780ME), and cisplatin (2780CP). Two additional cell lines, NIH:OVCAR-3nu(Ag+) and NIH:OVCAR-4(Ag+), were established from drug-refractory patients. 2780ME, 2780CP, OVCAR-3nu(Ag+), and OVCAR-4(Ag+) are all cross-resistant to irradiation, with DOS of 146, 187, 143, and 203, respectively. However, 2780AD remains sensitive to radiation, with a DO of 111, which is similar to that of A2780 (101). Glutathione (GSH) levels are elevated in 2780ME, 2780CP, OVCAR-3nu(Ag+), and OVCAR-4(Ag+) to 4.58, 6.13, 12.10, and 15.14 nmol/10(6) cells as compared to A2780, with 1.89 nmol/10(6) cells. However, the GSH level in 2780AD is only minimally higher than that in A2780 (2.94 nmol/10(6) cells). Buthionine sulfoximine, a specific inhibitor of GSH synthesis, significantly increases the radiation sensitivity of 2780ME (changing the DO from 143 to 95) and 2780CP to a lesser extent, suggesting that intracellular GSH levels may play an important role in the radiation response of certain neoplastic cells

  2. Decreased agonist sensitivity of human GABA(A) receptors by an amino acid variant, isoleucine to valine, in the alpha1 subunit.

    Science.gov (United States)

    Westh-Hansen, S E; Rasmussen, P B; Hastrup, S; Nabekura, J; Noguchi, K; Akaike, N; Witt, M R; Nielsen, M

    1997-06-25

    Recombinant human GABA(A) receptors were investigated in vitro by coexpression of cDNAs coding for alpha1, beta2, and gamma2 subunits in the baculovirus/Sf-9 insect cell system. We report that a single amino acid exchange (isoleucine 121 to valine 121) in the N-terminal, extracellular part of the alpha1 subunit induces a marked decrease in agonist GABA(A) receptor ligand sensitivity. The potency of muscimol and GABA to inhibit the binding of the GABA(A) receptor antagonist [3H]SR 95531 (2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl)pyridazinium bromide) was higher in receptor complexes of alpha1(ile 121) beta2gamma2 than in those of alpha1(val 121) beta2gamma2 (IC50 values were 32-fold and 26-fold lower for muscimol and GABA, respectively). The apparent affinity of the GABA(A) receptor antagonist bicuculline methiodide to inhibit the binding of [3H]SR 95531 did not differ between the two receptor complex variants. Electrophysiological measurements of GABA induced whole-cell Cl- currents showed a ten-fold decrease in the GABA(A) receptor sensitivity of alpha1 (val 121) beta2gamma2 as compared to alpha1(ile 121) beta2gamma2 receptor complexes. Thus, a relatively small change in the primary structure of the alpha1 subunit leads to a decrease selective for GABA(A) receptor sensitivity to agonist ligands, since no changes were observed in a GABA(A) receptor antagonist affinity and benzodiazepine receptor binding.

  3. Effect of 12 months of recombinant human growth hormone replacement therapy on insulin sensitivity in GH-deficient adults as determined by different methods.

    Directory of Open Access Journals (Sweden)

    Micic D

    2002-10-01

    Full Text Available BACKGROUND: Controversial results have been obtained in measuring insulin sensitivity (S(I during recombinant human growth hormone (rhGH treatment in adult growth hormone deficient (GH-deficient patients. AIMS: The aim of our study was to estimate S(I before and during treatment using three different methods for quantifying insulin sensitivity in GH-deficient adults treated with rhGH. SETTINGS AND DESIGN: Twenty-one GH-deficient adults were treated with rhGH during 12 months. S(I was estimated using Minimal model analysis, Homeostatic Model of Assessment (HOMA and Quantitative Insulin Sensitivity Check Index (QUICKI before and after 3, 6, 9 and 12 months of rhGH therapy. MATERIAL AND METHODS: Oral Glucose Tolerance Test (OGTT and Frequently Sampled Intravenous Glucose Tolerance Test (FSIGT were performed in each patient at respective time intervals. QUICKI and HOMA were calculated using basal values of glucose and insulin from FSIGT. Minimal model computer analysis was calculated from glucose and insulin data obtained during FSIGT. STATISTICAL ANALYSIS: Area under the curve for glucose, insulin and C-peptide were calculated using trapezoidal rule from OGTT data. Differences and correlations were tested using ANOVA for repeated measures, Wilcoxon′s matched-paired test, paired t-test, Pearson′s correlation and Bland Altman plot. RESULTS: There were no significant changes in S(I using Minimal model analysis and QUICKI during rhGH treatment. On the contrary, HOMA analysis indicated significant deterioration in S(I after 12 months of therapy. CONCLUSION: Our study did not demonstrate any changes in S(I using Minimal model and QUICKI analysis, while there was significant increase in insulin resistance using HOMA model. We suggest that the choice of method for the determination of S(I may influence the interpretation of results concerning the effect of rhGH therapy on S(I in GH-deficient adults.

  4. Quantitation of donepezil and its active metabolite 6-O-desmethyl donepezil in human plasma by a selective and sensitive liquid chromatography-tandem mass spectrometric method

    Energy Technology Data Exchange (ETDEWEB)

    Patel, Bhavin N. [Chemistry Department, School of Sciences, Gujarat University, Navrangpura, Ahmedabad 380 009, Gujarat (India); Analytical Laboratory, BA Research India Ltd., Bodakdev, Ahmedabad 380 054, Gujarat (India); Sharma, Naveen [Analytical Laboratory, BA Research India Ltd., Bodakdev, Ahmedabad 380 054, Gujarat (India); Sanyal, Mallika [Chemistry Department, St. Xaviers' College, Navrangpura, Ahmedabad 380 009, Gujarat (India); Shrivastav, Pranav S. [Chemistry Department, School of Sciences, Gujarat University, Navrangpura, Ahmedabad 380 009, Gujarat (India)], E-mail: pranav_shrivastav@yahoo.com

    2008-11-23

    A sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the simultaneous determination of donepezil (D) and its pharmacologically active metabolite, 6-O-desmethyl donepezil (6-ODD) in human plasma is developed using galantamine as internal standard (IS). The analytes and IS were extracted from 500 {mu}L aliquots of human plasma via solid-phase extraction (SPE) on Waters Oasis HLB cartridges. Chromatographic separation was achieved in a run time of 6.0 min on a Waters Novapak C18 (150 mm x 3.9 mm, 4 {mu}m) column under isocratic conditions. Detection of analytes and IS was done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The protonated precursor to product ion transitions monitored for D, 6-ODD and IS were at m/z 380.1 {yields} 91.2, 366.3 {yields} 91.3 and 288.2 {yields} 213.2, respectively. The method was fully validated for its selectivity, interference check, sensitivity, linearity, precision and accuracy, recovery, matrix effect, ion suppression/enhancement, cross-specificity, stability and dilution integrity. A linear dynamic range of 0.10-50.0 ng mL{sup -1} for D and 0.02-10.0 ng mL{sup -1} for 6-ODD was evaluated with mean correlation coefficient (r) of 0.9975 and 0.9985, respectively. The intra-batch and inter-batch precision (%CV, coefficient of variation) across five quality control levels was less than 7.5% for both the analytes. The method was successfully applied to a bioequivalence study of 10 mg donepezil tablet formulation in 24 healthy Indian male subjects under fasting condition.

  5. Predicting skin sensitization potential and inter-laboratory reproducibility of a human Cell Line Activation Test (h-CLAT) in the European Cosmetics Association (COLIPA) ring trials.

    Science.gov (United States)

    Sakaguchi, Hitoshi; Ryan, Cindy; Ovigne, Jean-Marc; Schroeder, Klaus R; Ashikaga, Takao

    2010-09-01

    Regulatory policies in Europe prohibited the testing of cosmetic ingredients in animals for a number of toxicological endpoints. Currently no validated non-animal test methods exist for skin sensitization. Evaluation of changes in cell surface marker expression in dendritic cell (DC)-surrogate cell lines represents one non-animal approach. The human Cell Line Activation Test (h-CLAT) examines the level of CD86 and CD54 expression on the surface of THP-1 cells, a human monocytic leukemia cell line, following 24h of chemical exposure. To examine protocol transferability, between-lab reproducibility, and predictive capacity, the h-CLAT has been evaluated by five independent laboratories in several ring trials (RTs) coordinated by the European Cosmetics Association (COLIPA). The results of the first and second RTs demonstrated that the protocol was transferable and basically had good between-lab reproducibility and predictivity, but there were some false negative data. To improve performance, protocol and prediction model were modified. Using the modified prediction model in the first and second RT, accuracy was improved. However, about 15% of the outcomes were not correctly identified, which exposes some of the limitations of the assay. For the chemicals evaluated, the limitation may due to chemical being a weak allergen or having low solubility (ex. alpha-hexylcinnamaldehyde). The third RT evaluated the modified prediction model and satisfactory results were obtained. From the RT data, the feasibility of utilizing cell lines as surrogate DC in development of in vitro skin sensitization methods shows promise. The data also support initiating formal pre-validation of the h-CLAT in order to fully understand the capabilities and limitations of the assay. Copyright 2010 Elsevier Ltd. All rights reserved.

  6. A simple and sensitive HPLC method for analysis of imipramine in human plasma with UV detection and liquid-liquid extraction: Application in bioequivalence studies.

    Science.gov (United States)

    Rezazadeh, Mahboubeh; Emami, Jaber

    2016-01-01

    High-performance liquid chromatography (HPLC) methods employing ultraviolet (UV) detector are not sufficiently sensitive to measure the low plasma concentrations following single oral dose of imipramine. Therefore, in the present study a simple, rapid and yet sensitive HPLC method with UV detection was developed and validated for quantitation of imipramine in human plasma samples. An efficient liquid-liquid extraction (LLE) of imipramine from plasma with the mixture of hexane/isoamyl alcohol (98:2) and back extraction of the drug in acidic medium concomitant with evaporation of organic phase allowed the use of UV detector to conveniently measure plasma levels of this compound as low level as 3 ng/ml. Separation was achieved on a μ-Bondapak C18 HPLC column using sodium hydrogen phosphate solution (0.01 M)/acetonitrile (60/40 v/v) at pH 3.5 ± 0.1 at 1.5 ml/min. Trimipramine was used as the internal standard for analysis of plasma samples. The retention times for imipramine and trimipramine were 4.3 and 5.2 min, respectively. Calibration curve was linear in the range of 3-40 ng/ml using human plasma with the average extraction recovery of 85 ± 5%. Imipramine was found to be stable in plasma samples with no evidence of degradation during three freeze-thaw cycles and three months storage at -70°C. The current validated method was finally applied in bioequivalence studies of two different imipramine products according to a standard two-way crossover design with a two weeks washout period.

  7. Development and validation of sensitive LC-MS/MS assays for quantification of HP-β-CD in human plasma and CSF.

    Science.gov (United States)

    Jiang, Hui; Sidhu, Rohini; Fujiwara, Hideji; De Meulder, Marc; de Vries, Ronald; Gong, Yong; Kao, Mark; Porter, Forbes D; Yanjanin, Nicole M; Carillo-Carasco, Nuria; Xu, Xin; Ottinger, Elizabeth; Woolery, Myra; Ory, Daniel S; Jiang, Xuntian

    2014-07-01

    2-Hydroxypropyl-β-cyclodextrin (HP-β-CD), a widely used excipient for drug formulation, has emerged as an investigational new drug for the treatment of Niemann-Pick type C1 (NPC1) disease, a neurodegenerative cholesterol storage disorder. Development of a sensitive quantitative LC-MS/MS assay to monitor the pharmacokinetics (PKs) of HP-β-CD required for clinical trials has been challenging owing to the dispersity of the HP-β-CD. To support a phase 1 clinical trial for ICV delivery of HP-β-CD in NPC1 patients, novel methods for quantification of HP-β-CD in human plasma and cerebrospinal fluid (CSF) using LC-MS/MS were developed and validated: a 2D-LC-in-source fragmentation-MS/MS (2D-LC-IF-MS/MS) assay and a reversed phase ultra performance LC-MS/MS (RP-UPLC-MS/MS) assay. In both assays, protein precipitation and "dilute and shoot" procedures were used to process plasma and CSF, respectively. The assays were fully validated and in close agreement, and allowed determination of PK parameters for HP-β-CD. The LC-MS/MS methods are ∼100-fold more sensitive than the current HPLC assay, and were successfully employed to analyze HP-β-CD in human plasma and CSF samples to support the phase 1 clinical trial of HP-β-CD in NPC1 patients. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

  8. Sustained Brown Fat Stimulation and Insulin Sensitization by a Humanized Bispecific Antibody Agonist for Fibroblast Growth Factor Receptor 1/βKlotho Complex

    Directory of Open Access Journals (Sweden)

    Ganesh Kolumam

    2015-07-01

    Full Text Available Dissipating excess calories as heat through therapeutic stimulation of brown adipose tissues (BAT has been proposed as a potential treatment for obesity-linked disorders. Here, we describe the generation of a humanized effector-less bispecific antibody that activates fibroblast growth factor receptor (FGFR 1/βKlotho complex, a common receptor for FGF21 and FGF19. Using this molecule, we show that antibody-mediated activation of FGFR1/βKlotho complex in mice induces sustained energy expenditure in BAT, browning of white adipose tissue, weight loss, and improvements in obesity-associated metabolic derangements including insulin resistance, hyperglycemia, dyslipidemia and hepatosteatosis. In mice and cynomolgus monkeys, FGFR1/βKlotho activation increased serum high-molecular-weight adiponectin, which appears to contribute over time by enhancing the amplitude of the metabolic benefits. At the same time, insulin sensitization by FGFR1/βKlotho activation occurs even before the onset of weight loss in a manner that is independent of adiponectin. Together, selective activation of FGFR1/βKlotho complex with a long acting therapeutic antibody represents an attractive approach for the treatment of type 2 diabetes and other obesity-linked disorders through enhanced energy expenditure, insulin sensitization and induction of high-molecular-weight adiponectin.

  9. Simple and sensitive analysis of blonanserin and blonanserin C in human plasma by liquid chromatography tandem mass spectrometry and its application.

    Science.gov (United States)

    Zheng, Yunliang; Hu, Xingjiang; Liu, Jian; Wu, Guolan; Zhou, Huili; Zhu, Meixiang; Zhai, You; Wu, Lihua; Shentu, Jianzhong

    2014-01-01

    A highly sensitive, simple, and rapid liquid chromatography tandem mass spectrometry method to simultaneously determine blonanserin and blonanserin C in human plasma with AD-5332 as internal standard (IS) was established. A simple direct protein precipitation method was used for the sample pretreatment, and chromatographic separation was performed on a Waters XBridge C8 (4.6 × 150 mm, 3.5  μ m) column. The mobile phase consists of a mixture of 10 mM ammonium formate and 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B). To quantify blonanserin, blonanserin C, and IS, multiple reaction monitoring (MRM) was performed in positive ESI mode. The calibration curve was linear in the concentration range of 0.012-5.78 ng·mL(-1) for blonanserin and 0.023-11.57 ng·mL(-1) for blonanserin C (r (2) > 0.9990). The intra- and interday precision of three quality control (QC) levels in plasma were less than 7.5%. Finally, the current simple, sensitive, and accurate LC-MS/MS method was successfully applied to investigate the pharmacokinetics of blonanserin and blonanserin C in healthy Chinese volunteers.

  10. Simple and Sensitive Analysis of Blonanserin and Blonanserin C in Human Plasma by Liquid Chromatography Tandem Mass Spectrometry and Its Application

    Directory of Open Access Journals (Sweden)

    Yunliang Zheng

    2014-01-01

    Full Text Available A highly sensitive, simple, and rapid liquid chromatography tandem mass spectrometry method to simultaneously determine blonanserin and blonanserin C in human plasma with AD-5332 as internal standard (IS was established. A simple direct protein precipitation method was used for the sample pretreatment, and chromatographic separation was performed on a Waters XBridge C8 (4.6×150 mm, 3.5 μm column. The mobile phase consists of a mixture of 10 mM ammonium formate and 0.1% formic acid in water (A and 0.1% formic acid in methanol (B. To quantify blonanserin, blonanserin C, and IS, multiple reaction monitoring (MRM was performed in positive ESI mode. The calibration curve was linear in the concentration range of 0.012–5.78 ng·mL−1 for blonanserin and 0.023–11.57 ng·mL−1 for blonanserin C (r2>0.9990. The intra- and interday precision of three quality control (QC levels in plasma were less than 7.5%. Finally, the current simple, sensitive, and accurate LC-MS/MS method was successfully applied to investigate the pharmacokinetics of blonanserin and blonanserin C in healthy Chinese volunteers.

  11. Local temperature-sensitive mechanisms are important mediators of limb tissue hyperemia in the heat-stressed human at rest and during small muscle mass exercise.

    Science.gov (United States)

    Chiesa, Scott T; Trangmar, Steven J; Kalsi, Kameljit K; Rakobowchuk, Mark; Banker, Devendar S; Lotlikar, Makrand D; Ali, Leena; González-Alonso, José

    2015-07-15

    Limb tissue and systemic blood flow increases with heat stress, but the underlying mechanisms remain poorly understood. Here, we tested the hypothesis that heat stress-induced increases in limb tissue perfusion are primarily mediated by local temperature-sensitive mechanisms. Leg and systemic temperatures and hemodynamics were measured at rest and during incremental single-legged knee extensor exercise in 15 males exposed to 1 h of either systemic passive heat-stress with simultaneous cooling of a single leg (n = 8) or isolated leg heating or cooling (n = 7). Systemic heat stress increased core, skin and heated leg blood temperatures (Tb), cardiac output, and heated leg blood flow (LBF; 0.6 ± 0.1 l/min; P 0.05). Increased heated leg deep tissue blood flow was closely related to Tb (R(2) = 0.50; P 0.05), despite unchanged systemic temperatures and hemodynamics. During incremental exercise, heated LBF was consistently maintained ∼ 0.6 l/min higher than that in the cooled leg (P conductance in both legs showing a strong correlation with their respective local Tb (R(2) = 0.85 and 0.95, P < 0.05). We conclude that local temperature-sensitive mechanisms are important mediators in limb tissue perfusion regulation both at rest and during small-muscle mass exercise in hyperthermic humans. Copyright © 2015 the American Physiological Society.

  12. Relationships between structure, ionization profile and sensitivity of exogenous anabolic steroids under electrospray ionization and analysis in human urine using liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Cha, Eunju; Kim, Sohee; Kim, Hee Won; Lee, Kang Mi; Kim, Ho Jun; Kwon, Oh-Seung; Lee, Jaeick

    2016-04-01

    The relationships between the ionization profile, sensitivity, and structures of 64 exogenous anabolic steroids (groups I-IV) was investigated under electrospray ionization (ESI) conditions. The target analytes were ionized as [M + H](+) or [M + H-nH2 O](+) in the positive mode, and these ions were used as precursor ions for selected reaction monitoring analysis. The collision energy and Q3 ions were optimized based on the sensitivity and selectivity. The limits of detection (LODs) were 0.05-20 ng/mL for the 64 steroids. The LODs for 38 compounds, 14 compounds and 12 compounds were in the range of 0.05-1, 2-5 and 10-20 ng/mL, respectively. Steroids including the conjugated keto-functional group at C3 showed good proton affinity and stability, and generated the [M + H](+) ion as the most abundant precursor ion. In addition, the LODs of steroids using the [M + H](+) ion as the precursor ion were mostly distributed at low concentrations. In contrast, steroids containing conjugated/unconjugated hydroxyl functional groups at C3 generated [M + H - H2 O](+) or [M + H - 2H2 O](+) ions, and these steroids showed relatively high LODs owing to poor stability and multiple ion formation. An LC-MS/MS method based on the present ionization profile was developed and validated for the determination of 78 steroids (groups I-V) in human urine. Copyright © 2015 John Wiley & Sons, Ltd.

  13. Glutathione export by human lymphoid cells: depletion of glutathione by inhibition of its synthesis decreases export and increases sensitivity to irradiation.

    Science.gov (United States)

    Dethmers, J K; Meister, A

    1981-12-01

    Glutathione (in the form of GSH) is transported out of cultured human lymphoid cells at rates proportional to the intracellular glutathione levels. Inhibition of glutathione synthesis by buthionine sulfoximine, a potent selective inhibitor of gamma-glutamylcysteine synthetase, leads to exponential decrease in intracellular glutathione, a large fraction of which appears extracellularly, indicating that glutathione turnover is associated with its export. Although cells with 0.09 mM glutathione (4% of controls) were 85% viable, further decrease was associated with marked loss of viability. Cells with 4-5% of control glutathione levels were much more sensitive than control cells to the effects of gamma radiation and of 5,5'-dithiobis(2-nitrobenzoate). Depletion of glutathione by use of buthionine sulfoximine has advantages over other reagents (such as diamide, other oxidizing agents, and diethylmaleate, which affect other cellular components and may increase glutathione disulfide levels) and therefore has potential usefulness in sensitizing cells to the effects of radiation and to therapeutic agents that are detoxified by reactions involving glutathione.

  14. Generation of glucose-sensitive insulin-secreting beta-like cells from human embryonic stem cells by incorporating a synthetic lineage-control network.

    Science.gov (United States)

    Saxena, Pratik; Bojar, Daniel; Zulewski, Henryk; Fussenegger, Martin

    2017-10-10

    We previously reported novel technology to differentiate induced pluripotent stem cells (IPSCs) into glucose-sensitive insulin-secreting beta-like cells by engineering a synthetic lineage-control network regulated by the licensed food additive vanillic acid. This genetic network was able to program intricate expression dynamics of the key transcription factors Ngn3 (neurogenin 3, OFF-ON-OFF), Pdx1 (pancreatic and duodenal homeobox 1, ON-OFF-ON) and MafA (V-maf musculoaponeurotic fibrosarcoma oncogene homologue A, OFF-ON) to guide the differentiation of IPSC-derived pancreatic progenitor cells to beta-like cells. In the present study, we show for the first time that this network can also program the expression dynamics of Ngn3, Pdx1 and MafA in human embryonic stem cell (hESC)-derived pancreatic progenitor cells and drive differentiation of these cells into glucose-sensitive insulin-secreting beta-like cells. Therefore, synthetic lineage-control networks appear to be a robust methodology for differentiating pluripotent stem cells into somatic cell types for basic research and regenerative medicine. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Reactivity of human sera in a sensitive, high-throughput pseudovirus-based papillomavirus neutralization assay for HPV16 and HPV18

    International Nuclear Information System (INIS)

    Pastrana, Diana V.; Buck, Christopher B.; Pang, Y.-Y. S.; Thompson, Cynthia D.; Castle, Philip E.; FitzGerald, Peter C.; Krueger Kjaer, Susanne; Lowy, Douglas R.; Schiller, John T.

    2004-01-01

    Sensitive high-throughput neutralization assays, based upon pseudoviruses carrying a secreted alkaline phosphatase (SEAP) reporter gene, were developed and validated for human papillomavirus (HPV)16, HPV18, and bovine papillomavirus 1 (BPV1). SEAP pseudoviruses were produced by transient transfection of codon-modified papillomavirus structural genes into an SV40 T antigen expressing line derived from 293 cells, yielding sufficient pseudovirus from one flask for thousands of titrations. In a 96-well plate format, in this initial characterization, the assay was reproducible and appears to be as sensitive as, but more specific than, a standard papillomavirus-like particle (VLP)-based enzyme-linked immunosorbent assay (ELISA). The neutralization assay detected type-specific HPV16 or HPV18 neutralizing antibodies (titers of 160-10240) in sera of the majority of a group of women infected with the corresponding HPV type, but not in virgin women. Sera from HPV16 VLP vaccinees had high anti-HPV16 neutralizing titers (mean: 45000; range: 5120-163840), but no anti-HPV18 neutralizing activity. The SEAP pseudovirus-based neutralization assay should be a practical method for quantifying potentially protective antibody responses in HPV natural history and prophylactic vaccine studies

  16. Validity and sensitivity of a human cranial finite element model: implications for comparative studies of biting performance.

    Science.gov (United States)

    Toro-Ibacache, Viviana; Fitton, Laura C; Fagan, Michael J; O'Higgins, Paul

    2016-01-01

    Finite element analysis (FEA) is a modelling technique increasingly used in anatomical studies investigating skeletal form and function. In the case of the cranium this approach has been applied to both living and fossil taxa to (for example) investigate how form relates to function or infer diet or behaviour. However, FE models of complex musculoskeletal structures always rely on simplified representations because it is impossible completely to image and represent every detail of skeletal morphology, variations in material properties and the complexities of loading at all spatial and temporal scales. The effects of necessary simplifications merit investigation. To this end, this study focuses on one aspect, model geometry, which is particularly pertinent to fossil material where taphonomic processes often destroy the finer details of anatomy or in models built from clinical CTs where the resolution is limited and anatomical details are lost. We manipulated the details of a finite element (FE) model of an adult human male cranium and examined the impact on model performance. First, using digital speckle interferometry, we directly measured strains from the infraorbital region and frontal process of the maxilla of the physical cranium under simplified loading conditions, simulating incisor biting. These measured strains were then compared with predicted values from FE models with simplified geometries that included modifications to model resolution, and how cancellous bone and the thin bones of the circum-nasal and maxillary regions were represented. Distributions of regions of relatively high and low principal strains and principal strain vector magnitudes and directions, predicted by the most detailed FE model, are generally similar to those achieved in vitro. Representing cancellous bone as solid cortical bone lowers strain magnitudes substantially but the mode of deformation of the FE model is relatively constant. In contrast, omitting thin plates of bone in

  17. Contribution of aquaporin 9 and multidrug resistance-associated protein 2 to differential sensitivity to arsenite between primary cultured chorion and amnion cells prepared from human fetal membranes

    International Nuclear Information System (INIS)

    Yoshino, Yuta; Yuan, Bo; Kaise, Toshikazu; Takeichi, Makoto; Tanaka, Sachiko; Hirano, Toshihiko; Kroetz, Deanna L.; Toyoda, Hiroo

    2011-01-01

    Arsenic trioxide (arsenite, As III ) has shown a remarkable clinical efficacy, whereas its side effects are still a serious concern. Therefore, it is critical to understand the effects of As III on human-derived normal cells for revealing the mechanisms underlying these side effects. We examined the effects of As III on primary cultured chorion (C) and amnion (A) cells prepared from human fetal membranes. A significant dose-dependent As III -mediated cytotoxicity was observed in the C-cells accompanied with an increase of lactate dehydrogenase (LDH) release. Higher concentrations of As III were required for the A-cells to show cytotoxicity and LDH release, suggesting that the C-cells were more sensitive to As III than the A-cells. The expression levels of aquaporin 9 (AQP9) were approximately 2 times higher in the C-cells than those in the A-cells. Both intracellular arsenic accumulation and its cytotoxicity in the C-cells were significantly abrogated by sorbitol, a competitive AQP9 inhibitor, in a dose-dependent manner. The protein expression levels of multidrug resistance-associated protein (MRP) 2 were downregulated by As III in the C-cells, but not in the A-cells. No significant differences in the expression levels of MRP1 were observed between C- and A-cells. The protein expression of P-glycoprotein (P-gp) was hardly detected in both cells, although a detectable amount of its mRNA was observed. Cyclosporine A, a broad-spectrum inhibitor for ABC transporters, and MK571, a MRP inhibitor, but not PGP-4008, a P-gp specific inhibitor, potently sensitized both cells to As III -mediated cytotoxicity. These results suggest that AQP9 and MRP2 are involved in controlling arsenic accumulation in these normal cells, which then contribute to differential sensitivity to As III cytotoxicity between these cells. -- Highlights: ► Examination of effect of As III on primary cultured chorion (C) and amnion (A) cells. ► Dose-dependent As III -mediated cytotoxicity in C

  18. Contribution of aquaporin 9 and multidrug resistance-associated protein 2 to differential sensitivity to arsenite between primary cultured chorion and amnion cells prepared from human fetal membranes

    Energy Technology Data Exchange (ETDEWEB)

    Yoshino, Yuta [Department of Clinical Molecular Genetics, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan); Yuan, Bo, E-mail: yuanbo@toyaku.ac.jp [Department of Clinical Molecular Genetics, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan); Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, 1550 4th St, RH584E Box 2911 San Francisco, CA 94158-2911 (United States); Kaise, Toshikazu [Laboratory of Environmental Chemodynamics, School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan); Takeichi, Makoto [Yoneyama Maternity Hospital, 2-12 Shin-machi, Hachioji, Tokyo 192-0065 (Japan); Tanaka, Sachiko; Hirano, Toshihiko [Department of Clinical Pharmacology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan); Kroetz, Deanna L. [Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, 1550 4th St, RH584E Box 2911 San Francisco, CA 94158-2911 (United States); Toyoda, Hiroo [Department of Clinical Molecular Genetics, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan)

    2011-12-15

    Arsenic trioxide (arsenite, As{sup III}) has shown a remarkable clinical efficacy, whereas its side effects are still a serious concern. Therefore, it is critical to understand the effects of As{sup III} on human-derived normal cells for revealing the mechanisms underlying these side effects. We examined the effects of As{sup III} on primary cultured chorion (C) and amnion (A) cells prepared from human fetal membranes. A significant dose-dependent As{sup III}-mediated cytotoxicity was observed in the C-cells accompanied with an increase of lactate dehydrogenase (LDH) release. Higher concentrations of As{sup III} were required for the A-cells to show cytotoxicity and LDH release, suggesting that the C-cells were more sensitive to As{sup III} than the A-cells. The expression levels of aquaporin 9 (AQP9) were approximately 2 times higher in the C-cells than those in the A-cells. Both intracellular arsenic accumulation and its cytotoxicity in the C-cells were significantly abrogated by sorbitol, a competitive AQP9 inhibitor, in a dose-dependent manner. The protein expression levels of multidrug resistance-associated protein (MRP) 2 were downregulated by As{sup III} in the C-cells, but not in the A-cells. No significant differences in the expression levels of MRP1 were observed between C- and A-cells. The protein expression of P-glycoprotein (P-gp) was hardly detected in both cells, although a detectable amount of its mRNA was observed. Cyclosporine A, a broad-spectrum inhibitor for ABC transporters, and MK571, a MRP inhibitor, but not PGP-4008, a P-gp specific inhibitor, potently sensitized both cells to As{sup III}-mediated cytotoxicity. These results suggest that AQP9 and MRP2 are involved in controlling arsenic accumulation in these normal cells, which then contribute to differential sensitivity to As{sup III} cytotoxicity between these cells. -- Highlights: Black-Right-Pointing-Pointer Examination of effect of As{sup III} on primary cultured chorion (C) and amnion

  19. Sensitization of human cancer cells to gemcitabine by the Chk1 inhibitor MK-8776: cell cycle perturbation and impact of administration schedule in vitro and in vivo

    International Nuclear Information System (INIS)

    Montano, Ryan; Thompson, Ruth; Chung, Injae; Hou, Huagang; Khan, Nadeem; Eastman, Alan

    2013-01-01

    Chk1 inhibitors have emerged as promising anticancer therapeutic agents particularly when combined with antimetabolites such as gemcitabine, cytarabine or hydroxyurea. Here, we address the importance of appropriate drug scheduling when gemcitabine is combined with the Chk1 inhibitor MK-8776, and the mechanisms involved in the schedule dependence. Growth inhibition induced by gemcitabine plus MK-8776 was assessed across multiple cancer cell lines. Experiments used clinically relevant “bolus” administration of both drugs rather than continuous drug exposures. We assessed the effect of different treatment schedules on cell cycle perturbation and tumor cell growth in vitro and in xenograft tumor models. MK-8776 induced an average 7-fold sensitization to gemcitabine in 16 cancer cell lines. The time of MK-8776 administration significantly affected the response of tumor cells to gemcitabine. Although gemcitabine induced rapid cell cycle arrest, the stalled replication forks were not initially dependent on Chk1 for stability. By 18 h, RAD51 was loaded onto DNA indicative of homologous recombination. Inhibition of Chk1 at 18 h rapidly dissociated RAD51 leading to the collapse of replication forks and cell death. Addition of MK-8776 from 18–24 h after a 6-h incubation with gemcitabine induced much greater sensitization than if the two drugs were incubated concurrently for 6 h. The ability of this short incubation with MK-8776 to sensitize cells is critical because of the short half-life of MK-8776 in patients’ plasma. Cell cycle perturbation was also assessed in human pancreas tumor xenografts in mice. There was a dramatic accumulation of cells in S/G 2 phase 18 h after gemcitabine administration, but cells had started to recover by 42 h. Administration of MK-8776 18 h after gemcitabine caused significantly delayed tumor growth compared to either drug alone, or when the two drugs were administered with only a 30 min interval. There are two reasons why delayed

  20. Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP kit prototype for detection of Trypanosoma cruzi DNA in human blood samples.

    Directory of Open Access Journals (Sweden)

    Susana A Besuschio

    2017-07-01

    Full Text Available This study aimed to assess analytical parameters of a prototype LAMP kit that was designed for detection of Trypanosoma cruzi DNA in human blood. The prototype is based on the amplification of the highly repetitive satellite sequence of T.cruzi in microtubes containing dried reagents on the inside of the caps. The reaction is carried out at 65°C during 40 minutes. Calcein allows direct detection of amplified products with the naked eye. Inclusivity and selectivity were tested in purified DNA from Trypanosoma cruzi stocks belonging to the six discrete typing units (DTUs, in DNA from other protozoan parasites and in human DNA. Analytical sensitivity was estimated in serial dilutions of DNA samples from Sylvio X10 (Tc I and CL Brener (Tc VI stocks, as well as from EDTA-treated or heparinized blood samples spiked with known amounts of cultured epimastigotes (CL Brener. LAMP sensitivity was compared after DNA extraction using commercial fiberglass columns or after "Boil & Spin" rapid preparation. Moreover, the same DNA and EDTA-blood spiked samples were subjected to standardized qPCR based on the satellite DNA sequence for comparative purposes. A panel of peripheral blood specimens belonging to Chagas disease patients, including acute, congenital, chronic and reactivated cases (N = 23, as well as seronegative controls (N = 10 were evaluated by LAMP in comparison to qPCR. LAMP was able to amplify DNAs from T. cruzi stocks representative of the six DTUs, whereas it did not amplify DNAs from Leishmania sp, T. brucei sp, T. rangeli KPN+ and KPN-, P. falciparum and non-infected human DNA. Analytical sensitivity was 1x10-2 fg/μL of both CL Brener and Sylvio X10 DNAs, whereas qPCR detected up to 1x 10-1 fg/μL of CL Brener DNA and 1 fg/μl of Sylvio X10 DNA. LAMP detected 1x10-2 parasite equivalents/mL in spiked EDTA blood and 1x10-1 par.eq/mL in spiked heparinized blood using fiberglass columns for DNA extraction, whereas qPCR detected 1x10-2 par

  1. Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) kit prototype for detection of Trypanosoma cruzi DNA in human blood samples.

    Science.gov (United States)

    Besuschio, Susana A; Llano Murcia, Mónica; Benatar, Alejandro F; Monnerat, Severine; Cruz, Israel; Picado, Albert; Curto, María de Los Ángeles; Kubota, Yutaka; Wehrendt, Diana P; Pavia, Paula; Mori, Yasuyoshi; Puerta, Concepción; Ndung'u, Joseph M; Schijman, Alejandro G

    2017-07-01

    This study aimed to assess analytical parameters of a prototype LAMP kit that was designed for detection of Trypanosoma cruzi DNA in human blood. The prototype is based on the amplification of the highly repetitive satellite sequence of T.cruzi in microtubes containing dried reagents on the inside of the caps. The reaction is carried out at 65°C during 40 minutes. Calcein allows direct detection of amplified products with the naked eye. Inclusivity and selectivity were tested in purified DNA from Trypanosoma cruzi stocks belonging to the six discrete typing units (DTUs), in DNA from other protozoan parasites and in human DNA. Analytical sensitivity was estimated in serial dilutions of DNA samples from Sylvio X10 (Tc I) and CL Brener (Tc VI) stocks, as well as from EDTA-treated or heparinized blood samples spiked with known amounts of cultured epimastigotes (CL Brener). LAMP sensitivity was compared after DNA extraction using commercial fiberglass columns or after "Boil & Spin" rapid preparation. Moreover, the same DNA and EDTA-blood spiked samples were subjected to standardized qPCR based on the satellite DNA sequence for comparative purposes. A panel of peripheral blood specimens belonging to Chagas disease patients, including acute, congenital, chronic and reactivated cases (N = 23), as well as seronegative controls (N = 10) were evaluated by LAMP in comparison to qPCR. LAMP was able to amplify DNAs from T. cruzi stocks representative of the six DTUs, whereas it did not amplify DNAs from Leishmania sp, T. brucei sp, T. rangeli KPN+ and KPN-, P. falciparum and non-infected human DNA. Analytical sensitivity was 1x10-2 fg/μL of both CL Brener and Sylvio X10 DNAs, whereas qPCR detected up to 1x 10-1 fg/μL of CL Brener DNA and 1 fg/μl of Sylvio X10 DNA. LAMP detected 1x10-2 parasite equivalents/mL in spiked EDTA blood and 1x10-1 par.eq/mL in spiked heparinized blood using fiberglass columns for DNA extraction, whereas qPCR detected 1x10-2 par.eq./mL in EDTA blood

  2. A simple, rapid and sensitive liquid chromatography-tandem mass spectrometry method for the determination of dienogest in human plasma and its pharmacokinetic applications under fasting.

    Science.gov (United States)

    Pallapothu, Leela Mohan Kumar; Batta, Neelima; Pigili, Ravi Kumar; Yejella, Rajendra Prasad

    2015-02-01

    A simple, rapid and sensitive analytical method using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) detection with positive ion electrospray ionization was developed for the determination of dienogest in human K2 EDTA plasma using levonorgestrel d6 as an internal standard (IS). Dienogest and IS were extracted from human plasma using simple liquid-liquid extraction. Chromatographic separation was achieved on a Zorbax XDB-Phenyl column (4.6 × 75 mm, 3.5 µm) under isocratic conditions using acetonitrile-5 mm ammonium acetate (70:30, v/v) at a flow rate of 0.60 mL/min. The protonated precursor to product ion transitions monitored for dienogest and IS were at m/z 312.30 → 135.30 and 319.00 → 251.30, respectively. The method was validated with a linearity range of 1.003-200.896 ng/mL having a total analysis time for each chromatograph of 3.0 min. The method has shown tremendous reproducibility with intra- and inter-day precision (coefficient of variation) dienogest tablets to healthy female volunteers and was proved to be highly reliable for the analysis of clinical samples. Copyright © 2014 John Wiley & Sons, Ltd.

  3. In vivo polarization-sensitive optical coherence tomography of human bu