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Sample records for lysate antigen ortho

  1. [The lysate and recombinant antigens in ELISA-test-systems for diagnostic of herpes simplex].

    PubMed

    Ganova, L A; Kovtoniuk, G V; Korshun, L N; Kiseleva, E K; Tereshchenko, M I; Vudmaska, M I; Moĭsa, L N; Shevchuk, V A; Spivak, N Ia

    2014-08-01

    The lysate and recombinant antigens of various production included informula of ELISA-test-systems were analyzed. The ELISA-test-systems are used for detection of IgG to Herpes simplex virus type I and II. For testing the panel of serums PTH 201 (BBI Inc.) were used. The samples of this panel contain antibodies to Herpes simplex virus type I and II in mixed titers. The 69 serums of donors were used too (17 samples had IgG to Herpes simplex virus type I, 23 samples to Herpes simplex virus type II and 29 samples had no antibodies to Herpes simplex virus). The diagnostic capacity of mixture of recombinant antigens gG1 Herpes simplex virus type I and gG2 Herpes simplex virus type II (The research-and-production complex "DiaprofMed") was comparable with mixture of lysate antigen Herpes simplex virus type I and II (Membrane) EIE Antigen ("Virion Ltd."). In the test-systems for differentiation of IgG to Herpes simplex virus type I the recombinant antigen gG1 Herpes simplex virus type I proved to be comparable with commercial analogue Herpes simplex virus-1 gG1M ("Viral Therapeutics Inc."'). At the same time, capacity to detect IgG to Herpes simplex virus type II in recombinant protein gG2 Herpes simplex virus type II is significantly higher than in its analogue Herpes simplex virus-2 gG2c ("Viral Therapeutics Inc."). PMID:25552056

  2. Enhanced target-specific signal detection using an Escherichia coli lysate in multiplex microbead immunoassays with E. coli-derived recombinant antigens.

    PubMed

    Crestani, Sandra; Leitolis, Amanda; Lima, Lucianna Freitas Oliveira; Krieger, Marco A; Foti, Leonardo

    2016-08-01

    Diverse techniques have been developed to analyze antibody-mediated responses to infections. However, the most common tests, i.e., enzyme-linked immunosorbent assays, require separate reactions for each antigen and consequently necessitate large sample volumes. Luminex technology allows the detection of multiple antibodies in a single experiment, but nonspecific binding can impair the results. Therefore, we examined the use of Escherichia coli lysates to reduce nonspecific binding and improve the results of liquid microarrays based on Luminex technology. Anti-bacteria antibodies were detected in human serum samples, as evidenced by high median fluorescence intensity (MFI) in assays performed with paramagnetic microspheres coupled with E. coli lysates. Moreover, the addition of an E. coli lysate as a blocker reduced the nonspecific binding of antigens produced by E. coli in a concentration-dependent manner. Tris-HCl reduced MFI values in negative samples, but did not affect MFI for positive samples. For microspheres coupled with different antigens, an E. coli lysate blocker significantly improved the fluorescence signals from positive samples. The addition of Tris-HCl and the E. coli lysate induced antigen-specific differences in MFI. This combination of the E. coli lysate blocker and Tris-HCl yielded a statistically significant improvement in MFI in the assays for Chagas disease and hepatitis C virus samples. However, for the Treponema pallidum p47 antigen improvement in MFI was only observed for the preparation with the E. coli blocker at a concentration of 3%. In conclusion, the addition of an E. coli lysate and Tris-HCl to the microarray assay reduced the nonspecific binding of human anti-bacteria antibodies and, therefore, increased the specific MFI. PMID:27156997

  3. Suppressed CD31 Expression in Sarcoma-180 Tumors after Injection with Toxoplasma gondii Lysate Antigen in BALB/c Mice

    PubMed Central

    Pyo, Kyoung-Ho; Jung, Bong-Kwang; Chai, Jong-Yil

    2010-01-01

    The anti-tumorigenic effects of Toxoplasma gondii (RH) antigens were studied in a murine sarcoma-180 tumor model. To determine the anti-tumor effects, the reduction in tumor size and expression of CD31 (an angiogenesis marker in the tumor tissue) were examined after injection of BALB/c mice with T. gondii lysate antigen (TLA) or formalin-fixed, proliferation-inhibited, T. gondii tachyzoites. Tumors were successfully produced by an intradermal injection of sarcoma-180 cells with plain Matrigel in the mid-backs of mice. After injection with TLA or formalin-fixed T. gondii tachyzoites, the increase in tumor size and weight nearly stopped while tumor growth continued in control mice that were injected with PBS. CD31 expression in TLA-treated or formalin-fixed T. gondii-injected mice was lower than the control mice. Accordingly, the present study shows that the treatment of mice with formalin-fixed T. gondii or TLA in the murine sarcoma-180 tumor model results in a decrease of both tumor size and CD31 expression. PMID:20585536

  4. Use of viral lysate antigen combined with recombinant protein in Western immunoblot assay as confirmatory test for serodiagnosis of severe acute respiratory syndrome.

    PubMed

    Guan, Ming; Chen, Hsiao Ying; Tan, Phuay Heng; Shen, Shuo; Goh, Phuay-Yee; Tan, Yee-Joo; Pang, Peow Hoon; Lu, Yang; Fong, Priscilla Yiquan; Chin, Daria

    2004-11-01

    A Western immunoblot assay for confirmatory serodiagnosis of severe acute respiratory syndrome (SARS) was developed utilizing viral lysate antigens combined with a recombinant nucleocapsid protein, GST-N (glutathione S-transferase-nucleocapsid) of the SARS coronavirus (SARS-CoV). The viral lysate antigens were separated by electrophoresis and transblotted onto nitrocellulose membranes. The resultant membrane was subsequently added with the GST-N recombinant protein at a specific location. The positions of bands corresponding to some of the structural proteins immobilized on the membrane were then located and verified with mouse or rabbit antisera specific to the respective proteins. The Western immunoblot assay was able to detect antibodies to SARS-CoV in all 40 serum specimens from SARS patients and differentiate the SARS-positive samples from those of the healthy donor or non-SARS patient controls (150 samples) when set criteria were followed. In addition, when the immunoblot was used to test samples considered falsely positive by an in-house-developed SARS-specific enzyme-linked immunosorbent assay, band patterns different from those with samples from SARS patients were obtained. PMID:15539520

  5. Effect of bacterial antigen lysate on IgG and IgA levels in children with recurrent infections and hypogammaglobulinemia.

    PubMed

    Quezada, A; Maggi, L; Pérez, M A; Rodríguez, J

    1999-01-01

    To evaluate the effect of bacterial antigen lysate on serum immunoglobulin (Ig) levels, we studied 14 children with recurrent infections and hypogammaglobulinemia (IgG and IgA levels below 2 standard deviations for age). Patients were treated for a 60-90 day period with OM-85 BV and reevaluated both clinically and by measuring serum Ig levels at the end of follow-up. The control group consisted of 10 children with recurrent infections who received a placebo. Serum Ig levels were also compared with the reference values for age. The Wilcoxon and Mann-Whitney tests were used for statistical analysis. In the study group, IgG (pretreatment: 707 mg/dl; post-treatment: 1,022 mg/dl; p < 0.004) and IgA levels (pretreatment: 41 mg/dl; post-treatment: 83 mg/dl; p < 0.018) increased significantly. Furthermore, 13/14 children reached normal IgG levels, and 12/14 children reached normal age levels for serum IgA. Similarly, when comparing the pre- and post-treatment levels in the study group with the levels in the control group, they were significant for IgG (p < 0.002) as well as IgA levels (p < 0.04). The overall clinical response was favorable in all patients in the treated group. These results suggest an immunostimulant effect of OM-85 BV, both improving Ig levels and reducing recurrent infections. PMID:10412680

  6. Prime-Boost Vaccination with Toxoplasma Lysate Antigen, but Not with a Mixture of Recombinant Protein Antigens, Leads to Reduction of Brain Cyst Formation in BALB/c Mice

    PubMed Central

    Wagner, Angelika; Schabussova, Irma; Ruttkowski, Bärbel; Peschke, Roman; Kur, Józef; Kundi, Michael; Joachim, Anja; Wiedermann, Ursula

    2015-01-01

    Introduction Infection with the ubiquitous parasite Toxoplasma gondii is a threat for immunocompromised patients and pregnant women and effective immune-prophylaxis is still lacking. Methods Here we tested a mixture of recombinant T. gondii antigens expressed in different developmental stages, i.e., SAG1, MAG1 and GRA7 (SMG), and a lysate derived from T. gondii tachyzoites (TLA) for prophylactic vaccination against cyst formation. Both vaccine formulations were applied systemically followed by an oral TLA-booster in BALB/c mice. Results Systemic priming with SMG and oral TLA-booster did not show significant induction of protective immune responses. In contrast, systemic priming and oral booster with TLA induced higher levels of Toxoplasma-specific IgG, IgG1 and IgG2a in sera as well as high levels of Toxoplasma-specific IgG1 in small intestines. Furthermore, high levels of Toxoplasma-specific Th1-, Th17- and Th2-associated cytokines were only detected in restimulated splenocytes of TLA-vaccinated mice. Importantly, in mice orally infected with T. gondii oocysts, only TLA-vaccination and booster reduced brain cysts. Furthermore, sera from these mice reduced tachyzoites invasion of Vero cells in vitro, indicating that antibodies may play a critical role for protection against Toxoplasma infection. Additionally, supernatants from splenocyte cultures of TLA-vaccinated mice containing high levels of IFN-γ lead to substantial production of nitric oxide (NO) after incubation with macrophages in vitro. Since NO is involved in the control of parasite growth, the high levels of IFN-γ induced by vaccination with TLA may contribute to the protection against T. gondii. Conclusion In conclusion, our data indicate that prime-boost approach with TLA, but not with the mixture of recombinant antigens SMG, induces effective humoral and cellular Toxoplasma-specific responses and leads to significant reduction of cerebral cysts, thereby presenting a viable strategy for further

  7. Tumor cell lysates as immunogenic sources for cancer vaccine design

    PubMed Central

    González, Fermín E; Gleisner, Alejandra; Falcón-Beas, Felipe; Osorio, Fabiola; López, Mercedes N; Salazar-Onfray, Flavio

    2015-01-01

    Autologous dendritic cells (DCs) loaded with tumor-associated antigens (TAAs) are a promising immunological tool for cancer therapy. These stimulate the antitumor response and immunological memory generation. Nevertheless, many patients remain refractory to DC approaches. Antigen (Ag) delivery to DCs is relevant to vaccine success, and antigen peptides, tumor-associated proteins, tumor cells, autologous tumor lysates, and tumor-derived mRNA have been tested as Ag sources. Recently, DCs loaded with allogeneic tumor cell lysates were used to induce a potent immunological response. This strategy provides a reproducible pool of almost all potential Ags suitable for patient use, independent of MHC haplotypes or autologous tumor tissue availability. However, optimizing autologous tumor cell lysate preparation is crucial to enhancing efficacy. This review considers the role of cancer cell-derived lysates as a relevant source of antigens and as an activating factor for ex vivo therapeutic DCs capable of responding to neoplastic cells. These promising therapies are associated with the prolonged survival of advanced cancer patients. PMID:25625929

  8. Tumor cell lysates as immunogenic sources for cancer vaccine design.

    PubMed

    González, Fermín E; Gleisner, Alejandra; Falcón-Beas, Felipe; Osorio, Fabiola; López, Mercedes N; Salazar-Onfray, Flavio

    2014-01-01

    Autologous dendritic cells (DCs) loaded with tumor-associated antigens (TAAs) are a promising immunological tool for cancer therapy. These stimulate the antitumor response and immunological memory generation. Nevertheless, many patients remain refractory to DC approaches. Antigen (Ag) delivery to DCs is relevant to vaccine success, and antigen peptides, tumor-associated proteins, tumor cells, autologous tumor lysates, and tumor-derived mRNA have been tested as Ag sources. Recently, DCs loaded with allogeneic tumor cell lysates were used to induce a potent immunological response. This strategy provides a reproducible pool of almost all potential Ags suitable for patient use, independent of MHC haplotypes or autologous tumor tissue availability. However, optimizing autologous tumor cell lysate preparation is crucial to enhancing efficacy. This review considers the role of cancer cell-derived lysates as a relevant source of antigens and as an activating factor for ex vivo therapeutic DCs capable of responding to neoplastic cells. These promising therapies are associated with the prolonged survival of advanced cancer patients. PMID:25625929

  9. Antigen

    MedlinePlus

    An antigen is any substance that causes your immune system to produce antibodies against it. This means your immune ... and is trying to fight it off. An antigen may be a substance from the environment, such ...

  10. Protective immune-response of aluminium hydroxide gel adjuvanted phage lysate of Brucella abortus S19 in mice against direct virulent challenge with B. abortus 544.

    PubMed

    Jain, Lata; Rawat, Mayank; Prajapati, Awadhesh; Tiwari, Ashok Kumar; Kumar, Bablu; Chaturvedi, V K; Saxena, H M; Ramakrishnan, Sarvanan; Kumar, Jatin; Kerketta, Priscilla

    2015-09-01

    The prophylactic efficacies of plain and alum adsorbed lysate were evaluated by direct virulent challenge in mice model. A recently isolated brucellaphage 'ϕLd' was used for generation of lysates. Twenty four h incubated Brucella abortus S19 broth cultures standardized to contain approximately 10(8) CFU/ml were found suitable for generation of lysates. Three lysate batches produced through separate cycles did not show any significant variation with respect to protein and polysaccharide contents, endotoxin level and phage counts, indicating that compositionally stable lysate preparations can be generated through an optimized production process. Three polypeptides of ∼16, 19 and 23 kDa could be identified as immuno-dominant antigens of the lysate which induced both humoral and cell-mediated immune responses in a dose dependent manner. Results of efficacy evaluation trial confirmed dose-dependent protective potencies of lysate preparation. The lysate with an antigenic dose of 0.52 μg protein and 60 μg CHO adsorbed on aluminium gel (0.1 percent aluminium concentration) exhibited the highest protective potency which was greater than that induced by standard S19 vaccine. Phage lysate methodology provides a very viable option through which an improved immunizing preparation with all desirable traits can be developed against brucellosis, and integrated with immunization programmes in a more efficient manner. PMID:26156404

  11. ortho-Anisidine

    Integrated Risk Information System (IRIS)

    ortho - Anisidine ; CASRN 90 - 04 - 0 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic

  12. [Ortho-molecular nutrition].

    PubMed

    Martínez Bradshaw, Alejandro

    2005-03-01

    Ortho-molecular nutrition contemplates the deficiency of certain nutrients, not their deprivation, as the generator of short-term and long-term pathologies. By means of supplying these nutrients, an organism recovers. This method consists in building up an organism's functions by following the guides and indications provided by the organism itself. PMID:15871343

  13. Compaction agent clarification of microbial lysates

    NASA Technical Reports Server (NTRS)

    DeWalt, Brad W.; Murphy, Jason C.; Fox, George E.; Willson, Richard C.

    2003-01-01

    Recombinant proteins are often purified from microbial lysates containing high concentrations of nucleic acids. Pre-purification steps such as nuclease addition or precipitation with polyethyleneimine or ammonium sulfate are normally required to reduce viscosity and to eliminate competing polyanions before anion exchange chromatography. We report that small polycationic compaction agents such as spermine selectively precipitate nucleic acids during or after Escherichia coli lysis, allowing DNA and RNA to be pelleted with the insoluble cell debris. Analysis by spectrophotometry and protein assay confirmed a significant reduction in the concentration of nucleic acids present, with preservation of protein. Lysate viscosity is greatly reduced, facilitating subsequent processing. We have used 5mM spermine to remove nucleic acids from E. coli lysate in the purification of a hexahistidine-tagged HIV reverse transcriptase.

  14. The Microsoft Global Ortho Program

    NASA Astrophysics Data System (ADS)

    Walcher, W.; Leberl, F.; Gruber, M.

    2012-07-01

    Wide area and thus continental mapping extending beyond national borders is a novel concept in civilian photogrammetry. The Microsoft Global Ortho Program was launched in the Spring of 2009 as a result of Microsoft's need for global geo-data at a high geometric resolution and radiometric excellence. By fall of 2012 more than 10 million km2 of the USA and 14 European countries will have been covered by seamless 30 cm GSD color-, 60 cm GSD false-color infrared ortho-mosaics and a 1 meter GSD digital surface model. The ortho-maps are being published to Microsoft's Bing Maps Internet mapping portal. The Global Ortho Program was designed for highly and unprecedented automated mapping of essentially entire continents. In 2011, exclusive of flight operations, the product output per person has been measured in excess of 275,000 square km per year. We describe research efforts that made this achievement possible. Those include a specially designed aerial sensor (Ultracam G), logistics simulation for fight planning and optimization, in-flight blur detection and subsequent automatic blur removal, modeling and removal of atmospheric and environmental conditions, automated shear detection and DTM refinement, an IT architecture to process >200,000 aerial images/day, and for creating over 1,000,000 km2 ortho-imagery and DSM data in 24 hours. While addressing these issues, we provide ideas how this might affect the future of spatial infrastructure initiatives.

  15. Murine Dendritic Cells Pulsed with Whole Tumor Lysates Mediate Potent Antitumor Immune Responses in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Fields, R. C.; Shimizu, K.; Mule, J. J.

    1998-08-01

    The highly efficient nature of dendritic cells (DC) as antigen-presenting cells raises the possibility of uncovering in tumor-bearing hosts very low levels of T cell reactivity to poorly immunogenic tumors that are virtually undetectable by other means. Here, we demonstrate the in vitro and in vivo capacities of murine bone marrow-derived, cytokine-driven DC to elicit potent and specific anti-tumor responses when pulsed with whole tumor lysates. Stimulation of naive spleen-derived T cells by tumor lysate-pulsed DC generated tumor-specific proliferative cytokine release and cytolytic reactivities in vitro. In addition, in two separate strains of mice with histologically distinct tumors, s.c. injections of DC pulsed with whole tumor lysates effectively primed these animals to reject subsequent lethal challenges with viable parental tumor cells and, important to note, also mediated significant reductions in the number of metastases established in the lungs. Tumor rejection depended on host-derived CD8+ T cells and, to a lesser extent, CD4+ T cells. Spleens from mice that had rejected their tumors contained specific precursor cytotoxic T lymphocytes. The use of whole tumor lysates as a source of tumor-associated antigen(s) for pulsing of DC circumvents several limitations encountered with other methods as well as provides certain distinct advantages, which are discussed. These data serve as rationale for our recent initiation of a phase I clinical trial of immunization with autologous tumor lysate-pulsed DC in adult and pediatric cancer patients.

  16. Immunological Characterization of Whole Tumour Lysate-Loaded Dendritic Cells for Cancer Immunotherapy

    PubMed Central

    Ottobrini, Luisa; Biasin, Mara; Borelli, Manuela; Lucignani, Giovanni; Trabattoni, Daria; Clerici, Mario

    2016-01-01

    Introduction Dendritic cells play a key role as initiators of T-cell responses, and even if tumour antigen-loaded dendritic cells can induce anti-tumour responses, their efficacy has been questioned, suggesting a need to enhance immunization strategies. Matherials & Methods We focused on the characterization of bone marrow-derived dendritic cells pulsed with whole tumour lysate (TAA-DC), as a source of known and unknown antigens, in a mouse model of breast cancer (MMTV-Ras). Dendritic cells were evaluated for antigen uptake and for the expression of MHC class I/II and costimulatory molecules and markers associated with maturation. Results Results showed that antigen-loaded dendritic cells are characterized by a phenotypically semi-mature/mature profile and by the upregulation of genes involved in antigen presentation and T-cell priming. Activated dendritic cells stimulated T-cell proliferation and induced the production of high concentrations of IL-12p70 and IFN-γ but only low levels of IL-10, indicating their ability to elicit a TH1-immune response. Furthermore, administration of Antigen loaded-Dendritic Cells in MMTV-Ras mice evoked a strong anti-tumour response in vivo as demonstrated by a general activation of immunocompetent cells and the release of TH1 cytokines. Conclusion Data herein could be useful in the design of antitumoral DC-based therapies, showing a specific activation of immune system against breast cancer. PMID:26795765

  17. A Dendritic Cell Vaccine Pulsed with Autologous Hypochlorous Acid-Oxidized Ovarian Cancer Lysate Primes Effective Broad Antitumor Immunity: From Bench to Bedside

    PubMed Central

    Chiang, Cheryl Lai-Lai; Kandalaft, Lana E.; Tanyi, Janos; Hagemann, Andrea R.; Motz, Gregory T.; Svoronos, Nikolaos; Montone, Kathleen; Mantia-Smaldone, Gina M.; Nisenbaum, Harvey L.; Levine, Bruce L.; Kalos, Michael; Czerniecki, Brian J.; Torigian, Drew A.; Powell, Daniel J.; Mick, Rosemarie; Smith, Lori; Coukos, George

    2013-01-01

    Purpose Whole tumor lysates are promising antigen sources for dendritic cell (DC) therapy for they contain many relevant immunogenic epitopes to help prevent tumor escape. Two common methods of tumor lysate preparations are freeze-thaw processing and UVB-irradiation to induce necrosis and apoptosis, respectively. Hypochlorous acid (HOCl)-oxidation is a new method for inducing primary necrosis and enhancing the immunogenicity of tumor cells. Experimental Design We compared DCs’ ability to engulf three different tumor lysate preparations, produce Th1-priming cytokines and chemokines, stimulate mixed leukocyte reactions (MLR), and finally elicit T-cell responses capable of controlling tumor growth in vivo. Results We demonstrated that DCs engulfed HOCl-oxidized lysate most efficiently, stimulated robust MLRs and elicited strong tumor-specific IFN-γ secretions in autologous T-cells. These DCs produced the highest levels of Th1-priming cytokines and chemokines, including IL-12. Mice vaccinated with HOCl-oxidized ID8-ova lysate pulsed DCs developed T-cell responses that effectively controlled tumor growth. Safety, immunogenicity of autologous DCs pulsed with HOCl-oxidized autologous tumor lysate (OCDC vaccine), clinical efficacy and progression free survival (PFS) were evaluated in a pilot study of five subjects with recurrent ovarian cancer. OCDC vaccination produced few grade 1 toxicities and elicited potent T-cell responses against known ovarian tumor antigens. Circulating T regulatory cells and serum IL-10 were also reduced. Two subjects experienced durable PFS of ≥24 months after OCDC. Conclusions This is the first study demonstrating the potential efficacy of a DC vaccine pulsed with HOCl-oxidized tumor lysate, a novel approach in preparing DC vaccine that is potentially applicable to many cancers. PMID:23838316

  18. Metabolism of Peptide Reporters in Cell Lysates and Single Cells

    PubMed Central

    Proctor, Angela; Wang, Qunzhao; Lawrence, David S.; Allbritton, Nancy L.

    2013-01-01

    The stability of an Abl kinase substrate peptide in a cytosolic lysate and in single cells was characterized. In the cytosolic lysate, the starting peptide was metabolized at an average initial rate of 1.7 ± 0.3 zmol pg−;1 s−;1 with a t1/2 of 1.3 min. Five different fragments formed over time; however, a dominant cleavage site was identified. Multiple rational design cycles were utilized to develop a lead peptide with a phenylalanine and alanine replaced by an (N-methyl)phenylalanine and isoleucine, respectively, to attain cytosolic peptidase resistance while maintaining Abl substrate efficacy. This lead peptide possessed a 15-fold greater lifetime in the cytosolic lysate while attaining a 7-fold improvement in kcat as an Abl kinase substrate compared to the starting peptide. However, when loaded into single cells, the starting peptide and lead peptide possessed nearly identical degradation rates and an altered pattern of fragmentation relative to that in cell lysates. Preferential accumulation of a fragment with cleavage at an Ala-Ala bond in single cells suggested that dissimilar peptidases act on the peptides in the lysate versus single cells. A design strategy for peptide stabilization, analogous to that demonstrated for the lysate, should be effective for stabilization in single cells. PMID:22314840

  19. Separating para and ortho water.

    PubMed

    Horke, Daniel A; Chang, Yuan-Pin; Długołęcki, Karol; Küpper, Jochen

    2014-10-27

    Water exists as two nuclear-spin isomers, para and ortho, determined by the overall spin of its two hydrogen nuclei. For isolated water molecules, the conversion between these isomers is forbidden and they act as different molecular species. Yet, these species are not readily separated, and no pure para sample has been produced. Accordingly, little is known about their specific physical and chemical properties, conversion mechanisms, or interactions. The production of isolated samples of both spin isomers is demonstrated in pure beams of para and ortho water in their respective absolute ground state. These single-quantum-state samples are ideal targets for unraveling spin-conversion mechanisms, for precision spectroscopy and fundamental symmetry-breaking studies, and for spin-enhanced applications, for example laboratory astrophysics and astrochemistry or hypersensitized NMR experiments. PMID:25196938

  20. Immune response in the lungs following oral immunization with bacterial lysates of respiratory pathogens.

    PubMed Central

    Ruedl, C; Frühwirth, M; Wick, G; Wolf, H

    1994-01-01

    We have investigated the local immune response of the BALB/c mouse respiratory tract after oral immunization with a bacterial lysate of seven common respiratory pathogens. After two immunization on five consecutive days, we examined the immunoglobulin (immunoglobulin G [IgG], IgM, and IgA) secretion rates of cells isolated from the lungs and compared them with those of spleen cells of orally immunized and nonimmunized animals by using a new test system based on time-resolved fluorescence. The procedure followed the principle of the classical ELISPOT test with nitrocellulose-bottomed microtiter plates, but europium (Eu3+)-linked streptavidin rather than enzyme-conjugated streptavidin was used, with the advantage of quantifying secreted immunoglobulins instead of detecting single antibody-secreting cells. Lymphocytes isolated from the lungs of treated animals revealed significant increases in total and antigen-specific IgA synthesis compared with the rates of the controls, whereas IgG and IgM production rates showed no remarkable differences. In addition, the sera of treated mice revealed higher antigen-specific IgA titers but not increased IgM and IgG levels. We conclude that priming the gut-associated lymphoid tissue with bacterial antigens of pneumotropic microorganisms can elicit an enhanced IgA response in a distant mucosal effector site, such as the respiratory tract, according to the concept of a common mucosa-associated immune system. PMID:7496936

  1. Construction and hyperspectral imaging of quantum dot lysate arrays.

    PubMed

    Rosenblatt, Kevin P; Huebschman, Michael L; Garner, Harold R

    2012-01-01

    The emerging field of proteomic molecular profiling will be driven by new technologies that can measure dozens to hundreds of proteins from a small sample input from a patient's biopsy. Lysate arrays, or reverse-phase protein microarrays, provide a platform for complex mixtures of proteins extracted from cells and tissues to be directly immobilized onto a solid support (such as a biochip with protein binding capacity) in diminutive volumes (picoliter-to-nanoliter). The proteins are spotted using precision robotics and then quantitatively assayed using primary antibodies; important posttranslational modifications, such as phosphorylations that are important for protein activation, may also be assayed to provide an estimate of the regulation of cellular signaling. Until recently, chromogenic signals and fluorescence (using organic fluorophores) detection were two strategies relied upon for signal detection. Emerging regents such as quantum dots (Qdot® nanocrystals; QD) are now employed for improved performance. QD embody a more versatile detection system because the robust signals may be time averaged and the narrow spectral emissions enable many protein targets to be quantified within the same lysate spot. Previously, we found that commercially available pegylated, streptavidin-conjugated QD were effective detection agents, with low-background affinities to spurious components within heterogeneous protein mixtures. Hyperspectral imaging allows the simultaneous detection of the different colored QD reagents within a single lysate spot. Here, we described the construction and imaging of QD lysate arrays. This technology is an emerging, enabling tool within the exciting, clinically oriented field of clinical tissue proteomics. PMID:22081354

  2. May We Strengthen the Human Natural Defenses with Bacterial Lysates?

    PubMed Central

    2010-01-01

    During the last twenty years bacterial lysates have gained a new interest and their use has obtained a progressively larger consensus in the medical practice. They are commonly used as immunomodulators, in order to up-regulate immune responses against infectious damages. As a matter of fact, the role of these lysate seems relevant in upper and lower respiratory tract infections prevention, frequently observed both in paediatric and elder ages, and which represent a relevant problem also in terms of socio-economical implications. The effects of bacterial lysates as immunostimulatory agents have become the central point of many studies. The aim of those in vivo and in vitro studies was to understand and evaluate the capacity of this kind of treatments to create a better answer of the immune system against microbial infections, eventually leading to a reduction in their number. All the in vivo and in vitro findings analyzed support the evidence that bacterial lysates are powerful inducers of a specific immune response against bacterial infections. Both in paediatric and adult clinical trials, a positive trend has been found in terms of overall reduction of infection rates and duration, beneficial effect on symptoms, reduction in antibiotics use and possibility to improve the patient's quality of life in several diseases. Further well-designed trials in terms of blinding and randomization procedures and including a higher number of patients, selected according to the disease and its severity, are needed. PMID:23282746

  3. Molecularly engineered poly(ortho ester) microspheres for enhanced delivery of DNA vaccines

    NASA Astrophysics Data System (ADS)

    Wang, Chun; Ge, Qing; Ting, David; Nguyen, David; Shen, Hui-Rong; Chen, Jianzhu; Eisen, Herman N.; Heller, Jorge; Langer, Robert; Putnam, David

    2004-03-01

    Genetic vaccination using plasmid DNA presents a unique opportunity for achieving potent immune responses without the potential limitations of many conventional vaccines. Here we report the design of synthetic biodegradable polymers specifically for enhancing DNA vaccine efficacy in vivo. We molecularly engineered poly(ortho ester) microspheres that are non-toxic to cells, protect DNA from degradation, enable uptake by antigen-presenting cells, and release DNA rapidly in response to phagosomal pH. One type of microsphere of poly(ortho esters) that releases DNA vaccines in synchrony with the natural development of adaptive immunity, elicited distinct primary and secondary humoral and cellular immune responses in mice, and suppressed the growth of tumour cells bearing a model antigen. This polymer microparticulate system could, with further study, have implications for advancing the clinical utility of DNA vaccines as well as other nucleic-acid-based therapeutics against viral infections and cancer.

  4. Direct ortho-arylation of ortho-substituted benzoic acids: overriding Pd-catalyzed protodecarboxylation.

    PubMed

    Arroniz, Carlos; Ironmonger, Alan; Rassias, Gerry; Larrosa, Igor

    2013-02-15

    ortho-Arylation of ortho-substituted benzoic acids is a challenging process due to the tendency of the reaction products toward Pd-catalyzed protodecarboxylation. A simple method for preventing decarboxylation in sterically hindered benzoic acids is reported. The method described represents a reliable and broadly applicable entry to 2-aryl-6-substituted benzoic acids. PMID:23373630

  5. The effect of subarachnoid erythrocyte lysate on brain injury: a preliminary study

    PubMed Central

    Zhang, Zi-Huan; Han, Yan-Ling; Wang, Chun-Xi; Zhou, Chen-Hui; Wu, Ling-Yun; Zhang, Hua-Sheng; Chen, Qiang; Fan, Jie-Mei; Zhou, Meng-Liang; Hang, Chun-Hua

    2016-01-01

    Abundant erythrocytes remain and lyse partially in the subarachnoid space after severe subarachnoid haemorrhage (SAH). But the effect of subarachnoid erythrocyte lysate on brain injury is still not completely clear. In this study, autologous erythrocytes (the non-lysate group) and their lysate (the lysate group) were injected separately into the cistern magna of rabbits to induce a model of experimental SAH, although the control group received isotonic sodium chloride solution instead of erythrocyte solution. Results showed that vasospasm of the basilar artery was observed at 72 h after experimental SAH, but there was no significant difference between the non-lysate group and the lysate group. Brain injury was more severe in the lysate group than in the non-lysate group. Meanwhile, the levels of peroxiredoxin 2 (Prx2), IL-6 and TNF-α in brain cortex and in CSF were significantly higher in the lysate group than those in the non-lysate group. These results demonstrated that brain injury was more likely to be caused by erythrocyte lysate than by intact erythrocytes in subarachnoid space, and inflammation response positively correlated with Prx2 expression might be involved in mechanism of brain injury after SAH. PMID:27279653

  6. The effect of subarachnoid erythrocyte lysate on brain injury: a preliminary study.

    PubMed

    Zhang, Zi-Huan; Han, Yan-Ling; Wang, Chun-Xi; Zhou, Chen-Hui; Wu, Ling-Yun; Zhang, Hua-Sheng; Chen, Qiang; Fan, Jie-Mei; Zhou, Meng-Liang; Hang, Chun-Hua

    2016-08-01

    Abundant erythrocytes remain and lyse partially in the subarachnoid space after severe subarachnoid haemorrhage (SAH). But the effect of subarachnoid erythrocyte lysate on brain injury is still not completely clear. In this study, autologous erythrocytes (the non-lysate group) and their lysate (the lysate group) were injected separately into the cistern magna of rabbits to induce a model of experimental SAH, although the control group received isotonic sodium chloride solution instead of erythrocyte solution. Results showed that vasospasm of the basilar artery was observed at 72 h after experimental SAH, but there was no significant difference between the non-lysate group and the lysate group. Brain injury was more severe in the lysate group than in the non-lysate group. Meanwhile, the levels of peroxiredoxin 2 (Prx2), IL-6 and TNF-α in brain cortex and in CSF were significantly higher in the lysate group than those in the non-lysate group. These results demonstrated that brain injury was more likely to be caused by erythrocyte lysate than by intact erythrocytes in subarachnoid space, and inflammation response positively correlated with Prx2 expression might be involved in mechanism of brain injury after SAH. PMID:27279653

  7. Variation of proteins and proteinases in Entamoeba histolytica lysates containing a protease inhibitor.

    PubMed

    López-Revilla, R; Jiménez-Delgadillo, B; Canto-Ortiz, L; Chávez-Dueñas, L

    1992-01-01

    Sodium dodecyl sulfate (SDS)-lysates of E. histolytica trophozoites were analyzed by electrophoresis in simple and gelatin-containing ("substrate") SDS-polyacrylamide gels. In simple gels, boiled lysates with para hydroxymercuribenzoate (pHMB) had a complex pattern of apparently undegraded proteins; boiled lysates without pHMB showed a major 30 kDa and four minor (43, 46, 63 and 117 kDa) proteins, whereas unheated lysates displayed only the 117 kDa protein. Using substrate gels no gelatinases were detected in heated lysates; unheated lysates without pHMB showed a major 30 kDa and three minor (33, 46 and 68 kDa) gelatinases, whereas those with pHMB presented a major 56 kDa and two minor (70 and 105 kDa) gelatinases. Three caseinase peaks were separated by Sephadex G-75 chromatography from unheated lysates: peak I contained 46, 56 and 117 kDa pHMB-sensitive gelatinases and peaks II and III contained smaller pHMB-resistant caseinases. We conclude that proteins remaining in lysates after SDS-induced proteolysis appear to be mainly proteases relatively resistant to self-digestion whose type and amount changes with the conditions of lysis and the presence of inhibitors; this is exemplified by the finding of the major gelatinase of lysates with pHMB being larger (56 kDa) than in lysates lacking the inhibitor (30 kDa). PMID:1340329

  8. In vitro nonsense suppression in [psi+] and [psi-] cell-free lysates of Saccharomyces cerevisiae.

    PubMed Central

    Tuite, M F; Cox, B S; McLaughlin, C S

    1983-01-01

    An homologous in vitro assay for yeast nonsense suppressors was used to examine the effect of the cytoplasmically inherited genetic determinant [psi] on the efficiency of in vitro nonsense suppression. The efficiency of all three types of yeast tRNA-mediated nonsense suppressor (ochre, amber, and UGA) is much greater in cell-free lysates prepared from a sup+ [psi+] strain than in lysates prepared from an isogeneic sup+ [psi-] strain. Lysates prepared from a [psi-] strain, into which the [psi+] determinant was reintroduced by kar1-mediated cytoduction, support efficient suppression. Evidence is also presented that [psi-] lysates contain an inhibitor of in vitro nonsense suppression. Images PMID:6344070

  9. Probing small molecule microarrays with tagged proteins in cell lysates

    PubMed Central

    Pop, Marius S.; Wassaf, Dina; Koehler, Angela N.

    2014-01-01

    The technique of small-molecule microarray (SMM) screening is based on the ability of small molecules to bind to various soluble proteins. This type of interaction is easily detected by the presence of a fluorescence signal produced by labeled antibodies that specifically recognize a unique sequence (tag) present on the target protein. The fluorescent signal intensity values are determined based on signal-to-noise ratios (SNR). SMM screening is a high throughput, unbiased method that can rapidly identify novel direct ligands for various protein targets. This binding-based assay format is generally applicable to most proteins, but it is especially useful for protein targets that do not possess an enzymatic activity. SMMs enable screening a protein in a purified form or in the context of a cellular lysate, likely providing a more physiologically relevant screening environment. PMID:25445177

  10. Bifidobacterium longum lysate, a new ingredient for reactive skin.

    PubMed

    Guéniche, Audrey; Bastien, Philippe; Ovigne, Jean Marc; Kermici, Michel; Courchay, Guy; Chevalier, Veronique; Breton, Lionel; Castiel-Higounenc, Isabelle

    2010-08-01

    Reactive skin is characterized by marked sensitivity to physical (heat, cold, wind) or chemical (topically applied products) stimuli and by the impairment of the skin barrier's ability to repair itself. Several lines of evidence suggest that beyond their capacity to positively influence the composition of intestinal microbiota, some probiotic bacteria can modulate the immune system both at local and systemic levels, thereby improving immune defense mechanisms and/or down-regulating immune disorders such as allergies and intestinal inflammation. Several recent human clinical trials clearly suggest that probiotic supplementation might be beneficial to the skin. Using a probiotic lysate, Bifidobacterium longum sp. extract (BL), we demonstrated first in vitro, and then in a clinical trial, that this non-replicating bacteria form applied to the skin was able to improve sensitive skin. The effect of BL were evaluated first on two different models. Using ex vivo human skin explant model we found a statistically significant improvement versus placebo in various parameters associated with inflammation such as a decrease in vasodilation, oedema, mast cell degranulation and TNF-alpha release. Moreover, using nerve cell cultures in vitro, we showed that after 6 h of incubation in culture medium (0.3-1%), the probiotic lysate significantly inhibited capsaicin-induced CGRP release by neurones. Then, a topical cream containing the active extract was tested in a randomized, double-blind, placebo-controlled trial. Sixty-six female volunteers with reactive skin were randomly given either the cream with the bacterial extract at 10% (n = 33) or the control cream (n = 33). The volunteers applied the cream to the face, arms and legs twice a day for two months. Skin sensitivity was assessed by stinging test (lactic acid) and skin barrier recovery was evaluated by measuring trans-epidermal water loss following barrier disruption induced by repeated tape-stripping at D1, D29 and D57. The

  11. Peroxide-Free Pd(II)-Catalyzed Ortho Aroylation and Ortho Halogenation of Directing Arenes.

    PubMed

    Santra, Sourav Kumar; Banerjee, Arghya; Mohanta, Prakash Ranjan; Patel, Bhisma K

    2016-07-15

    A Pd(II)-catalyzed peroxide-free ortho aroylation of directing arenes has been developed via cross dehydrogenative coupling (CDC) in the presence of the terminal oxidant Cu(OAc)2·H2O. Ortho aroylation of directing arenes proceeds via decarbonylation of the in situ generated phenyl glyoxal, which is obtained from 2-acetoxyacetophenone in the presence of the oxidant Cu(OAc)2·H2O. However, changing the oxidant to CuX2 (X = Cl, Br) provided exclusive di-ortho-halogenated 2-arylbenzothiazoles. During the halogenation, CuX2 served the dual role of a halogen source as well as a co-oxidant. PMID:27314648

  12. Monocyte Caspase-1 Is Released in a Stable, Active High Molecular Weight Complex Distinct from the Unstable Cell Lysate-Activated Caspase-1

    PubMed Central

    Shamaa, Obada R.; Mitra, Srabani; Gavrilin, Mikhail A.; Wewers, Mark D.

    2015-01-01

    Mononuclear phagocytes utilize caspase-1 activation as a means to respond to danger signals. Although caspase-1 was discovered using highly concentrated cell extracts that spontaneously activate caspase-1, it is now clear that in live cell models caspase-1 activation occurs in the process of its cellular release and is not an intracellular event. Therefore, we compared the characteristics of caspase-1 activation in the cell lysate model to that of caspase-1 that is released in response to exogenous inflammasome activation. Whereas both models generated active caspase-1, the cell-lysate induced caspase-1 required highly concentrated cell lysates and had a short half-life (~15 min) whereas, the activation induced released caspase-1 required 2–3 log fold fewer cells and was stable for greater than 12 h. Both forms were able to cleave proIL-1beta but unexpectedly, the released activity was unable to be immunodepleted by caspase-1 antibodies. Size exclusion chromatography identified two antigenic forms of p20 caspase-1 in the activation induced released caspase-1: one at the predicted size of tetrameric, p20/p10 caspase-1 and the other at >200 kDa. However, only the high molecular weight form had stable functional activity. These results suggest that released caspase-1 exists in a unique complex that is functionally stable and protected from immunodepletion whereas cell-extract generated active caspase-1 is rapidly inhibited in the cytosolic milieu. PMID:26599267

  13. Role of ortho-retronasal olfaction in mammalian cortical evolution.

    PubMed

    Rowe, Timothy B; Shepherd, Gordon M

    2016-02-15

    Fossils of mammals and their extinct relatives among cynodonts give evidence of correlated transformations affecting olfaction as well as mastication, head movement, and ventilation, and suggest evolutionary coupling of these seemingly separate anatomical regions into a larger integrated system of ortho-retronasal olfaction. Evidence from paleontology and physiology suggests that ortho-retronasal olfaction played a critical role at three stages of mammalian cortical evolution: early mammalian brain development was driven in part by ortho-retronasal olfaction; the bauplan for neocortex had higher-level association functions derived from olfactory cortex; and human cortical evolution was enhanced by ortho-retronasal smell. PMID:25975561

  14. Exosomes: novel effectors of human platelet lysate activity.

    PubMed

    Torreggiani, E; Perut, F; Roncuzzi, L; Zini, N; Baglìo, S R; Baldini, N

    2014-01-01

    Despite the popularity of platelet-rich plasma (PRP) and platelet lysate (PL) in orthopaedic practice, the mechanism of action and the effectiveness of these therapeutic tools are still controversial. So far, the activity of PRP and PL has been associated with different growth factors (GF) released during platelet degranulation. This study, for the first time, identifies exosomes, nanosized vesicles released in the extracellular compartment by a number of elements, including platelets, as one of the effectors of PL activity. Exosomes were isolated from human PL by differential ultracentrifugation, and analysed by electron microscopy and Western blotting. Bone marrow stromal cells (MSC) treated with three different exosome concentrations (0.6 μg, 5 μg and 50 μg) showed a significant, dose-dependent increase in cell proliferation and migration compared to the control. In addition, osteogenic differentiation assays demonstrated that exosome concentration differently affected the ability of MSC to deposit mineralised matrix. Finally, the analysis of exosome protein content revealed a higher amount of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF-BB) and transforming growth factor beta 1 (TGF-β1) as compared to PL. In regards to RNA content, an enrichment of small RNAs in exosomes as compared to donor platelets has been found. These results suggest that exosomes consistently contribute to PL activity and could represent an advantageous nanodelivery system for cell-free regeneration therapies. PMID:25241964

  15. Oral immunization with bacterial lysate against infection with Streptococcus pneumoniae in mice.

    PubMed

    van Daal, G J; de Jong, P T; Tenbrinck, R; Mouton, J W; Petzoldt, K; Bergmann, K C; Lachmann, B

    1990-01-01

    The protective effect of oral immunization against infection with Streptococcus pneumoniae was investigated in mice. Two bacterial lysates, one with an additional lysate of Candida albicans, were investigated. Intranasal inoculation of adult Balb-C mice with a S. pneumoniae type I strain resulted in a lethal infection, with deaths occurring from the 2nd until the 6th day after infection. Oral immunization resulted in a significant decrease in mortality rate (18-48% reduction). No significant difference in mortality rates was observed between the groups immunized with different lysates in the same concentrations. PMID:2095604

  16. ORTHO- ELIMINATION OF TRACKING SYSTEM CLOCK ERRORS

    NASA Technical Reports Server (NTRS)

    Wu, J. T.

    1994-01-01

    ORTHO is part of the Global Positioning System (GPS) being developed by the U.S. Air Force, a navigational system that will use 18 NAVSTAR satellites to broadcast navigation messages and achieve worldwide coverage. The normal positioning technique uses one receiver which receives signals from at least four GPS satellites. For higher accuracy work it is often necessary to use a differential technique in which more than one receiver is used. The geodetic measurement has all receivers on the ground and allows the determination of the relative locations of the ground sites. The main application of the ORTHO program is in the elimination of clock errors in a GPS based tracking system. The measured distance (pseudo-range) from a GPS receiver contains errors due to differences in the receiver and satellite clocks. The conventional way of eliminating clock errors is to difference pseudo-ranges between different GPS satellites and receivers. The Householder transformation used in this program performs a function similar to the conventional single differencing or double differencing. This method avoids the problem of redundancy and correlation encountered in a differencing scheme. It is able to keep all information contained in the measurements within the scope of a least square estimation. For multiple transmitter and receiver GPS tracking network, this method is in general more accurate than the differencing technique. This program assumes that the non-clock measurement partial derivatives for the particular application are computed earlier by another program. With the partial derivatives and information to identify the transmitters and receivers as the input, the program performs the Householder transformation on the partial derivatives. The transformed partials are output by the program and may be used as an input to the filter program in the subsequent estimation process. Clock partial derivatives are generated internally and are not part of the input to the program

  17. A Phase I vaccine trial using dendritic cells pulsed with autologous oxidized lysate for recurrent ovarian cancer

    PubMed Central

    2013-01-01

    Purpose Ovarian cancer, like most solid tumors, is in dire need of effective therapies. The significance of this trial lies in its promise to spearhead the development of combination immunotherapy and to introduce novel approaches to therapeutic immunomodulation, which could enable otherwise ineffective vaccines to achieve clinical efficacy. Rationale Tumor-infiltrating T cells have been associated with improved outcome in ovarian cancer, suggesting that activation of antitumor immunity will improve survival. However, molecularly defined vaccines have been generally disappointing. Cancer vaccines elicit a modest frequency of low-to-moderate avidity tumor-specific T-cells, but powerful tumor barriers dampen the engraftment, expansion and function of these effector T-cells in the tumor, thus preventing them from reaching their full therapeutic potential. Our work has identified two important barriers in the tumor microenvironment: the blood-tumor barrier, which prevents homing of effector T cells, and T regulatory cells, which inactivate effector T cells. We hypothesize that cancer vaccine therapy will benefit from combinations that attenuate these two barrier mechanisms. Design We propose a three-cohort sequential study to investigate a combinatorial approach of a new dendritic cell (DC) vaccine pulsed with autologous whole tumor oxidized lysate, in combination with antiangiogenesis therapy (bevacizumab) and metronomic cyclophosphamide, which impacts Treg cells. Innovation This study uses a novel autologous tumor vaccine developed with 4-day DCs pulsed with oxidized lysate to elicit antitumor response. Furthermore, the combination of bevacizumab with a whole tumor antigen vaccine has not been tested in the clinic. Finally the combination of bevacizumab and metronomic cyclophosphamide in immunotherapy is novel. PMID:23777306

  18. Two new ortho benzoquinones from Uncaria rhynchophylla.

    PubMed

    Zhang, Qian; Chen, Lei; Hu, Le-Jian; Liu, Wen-Yuan; Feng, Feng; Qu, Wei

    2016-03-01

    The present study was designed to determine the chemical constituents of the stems and hooks of Uncaria rhynchophylla. The chemical constituents were isolated and purified from CH2Cl2 fraction by chromatography. Their structures were elucidated by spectroscopic analyses. Their cytotoxicity was tested using MTT method. Two new ortho benzoquinones, 3-diethylamino-5-methoxy-1, 2-benzoquinone (1) and 3-ethylamino-5-methoxy-1, 2-benzoquinone (2), together with a known compound isorhynchophyllic acid (3) were isolated from U. rhynchophylla. These compounds were evaluated for their cytotoxicity against cancer cells A549, HepG2 and A2780. Compounds 1 and 2 were new ortho benzoquinones and showed weak antiproliferative activities on A549, HepG2 and A2780 cells. Compound 3 significantly inhibited the proliferation of A549, HepG2 and A2780 cells with IC50 values being 5.8, 12.8 and 11.8 µmol·L(-1), respectively. PMID:27025371

  19. Aflatoxin B1 degradation by liquid cultures and lysates of three bacterial strains.

    PubMed

    Adebo, Oluwafemi Ayodeji; Njobeh, Patrick Berka; Sidu, Sibusiso; Tlou, Matsobane Godfrey; Mavumengwana, Vuyo

    2016-09-16

    Aflatoxin contamination remains a daunting issue to address in food safety. In spite of the efforts geared towards prevention and elimination of this toxin, it still persists in agricultural commodities. This has necessitated the search for other measures such as microbial degradation to combat this hazard. In this study, we investigated the biodegradation of aflatoxin B1 (AFB1), using lysates of three bacterial strains (Pseudomonas anguilliseptica VGF1, Pseudomonas fluorescens and Staphylococcus sp. VGF2) isolated from a gold mine aquifer. The bacterial cells were intermittently lysed in the presence and absence of protease inhibitors to obtain protease free lysates, subsequently incubated with AFB1 for 3, 6, 12, 24, and 48h to investigate whether any possible AFB1 degradation occurred using high performance liquid chromatography (HPLC) for detection. Results obtained revealed that after 6h of incubation, protease inhibited lysates of Staphylococcus sp. VGF2 demonstrated the highest degradation capacity of 100%, whereas P. anguilliseptica VGF1 and P. fluorescens lysates degraded AFB1 by 66.5 and 63%, respectively. After further incubation to 12h, no residual AFB1 was detected for all the lysates. Lower degrading ability was however observed for liquid cultures and uninhibited lysates. Data on cytotoxicity studies against human lymphocytes showed that the degraded products were less toxic than the parent AFB1. From this study, it can thus be deduced that the mechanism of degradation by these bacterial lysates is enzymatic. This study shows the efficacy of crude bacterial lysates for detoxifying AFB1 indicating potential for application in the food and feed industry. PMID:27294556

  20. Metal based nanoparticles as cancer antigen delivery vehicles for macrophage based antitumor vaccine.

    PubMed

    Chattopadhyay, Sourav; Dash, Sandeep Kumar; Mandal, Debasis; Das, Balaram; Tripathy, Satyajit; Dey, Aditi; Pramanik, Panchanan; Roy, Somenath

    2016-02-10

    In the present study, we would like to evaluate the efficacy of modified metal oxide nanoparticles (NPs) as cancer antigen delivery vehicles for macrophage (MФs) based antitumor vaccine. The cobalt oxide nanoparticles (CoO NPs) were promising tools for delivery of antigens to antigen presenting cells and have induced an antitumor immune response. Synthesized CoO NPs were modified by N-phosphonomethyliminodiacetic acid (PMIDA), facilitated the conjugation of lysate antigen, i.e. cancer antigen derived from lysis of cancer cells. The cancer cell lysate antigen conjugated PMIDA-CoO NPs (Ag-PMIDA-CoO NPs) successfully activated macrophage (MФ) evident by the increasing the serum IFN-γ and TNF-α level. Immunization of mice with the Ag-PMIDA-CoO NPs constructed an efficient immunological adjuvant induced anticancer IgG responses, and increased the antibody dependent cellular cytotoxicity (ADCC) response than only lysate antigen treated group to combat the cancer cell. The nanocomplexes enhanced the anticancer CD4(+)T cell response in mice. The result showed that Ag-PMIDA-CoO NPs can stimulate the immune responses over only lysate antigens, which are the most important findings in this study. These NP-mediated Ag deliveries may significantly improve the anticancer immune response by activating MФs and may act as adjuvant and will balance the pro-inflammatory and anti-inflammatory immunoresponse. The crosstalk between the activated MФ with other immune competent cells will be monitored by measuring the cytokines which illustrate the total immunological network setups. PMID:26772632

  1. Effect of cell lysates on retroviral transduction efficiency of cells in suspension culture.

    PubMed

    Beauchesne, Pascal R; Bruce, Katherine J; Bowen, Bruce D; Piret, James M

    2010-04-15

    Recombinant retroviruses are effective vectors able to integrate transgenes into the target cell's genome to achieve longer-term expression. This study investigates the effect of cell lysis products, a common cell culture by-product, on the transduction of suspension cells by gammaretroviral vectors. Cell lysates derived from human and murine suspension cell lines significantly increased the transduction of human TF-1 and K-562 cell lines by gibbon ape leukemia virus-pseudotyped retroviral vectors without altering tropism. The transduction efficiency of TF-1 cells increased as a function of lysate concentration and decreased with increasing target cell concentrations. This was adequately predicted using a saturation equation based on the lysed-to-target cell concentration ratio, R, where: Fold increase = 1+Fold_(Max) (R/(K_(L)+R)). Lysate completely masked the effects of fibronectin when the two were added in combination. With protamine sulfate, the transduction efficiency was increased by lysate to 58% from 20% for protamine sulfate alone. Overall, the presence of cell lysate significantly influenced the outcome of the transduction process, either alone or in the presence of protamine sulfate or fibronectin. PMID:20014140

  2. Detection of embryonic stem cell lysate biomarkers by surface plasmon resonance with reduced nonspecific adsorption.

    PubMed

    Tyagi, Deependra; Perez, Javier Batista; Nand, Amita; Zhiqiang, Cheng; Wang, Peizhe; Na, Jie; Zhu, Jingsong

    2015-02-15

    Surface plasmon resonance imaging (SPRi) has emerged as a versatile biosensor to detect a wide range of biomolecular interactions with divergent potential applications. However, the use of this advanced-level technology for stem cell lysate study is still not much explored. Cell lysates are significant biological analytes used for disease diagnostics and proteomic studies, but their complex nature limits their use as an analyte for SPRi biosensors. Here, we review the problems associated with the use of SPRi for stem cell lysate study and examine the role of surface chemistry, running buffer, and blocking solution in order to minimize nonspecific adsorption (NSA). We detect the expression of Oct4, Sox2, Nanog, Rex1, and Lin28 biomarkers present in mouse embryonic stem cell (mESC) lysate against their corresponding antibodies immobilized on the sensor surface with reduced NSA. The current study shows that the conjunction of SPRi and microarray can be used as a label-free, high-throughput, and rapid technique for detection of biomarkers and their relative abundance in stem cell lysate study. PMID:25447493

  3. Cooling by conversion of para to ortho-hydrogen

    NASA Technical Reports Server (NTRS)

    Sherman, A. (Inventor)

    1983-01-01

    The cooling capacity of a solid hydrogen cooling system is significantly increased by exposing vapor created during evaporation of a solid hydrogen mass to a catalyst and thereby accelerating the endothermic para-to-ortho transition of the vapor to equilibrium hydrogen. Catalyst such as nickel, copper, iron or metal hydride gels of films in a low pressure drop catalytic reactor are suitable for accelerating the endothermic para-to-ortho conversion.

  4. Investigating Birth Control: Comparing Oestrogen Levels in Patients Using the Ortho Evra[R] Patch versus the Ortho-Cyclen[R] Pill

    ERIC Educational Resources Information Center

    Laurent, Theresa A.

    2008-01-01

    Recent drug studies have investigated the incidence of blood clots among patients using the Ortho Evra[R] birth control patch. In this article, the author describes an investigation of oestrogen levels in the body resulting from the application of the Ortho Evra[R] birth control patch versus daily use of Ortho-Cyclen[R] birth control pills.…

  5. The Domestication of ortho-Quinone Methides

    PubMed Central

    2015-01-01

    Conspectus An ortho-quinone methide (o-QM) is a highly reactive chemical motif harnessed by nature for a variety of purposes. Given its extraordinary reactivity and biological importance, it is surprising how few applications within organic synthesis exist. We speculate that their widespread use has been slowed by the complications that surround the preparation of their precursors, the harsh generation methods, and the omission of this stratagem from computer databases due to its ephemeral nature. About a decade ago, we discovered a mild anionic triggering procedure to generate transitory o-QMs at low temperature from readily available salicylaldehydes, particularly OBoc derivatives. This novel reaction cascade included both the o-QM formation and the subsequent consumption reaction. The overall transformation was initiated by the addition of the organometallic reagent, usually a Grignard reagent, which resulted in the formation of a benzyloxy alkoxide. Boc migration from the neighboring phenol produced a magnesium phenoxide that we supposed underwent β-elimination of the transferred Boc residue to form an o-QM for immediate further reactions. Moreover, the cascade proved controllable through careful manipulation of metallic and temperature levers so that it could be paused, stopped, or restarted at various intermediates and stages. This new level of domestication enabled us to deploy o-QMs for the first time in a range of applications including diastereocontrolled reactions. This sequence ultimately could be performed in either multipot or single pot processes. The subsequent reaction of the fleeting o-QM intermediates included the 1,4-conjugate additions that led to unbranched or branched ortho-alkyl substituted phenols and Diels–Alder reactions that provided 4-unsubstituted or 4-substituted benzopyrans and chroman ketals. The latter cycloadducts were obtained for the first time with outstanding diastereocontrol. In addition, the steric effects of the newly

  6. Enhanced Stimulation of Anti-Ovarian Cancer CD8+ T Cells by Dendritic Cells Loaded with Nanoparticle Encapsulated Tumor Antigen

    PubMed Central

    Hanlon, Douglas J; Aldo, Paulomi B.; Devine, Lesley; Alvero, Ayesha B.; Engberg, Anna K.; Edelson, Richard; Mor, Gil

    2011-01-01

    Problem Dendritic cell (DC)-based cancer therapies are favored approaches to stimulate anti-tumor T cells responses. Unfortunately, tolerance to tumor antigens is difficult to overcome. Biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP) are effective reagents in the delivery of drugs and tumor-associated antigens (TAA). In this study, we assessed the capacity of a PLGA NP-based delivery system to augment CD8 T cell responses to ovarian cancer TAA. Method of Study Human DC were generated from blood monocytes by conventional in vitro differentiation and loaded with either soluble tumor lysate or NP/lysate conjugates (NPL). These antigen-loaded DC were then used to stimulate autologous CD8+ T cells. Cytokine production and activation markers were evaluated in the CD8+T cells. Results DC loading with NPL increased cytokine production by stimulated CD8 T cells and induced T cell expression of cell surface co-stimulatory molecules, typical of anti-tumor immune responses. In contrast, delivery of naked tumor lysate antigens preferentially induced a T cell profile characteristic of tolerization/exhaustion. Conclusion These findings indicate that delivery of TAA in NP enables DC to efficiently activate anti-tumor CD8+ T cells. PLGA NP encapsulation of tumor-derived lysate protein antigens is an encouraging new preparative methodology for DC-based vaccination meriting clinical testing. PMID:21241402

  7. High throughput ranking of recombinant avian scFv antibody fragments from crude lysates using the Biacore A100.

    PubMed

    Leonard, Paul; Säfsten, Pär; Hearty, Stephen; McDonnell, Barry; Finlay, William; O'Kennedy, Richard

    2007-06-30

    Advances in molecular evolution strategies have made it possible to identify antibodies with exquisite specificities and also to fine-tune their biophysical properties for practically any specified application. Depending on the desired function, antibody/antigen interactions can be long-lived or short-lived and, therefore, particular attention is needed when seeking to identify antibodies with specific reaction-rate and affinity properties. Surface plasmon resonance (SPR) biosensors routinely generate sensitive and reliable kinetic data from antibody/antigen interactions for both therapeutic and diagnostic applications. However, many kinetic-based screening assays require rigorous sample preparation and purification prior to analysis. To ameliorate this problem, we developed a rapid and reliable assay for characterising recombinant scFv antibody fragments, directly from crude bacterial lysates. Ninety-six scFv antibodies derived from chickens immunised with C-reactive protein (CRP) were selected by phage display and evaluated using the Biacore A100 protein interaction array system. Antibodies were captured from crude bacterial extracts on the sensor chip surface and ranked based on the percentage of the complex left (% left) after dissociation in buffer. Kinetic rate constants (k(a) and k(d)) and affinity (K(D)) data were obtained for six clones that bound monomeric CRP across a broad affinity range (2.54 x 10(-8) to 3.53 x 10(-10) M). Using this assay format the A100 biosensor yielded high quality kinetic data, permitting the screening of nearly 400 antibody clones per day. PMID:17532001

  8. Strong and oriented immobilization of single domain antibodies from crude bacterial lysates for high-throughput compatible cost-effective antibody array generation

    PubMed Central

    Even-Desrumeaux, Klervi; Baty, Daniel; Chames, Patrick

    2010-01-01

    Antibodies microarrays are among the novel class of rapidly emerging proteomic technologies that will allow us to efficiently perform specific diagnosis and proteome analysis. Recombinant antibody fragments are especially suited for this approach but their stability is often a limiting factor. Camelids produce functional antibodies devoid of light chains (HCAbs) of which the single N-terminal domain is fully capable of antigen binding. When produced as an independent domain, these so-called single domain antibody fragments (sdAbs) have several advantages for biotechnological applications thanks to their unique properties of size (15 kDa), stability, solubility, and expression yield. These features should allow sdAbs to outperform other antibody formats in a number of applications, notably as capture molecule for antibody arrays. In this study, we have produced antibody microarrays using direct and oriented immobilization of sdAbs produced in crude bacterial lysates to generate proof-of-principle of a high-throughput compatible array design. Several sdAb immobilization strategies have been explored. Immobilization of in vivo biotinylated sdAbs by direct spotting of bacterial lysate on streptavidin and sandwich detection was developed to achieve high sensitivity and specificity, whereas immobilization of “multi-tagged” sdAbs via anti-tag antibodies and direct labeled sample detection strategy was optimized for the design of high-density antibody arrays for high-throughput proteomics and identification of potential biomarkers. PMID:20859568

  9. Th2 Allergic Immune Response to Inhaled Fungal Antigens is Modulated By TLR-4-Independent Bacterial Products

    PubMed Central

    Allard, Jenna B.; Rinaldi, Lisa; Wargo, Matt; Allen, Gilman; Akira, Shizuo; Uematsu, Satoshi; Poynter, Matthew E.; Hogan, Deborah A.; Rincon, Mercedes; Whittaker, Laurie A.

    2009-01-01

    SUMMARY Allergic airway disease is characterized by eosinophilic inflammation, mucus hypersecretion and increased airway resistance. Fungal antigens are ubiquitous within the environment and are well know triggers of allergic disease. Bacterial products are also frequently encountered within the environment and may alter the immune response to certain antigens. The consequence of simultaneous exposure to bacterial and fungal products on the lung adaptive immune response has not been explored. Here we show that oropharyngeal aspiration of fungal lysates (Candida albicans, Aspergillus fumigatus) promotes airway eosinophilia, secretion of Th2 cytokines and mucus cell metaplasia. In contrast, oropharyngeal exposure to bacterial lysates (Pseudomonas aeruginosa) promotes airway inflammation characterized by neutrophils, Th1 cytokine secretion and no mucus production. More importantly, administration of bacterial lysates together with fungal lysates deviates the adaptive immune response to a Th1 type associated with neutrophilia and diminished mucus production. The immunomodulatory effect that bacterial lysates have on the response to fungi is TLR4-independent but MyD88 dependent. Thus, different types of microbial products within the airway can alter the host's adaptive immune response, and potentially impact the development of allergic airway disease to environmental fungal antigens. PMID:19224641

  10. Synthesis of Unsymmetrical ortho-Biphenols and ortho-Binaphthols via Silicon-Tethered Pd-Catalyzed C–H Arylation

    PubMed Central

    Huang, Chunhui; Gevorgyan, Vladimir

    2010-01-01

    A mild, practical, and efficient method for the synthesis of unsymmetrical ortho-biphenols (including ortho-phenol-naphthols and ortho-binaphthols) has been developed. Unsymmetrical bis-aryloxy silanes, which were readily prepared in a semi-one-pot fashion, underwent the Pd-catalyzed intramolecular arylation followed by a routine TBAF desilylation step to furnish valuable unsymmetrical biphenols without necessity of isolation of 7-membered intermediates. The excellent functional group tolerance allows for synthesis of a variety of functionalized ortho-biphenols and ortho-binaphthols from easily available staring materials. PMID:20423110

  11. Isolation, bioactivity, and production of ortho-hydroxydaidzein and ortho-hydroxygenistein.

    PubMed

    Chang, Te-Sheng

    2014-01-01

    Daidzein and genistein are two major components of soy isoflavones. They exist abundantly in plants and possess multiple bioactivities. In contrast, ortho-hydroxydaidzein (OHD) and ortho-hydroxygenistein (OHG), including 6-hydroxydaidzein (6-OHD), 8-hydroxydaidzein (8-OHD), 3'-hydroxydaidzein (3'-OHD), 6-hydroxygenistein (6-OHG), 8-hydroxygenistein (8-OHG), and 3'-hydroxygenistein (3'-OHG), are rarely found in plants. Instead, they are usually isolated from fermented soybean foods or microbial fermentation broth feeding with soybean meal. Accordingly, the bioactivity of OHD and OHG has been investigated less compared to that of soy isoflavones. Recently, OHD and OHG were produced by genetically engineering microorganisms through gene cloning of cytochrome P450 (CYP) enzyme systems. This success opens up bioactivity investigation and industrial applications of OHD and OHG in the future. This article reviews isolation of OHD and OHG from non-synthetic sources and production of the compounds by genetically modified microorganisms. Several bioactivities, such as anticancer and antimelanogenesis-related activities, of OHD and OHG, are also discussed. PMID:24705463

  12. INTERFERON-GAMMA STIMULATING ACTIVITIES OF THE FRACTIONATED NEOSPORA CANINUM TACHYZOITE LYSATE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Neospora caninum is an obligate intracellular protozoan parasite, causing bovine abortion worldwide. Our recent research showed that N. caninum tachyzoite lysate elicits production of the T cell cytokine interferon-gamma (IFN-g) by both bovine and murine T cells, which may be critical to host protec...

  13. Lactobacillus rhamnosus GG Lysate Increases Re-Epithelialization of Keratinocyte Scratch Assays by Promoting Migration

    PubMed Central

    Mohammedsaeed, Walaa; Cruickshank, Sheena; McBain, Andrew J.; O’Neill, Catherine A.

    2015-01-01

    A limited number of studies have investigated the potential of probiotics to promote wound healing in the digestive tract. The aim of the current investigation was to determine whether probiotic bacteria or their extracts could be beneficial in cutaneous wound healing. A keratinocyte monolayer scratch assay was used to assess re-epithelialization; which comprises keratinocyte proliferation and migration. Primary human keratinocyte monolayers were scratched then exposed to lysates of Lactobacillus (L) rhamnosus GG, L. reuteri, L. plantarum or L. fermentum. Re-epithelialization of treated monolayers was compared to that of untreated controls. Lysates of L. rhamnosus GG and L. reuteri significantly increased the rate of re-epithelialization, with L. rhamnosus GG being the most efficacious. L. reuteri increased keratinocyte proliferation while L. rhamnosus GG lysate significantly increased proliferation and migration. Microarray analysis of L. rhamnosus GG treated scratches showed increased expression of multiple genes including the chemokine CXCL2 and its receptor CXCR2. These are involved in normal wound healing where they stimulate keratinocyte proliferation and/or migration. Increased protein expression of both CXCL2 and CXCR2 were confirmed by ELISA and immunoblotting. These data demonstrate that L. rhamnosus GG lysate accelerates re-epithelialization of keratinocyte scratch assays, potentially via chemokine receptor pairs that induce keratinocyte migration. PMID:26537246

  14. Lactobacillus rhamnosus GG Lysate Increases Re-Epithelialization of Keratinocyte Scratch Assays by Promoting Migration.

    PubMed

    Mohammedsaeed, Walaa; Cruickshank, Sheena; McBain, Andrew J; O'Neill, Catherine A

    2015-01-01

    A limited number of studies have investigated the potential of probiotics to promote wound healing in the digestive tract. The aim of the current investigation was to determine whether probiotic bacteria or their extracts could be beneficial in cutaneous wound healing. A keratinocyte monolayer scratch assay was used to assess re-epithelialization; which comprises keratinocyte proliferation and migration. Primary human keratinocyte monolayers were scratched then exposed to lysates of Lactobacillus (L) rhamnosus GG, L. reuteri, L. plantarum or L. fermentum. Re-epithelialization of treated monolayers was compared to that of untreated controls. Lysates of L. rhamnosus GG and L. reuteri significantly increased the rate of re-epithelialization, with L. rhamnosus GG being the most efficacious. L. reuteri increased keratinocyte proliferation while L. rhamnosus GG lysate significantly increased proliferation and migration. Microarray analysis of L. rhamnosus GG treated scratches showed increased expression of multiple genes including the chemokine CXCL2 and its receptor CXCR2. These are involved in normal wound healing where they stimulate keratinocyte proliferation and/or migration. Increased protein expression of both CXCL2 and CXCR2 were confirmed by ELISA and immunoblotting. These data demonstrate that L. rhamnosus GG lysate accelerates re-epithelialization of keratinocyte scratch assays, potentially via chemokine receptor pairs that induce keratinocyte migration. PMID:26537246

  15. Platelet lysate and chondroitin sulfate loaded contact lenses to heal corneal lesions.

    PubMed

    Sandri, Giuseppina; Bonferoni, Maria Cristina; Rossi, Silvia; Delfino, Alessio; Riva, Federica; Icaro Cornaglia, Antonia; Marrubini, Giorgio; Musitelli, Giorgio; Del Fante, Claudia; Perotti, Cesare; Caramella, Carla; Ferrari, Franca

    2016-07-25

    Hemoderivative tear substitutes contain various ephiteliotrophic factors, such as growth factors (GF), involved in ocular surface homeostasis without immunogenic properties. The aim of the present work was the loading of platelet lysate into contact lenses to improve the precorneal permanence of platelet lysate growth factors on the ocular surface to enhance the treatment of corneal lesions. To this purpose, chondroitin sulfate, a sulfated glycosaminoglycan, which is normally present in the extracellular matrix, was associated with platelet lysate. In fact, chondroitin sulfate is capable of electrostatic interaction with positively charged growth factors, in particular, with bFGF, IGF, VEGF, PDGF and TGF-β, resulting in their stabilization and reduced degradation in solution. In the present work, various types of commercially available contact lenses have been loaded with chondroitin sulfate or chondroitin sulfate in association with platelet lysate to achieve a release of growth factors directly onto the corneal surface lesions. One type of contact lenses (PureVision(®)) showed in vitro good proliferation properties towards corneal cells and were able to enhance cut closure in cornea constructs. PMID:27234702

  16. Human platelet lysate as a promising growth-stimulating additive for culturing of stem cells and other cell types.

    PubMed

    Shanskii, Ya D; Sergeeva, N S; Sviridova, I K; Kirakozov, M S; Kirsanova, V A; Akhmedova, S A; Antokhin, A I; Chissov, V I

    2013-11-01

    We compared the composition and biological activity of fetal calf serum and platelet lysate from donor platelet concentrate. In platelet lysate, the concentrations of alkaline phosphatase, lactate dehydrogenase, creatinine, and mineral metabolism parameters were lower, while parameters of lipid and protein metabolism were higher than in fetal calf serum. The concentrations of growth factors (platelet-derived (AA, AB, BB), vascular endothelial, insulin-like, and transforming growth factor β) in platelet lysate 1.7-148.7-fold surpassed the corresponding parameters in fetal calf serum. After replacement of fetal calf serum with platelet lysate in the culture medium (0, 25, 50, 75, and 100%), the count of multipotent mesenchymal stromal cells on day 7 (in comparison with day 1) increased by 154.8, 206.6, 228.2, 367.7, and 396.5%, respectively. Thus, platelet lysate can be an adequate non-xenogenic alternative for fetal calf serum. PMID:24319712

  17. Analysis of culture filtrate and cell wall-associated antigens of Mycobacterium paratuberculosis with monoclonal antibodies.

    PubMed Central

    Mutharia, L M; Moreno, W; Raymond, M

    1997-01-01

    Proteins secreted by Mycobacterium species have been suggested as major immune targets in the early phase of infection. In this study, we sought to identify specific antigens in culture filtrates and in soluble cell extracts of Mycobacterium paratuberculosis. The release of antigens into the culture medium during growth of the bacilli and the distribution of specific epitopes within the Mycobacterium species were investigated by immunoblot analysis with monoclonal antibodies (MAbs) raised against M. paratuberculosis antigens. MAb B6A interacted with a cellular antigen with an apparent molecular mass of 34.5 kDa in lysates of M. paratuberculosis. MAb B6A did not interact with lysates from any other mycobacterial species, suggesting recognition of an M. paratuberculosis species-specific epitope. MAb FL1-A1 reacted with an antigen of 44.3 kDa in M. paratuberculosis and a 9-kDa antigen in Mycobacterium kansasii. MAb PII-B1 reacted with concanavalin A (ConA)-binding cellular and filtrate molecules of M. paratuberculosis and with lysates of Mycobacterium kansasii and Mycobacterium avium 18. The affinity-purified glycosylated antigens migrated as a diffuse band of between 35 and 45.6 kDa and reacted strongly with ovine and bovine paratuberculosis serum and polyclonal serum against M. tuberculosis lipoarabinomannan antigens. These glycoconjugates were the earliest antigens detected in culture filtrates of M. paratuberculosis. Deglycosylation of the ConA-binding molecules with alpha-mannosidase enzyme abolished the reaction with MAb PII-B1 and with bovine but not ovine paratuberculosis serum, suggesting selective immunogenicity in the different animal species. PMID:9009287

  18. Human humoral responses to antigens of Mycobacterium tuberculosis: immunodominance of high-molecular-mass antigens.

    PubMed Central

    Laal, S; Samanich, K M; Sonnenberg, M G; Zolla-Pazner, S; Phadtare, J M; Belisle, J T

    1997-01-01

    The selection of antigens of Mycobacterium tuberculosis for most studies of humoral responses in tuberculosis patients has been restricted to molecules that were either immunodominant in immunized animals or amenable to biochemical purification rather than those that were reactive with the human immune system. Delineation of antigens that elicit humoral responses during the natural course of disease progression in humans has been hindered by the presence of cross-reactive antibodies to conserved regions on ubiquitous prokaryotic antigens in sera from healthy individuals and tuberculosis patients. The levels of cross-reactive antibodies in the sera were reduced by preadsorption with Escherichia coli lysates, prior to studying their reactivity against a large panel of M. tuberculosis antigens to which the human immune system may be exposed during natural infection and disease. Thus, reactivity against pools of secreted, cellular, and cell wall-associated antigens of M. tuberculosis was assessed by an enzyme-linked immunosorbent assay (ELISA). Initial results suggested that the secreted protein preparation contained antigens most frequently recognized by the humoral responses of pulmonary tuberculosis patients. The culture filtrate proteins were subsequently size fractionated by preparative polyacrylamide gel electrophoresis, characterized by reaction with murine monoclonal antibodies to known antigens of M. tuberculosis by an ELISA, and assessed for reactivity with tuberculous and nontuberculous sera. Results show that a secreted antigen of 88 kDa elicits a strong antibody response in a high percentage of patients with pulmonary tuberculosis. This and other antigens identified on the basis of their reactivity with patient sera may prove useful for developing serodiagnosis for tuberculosis. PMID:9008280

  19. Fluorescence-linked Antigen Quantification (FLAQ) Assay for Fast Quantification of HIV-1 p24Gag

    PubMed Central

    Gesner, Marianne; Maiti, Mekhala; Grant, Robert; Cavrois, Marielle

    2016-01-01

    The fluorescence-linked antigen quantification (FLAQ) assay allows a fast quantification of HIV-1 p24Gag antigen. Viral supernatant are lysed and incubated with polystyrene microspheres coated with polyclonal antibodies against HIV-1 p24Gag and detector antibodies conjugated to fluorochromes (Figure 1). After washes, the fluorescence of microspheres is measured by flow cytometry and reflects the abundance of the antigen in the lysate. The speed, simplicity, and wide dynamic range of the FLAQ assay are optimum for many applications performed in HIV-1 research laboratories.

  20. Expression of Plasmodium falciparum surface antigens in Escherichia coli.

    PubMed Central

    Ardeshir, F; Flint, J E; Reese, R T

    1985-01-01

    The asexual blood stages of the human malarial parasite Plasmodium falciparum produce many antigens, only some of which are important for protective immunity. Most of the putative protective antigens are believed to be expressed in schizonts and merozoites, the late stages of the asexual cycle. With the aim of cloning and characterizing genes for important parasite antigens, we used late-stage P. falciparum mRNA to construct a library of cDNA sequences inserted in the Escherichia coli expression vector pUC8. Nine thousand clones from the expression library were immunologically screened in situ with serum from Aotus monkeys immune to P. falciparum, and 95 clones expressing parasite antigens were identified. Mice were immunized with lysates from 49 of the bacterial clones that reacted with Aotus sera, and the mouse sera were tested for their reactivity with parasite antigens by indirect immunofluorescence, immunoprecipitation, and immunoblotting assays. Several different P. falciparum antigens were identified by these assays. Indirect immunofluorescence studies of extracellular merozoites showed that three of these antigens appear to be located on the merozoite surface. Thus, we have identified cDNA clones to three different P. falciparum antigens that may be important in protective immunity. Images PMID:3887406

  1. Activity profiling of aminopeptidases in cell lysates using a fluorogenic substrate library.

    PubMed

    Byzia, Anna; Szeffler, Agata; Kalinowski, Leszek; Drag, Marcin

    2016-03-01

    Aminopeptidases are exopeptidases that process peptide bonds at the N-terminus of protein substrates, and they are involved in controlling several metabolic pathways. Due to their involvement in diseases such as cancer or rheumatoid arthritis, their presence can also be used as a predictive biomarker. Here, we used a library of fluorogenic substrates containing natural and unnatural amino acids to reliably measure the aminopeptidase N (APN) activity in cell lysates obtained from human, pig and rat kidneys. We compared our results to the substrate specificity profile of isolated APN. Our data strongly support the observation that fluorogenic substrates can be successfully used to identify aminopeptidases and to measure their activity in cell lysates. Moreover, in contrast to assays using single substrates, which can result in overlapping specificity due to cleavage by several aminopeptidases, our library fingerprint can provide information about single enzymes. PMID:26449746

  2. Occurrence of non-ortho-, mono-ortho- and di-ortho- substituted PCB congeners in different organs and tissues of polecats (Mustela putorius L. ) from the Netherlands

    SciTech Connect

    Leonards, P.E.G.; Hattum, B. Van; Cofino, W.P. . Inst. for Environmental Studies); Brinkman, U.A. . Dept. of Analytical Chemistry)

    1994-01-01

    The presence and concentrations of non-ortho-, mono-ortho-, and di-ortho--substituted PCB congeners in the pole-cat (n = 7), were investigated. PCBs were extracted with a Soxhlet apparatus. After cleanup the non-ortho-substituted PCB congeners were separated from the other PCBs by HPLC. Determinations were accomplished with GC-ECD or GC-MSD. Patterns of PCBs were examined in different organs and tissues: liver, kidney, muscle, anal gland secretion, mesenteric fat, and subcutaneous fat. Using a multivariate statistical method for data analysis (SIMCA), a significant difference of PCB patterns between anal gland secretion and the other organs and tissues was revealed. Lesser concentrations of congeners with seven and eight chlorine atoms in anal gland secretion were mainly responsible for this phenomenon. A more or less organ- and tissue-specific PCB pattern was observed in all animals. PCB patterns were not dependent on prey choice, which ranged from terrestrial (small rodents) to aquatic (amphibians). This finding implies that PCB patterns in the pole-cat seem to be controlled by metabolic processes rather than diet factors. The total concentration of PCBs in polecats varies widely, two orders of magnitude, from 1 to 3,700 [mu]g/g lipid. In some animals, PCBs exceeded the experimentally determined reproduction effect concentrations of mink and ferrets. Using the toxic equivalent approach, it was observed that planar PCB 126 accounts for 63 to 98% of the toxic equivalents. The results showed that juvenile animals contain greater PCB levels than adult males and females, which might be related to an increased elimination of PCBs in adult animals due to anal gland secretion. High concentrations of PCBs were observed in such secretion. A preliminary model for concentration of PCBs in polecats including this effect is proposed.

  3. Silver-catalysed protodecarboxylation of ortho-substituted benzoic acids.

    PubMed

    Cornella, Josep; Sanchez, Carolina; Banawa, David; Larrosa, Igor

    2009-12-14

    Catalytic amounts of Ag(I) salts in DMSO have been found to promote the protodecarboxylation of a wide variety of ortho-substituted benzoic acids under mild conditions and in excellent yields, highlighting a possible role for silver in decarboxylative cross-couplings. PMID:19921021

  4. Improved Preparation of Halopropyl Bridged Carboxylic Ortho Esters

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protection of a carboxylic acid function as a bridged ortho ester derivative enables the use of strongly basic conditions in the synthetic strategy because the protons, alpha to the previous carbonyl carbon, are less acidic. Protected 3-halopropionic acid can behave like an alkyl halide making them...

  5. Improved preparation of haloalkyl bridged carboxylic ortho esters

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protection of a carboxylic acid function as a bridged ortho ester derivative enables the use of strong basic conditions in the synthetic strategy. For example, a protected 3-halopropionic acid can behave like an alkyl halide because the protons, alpha to the halide function, are less acidic. Ester...

  6. Cooling by Para-to-Ortho-Hydrogen Conversion

    NASA Technical Reports Server (NTRS)

    Sherman, A.; Nast, T.

    1983-01-01

    Catalyst speeds conversion, increasing capacity of solid hydrogen cooling system. In radial-flow catalytic converter, para-hydrogen is converted to equilibrium mixture of para-hydrogen and ortho-hydrogen as it passes through porous cylinder of catalyst. Addition of catalyst increases capacity of hydrogen sublimation cooling systems for radiation detectors.

  7. FipsOrtho: A Spell Checker for Learners of French

    ERIC Educational Resources Information Center

    L'Haire, Sebastien

    2007-01-01

    This paper presents FipsOrtho, a spell checker targeted at learners of French, and a corpus of learners' errors which has been gathered to test the system and to get a sample of specific language learners' errors. Spell checkers are a standard feature of many software products, however they are not designed for specific language learners' errors.…

  8. Bacterial lysate in the prevention of acute exacerbation of COPD and in respiratory recurrent infections

    PubMed Central

    Braido, F; Tarantini, F; Ghiglione, V; Melioli, G; Canonica, G W

    2007-01-01

    Respiratory tract infections (RTIs) represent a serious problem because they are one of the most common cause of human death by infection. The search for the treatment of those diseases has therefore a great importance. In this study we provide an overview of the currently available treatments for RTIs with particular attention to chronic obstructive pulmonary diseases exacerbations and recurrent respiratory infections therapy and a description of bacterial lysate action, in particular making reference to the medical literature dealing with its clinical efficacy. Those studies are based on a very large number of clinical trials aimed to evaluate the effects of this drug in maintaining the immune system in a state of alert, and in increasing the defences against microbial infections. From this analysis it comes out that bacterial lysates have a protective effect, which induce a significant reduction of the symptoms related to respiratory infections. Those results could be very interesting also from an economic point of view, because they envisage a reduction in the number of acute exacerbations and a shorter duration of hospitalization. The use of bacterial lysate could therefore represent an important means to achieve an extension of life duration in patients affected by respiratory diseases. PMID:18229572

  9. Human platelet lysate: Replacing fetal bovine serum as a gold standard for human cell propagation?

    PubMed

    Burnouf, Thierry; Strunk, Dirk; Koh, Mickey B C; Schallmoser, Katharina

    2016-01-01

    The essential physiological role of platelets in wound healing and tissue repair builds the rationale for the use of human platelet derivatives in regenerative medicine. Abundant growth factors and cytokines stored in platelet granules can be naturally released by thrombin activation and clotting or artificially by freeze/thaw-mediated platelet lysis, sonication or chemical treatment. Human platelet lysate prepared by the various release strategies has been established as a suitable alternative to fetal bovine serum as culture medium supplement, enabling efficient propagation of human cells under animal serum-free conditions for a multiplicity of applications in advanced somatic cell therapy and tissue engineering. The rapidly increasing number of studies using platelet derived products for inducing human cell proliferation and differentiation has also uncovered a considerable variability of human platelet lysate preparations which limits comparability of results. The main variations discussed herein encompass aspects of donor selection, preparation of the starting material, the possibility for pooling in plasma or additive solution, the implementation of pathogen inactivation and consideration of ABO blood groups, all of which can influence applicability. This review outlines the current knowledge about human platelet lysate as a powerful additive for human cell propagation and highlights its role as a prevailing supplement for human cell culture capable to replace animal serum in a growing spectrum of applications. PMID:26561934

  10. Molecular reorientation in ortho-carborane studied by dielectric spectroscopy

    NASA Astrophysics Data System (ADS)

    Lunkenheimer, P.; Loidl, A.

    1996-03-01

    The dielectric properties of ortho-carborane have been investigated in a broad frequency range of 20 Hz≤ν≤1 GHz and at temperatures 10 K≤T≤380 K. At T≳275 K the ortho-carborane molecules undergo fast and isotropic reorientations with frequencies larger than 1 GHz. Below a phase transition at Tc≊275 K the reorientation is partly restricted and the dynamics of the remaining reorientational motion is reduced. At 160 K, the dielectric results reveal a small anomaly which we assign to a second phase transition. At temperatures 150 Kortho-carborane can be estimated as m≊20 characterizing it as a strong glass former. Ortho-carborane fits well into the correlation scheme of Böhmer et al. where polydispersivity and fragility are related to each other. At high temperatures and low frequencies conductivity processes (hopping) dominate the dielectric response of ortho-carborane.

  11. Effects of a mixture of non-ortho- and mono-ortho-polychlorinated biphenyls on reproduction in Fundulus heteroclitus (Linnaeus)

    SciTech Connect

    Black, D.E.; Gutjahr-Gobell, R.; Pruell, R.J.; Bergen, B.; McElroy, A.E.

    1998-07-01

    To assess the effects of polychlorinated biphenyls (PCBs) on reproduction, female Fundulus heteroclitus were exposed to a mixture of non-ortho- and mono-ortho-PCBs, mimicking the mixture found in fish collected from New Bedford Harbor, Massachusetts, USA, a PCB-contaminated estuary. Exposure was by intraperitoneal injection of the mixture dissolved in corn oil. Doses of 0.76, 3.8, and 19 {micro}g PCB mixture per gram of wet weight produced liver concentrations of 2.99, 12.2, and 32.8 {micro}g non-ortho- and mono-ortho-PCBs per gram of dry liver, with dioxin toxic equivalency concentrations (TEQs) of 0.0963, 0.409, and 0.720 ng/g, respectively. Female mortality was 58%, and egg production was reduced by 77% at the highest dose, compared to controls. Food consumption declined with increasing PCB concentration, suggesting that PCBs act indirectly to reduce fecundity through an energetic effect. Pituitary gonadotropin content appeared to be suppressed at the highest dose, but the ability of ovarian follicles to produce estradiol and testosterone in vitro was not impaired. Significant residue-effects linkages were found, with TEQ emerging as a potential indicator of adverse effects. Mortality was directly related, and egg production was inversely related to log{sub 10}TEQ. Multiple regression analysis indicated that egg production was directly related to pituitary gonadotropin content and food consumption.

  12. Effect of Conditioned Medium and Bone Marrow Stem Cell Lysate on the Course of Acetaminophen-Induced Liver Failure.

    PubMed

    Khubutiya, M Sh; Temnov, A A; Vagabov, V A; Sklifas, A N; Rogov, K A; Zhgutov, Yu A

    2015-05-01

    A composition containing culture medium conditioned by mesenchymal stem cells and mesenchymal stem cell lysate improves biochemical parameters, reduces inflammation, and stimulates regenerative processes in the liver. PMID:26033600

  13. Antigenic sites in carcinoembryonic antigen.

    PubMed

    Hammarstrom, S; Shively, J E; Paxton, R J; Beatty, B G; Larsson, A; Ghosh, R; Bormer, O; Buchegger, F; Mach, J P; Burtin, P

    1989-09-01

    The epitope reactivities of 52 well-characterized monoclonal antibodies (Mabs) against carcinoembryonic antigen from 11 different research groups were studied using competitive solid-phase immunoassays. About 60% of all possible combinations of Mabs as inhibitors and as the primary binding antibody were investigated. The inhibition data were analyzed by a specially developed computer program "EPITOPES" which measures concordance and discordance in inhibition patterns between Mabs. The analysis showed that 43 of the 52 Mabs (83%) could be classified into one of five essentially noninteracting epitope groups (GOLD 1-5) containing between four and 15 Mabs each. The epitopes recognized by the Mabs belonging to groups 1 to 5 were peptide in nature. With one or two possible exceptions non-classifiable Mabs were either directed against carbohydrate epitopes (4 Mabs) or were inactive in the tests used. Within epitope groups GOLD 1, 4, and 5 two partially overlapping subgroups were distinguished. Mabs with a high degree of carcinoembryonic antigen specificity generally belonged to epitope groups GOLD 1 and 3. PMID:2474375

  14. Precision cancer immunotherapy: optimizing dendritic cell-based strategies to induce tumor antigen-specific T-cell responses against individual patient tumors.

    PubMed

    Osada, Takuya; Nagaoka, Koji; Takahara, Masashi; Yang, Xiao Yi; Liu, Cong-Xiao; Guo, Hongtao; Roy Choudhury, Kingshuk; Hobeika, Amy; Hartman, Zachary; Morse, Michael A; Lyerly, H Kim

    2015-05-01

    Most dendritic cell (DC)-based vaccines have loaded the DC with defined antigens, but loading with autologos tumor-derived antigens would generate DCs that activate personalized tumor-specific T-cell responses. We hypothesized that DC matured with an optimized combination of reagents and loaded with tumor-derived antigens using a clinically feasible electroporation strategy would induce potent antitumor immunity. We first studied the effects on DC maturation and antigen presentation of the addition of picibanil (OK432) to a combination of zoledronic acid, tumor necrosis factor-α, and prostaglandin E2. Using DC matured with the optimized combination, we tested 2 clinically feasible sources of autologous antigen for electroloading, total tumor mRNA or total tumor lysate, to determine which stimulated more potent antigen-specific T cells in vitro and activated more potent antitumor immunity in vivo. The combination of tumor necrosis factor-α/prostaglandin E2/zoledronic acid/OK432 generated DC with high expression of maturation markers and antigen-specific T-cell stimulatory function in vitro. Mature DC electroloaded with tumor-derived mRNA [mRNA electroporated dendritic cell (EPDC)] induced greater expansion of antigen-specific T cells in vitro than DC electroloaded with tumor lysate (lysate EPDC). In a therapeutic model of MC38-carcinoembryonic antigen colon cancer-bearing mice, vaccination with mRNA EPDC induced the most efficient anti-carcinoembryonic antigen cellular immune response, which significantly suppressed tumor growth. In conclusion, mature DC electroloaded with tumor-derived mRNA are a potent cancer vaccine, especially useful when specific tumor antigens for vaccination have not been identified, allowing autologous tumor, and if unavailable, allogeneic cell lines to be used as an unbiased source of antigen. Our data support clinical testing of this strategy. PMID:25839441

  15. Isolation of ortho- and paramyxoviruses from wild birds in Western Australia, and the characterization of novel influenza A viruses.

    PubMed

    Mackenzie, J S; Edwards, E C; Holmes, R M; Hinshaw, V S

    1984-02-01

    As part of the World Health Organization's international programme on the ecology of influenza, cloacal swabs were collected from 3,736 birds belonging to 67 species over a 3-year period in Western Australia for the isolation of ortho- and paramyxoviruses. A total of 24 influenza A viruses were isolated from various species of ducks, shearwaters , noddies , terns and a coot , and were subtyped as H1N9 , H3N8 , H4N4 , H4N6 , H6N2 , H6N4 , H?N2, H?N6 and H? N9 . The H? haemagglutinins did not react in tests with reference antisera. Whether they represent a novel haemagglutinin subtype or atypical members of an established subtype remains to be determined, although preliminary results indicate that they may be atypical members of the H7 subtype. The H1N9 isolate is the first reported isolate of this particular antigenic combination. A total of 17 Newcastle disease viruses was isolated from ducks, noddies , terns and a black- fronted plover : preliminary results suggest that they are avirulent for domestic chickens. This study indicates that ortho- and paramyxoviruses are present in a variety of wild birds in Australia. PMID:6430260

  16. Proteomics as a Quality Control Tool of Pharmaceutical Probiotic Bacterial Lysate Products.

    PubMed

    Klein, Günter; Schanstra, Joost P; Hoffmann, Janosch; Mischak, Harald; Siwy, Justyna; Zimmermann, Kurt

    2013-01-01

    Probiotic bacteria have a wide range of applications in veterinary and human therapeutics. Inactivated probiotics are complex samples and quality control (QC) should measure as many molecular features as possible. Capillary electrophoresis coupled to mass spectrometry (CE/MS) has been used as a multidimensional and high throughput method for the identification and validation of biomarkers of disease in complex biological samples such as biofluids. In this study we evaluate the suitability of CE/MS to measure the consistency of different lots of the probiotic formulation Pro-Symbioflor which is a bacterial lysate of heat-inactivated Escherichia coli and Enterococcus faecalis. Over 5000 peptides were detected by CE/MS in 5 different lots of the bacterial lysate and in a sample of culture medium. 71 to 75% of the total peptide content was identical in all lots. This percentage increased to 87-89% when allowing the absence of a peptide in one of the 5 samples. These results, based on over 2000 peptides, suggest high similarity of the 5 different lots. Sequence analysis identified peptides of both E. coli and E. faecalis and peptides originating from the culture medium, thus confirming the presence of the strains in the formulation. Ontology analysis suggested that the majority of the peptides identified for E. coli originated from the cell membrane or the fimbrium, while peptides identified for E. faecalis were enriched for peptides originating from the cytoplasm. The bacterial lysate peptides as a whole are recognised as highly conserved molecular patterns by the innate immune system as microbe associated molecular pattern (MAMP). Sequence analysis also identified the presence of soybean, yeast and casein protein fragments that are part of the formulation of the culture medium. In conclusion CE/MS seems an appropriate QC tool to analyze complex biological products such as inactivated probiotic formulations and allows determining the similarity between lots. PMID

  17. Proteomics as a Quality Control Tool of Pharmaceutical Probiotic Bacterial Lysate Products

    PubMed Central

    Klein, Günter; Schanstra, Joost P.; Hoffmann, Janosch; Mischak, Harald; Siwy, Justyna; Zimmermann, Kurt

    2013-01-01

    Probiotic bacteria have a wide range of applications in veterinary and human therapeutics. Inactivated probiotics are complex samples and quality control (QC) should measure as many molecular features as possible. Capillary electrophoresis coupled to mass spectrometry (CE/MS) has been used as a multidimensional and high throughput method for the identification and validation of biomarkers of disease in complex biological samples such as biofluids. In this study we evaluate the suitability of CE/MS to measure the consistency of different lots of the probiotic formulation Pro-Symbioflor which is a bacterial lysate of heat-inactivated Escherichia coli and Enterococcus faecalis. Over 5000 peptides were detected by CE/MS in 5 different lots of the bacterial lysate and in a sample of culture medium. 71 to 75% of the total peptide content was identical in all lots. This percentage increased to 87–89% when allowing the absence of a peptide in one of the 5 samples. These results, based on over 2000 peptides, suggest high similarity of the 5 different lots. Sequence analysis identified peptides of both E. coli and E. faecalis and peptides originating from the culture medium, thus confirming the presence of the strains in the formulation. Ontology analysis suggested that the majority of the peptides identified for E. coli originated from the cell membrane or the fimbrium, while peptides identified for E. faecalis were enriched for peptides originating from the cytoplasm. The bacterial lysate peptides as a whole are recognised as highly conserved molecular patterns by the innate immune system as microbe associated molecular pattern (MAMP). Sequence analysis also identified the presence of soybean, yeast and casein protein fragments that are part of the formulation of the culture medium. In conclusion CE/MS seems an appropriate QC tool to analyze complex biological products such as inactivated probiotic formulations and allows determining the similarity between lots. PMID

  18. Performance of two different Limulus amebocyte lysate assays for the quantitation of fungal glucan.

    PubMed

    Cherid, Hafsa; Foto, Mark; Miller, J David

    2011-09-01

    This study examined the response of various forms and sources of glucans toward two different Limulus amebocyte lysate (LAL) methods, the modified LAL, and Glucatell. The glucans studied were curdlan, laminarin, yeast glucan, barley glucan, paramylon, pullulan, pustulan, mannan, and pachyman (as part of the Glucatell kit). Both methods provided largely similar results for each of the glucans; however, the Glucatell method yielded slightly higher responses to certain structures that may not necessarily be of fungal origin, leading to falsely greater positive results. The performance of each method to measure fungal glucan concentration specifically was then assessed. PMID:21830869

  19. Comparative analysis of click chemistry mediated activity-based protein profiling in cell lysates.

    PubMed

    Yang, Yinliang; Yang, Xiaomeng; Verhelst, Steven H L

    2013-01-01

    Activity-based protein profiling uses chemical probes that covalently attach to active enzyme targets. Probes with conventional tags have disadvantages, such as limited cell permeability or steric hindrance around the reactive group. A tandem labeling strategy with click chemistry is now widely used to study enzyme targets in situ and in vivo. Herein, the probes are reacted in live cells, whereas the ensuing detection by click chemistry takes place in cell lysates. We here make a comparison of the efficiency of the activity-based tandem labeling strategy by using Cu(I)-catalyzed and strain-promoted click chemistry, different ligands and different lysis conditions. PMID:24126377

  20. Flow carbonylation of sterically hindered ortho-substituted iodoarenes.

    PubMed

    Mallia, Carl J; Walter, Gary C; Baxendale, Ian R

    2016-01-01

    The flow synthesis of ortho-substituted carboxylic acids, using carbon monoxide gas, has been studied for a number of substrates. The optimised conditions make use of a simple catalyst system compromising of triphenylphosphine as the ligand and palladium acetate as the pre-catalyst. Carbon monoxide was introduced via a reverse "tube-in-tube" flow reactor at elevated pressures to give yields of carboxylated products that are much higher than those obtained under normal batch conditions. PMID:27559403

  1. Direct Intermolecular Aniline ortho-Arylation via Benzyne Intermediates

    PubMed Central

    Truong, Thanh

    2012-01-01

    A method for direct, transition-metal-free ortho-arylation of anilines by aryl chlorides, bromides, fluorides, and triflates has been developed. This methodology provides the most direct approach to 2-arylanilines since no protecting or directing groups on nitrogen are required. The arylation is functional-group tolerant, with alkene, ether, trifluoromethyl, dimethylamino, carbonyl, chloro, and cyano functionalities tolerated. Phenylation of enantiopure binaphthyldiamine affords a product with >99% ee. PMID:23148679

  2. Flow carbonylation of sterically hindered ortho-substituted iodoarenes

    PubMed Central

    Mallia, Carl J; Walter, Gary C

    2016-01-01

    Summary The flow synthesis of ortho-substituted carboxylic acids, using carbon monoxide gas, has been studied for a number of substrates. The optimised conditions make use of a simple catalyst system compromising of triphenylphosphine as the ligand and palladium acetate as the pre-catalyst. Carbon monoxide was introduced via a reverse “tube-in-tube” flow reactor at elevated pressures to give yields of carboxylated products that are much higher than those obtained under normal batch conditions. PMID:27559403

  3. Biological and therapeutic effects of ortho-silicic acid and some ortho-silicic acid-releasing compounds: New perspectives for therapy

    PubMed Central

    2013-01-01

    Silicon (Si) is the most abundant element present in the Earth's crust besides oxygen. However, the exact biological roles of silicon remain unknown. Moreover, the ortho-silicic acid (H4SiO4), as a major form of bioavailable silicon for both humans and animals, has not been given adequate attention so far. Silicon has already been associated with bone mineralization, collagen synthesis, skin, hair and nails health atherosclerosis, Alzheimer disease, immune system enhancement, and with some other disorders or pharmacological effects. Beside the ortho-silicic acid and its stabilized formulations such as choline chloride-stabilized ortho-silicic acid and sodium or potassium silicates (e.g. M2SiO3; M= Na,K), the most important sources that release ortho-silicic acid as a bioavailable form of silicon are: colloidal silicic acid (hydrated silica gel), silica gel (amorphous silicon dioxide), and zeolites. Although all these compounds are characterized by substantial water insolubility, they release small, but significant, equilibrium concentration of ortho-silicic acid (H4SiO4) in contact with water and physiological fluids. Even though certain pharmacological effects of these compounds might be attributed to specific structural characteristics that result in profound adsorption and absorption properties, they all exhibit similar pharmacological profiles readily comparable to ortho-silicic acid effects. The most unusual ortho-silicic acid-releasing agents are certain types of zeolites, a class of aluminosilicates with well described ion(cation)-exchange properties. Numerous biological activities of some types of zeolites documented so far might probably be attributable to the ortho-silicic acid-releasing property. In this review, we therefore discuss biological and potential therapeutic effects of ortho-silicic acid and ortho-silicic acid -releasing silicon compounds as its major natural sources. PMID:23298332

  4. A microarray method for identifying tumor antigens by screening a tumor cDNA expression library against cancer sera

    PubMed Central

    Whittemore, Kurt; Sykes, Kathryn

    2013-01-01

    The immune system responds to tumor cells. The challenge has been how to effectively use these responses to treat or protect against cancer. Toward the goal of developing a cancer vaccine, we are pursuing methodologies for the discovery and testing of useful antigens. We present an array-based approach for discovering these B cell antigens by directly screening for specific host-sera reactivity to lysates from tumor-derived cDNA expression libraries. Several cancer-specific antigens were identified, and these are currently being validated as potential candidates. PMID:23851590

  5. OrthoInspector: comprehensive orthology analysis and visual exploration

    PubMed Central

    2011-01-01

    Background The accurate determination of orthology and inparalogy relationships is essential for comparative sequence analysis, functional gene annotation and evolutionary studies. Various methods have been developed based on either simple blast all-versus-all pairwise comparisons and/or time-consuming phylogenetic tree analyses. Results We have developed OrthoInspector, a new software system incorporating an original algorithm for the rapid detection of orthology and inparalogy relations between different species. In comparisons with existing methods, OrthoInspector improves detection sensitivity, with a minimal loss of specificity. In addition, several visualization tools have been developed to facilitate in-depth studies based on these predictions. The software has been used to study the orthology/in-paralogy relationships for a large set of 940,855 protein sequences from 59 different eukaryotic species. Conclusion OrthoInspector is a new software system for orthology/paralogy analysis. It is made available as an independent software suite that can be downloaded and installed for local use. Command line querying facilitates the integration of the software in high throughput processing pipelines and a graphical interface provides easy, intuitive access to results for the non-expert. PMID:21219603

  6. Effect of Different Adjuvants on Protection and Side-Effects Induced by Helicobacter suis Whole-Cell Lysate Vaccination

    PubMed Central

    Bosschem, Iris; Bayry, Jagadeesh; De Bruyne, Ellen; Van Deun, Kim; Smet, Annemieke; Vercauteren, Griet; Ducatelle, Richard; Haesebrouck, Freddy; Flahou, Bram

    2015-01-01

    Helicobacter suis (H. suis) is a widespread porcine gastric pathogen, which is also of zoonotic importance. The first goal of this study was to investigate the efficacy of several vaccine adjuvants (CpG-DNA, Curdlan, Freund’s Complete and Incomplete, Cholera toxin), administered either subcutaneously or intranasally along with H. suis whole-cell lysate, to protect against subsequent H. suis challenge in a BALB/c infection model. Subcutaneous immunization with Freund’s complete (FC)/lysate and intranasal immunization with Cholera toxin (CT)/lysate were shown to be the best options for vaccination against H. suis, as determined by the amount of colonizing H. suis bacteria in the stomach, although adverse effects such as post-immunization gastritis/pseudo-pyloric metaplasia and increased mortality were observed, respectively. Therefore, we decided to test alternative strategies, including sublingual vaccine administration, to reduce the unwanted side-effects. A CCR4 antagonist that transiently inhibits the migration of regulatory T cells was also included as a new adjuvant in this second study. Results confirmed that immunization with CT (intranasally or sublingually) is among the most effective vaccination protocols, but increased mortality was still observed. In the groups immunized subcutaneously with FC/lysate and CCR4 antagonist/lysate, a significant protection was observed. Compared to the FC/lysate immunized group, gastric pseudo-pyloric metaplasia was less severe or even absent in the CCR4 antagonist/lysate immunized group. In general, an inverse correlation was observed between IFN-γ, IL-4, IL-17, KC, MIP-2 and LIX mRNA expression and H. suis colonization density, whereas lower IL-10 expression levels were observed in partially protected animals. PMID:26115373

  7. ORTHO-PARA SELECTION RULES IN THE GAS-PHASE CHEMISTRY OF INTERSTELLAR AMMONIA

    SciTech Connect

    Faure, A.; Hily-Blant, P.; Le Gal, R.; Rist, C.

    2013-06-10

    The ortho-para chemistry of ammonia in the cold interstellar medium is investigated using a gas-phase chemical network. Branching ratios for the primary reaction chain involved in the formation and destruction of ortho- and para-NH{sub 3} were derived using angular momentum rules based on the conservation of the nuclear spin. We show that the 'anomalous' ortho-to-para ratio of ammonia ({approx}0.7) observed in various interstellar regions is in fact consistent with nuclear spin selection rules in a para-enriched H{sub 2} gas. This ratio is found to be independent of temperature in the range 5-30 K. We also predict an ortho-to-para ratio of {approx}2.3 for NH{sub 2}. We conclude that a low ortho-to-para ratio of H{sub 2} naturally drives the ortho-to-para ratios of nitrogen hydrides below the statistical values.

  8. Studies of dissolution inhibition mechanism of DNQ-novolak resist (II): effect of extended ortho-ortho bond in novolak

    NASA Astrophysics Data System (ADS)

    Honda, Kenji; Beauchemin, Bernard T., Jr.; Fitzgerald, Edward A., III; Jeffries, Alfred T., III; Tadros, Sobhy P.; Blakeney, Andrew J.; Hurditch, Rodney J.; Tan, Shiro; Sakaguchi, Shinji

    1991-06-01

    A p-cresol trimer sequence was incorporated into a polymeric chain of novolak by copolymerization with m-cresol of a reactive precursor which was prepared by attaching two units of m-cresol to the terminal ortho positions of p-cresol trimer. The resulting novolak was characterized by 13C NMR and FTIR in an attempt to correlate novolak structure with dissolution inhibition function based on physicochemical analysis of molecular interactions between novolak and DNQ-PAC in solid films.

  9. Phage infection of an environmentally relevant marine bacterium alters host metabolism and lysate composition

    PubMed Central

    Ankrah, Nana Yaw D; May, Amanda L; Middleton, Jesse L; Jones, Daniel R; Hadden, Mary K; Gooding, Jessica R; LeCleir, Gary R; Wilhelm, Steven W; Campagna, Shawn R; Buchan, Alison

    2014-01-01

    Viruses contribute to the mortality of marine microbes, consequentially altering biological species composition and system biogeochemistry. Although it is well established that host cells provide metabolic resources for virus replication, the extent to which infection reshapes host metabolism at a global level and the effect of this alteration on the cellular material released following viral lysis is less understood. To address this knowledge gap, the growth dynamics, metabolism and extracellular lysate of roseophage-infected Sulfitobacter sp. 2047 was studied using a variety of techniques, including liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based metabolomics. Quantitative estimates of the total amount of carbon and nitrogen sequestered into particulate biomass indicate that phage infection redirects ∼75% of nutrients into virions. Intracellular concentrations for 82 metabolites were measured at seven time points over the infection cycle. By the end of this period, 71% of the detected metabolites were significantly elevated in infected populations, and stable isotope-based flux measurements showed that these cells had elevated metabolic activity. In contrast to simple hypothetical models that assume that extracellular compounds increase because of lysis, a profile of metabolites from infected cultures showed that >70% of the 56 quantified compounds had decreased concentrations in the lysate relative to uninfected controls, suggesting that these small, labile nutrients were being utilized by surviving cells. These results indicate that virus-infected cells are physiologically distinct from their uninfected counterparts, which has implications for microbial community ecology and biogeochemistry. PMID:24304672

  10. The Effect of Autologous Platelet Lysate Eye Drops: An In Vivo Confocal Microscopy Study

    PubMed Central

    Fea, Antonio M.; Testa, Valeria; Machetta, Federica; Parisi, Simone; D'Antico, Sergio; Spinetta, Roberta; Fusaro, Enrico; Grignolo, Federico M.

    2016-01-01

    Purpose. To determine the effectiveness of autologous platelet lysate (APL) eye drops in patients with primary Sjögren syndrome (SS) dry eye, refractory to standard therapy, in comparison with patients treated with artificial tears. We focused on the effect of APL on cornea morphology with the in vivo confocal microscopy (IVCM). Methods. Patients were assigned to two groups: group A used autologous platelet lysate QID, and group B used preservative-free artificial tears QID, for 90 days. Ophthalmological assessments included ocular surface disease index (OSDI), best corrected visual acuity (BCVA), Schirmer test, fluorescein score, and breakup time (BUT). A subgroup of patients in group A underwent IVCM: corneal basal epithelium, subbasal nerves, Langerhans cells, anterior stroma activated keratocytes, and reflectivity were evaluated. Results. 60 eyes of 30 patients were enrolled; in group A (n = 20 patients) mean OSDI, fluorescein score, and BUT showed significant improvement compared with group B (n = 10 patients). The IVCM showed a significant increase in basal epithelium cells density and subbasal nerve plexus density and number and a decrease in Langerhans cells density (p < 0.05). Conclusion. APL was found effective in the treatment of SS dry eye. IVCM seems to be a useful tool to visualize cornea morphologic modifications. PMID:27200376

  11. The effect of chitosan nanospheres on the immunogenicity of Toxoplasma lysate vaccine in mice.

    PubMed

    El Temsahy, Mona M; El Kerdany, Eman D H; Eissa, Maha M; Shalaby, Thanaa I; Talaat, Iman M; Mogahed, Nermine M F H

    2016-09-01

    Toxoplasmosis, a zoonotic parasitic disease, is a huge challenge for which there is no effective vaccine up till now. In this study, chitosan nanospheres encapsulated with Toxoplasma lysate vaccine was evaluated for its ability to protect mice against both acute and chronic toxoplasmosis models of infection. Results showed that chitosan nanospheres were equally effective to Freund's incomplete adjuvant (FIA) in enhancing the efficacy of Toxoplasma lysate vaccine. The effectiveness was demonstrated by the delayed death of vaccinated mice following challenge either with virulent RH or avirulent Me49 strains, the significant decrease in parasite density in different organs, significant increase in the humoral and cellular immune response (IgG and IFN γ) with a marked reduction of pathological changes in the different organs. However chitosan nanospheres were superior to FIA due to their cost effective preparation and much less necrotic changes induced in the studied organs. The success of chitosan polymer as an alternative to commonly used adjuvants paves the way for the use of other newly developed polymers to be used in the field of vaccine development. PMID:27605755

  12. Affinity chromatography of chaperones based on denatured proteins: Analysis of cell lysates of different origin.

    PubMed

    Marchenko, N Yu; Sikorskaya, E V; Marchenkov, V V; Kashparov, I A; Semisotnov, G V

    2016-03-01

    Molecular chaperones are involved in folding, oligomerization, transport, and degradation of numerous cellular proteins. Most of chaperones are heat-shock proteins (HSPs). A number of diseases of various organisms are accompanied by changes in the structure and functional activity of chaperones, thereby revealing their vital importance. One of the fundamental properties of chaperones is their ability to bind polypeptides lacking a rigid spatial structure. Here, we demonstrate that affinity chromatography using sorbents with covalently attached denatured proteins allows effective purification and quantitative assessment of their bound protein partners. Using pure Escherichia coli chaperone GroEL (Hsp60), the capacity of denatured pepsin or lysozyme-based affinity sorbents was evaluated as 1 mg and 1.4 mg of GroEL per 1 ml of sorbent, respectively. Cell lysates of bacteria (E. coli, Thermus thermophilus, and Yersinia pseudotuberculosis), archaea (Halorubrum lacusprofundi) as well as the lysate of rat liver mitochondria were analyzed using affinity carrier with denatured lysozyme. It was found that, apart from Hsp60, other proteins with a molecular weight of about 100, 50, 40, and 20 kDa are able to interact with denatured lysozyme. PMID:26644295

  13. The Effect of Autologous Platelet Lysate Eye Drops: An In Vivo Confocal Microscopy Study.

    PubMed

    Fea, Antonio M; Aragno, Vittoria; Testa, Valeria; Machetta, Federica; Parisi, Simone; D'Antico, Sergio; Spinetta, Roberta; Fusaro, Enrico; Grignolo, Federico M

    2016-01-01

    Purpose. To determine the effectiveness of autologous platelet lysate (APL) eye drops in patients with primary Sjögren syndrome (SS) dry eye, refractory to standard therapy, in comparison with patients treated with artificial tears. We focused on the effect of APL on cornea morphology with the in vivo confocal microscopy (IVCM). Methods. Patients were assigned to two groups: group A used autologous platelet lysate QID, and group B used preservative-free artificial tears QID, for 90 days. Ophthalmological assessments included ocular surface disease index (OSDI), best corrected visual acuity (BCVA), Schirmer test, fluorescein score, and breakup time (BUT). A subgroup of patients in group A underwent IVCM: corneal basal epithelium, subbasal nerves, Langerhans cells, anterior stroma activated keratocytes, and reflectivity were evaluated. Results. 60 eyes of 30 patients were enrolled; in group A (n = 20 patients) mean OSDI, fluorescein score, and BUT showed significant improvement compared with group B (n = 10 patients). The IVCM showed a significant increase in basal epithelium cells density and subbasal nerve plexus density and number and a decrease in Langerhans cells density (p < 0.05). Conclusion. APL was found effective in the treatment of SS dry eye. IVCM seems to be a useful tool to visualize cornea morphologic modifications. PMID:27200376

  14. Hydrodynamic size-based separation and characterization of protein aggregates from total cell lysates

    PubMed Central

    Tanase, Maya; Zolla, Valerio; Clement, Cristina C; Borghi, Francesco; Urbanska, Aleksandra M; Rodriguez-Navarro, Jose Antonio; Roda, Barbara; Zattoni, Andrea; Reschiglian, Pierluigi; Cuervo, Ana Maria; Santambrogio, Laura

    2016-01-01

    Herein we describe a protocol that uses hollow-fiber flow field-flow fractionation (FFF) coupled with multiangle light scattering (MALS) for hydrodynamic size-based separation and characterization of complex protein aggregates. The fractionation method, which requires 1.5 h to run, was successfully modified from the analysis of protein aggregates, as found in simple protein mixtures, to complex aggregates, as found in total cell lysates. In contrast to other related methods (filter assay, analytical ultracentrifugation, gel electrophoresis and size-exclusion chromatography), hollow-fiber flow FFF coupled with MALS allows a flow-based fractionation of highly purified protein aggregates and simultaneous measurement of their molecular weight, r.m.s. radius and molecular conformation (e.g., round, rod-shaped, compact or relaxed). The polyethersulfone hollow fibers used, which have a 0.8-mm inner diameter, allow separation of as little as 20 μg of total cell lysates. In addition, the ability to run the samples in different denaturing and nondenaturing buffer allows defining true aggregates from artifacts, which can form during sample preparation. The protocol was set up using Paraquat-induced carbonylation, a model that induces protein aggregation in cultured cells. This technique will advance the biochemical, proteomic and biophysical characterization of molecular-weight aggregates associated with protein mutations, as found in many CNS degenerative diseases, or chronic oxidative stress, as found in aging, and chronic metabolic and inflammatory conditions. PMID:25521790

  15. Effect of the ortho alkylation of perylene bisimides on the alignment and self-assembly properties.

    PubMed

    Dasgupta, Debarshi; Kendhale, Amol M; Debije, Michael G; Ter Schiphorst, Jeroen; Shishmanova, Ivelina K; Portale, Giuseppe; Schenning, Albertus P H J

    2014-08-01

    The effect of the ortho alkylation of perylene bisimides on the alignment and self-assembly properties has been studied. It was found that the dichroic properties of perylene bisimides in a liquid crystal host can be reversed with a single synthetic step by ortho alkylation. Furthermore, a solvent-induced growth of ultralong organic n-type semiconducting fibrils from non-ortho-alkylated perylene bisimide was observed. Ortho substitution of the perylene bisimide core alters the mode of fibrillar growth, leading to isotropic crystallization. PMID:25478308

  16. Rhodium-Catalyzed Atroposelective [2 + 2 + 2] Cycloaddition of Ortho-Substituted Phenyl Diynes with Nitriles: Effect of Ortho Substituents on Regio- and Enantioselectivity.

    PubMed

    Kashima, Kenichi; Teraoka, Kota; Uekusa, Hidehiro; Shibata, Yu; Tanaka, Ken

    2016-05-01

    Axially chiral 3-(2-halophenyl)pyridines were successfully synthesized in high yields with excellent enantioselectivity by the cationic rhodium(I)/(S)-H8-BINAP complex-catalyzed atroposelective [2 + 2 + 2] cycloaddition of (o-halophenyl)diynes with nitriles. Interestingly, regio- and enantioselectivity highly depend on ortho substituents on the phenyl group of diynes. When the ortho substituents were methoxy and methoxycarbonyl groups, axially chiral 3-arylpyridines were obtained as a major product, while enantioselectivity was lowered significantly. On the other hand, when the ortho substituents were alkyl groups, regioselectivity was switched to give achiral 6-arylpyridines in high yields. PMID:27074498

  17. Ortho-para mixing hyperfine interaction in the H2O+ ion and nuclear spin equilibration.

    PubMed

    Tanaka, Keiichi; Harada, Kensuke; Oka, Takeshi

    2013-10-01

    The ortho to para conversion of water ion, H2O(+), due to the interaction between the magnetic moments of the unpaired electron and protons has been theoretically studied to calculate the spontaneous emission lifetime between the ortho- and para-levels. The electron spin-nuclear spin interaction term, Tab(SaΔIb + SbΔIa) mixes ortho (I = 1) and para (I = 0) levels to cause the "forbidden" ortho to para |ΔI| = 1 transition. The mixing term with Tab = 72.0 MHz is 4 orders of magnitude higher for H2O(+) than for its neutral counterpart H2O where the magnetic field interacting with proton spins is by molecular rotation rather than the free electron. The resultant 10(8) increase of ortho to para conversion rate possibly makes the effect of conversion in H2O(+) measurable in laboratories and possibly explains the anomalous ortho to para ratio recently reported by Herschel heterodyne instrument for the far-infrared (HIFI) observation. Results of our calculations show that the ortho ↔ para mixings involving near-degenerate ortho and para levels are high (∼10(-3)), but they tend to occur at high energy levels, ∼300 K. Because of the rapid spontaneous emission, such high levels are not populated in diffuse clouds unless the radiative temperature of the environment is very high. The low-lying 101 (para) and 111 (ortho) levels of H2O(+) are mixed by ∼10(-4) making the spontaneous emission lifetime for the para 101 → ortho 000 transition 520 years and 5200 years depending on the F value of the hyperfine structure. Thus the ortho ↔ para conversion due to the unpaired electron is not likely to seriously affect thermalization of interstellar H2O(+) unless either the radiative temperature is very high or number density of the cloud is very low. PMID:23530629

  18. ELISA-Based Crossmatching Allowing the Detection of Emerging Donor-Specific Anti-HLA Antibodies through the Use of Stored Donors' Cell Lysates

    PubMed Central

    Schlaf, G.; Stöhr, K.; Rothhoff, A.; Altermann, W.

    2015-01-01

    About forty years ago the complement-dependent crossmatch assay (CDC-CM) was developed as standard procedure in order to select recipients without donor-specific antibodies directed against human leukocyte antigens of their given donors since the negative outcome of pretransplant crossmatching represents one of the most important requirements for a successful kidney graft survival. However, as a functional assay the CDC-CM strongly depends on the availability of donors' isolated lymphocytes and in particular on their vitality highly limiting its applicability for recipients treated with special drugs and therapeutic antibodies or suffering from underlying autoimmune diseases. In the great majority of these cases ELISA-based crossmatching has been demonstrated to be an adequate alternative procedure nevertheless leading to valid results. With these case reports we show for the first time that ELISA-based crossmatching is suitable to demonstrate the upcoming donor-specific anti-HLA antibodies as a consequence of allografting using deep-frozen deceased donor's material such as blood or spleen detergent lysate. Thus, this ELISA-based procedure first provides the option to routinely perform crossmatching using stored material of deceased donors in order to substitute or at least to complement virtual crossmatching, that is, the comparison of the recipients' anti-HLA antibody specificities with the donors' historically identified HLA types. PMID:26634169

  19. Impact of a mixed bacterial lysate (OM-85 BV) on the immunogenicity, safety and tolerability of inactivated influenza vaccine in children with recurrent respiratory tract infection.

    PubMed

    Esposito, Susanna; Marchisio, Paola; Prada, Elisabetta; Daleno, Cristina; Porretti, Laura; Carsetti, Rita; Bosco, Annalisa; Ierardi, Valentina; Scala, Alessia; Principi, Nicola

    2014-05-01

    It is known that the immunogenicity and efficacy of conventional inactivated influenza vaccines (IIVs) are not completely satisfactory in children. The aim of this prospective, randomised, single-blind study was to compare the immune response to, and the effectiveness and safety of, an IIV (Fluarix, GlaxoSmithKline Biologicals, Rixensart, Belgium) administered to 68 children aged 36-59 months affected by recurrent respiratory tract infections (RRTIs) who were vaccinated with (n=33) or without (n=35) the mixed bacterial lysate OM-85 BV (Broncho-vaxom, Vifor Pharma, Geneva, Switzerland). OM-85 BV had no effect on seroconversion or seroprotection rates, geometric mean titres, or dendritic cells, which were not significantly different between the two groups. Moreover, OM-85 BV did not significantly increase the pool of the memory B cells that produce IgG and IgM antibodies against the influenza antigens. However, respiratory morbidity was significantly lower in the children treated with OM-85 BV (p<0.05), thus confirming its positive effect on the incidence of RRTIs. There was no difference in the incidence of adverse events between the two groups. These findings show that the immune response of children to influenza vaccine is not significantly influenced by the administration of OM-85 BV. However, the use of OM-85 before and at the same time as IIV seems to reduce respiratory morbidity, and seems to be safe and well tolerated. PMID:24681270

  20. [Structure and function of the T-lymphocyte antigen receptor and its role in infectious diseases].

    PubMed

    Peralta Zaragoza, O; Sánchez Magaña, T; Barrera Rodriguez, R; Madrid Marina, V

    1996-01-01

    The T lymphocytes recognize antigens through antigen receptors (TcRs) and the major histocompatibility complex (MHC) molecules: they lysate the cells that bear the antigen, or release cytokines that are mediators of the immune response. The TcRs recognize antigens in the form of short peptides bound to MHC molecules. So far, there are two isotypes of TcR: gamma/delta and alpha/beta, which appear in the sequence during T-cell ontogeny. The process of selection of TcRs during thymic ontogeny obeys to molecular mechanisms which generate intracellular events that will participate in the gene expression of the TcR. The aim of the present paper is to review the molecular, structural, and functional aspects of the TcRs, and their role in human autoimmune infectious disease. PMID:8815490

  1. Capillary electrophoresis chips for screening of endotoxin chemotypes from whole-cell lysates.

    PubMed

    Kilár, Anikó; Péterfi, Zoltán; Csorba, Eszter; Kilár, Ferenc; Kocsis, Béla

    2008-10-01

    A fast microchip electrophoresis method was developed to analyze and differentiate bacterial endotoxins directly from whole-cell lysates after removal of the proteinaceous components with proteinase K digestion and a precipitation of the endotoxin components. The partially purified endotoxin components were visualized by the interaction with dodecyl sulphate and then a fluorescent dye. The lipopolysaccharide (LPS) profiles can be directly evaluated from digested bacterial cells, and the electrophoresis patterns very closely resembled to those of pure LPSs, and the R and S chemotypes can be used to assign the strains. The method has been found to be useful in the screening of a large number of bacterial mutants and the structural characterization of endotoxins extracted only from 1 ml cultures. PMID:18692189

  2. Selective Protein Hyperpolarization in Cell Lysates Using Targeted Dynamic Nuclear Polarization.

    PubMed

    Viennet, Thibault; Viegas, Aldino; Kuepper, Arne; Arens, Sabine; Gelev, Vladimir; Petrov, Ognyan; Grossmann, Tom N; Heise, Henrike; Etzkorn, Manuel

    2016-08-26

    Nuclear magnetic resonance (NMR) spectroscopy has the intrinsic capabilities to investigate proteins in native environments. In general, however, NMR relies on non-natural protein purity and concentration to increase the desired signal over the background. We here report on the efficient and specific hyperpolarization of low amounts of a target protein in a large isotope-labeled background by combining dynamic nuclear polarization (DNP) and the selectivity of protein interactions. Using a biradical-labeled ligand, we were able to direct the hyperpolarization to the protein of interest, maintaining comparable signal enhancement with about 400-fold less radicals than conventionally used. We could selectively filter out our target protein directly from crude cell lysate obtained from only 8 mL of fully isotope-enriched cell culture. Our approach offers effective means to study proteins with atomic resolution in increasingly native concentrations and environments. PMID:27351143

  3. Platelet lysate gel and endothelial progenitors stimulate microvascular network formation in vitro: tissue engineering implications

    PubMed Central

    Fortunato, Tiago M.; Beltrami, Cristina; Emanueli, Costanza; De Bank, Paul A.; Pula, Giordano

    2016-01-01

    Revascularisation is a key step for tissue regeneration and complete organ engineering. We describe the generation of human platelet lysate gel (hPLG), an extracellular matrix preparation from human platelets able to support the proliferation of endothelial colony forming cells (ECFCs) in 2D cultures and the formation of a complete microvascular network in vitro in 3D cultures. Existing extracellular matrix preparations require addition of high concentrations of recombinant growth factors and allow only limited formation of capillary-like structures. Additional advantages of our approach over existing extracellular matrices are the absence of any animal product in the composition hPLG and the possibility of obtaining hPLG from patients to generate homologous scaffolds for re-implantation. This discovery has the potential to accelerate the development of regenerative medicine applications based on implantation of microvascular networks expanded ex vivo or the generation of fully vascularised organs. PMID:27141997

  4. Enhanced transcription rates in membrane-free protocells formed by coacervation of cell lysate.

    PubMed

    Sokolova, Ekaterina; Spruijt, Evan; Hansen, Maike M K; Dubuc, Emilien; Groen, Joost; Chokkalingam, Venkatachalam; Piruska, Aigars; Heus, Hans A; Huck, Wilhelm T S

    2013-07-16

    Liquid-liquid phase transitions in complex mixtures of proteins and other molecules produce crowded compartments supporting in vitro transcription and translation. We developed a method based on picoliter water-in-oil droplets to induce coacervation in Escherichia coli cell lysate and follow gene expression under crowded and noncrowded conditions. Coacervation creates an artificial cell-like environment in which the rate of mRNA production is increased significantly. Fits to the measured transcription rates show a two orders of magnitude larger binding constant between DNA and T7 RNA polymerase, and five to six times larger rate constant for transcription in crowded environments, strikingly similar to in vivo rates. The effect of crowding on interactions and kinetics of the fundamental machinery of gene expression has a direct impact on our understanding of biochemical networks in vivo. Moreover, our results show the intrinsic potential of cellular components to facilitate macromolecular organization into membrane-free compartments by phase separation. PMID:23818642

  5. Bacterial lysates and ribosomes as inducers of specific immune responses: a comparative study.

    PubMed

    Béné, M C; Kahl, L; Perruchet, A M; Hermes, H; Mösges, M; Normier, G; Binz, H; Faure, G C

    1993-11-01

    A bacterial lysate (OM-85 BV), a preparation of purified bacterial ribosomes (D53) and a placebo were tested for ability to induce the local appearance of specific antibody-containing cells. The three compounds were given orally to 90 children who required tonsillectomy. Surgery was carried out after 1 month of therapy. Frozen-cut sections of each tonsil were tested in indirect immunofluorescence. Cells containing antibodies directed to Streptococcus pneumoniae, Streptococcus pyogenes, Haemophilus influenzae or Klebsiella pneumoniae were enumerated. Lowest values were noted in the placebo group. Slightly higher numbers were observed after treatment with OM-85 BV, but significant increases were noted only for the elevated numbers of specific antibody-containing cells observed after D53 therapy. Bacterial ribosomal preparations thus contribute efficient induction of specific local immune responses in man. PMID:8235452

  6. Platelet lysate gel and endothelial progenitors stimulate microvascular network formation in vitro: tissue engineering implications.

    PubMed

    Fortunato, Tiago M; Beltrami, Cristina; Emanueli, Costanza; De Bank, Paul A; Pula, Giordano

    2016-01-01

    Revascularisation is a key step for tissue regeneration and complete organ engineering. We describe the generation of human platelet lysate gel (hPLG), an extracellular matrix preparation from human platelets able to support the proliferation of endothelial colony forming cells (ECFCs) in 2D cultures and the formation of a complete microvascular network in vitro in 3D cultures. Existing extracellular matrix preparations require addition of high concentrations of recombinant growth factors and allow only limited formation of capillary-like structures. Additional advantages of our approach over existing extracellular matrices are the absence of any animal product in the composition hPLG and the possibility of obtaining hPLG from patients to generate homologous scaffolds for re-implantation. This discovery has the potential to accelerate the development of regenerative medicine applications based on implantation of microvascular networks expanded ex vivo or the generation of fully vascularised organs. PMID:27141997

  7. Probing small-molecule microarrays with tagged proteins in cell lysates.

    PubMed

    Pop, Marius S; Wassaf, Dina; Koehler, Angela N

    2014-01-01

    The technique of small-molecule microarray (SMM) screening is based on the ability of small molecules to bind to various soluble proteins. This type of interaction is easily detected by the presence of a fluorescence signal produced by labeled antibodies that specifically recognize a unique sequence (tag) present on the target protein. The fluorescent signal intensity values are determined based on signal-to-noise ratios (SNRs). SMM screening is a high-throughput, unbiased method that can rapidly identify novel direct ligands for various protein targets. This binding-based assay format is generally applicable to most proteins, but it is especially useful for protein targets that do not possess an enzymatic activity. SMMs enable screening a protein in a purified form or in the context of a cellular lysate, likely providing a more physiologically relevant screening environment. PMID:25445177

  8. Cell-Free Production of Membrane Proteins in Escherichia coli Lysates for Functional and Structural Studies.

    PubMed

    Rues, Ralf-Bernhardt; Henrich, Erik; Boland, Coilin; Caffrey, Martin; Bernhard, Frank

    2016-01-01

    The complexity of membrane protein synthesis is largely reduced in cell-free systems and it results into high success rates of target expression. Protocols for the preparation of bacterial lysates have been optimized in order to ensure reliable efficiencies in membrane protein production that are even sufficient for structural applications. The open accessibility of the semisynthetic cell-free expression reactions allows to adjust membrane protein solubilization conditions according to the optimal folding requirements of individual targets. Two basic strategies will be exemplified. The post-translational solubilization of membrane proteins in detergent micelles is most straightforward for crystallization approaches. The co-translational integration of membrane proteins into preformed nanodiscs will enable their functional characterization in a variety of natural lipid environments. PMID:27485326

  9. Enhanced transcription rates in membrane-free protocells formed by coacervation of cell lysate

    PubMed Central

    Sokolova, Ekaterina; Spruijt, Evan; Hansen, Maike M. K.; Dubuc, Emilien; Groen, Joost; Chokkalingam, Venkatachalam; Piruska, Aigars; Heus, Hans A.; Huck, Wilhelm T. S.

    2013-01-01

    Liquid–liquid phase transitions in complex mixtures of proteins and other molecules produce crowded compartments supporting in vitro transcription and translation. We developed a method based on picoliter water-in-oil droplets to induce coacervation in Escherichia coli cell lysate and follow gene expression under crowded and noncrowded conditions. Coacervation creates an artificial cell-like environment in which the rate of mRNA production is increased significantly. Fits to the measured transcription rates show a two orders of magnitude larger binding constant between DNA and T7 RNA polymerase, and five to six times larger rate constant for transcription in crowded environments, strikingly similar to in vivo rates. The effect of crowding on interactions and kinetics of the fundamental machinery of gene expression has a direct impact on our understanding of biochemical networks in vivo. Moreover, our results show the intrinsic potential of cellular components to facilitate macromolecular organization into membrane-free compartments by phase separation. PMID:23818642

  10. Synthesis and Properties of Ortho-Nitro-Fe Complex

    SciTech Connect

    Mishra, A.; Mishra, Niyati; Sharma, R.; Jain, G.; Ninama, S.; Awate, R.

    2011-07-15

    Ortho-Nitro-Fe complex (Transition metal complex) has synthesized by chemical route method and properties of made complex has characterized by X-Ray diffraction (XRD), Moessbauer spectroscopy, Fourier transformation infra-red spectroscopy (FTIR) and X-Ray photoelectron spectroscopy (XPS). XRD analysis shows that sample is crystalline in nature and having particle size in the range of few nano meters. Moessbauer spectroscopy at room temperature shows the oxidation state of Iron (central metal ion) after complaxasion. FTIR spectra of the complex confirms the coordination of metal ion with ligand.

  11. Ortho stops marketing Lippes Loop; cites economic factors.

    PubMed

    1985-11-01

    Ortho Pharmaceutical Corporation has stopped marketing the Lippes Loop IUD, the only inert IUD currently available in the US. The firm cited "economic considerations" as its reason. Linda Organ, company spokeswoman, told Contraceptive Technology Update (CTU) that the number of women using IUDs has declined in the past few years and, as a result, Ortho's Lippes Loop sales dropped. Most physicians, according to Organ, currently prescribe copper-bearing IUDs. Few devices have been studied as thoroughly before marketing as the Lippes Loop, according to its developer, Dr. Jack Lippes. Lippes told CTU that the Population Council analyzed 40,000 women from 1962 to 1968 and "found no trouble with the Loop." Lippes attributes Ortho's recent decision to 2 factors: the IUD has been only "marginally profitable" and the problems of A.H. Robins with the Dalkon Shield has most likely had an effect; and the US Food and Drug Administration (FDA) published a proposed rule in August 1985 that would require any company wanting to manufacture and market IUDs like the Lippes Loop to submit a premarketing approval application to that agency. In effect, the FDA's rule would only apply to the Lippes Loop. Under the proposed rule, any company wanting to market Lippes Loops, or any nondrug IUD, would have to submit an application to the FDA with a detailed discussion and supporting clinical studies addressing the following concerns: pelvic actinomycosis; tubal infertility; duration that the IUD should remain in situ; and safety of leaving the IUD in situ when contraception is no longer indicated. According to Lillian Yin, FDA device evaluation, the clinical effectiveness and most of the safety issues regarding inert IUDs have been thoroughly covered in published data. She told CTU that "most of the information needed is straightforward, but the part that's new involves the long term use infection rate." Yin indicated that the FDA received a letter from Ortho advising the agency of the

  12. UCN Production With a Single Crystal of Ortho-Deuterium

    PubMed Central

    Utsuro, M.; Tanaka, M.; Mishima, K.; Nagai, Y.; Shima, T.; Fukuda, Y.; Kohmoto, T.; Momose, T.; Moriai, A.; Okumura, K.; Yoshino, H.

    2005-01-01

    The present paper reports on the preliminary experimental results concerning a new concept of ultracold neutron production with a single crystal converter of ortho-deuterium lying in the ground rotational state at the low temperature of about 10 K, which should make it possible to utilize a guided cold neutron beam instead of irradiating the converter material in the inside of high radiation fields. The successful observation of the clear Bragg scattering pattern from the single crystal converter and the reasonable results from the first experimental trial of the ultracold neutron production with the single crystal are shown. PMID:27308135

  13. UCN Production With a Single Crystal of Ortho-Deuterium.

    PubMed

    Utsuro, M; Tanaka, M; Mishima, K; Nagai, Y; Shima, T; Fukuda, Y; Kohmoto, T; Momose, T; Moriai, A; Okumura, K; Yoshino, H

    2005-01-01

    The present paper reports on the preliminary experimental results concerning a new concept of ultracold neutron production with a single crystal converter of ortho-deuterium lying in the ground rotational state at the low temperature of about 10 K, which should make it possible to utilize a guided cold neutron beam instead of irradiating the converter material in the inside of high radiation fields. The successful observation of the clear Bragg scattering pattern from the single crystal converter and the reasonable results from the first experimental trial of the ultracold neutron production with the single crystal are shown. PMID:27308135

  14. Cross-linking approach to affinity capture of protein complexes from chaotrope-solubilized cell lysates.

    PubMed

    Alloza, Iraide; Martens, Erik; Hawthorne, Susan; Vandenbroeck, Koen

    2004-01-01

    Affinity capture methods are widely used for isolation and analysis of protein complexes. Short peptide tags fused to the protein of interest normally facilitate straightforward purification and detection of interacting proteins. We investigated the suitability of applying C-terminally hexahistidine-tagged interleukin-12 (IL-12) alpha- and beta-chains as "bait" proteins for cocapturing novel binding partners using heterologous recombinant human embryonic kidney-293 (HEK-293) cell lines. The beta-chain, but not the alpha-chain, extracted from cell lysates was capable of binding to the Ni(2+)-nitrilotriacetic acid affinity resin under nondenaturing conditions. Retention of the alpha-chain on this matrix was dependent on treatment of cell lysates with high concentrations of chaotropes such as urea. Since under these conditions any noncovalent protein associations are destroyed, prior cross-linking of proteins interacting with the alpha-chain in intact cells was required. The use of the thiol-cleavable cross-linker 3,3'-dithiobis(succinimidyl proprionate) facilitated dissociation of alpha-chain-binding proteins by means of dithiothreitol following purification. Using this approach we were able to demonstrate a strong interaction between the endoplasmic reticulum chaperone calreticulin (CRT) and the IL-12 alpha-chain that was confirmed in a reciprocal anti-CRT immunoprecipitation assay. The assay presented here provides a simple approach to exposing concealed hexahistidine tags while retaining native noncovalent protein interactions and should be generally applicable in a range of pull-down or affinity capture methods aiming at analysis of protein complexes. PMID:14654056

  15. Natural assembly of platelet lysate-loaded nanocarriers into enriched 3D hydrogels for cartilage regeneration.

    PubMed

    Santo, Vítor E; Popa, Elena G; Mano, João F; Gomes, Manuela E; Reis, Rui L

    2015-06-01

    The role of Platelet Lysates (PLs) as a source of growth factors (GFs) and as main element of three-dimensional (3D) hydrogels has been previously described. However, the resulting hydrogels usually suffer from high degree of contraction, limiting their usefulness. This work describes the development of a stable biomimetic 3D hydrogel structure based on PLs, through the spontaneous assembling of a high concentration of chitosan-chondroitin sulfate nanoparticles (CH/CS NPs) with PLs loaded by adsorption. The interactions between the NPs and the lysates resemble the ones observed in the extracellular matrix (ECM) native environment between glycosaminoglycans and ECM proteins. In vitro release studies were carried out focusing on the quantification of PDGF-BB and TGF-β1 GFs. Human adipose derived stem cells (hASCs) were entrapped in these 3D hydrogels and cultured in vitro under chondrogenic stimulus, in order to assess their potential use for cartilage regeneration. Histological, immunohistological and gene expression analysis demonstrated that the PL-assembled constructs entrapping hASCs exhibited results similar to the positive control (hASCS cultured in pellets), concerning the levels of collagen II expression and immunolocalization of collagen type I and II and aggrecan. Moreover, the deposition of new cartilage ECM was detected by alcian blue and safranin-O positive stainings. This work demonstrates the potential of PLs to act simultaneously as a source/carrier of GFs and as a 3D structure of support, through the application of a "bottom-up" approach involving the assembly of NPs, resulting in an enriched construct for cartilage regeneration applications. PMID:25795623

  16. Blue native polyacrylamide gel electrophoresis (BN-PAGE) for analysis of multiprotein complexes from cellular lysates.

    PubMed

    Fiala, Gina J; Schamel, Wolfgang W A; Blumenthal, Britta

    2011-01-01

    Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Schägger and von Jagow 1991); (Schägger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes. PMID:21403626

  17. Excited-state properties of a triply ortho-metalated iridium(III) complex

    SciTech Connect

    King, K.A.; Spellane, P.J.; Watts, R.J.

    1985-03-06

    The characterization of the ground and luminescent excited states of a triply ortho-metalated complex of ppy, fac-Ir(ppy)/sub 3/ (ppy = 2-phenylpyridine) is effected. This complex, which is the first triply ortho-metalated ppy species to be characterized, is one of the strongest transition-metal photoreductants thus far reported. 20 references, 2 figures.

  18. Ortho-eugenol exhibits anti-nociceptive and anti-inflammatory activities.

    PubMed

    Fonsêca, Diogo V; Salgado, Paula R R; Aragão Neto, Humberto de C; Golzio, Adriana M F O; Caldas Filho, Marcelo R D; Melo, Cynthia G F; Leite, Fagner C; Piuvezam, Marcia R; Pordeus, Liana Clébia de Morais; Barbosa Filho, José M; Almeida, Reinaldo N

    2016-09-01

    Ortho-eugenol is a much used phenylpropanoid whose ability to reduce pain and inflammation has never been studied. Researching ortho-eugenol's antinociceptive and anti-inflammatory activity, and its possible mechanisms of action is therefore of interest. The administration of vehicle, ortho-eugenol (50, 75 and 100mg/kg i.p.), morphine (6mg/kg, i.p.) or dexamethasone (2mg/kg, s.c.) occurred 30min before the completion of pharmacological tests. Pretreatment with ortho-eugenol did not change motor coordination test results, but reduced the number of writhes and licking times in the writhing test and glutamate test, respectively. The reaction time from thermal stimulus was significantly increased in the hot plate test after administration of ortho-eugenol. Treatment with yohimbine reversed the antinociceptive effect of ortho-eugenol, suggesting involvement of the adrenergic system. In anti-inflammatory tests, ortho-eugenol inhibited acetic acid induced vascular permeability and leukocyte migration, reducing TNF-α and IL-1β by virtue of its suppression of NF-κB and p38 phosphorylated forms in the peritonitis test. From these results, ortho-eugenol antinociceptive effects mediated by the adrenergic system and anti-inflammatory activity through regulation of proinflammatory cytokines and phosphorylation of NF-kB and p38 become evident for the first time. PMID:27355133

  19. Surface-Controlled Mono/Diselective ortho C-H Bond Activation.

    PubMed

    Li, Qing; Yang, Biao; Lin, Haiping; Aghdassi, Nabi; Miao, Kangjian; Zhang, Junjie; Zhang, Haiming; Li, Youyong; Duhm, Steffen; Fan, Jian; Chi, Lifeng

    2016-03-01

    One of the most charming and challenging topics in organic chemistry is the selective C-H bond activation. The difficulty arises not only from the relatively large bond-dissociation enthalpy, but also from the poor reaction selectivity. In this work, Au(111) and Ag(111) surfaces were used to address ortho C-H functionalization and ortho-ortho couplings of phenol derivatives. More importantly, the competition between dehydrogenation and deoxygenation drove the diversity of reaction pathways of phenols on surfaces, that is, diselective ortho C-H bond activation on Au(111) surfaces and monoselective ortho C-H bond activation on Ag(111) surfaces. The mechanism of this unprecedented phenomenon was extensively explored by scanning tunneling microscopy, density function theory, and X-ray photoelectron spectroscopy. Our findings provide new pathways for surface-assisted organic synthesis via the mono/diselective C-H bond activation. PMID:26853936

  20. Construction of a Para-Ortho Hydrogen Test Cryostat

    NASA Astrophysics Data System (ADS)

    Essler, J.; Haberstroh, Ch.

    2010-04-01

    In a prospective hydrogen economy it is necessary to verify the para concentration of the employed hydrogen. In case of a short storage time of about a few days only it has been shown that a partial conversion into para-hydrogen gives an optimized overall efficiency. Hence, an easy and reliable method of measuring the para-hydrogen concentration is needed. In this paper, the concept and construction of a small test cryostat are described and first results are presented. The measuring principle is based on a catalytic induced adiabatic ortho-para conversion of a hydrogen gas flow starting from a known temperature. The operation of the system only requires a certain amount of liquid nitrogen as coolant. To determine the concentration of para-hydrogen it is only necessary to measure the temperature of the gas before and after the adiabatic catalyst cell. The measuring cryostat is used for further investigation of the spontaneous para-ortho conversion in the supercritical state. In addition, the design of the cryostat allows the investigation of different catalyst materials regarding the catalytic activity and possible degradation by using a known para concentration for the measurement.

  1. Structure and hydrogen bonding in ortho-hydroxy Ketimines

    NASA Astrophysics Data System (ADS)

    Filarowski, A.; Koll, A.; Głowiak, T.

    2003-01-01

    Two ortho-hydroxy Ketimines (2,2'-dihydroxybenzophenone- N-methyl-imine ( 1) and 2,2'-dihydroxy-4-methoxy-benzophenone- N-methyl-imine ( 2)) were synthesized with the hydrogen atom replaced in the azomethine group by the ortho-hydroxyphenyl substituent. The crystal structures were determined, which contain two types of hydrogen bonds; the intra-molecular O⋯N ( d(ON)=2.540 Å, d(ON)=2.502 Å for compound 1, d(ON)=2.559 Å for compound 2, and intermolecular O⋯O ( d(OO)=2.632 Å, d(OO)=2.582 Å for compound 1 and d(OO)=2.581 Å for compound 2. FT-IR spectra of compounds 1 and 2 in solid state as function of temperature were recorded. Relation between the intra-molecular and intermolecular hydrogen bonds was discussed. Influence of methoxy group substituted in phenol ring on the hydrogen bond properties has been investigated.

  2. Rotavirus antigen test

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003349.htm Rotavirus antigen test To use the sharing features on this page, please enable JavaScript. The rotavirus antigen test detects rotavirus in the feces. This ...

  3. Endotoxin detection--from limulus amebocyte lysate to recombinant factor C.

    PubMed

    Ding, Jeak Ling; Ho, Bow

    2010-01-01

    Gram negative bacterial endotoxin is a biological pyrogen that causes fever when introduced intravenously. The endotoxin, also known as lipopolysaccharide (LPS), is found in the outer membrane of Gram-negative bacteria. During Gram-negative sepsis, endotoxin stimulates host macrophages to release inflammatory cytokines. However, excessive inflammation causes multiple organ failure and death. Endotoxins, which are ubiquitous pathogenic molecules, are a bane to the pharmaceutical industry and healthcare community. Thus early and sensitive detection of endotoxin is crucial to prevent endotoxaemia. The limulus amebocyte lysate (LAL) has been widely used for ~30 years for the detection of endotoxin in the quality assurance of injectable drugs and medical devices. The LAL constitutes a cascade of serine proteases which are triggered by trace levels of endotoxin, culminating in a gel clot at the end of the reaction. The Factor C, which normally exists as a zymogen, is the primer of this coagulation cascade. In vivo, Factor C is the perfect biosensor, which alerts the horseshoe crab of the presence of a Gram-negative invader. The hemostatic end-point entraps the invader, killing it and limiting further infection. However, as an in vitro endotoxin detection tool, variations in the sensitivity and specificity of LAL to endotoxin, and the dwindling supply of horseshoe crabs are posing increasing challenges to the biotechnology industry. This has necessitated the innovation of an alternative test for endotoxin. Thus, Factor C became the obvious, albeit tricky target for the recombinant technology effort. This chapter documents the backwater of mining the natural blood lysate of the endangered species to the monumental effort of genetic engineering, to produce recombinant Factor C (rFC). The rFC is a 132 kDa molecule, which was produced as a proenzyme inducible by the presence of trace levels of endotoxin. The rFC forms the basis of the "PyroGene" kit, which is a novel micro

  4. Measurement of ortho-positronium properties in liquid scintillators

    NASA Astrophysics Data System (ADS)

    Perasso, S.; Consolati, G.; Franco, D.; Hans, S.; Jollet, C.; Meregaglia, A.; Tonazzo, A.; Yeh, M.

    2013-08-01

    Pulse shape discrimination in liquid scintillator detectors is a well-established technique for the discrimination of heavy particles from light particles. Nonetheless, it is not efficient in the separation of electrons and positrons, as they give rise to indistinguishable scintillator responses. This inefficiency can be overtaken through the exploitation of the formation of ortho-Positronium (o-Ps), which alters the time profile of light pulses induced by positrons. We characterized the o-Ps properties in the most commonly used liquid scintillators, i.e. PC, PXE, LAB, OIL and PC + PPO. In addition, we studied the effects of scintillator doping on the o-Ps properties for dopants currently used in neutrino experiments, Gd and Nd. Further measurements for Li-loaded and Tl-loaded liquid scintillators are foreseen. We found that the o-Ps properties are suitable for enhancing the electron-positron discrimination.

  5. Photophysical investigation of palladium(II) ortho-metalated complexes

    SciTech Connect

    Craig, C.A.; Watts, R.J. )

    1989-01-25

    Syntheses and structural characterizations of four complexes of Pd(II) with ortho-metalated 2-phenylpyridinate (ppy{sup minus}) are reported. These complexes include a parent dimer, (Pd(ppy)Cl){sub 2}, and three derivative monomers, (Pd(ppy)(bpy))Cl, (Pd(ppy)(en))Cl, and (Pd(ppy)(CO)Cl), whereby = 2,2'-bipyridine and en = ethylenediamine. Photophysical characterizations of these species indicate low-energy absorption bands ({approximately}360 nm) and emission bands at 77 K ({approximately}460 nm) that are assigned to transitions located on the ppy{sup {minus}} ligand. Some evidence for low-energy charge-transfer states is found in trends in luminescence lifetimes. 2 tabs., 5 figs., 21 refs.

  6. Measurement of ortho-positronium properties in liquid scintillators

    SciTech Connect

    Perasso, S.; Franco, D.; Tonazzo, A.; Consolati, G.; Hans, S.; Yeh, M.; Jollet, C.; Meregaglia, A.

    2013-08-08

    Pulse shape discrimination in liquid scintillator detectors is a well-established technique for the discrimination of heavy particles from light particles. Nonetheless, it is not efficient in the separation of electrons and positrons, as they give rise to indistinguishable scintillator responses. This inefficiency can be overtaken through the exploitation of the formation of ortho-Positronium (o-Ps), which alters the time profile of light pulses induced by positrons. We characterized the o-Ps properties in the most commonly used liquid scintillators, i.e. PC, PXE, LAB, OIL and PC + PPO. In addition, we studied the effects of scintillator doping on the o-Ps properties for dopants currently used in neutrino experiments, Gd and Nd. Further measurements for Li-loaded and Tl-loaded liquid scintillators are foreseen. We found that the o-Ps properties are suitable for enhancing the electron-positron discrimination.

  7. Culture of human cell lines by a pathogen-inactivated human platelet lysate.

    PubMed

    Fazzina, R; Iudicone, P; Mariotti, A; Fioravanti, D; Procoli, A; Cicchetti, E; Scambia, G; Bonanno, G; Pierelli, L

    2016-08-01

    Alternatives to the use of fetal bovine serum (FBS) have been investigated to ensure xeno-free growth condition. In this study we evaluated the efficacy of human platelet lysate (PL) as a substitute of FBS for the in vitro culture of some human cell lines. PL was obtained by pools of pathogen inactivated human donor platelet (PLT) concentrates. Human leukemia cell lines (KG-1, K562, JURKAT, HL-60) and epithelial tumor cell lines (HeLa and MCF-7) were cultured with either FBS or PL. Changes in cell proliferation, viability, morphology, surface markers and cell cycle were evaluated for each cell line. Functional characteristics were analysed by drug sensitivity test and cytotoxicity assay. Our results demonstrated that PL can support growth and expansion of all cell lines, although the cells cultured in presence of PL experienced a less massive proliferation compared to those grown with FBS. We found a comparable percentage of viable specific marker-expressing cells in both conditions, confirming lineage fidelity in all cultures. Functionality assays showed that cells in both FBS- and PL-supported cultures maintained their normal responsiveness to adriamycin and NK cell-mediated lysis. Our findings indicate that PL is a feasible serum substitute for supporting growth and propagation of haematopoietic and epithelial cell lines with many advantages from a perspective of process standardization, ethicality and product safety. PMID:25944665

  8. Interference of silica nanoparticles with the traditional Limulus amebocyte lysate gel clot assay.

    PubMed

    Kucki, Melanie; Cavelius, Christian; Kraegeloh, Annette

    2014-04-01

    Endotoxin contaminations of engineered nanomaterials can be responsible for observed biological responses, especially for misleading results in in vitro test systems, as well as in vivo studies. Therefore, endotoxin testing of nanomaterials is necessary to benchmark their influence on cells. Here, we tested the traditional Limulus amebocyte lysate gel clot assay for the detection of endotoxins in nanoparticle suspensions with a focus on possible interference of the particles with the test system. We systematically investigated the effects of nanomaterials made of, or covered by, the same material. Different types of bare or PEGylated silica nanoparticles, as well as iron oxide-silica core shell nanoparticles, were tested. Detailed inhibition/enhancement controls revealed enhanced activity in the Limulus coagulation cascade for all particles with bare silica surface. In comparison, PEGylation led to a lower degree of enhancement. These results indicate that the protein-particle interactions are the basis for the observed inhibition and enhancement effects. The enhancement activity of a particle type was positively related to the calculated particle surface area. For most silica particles tested, a dilution of the sample within the maximum valid dilution was sufficient to overcome non-valid enhancement, enabling semi-quantification of the endotoxin contamination. PMID:23884096

  9. Layer-by-layer assembled cell instructive nanocoatings containing platelet lysate.

    PubMed

    Oliveira, Sara M; Santo, Vítor E; Gomes, Manuela E; Reis, Rui L; Mano, João F

    2015-04-01

    Great efforts have been made to introduce growth factors (GFs) onto 2D/3D constructs in order to control cell behavior. Platelet lysate (PL) presents itself as a cost-effective source of multiple GFs and other proteins. The instruction given by a construct-PL combination will depend on how its instructive cues are presented to the cells. The content, stability and conformation of the GFs affect their instruction. Strategies for a controlled incorporation of PL are needed. Herein, PL was incorporated into nanocoatings by layer-by-layer assembling with polysaccharides presenting different sulfation degrees (SD) and charges. Heparin and several marine polysaccharides were tested to evaluate their PL and GF incorporation capability. The consequent effects of those multilayers on human adipose derived stem cells (hASCs) were assessed in short-term cultures. Both nature of the polysaccharide and SD were important properties that influenced the adsorption of PL, vascular endothelial growth factor (VEGF), fibroblast growth factor b (FGFb) and platelet derived growth factor (PDGF). The sulfated polysaccharides-PL multilayers showed to be efficient in the promotion of morphological changes, serum-free adhesion and proliferation of high passage hASCs (P > 5). These biomimetic multilayers promise to be versatile platforms to fabricate instructive devices allowing a tunable incorporation of PL. PMID:25701032

  10. Platelet Lysate-Modified Porous Silicon Microparticles for Enhanced Cell Proliferation in Wound Healing Applications.

    PubMed

    Fontana, Flavia; Mori, Michela; Riva, Federica; Mäkilä, Ermei; Liu, Dongfei; Salonen, Jarno; Nicoletti, Giovanni; Hirvonen, Jouni; Caramella, Carla; Santos, Hélder A

    2016-01-13

    The new frontier in the treatment of chronic nonhealing wounds is the use of micro- and nanoparticles to deliver drugs or growth factors into the wound. Here, we used platelet lysate (PL), a hemoderivative of platelets, consisting of a multifactorial cocktail of growth factors, to modify porous silicon (PSi) microparticles and assessed both in vitro and ex vivo the properties of the developed microsystem. PL-modified PSi was assessed for its potential to induce proliferation of fibroblasts. The wound closure-promoting properties of the microsystem were then assessed in an in vitro wound healing assay. Finally, the PL-modified PSi microparticles were evaluated in an ex vivo experiment over human skin. It was shown that PL-modified PSi microparticles were cytocompatible and enhanced the cell proliferation in different experimental settings. In addition, this microsystem promoted the closure of the gap between the fibroblast cells in the wound healing assay, in periods of time comparable with the positive control, and induced a proliferation and regeneration process onto the human skin in an ex vivo experiment. Overall, our results show that PL-modified PSi microparticles are suitable microsystems for further development toward applications in the treatment of chronic nonhealing wounds. PMID:26652045

  11. Black cobra (Naja naja karachiensis) lysates exhibit broad-spectrum antimicrobial activities

    PubMed Central

    Sagheer, Mehwish; Siddiqui, Ruqaiyyah; Iqbal, Junaid; Khan, Naveed Ahmed

    2014-01-01

    It is hypothesized that animals living in polluted environments possess antimicrobials to counter pathogenic microbes. The fact that snakes feed on germ-infested rodents suggests that they encounter pathogenic microbes and likely possess antimicrobials. The venom is used only to paralyze the rodent, but the ability of snakes to counter potential infections in the gut due to disease-ridden rodents requires robust action of the immune system against a broad range of pathogens. To test this hypothesis, crude lysates of different organs of Naja naja karachiensis (black cobra) were tested for antimicrobial properties. The antimicrobial activities of extracts were tested against selected bacterial pathogens (neuropathogenic Escherichia coli K1, methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Streptococcus pneumonia), protist (Acanthamoeba castellanii), and filamentous fungus (Fusarium solani). The findings revealed that plasma and various organ extracts of N. n. karachiensis exhibited antimicrobial activity against E. coli K1, MRSA, P. aeruginosa, S. pneumoniae, A. castellanii, and F. solani in a concentration-dependent manner. The results of this study are promising for the development of new antimicrobials. PMID:24625321

  12. Development of an electrochemical Limulus amebocyte lysate assay technique for portable and highly sensitive endotoxin sensor.

    PubMed

    Inoue, Kumi Y; Takahashi, Satoko; Ino, Kosuke; Shiku, Hitoshi; Matsue, Tomokazu

    2012-04-01

    Here, we report the development of an electrochemical detection method for endotoxin based on the Limulus amebocyte lysate (LAL) assay. A mixture of LAL reagent and endotoxin sample solution was incubated for 1 h. The endotoxin activated a cascade reaction of zymogens contained in the LAL to generate p-nitroaniline (pNA) which was then electrochemically detected by differential pulse voltammetry (DPV). The generated pNA gave a clear peak at -0.75 V vs. silver/silver chloride (Ag/AgCl), which increased with the concentration of endotoxin in the LAL assay solution. This DPV detection was performed using an electrode chip device fabricated from a diamond-like carbon-coated glass substrate. This chip device could detect as low as 10 endotoxin units l(-1) at room temperature within 1 h. This novel electrochemical method for the detection of endotoxin appears promising for the development of compact, low-cost and easy-to-use sensors for on-site monitoring of potentially contaminated medical supplies, including dialysis fluid, transplanted tissue and culture medium for assisted reproduction. PMID:21844129

  13. Active, soluble recombinant melittin purified by extracting insoluble lysate of Escherichia coli without denaturation

    PubMed Central

    Buhrman, Jason S.; Cook, Laura C.; Rayahin, Jamie E.; Federle, Michael J.; Gemeinhart, Richard A.

    2013-01-01

    Cell lytic peptides are a class of drugs that can be used to selectively kill invading organisms or diseased cells. Several of these peptides have been identified as potential therapeutics. Herein, we report a novel process for purifying recombinant melittin, a cell lytic peptide that inserts into the membranes of cells causing cell lysis, from Escherichia coli. The process involves surfactant and low pH to solubilize melittin fusion proteins from the insoluble fraction of bacterial lysates. We are able to significantly improve purity of the final product and confirm the activity of the peptide. The process yields recombinant melittin that is effective when used to treat U-87 MG glioma cells and inhibits growth of the Gram-positive pathogenic bacterium Streptococcus pyogenes. We demonstrate a method of repeated extraction of the insoluble protein fraction with mild detergent at a low pH that is able to generate a yield of pure, soluble melittin of approximately 0.5 to 1 mg/L of E. coli culture. PMID:23926061

  14. Gene transcript amplification from cell lysates in continuous-flow microfluidic devices.

    PubMed

    Gonzalez, Asensio; Ciobanu, Doina; Sayers, Michael; Sirr, Noel; Dalton, Tara; Davies, Mark

    2007-10-01

    Continuous-flow analysis, where samples circulate encapsulated in a carrier fluid is an attractive alternative to batch processing for high-throughput devices that use the polymerase chain reaction (PCR). Challenges of continuous-flow prototypes include the hydrodynamic and biological incompatibility of the carrier fluid, microchannel fouling, sample carryover and the integration of a nucleic acid extraction and reverse transcription step. We tested two homemade, continuous-flow thermocycler microdevices for amplification of reverse-transcribed messages from cell lysates without nucleic acid extraction. Amplification yield and specificity were assessed with state-of-the-art, real-time quantitative equipment. Carryover contamination between consecutive samples was absent. Amplification specificity and interference by genomic DNA were optimized by primer design. Robust detection of the low-copy transcript CLIC5 from 18 cells per microliter is demonstrated in cultured lymphoblasts. The results prove the concept that the development of micro-total analysis systems (micro-TAS) for continuous gene expression directly from cell suspensions is viable with current technology. PMID:17492382

  15. Platelet-Rich Fibrin Lysate Can Ameliorate Dysfunction of Chronically UVA-Irradiated Human Dermal Fibroblasts.

    PubMed

    Wirohadidjojo, Yohanes Widodo; Budiyanto, Arief; Soebono, Hardyanto

    2016-09-01

    To determine whether platelet-rich fibrin lysate (PRF-L) could restore the function of chronically ultraviolet-A (UVA)-irradiated human dermal fibroblasts (HDFs), we isolated and sub-cultured HDFs from six different human foreskins. HDFs were divided into two groups: those that received chronic UVA irradiation (total dosages of 10 J cm⁻²) and those that were not irradiated. We compared the proliferation rates, collagen deposition, and migration rates between the groups and between chronically UVA-irradiated HDFs in control and PRF-L-treated media. Our experiment showed that chronic UVA irradiation significantly decreased (p<0.05) the proliferation rates, migration rates, and collagen deposition of HDFs, compared to controls. Compared to control media, chronically UVA-irradiated HDFs in 50% PRF-L had significantly increased proliferation rates, migration rates, and collagen deposition (p<0.05), and the migration rates and collagen deposition of chronically UVA-irradiated HDFs in 50% PRF-L were equal to those of normal fibroblasts. Based on this experiment, we concluded that PRF-L is a good candidate material for treating UVA-induced photoaging of skin, although the best method for its clinical application remains to be determined. PMID:27401663

  16. Purification and preconcentration of genomic DNA from whole cell lysates using photoactivated polycarbonate (PPC) microfluidic chips

    PubMed Central

    Witek, Małgorzata A.; Llopis, Shawn D.; Wheatley, Abigail; McCarley, Robin L.; Soper, Steven A.

    2006-01-01

    We discuss the use of a photoactivated polycarbonate (PPC) microfluidic chip for the solid-phase, reversible immobilization (SPRI) and purification of genomic DNA (gDNA) from whole cell lysates. The surface of polycarbonate was activated by UV radiation resulting in a photo-oxidation reaction, which produced a channel surface containing carboxylate groups. The gDNA was selectively captured on this photoactivated surface in an immobilization buffer, which consisted of 3% polyethylene glycol, 0.4 M NaCl and 70% ethanol. The methodology reported herein is similar to conventional SPRI in that surface-confined carboxylate groups are used for the selective immobilization of DNA; however, no magnetic beads or a magnetic field are required. As observed by UV spectroscopy, a load of ∼7.6 ± 1.6 µg/ml of gDNA was immobilized onto the PPC bed. The recovery of DNA following purification was estimated to be 85 ± 5%. The immobilization and purification assay using this PPC microchip could be performed within ∼25 min as follows: (i) DNA immobilization ∼6 min, (ii) chip washout with ethanol 10 min, and (iii) drying and gDNA desorption ∼6 min. The PPC microchip could also be used for subsequent assays with no substantial loss in recovery, no observable carryover and no need for ‘reactivation’ of the PC surface with UV light. PMID:16757572

  17. Modification of the Limulus amebocyte lysate assay for the analysis of glucan in indoor environments.

    PubMed

    Foto, Mark; Plett, Jonathan; Berghout, Joanne; Miller, J David

    2004-05-01

    beta-1,3- D-Glucan is a biologically active component mainly from fungi that has been shown in several studies to be related to respiratory health outcomes from damp building exposures. Here, we report the development and application of a method for the analysis of the glucan extracted in 0.5 N NaOH solution making use of an available preparation of Limulus amebocyte lysate (LAL). The method yields reproducible beta-1,3- D-glucan measurements from samples of outdoor air, yeast cells, fungal spore preparations and ragweed pollen, and is more sensitive than competing measurements. The LAL-based measurement compared favourably to that based on size-exclusion chromatography using UV and refractive index detection. Growth conditions of the fungi did not materially change the concentrations of glucan in spores indicating that this is a stable property. Glucan content was proportional to spore surface area; however, some species contain higher relative spore glucan contents. PMID:15042272

  18. Identification of Yeast V-ATPase Mutants by Western Blots Analysis of Whole Cell Lysates

    NASA Astrophysics Data System (ADS)

    Parra-Belky, Karlett

    2002-11-01

    A biochemistry laboratory was designed for an undergraduate course to help students better understand the link between molecular engineering and biochemistry. Students identified unknown yeast strains with high specificity using SDS-PAGE and Western blot analysis of whole cell lysates. This problem-solving exercise is a common application of biochemistry in biotechnology research. Three different strains were used: a wild-type and two mutants for the proton pump vacuolar ATPase (V-ATPase). V-ATPases are multisubunit enzymes and the mutants used were deletion mutants; each lacked one structural gene of the complex. After three, three-hour labs, mutant strains were easily identified by the students and distinguished from wild-type cells analyzing the pattern of SDS-PAGE distribution of proteins. Identifying different subunits of one multimeric protein allowed for discussion of the structure and function of this metabolic enzyme, which captured the interest of the students. The experiment can be adapted to other multimeric protein complexes and shows improvement of the described methodology over previous reports, perhaps because the problem and its solution are representative of the type of techniques currently used in research labs.

  19. Platelet lysate as replacement for fetal bovine serum in mesenchymal stromal cell cultures.

    PubMed

    Bieback, Karen

    2013-10-01

    Mesenchymal stromal cells (MSC) emerged as highly attractive in cell-based regenerative medicine. Initially thought to provide cells capable of differentiation towards mesenchymal cell types (osteoblasts, chondrocytes, adipocytes etc.), by and by potent immunoregulatory and pro-regenerative activities have been discovered, broadening the field of potential applications from bone and cartilage regeneration to wound healing and treatment of autoimmune diseases. Due to the limited frequency in most tissue sources, ex vivo expansion of MSC is required compliant with good manufacturing practice (GMP) guidelines to yield clinically relevant cell doses. Though, still most manufacturing protocols use fetal bovine serum (FBS) as cell culture supplement to isolate and to expand MSC. However, the high lot-to-lot variability as well as risk of contamination and immunization call for xenogenic-free culture conditions. In terms of standardization, chemically defined media appear as the ultimate achievement. Since these media need to maintain all key cellular and therapy-relevant features of MSC, the development of chemically defined media is still - albeit highly investigated - only in its beginning. The current alternatives to FBS rely on human blood-derived components: plasma, serum, umbilical cord blood serum, and platelet derivatives like platelet lysate. Focusing on quality aspects, the latter will be addressed within this review. PMID:24273486

  20. Measurement of the formaldehyde ortho to para ratio in three molecular clouds

    NASA Technical Reports Server (NTRS)

    Kahane, C.; Lucas, R.; Frerking, M. A.; Langer, W. D.; Encrenaz, P.

    1984-01-01

    Observations of ortho and para H2CO in two types of clouds, a warm cloud (Orion A) and two cold clouds (L183 and TMC1), are presented. The ortho to para ratio in Orion deduced from the H2(C-13)O data is about three, while that for TMC1 is about one and that for L183 is 1-2. The former value is in agreement with the value calculated from chemical models of ortho and para H2CO production. The values for the cold clouds are consistent with thermal equilibrium at a temperature slightly smaller than 10 K.

  1. Quantum rotation of ortho and para-water encapsulated in a fullerene cage

    PubMed Central

    Beduz, Carlo; Carravetta, Marina; Chen, Judy Y.-C.; Concistrè, Maria; Denning, Mark; Frunzi, Michael; Horsewill, Anthony J.; Johannessen, Ole G.; Lawler, Ronald; Lei, Xuegong; Levitt, Malcolm H.; Li, Yongjun; Mamone, Salvatore; Murata, Yasujiro; Nagel, Urmas; Nishida, Tomoko; Ollivier, Jacques; Rols, Stéphane; Rõõm, Toomas; Sarkar, Riddhiman; Turro, Nicholas J.; Yang, Yifeng

    2012-01-01

    Inelastic neutron scattering, far-infrared spectroscopy, and cryogenic nuclear magnetic resonance are used to investigate the quantized rotation and ortho–para conversion of single water molecules trapped inside closed fullerene cages. The existence of metastable ortho-water molecules is demonstrated, and the interconversion of ortho-and para-water spin isomers is tracked in real time. Our investigation reveals that the ground state of encapsulated ortho water has a lifted degeneracy, associated with symmetry-breaking of the water environment. PMID:22837402

  2. Grain and Bean Lysates Improve Function of Endothelial Progenitor Cells from Human Peripheral Blood: Involvement of the Endogenous Antioxidant Defenses

    PubMed Central

    Lucchesi, Daniela; Russo, Rossella; Gabriele, Morena; Longo, Vincenzo; Del Prato, Stefano; Penno, Giuseppe; Pucci, Laura

    2014-01-01

    Increased oxidative stress contributes to the functional impairment of endothelial progenitor cells (EPCs), the pivotal players in the servicing of the endothelial cell lining. Several evidences suggest that decreasing oxidative stress by natural compounds with antioxidant properties may improve EPCs bioactivity. Here, we investigated the effects of Lisosan G (LG), a Triticum Sativum grain powder, and Lady Joy (LJ), a bean lysate, on function of EPCs exposed to oxidative stress. Peripheral blood mononuclear cells were isolated and plated on fibronectin-coated culture dishes; adherent cells, identified as early EPCs, were pre-treated with different concentrations of LG and LJ and incubated with hydrogen peroxide (H2O2). Viability, senescence, adhesion, ROS production and antioxidant enzymes gene expression were evaluated. Lysate-mediated Nrf-2 (nuclear factor (erythroid-derived 2)-like 2)/ARE (antioxidant response element) activation, a modulator of oxidative stress, was assessed by immunocytochemistry. Lady Joy 0.35–0.7 mg/ml increases EPCs viability; pre-treatment with either LG 0.7 mg/ml and LJ 0.35–0.7 mg/ml protect EPCs viability against H2O2-induced injury. LG 0.7 and LJ 0.35–0.7 mg/ml improve EPCs adhesion; pre-treatment with either LG 0.35 and 0.7 mg/ml or LJ 0.35, 0.7 and 1.4 mg/ml preserve adhesiveness of EPCs exposed to H2O2. Senescence is attenuated in EPCs incubated with lysates 0.35 mg/ml. After exposure to H2O2, LG pre-treated cells show a lower senescence than untreated EPCs. Lysates significantly decrease H2O2-induced ROS generation. Both lysates increase glutathione peroxidase-1 and superoxide dismutase-2 (SOD-2) expression; upon H2O2 exposure, pre-treatment with LJ allows higher SOD-2 expression. Heme oxigenase-1 increases in EPCs pre-treated with LG even upon H2O2 exposure. Finally, incubation with LG 0.7 mg/ml results in Nrf-2 translocation into the nucleus both at baseline and after the oxidative challenge. Our data suggest a protective

  3. Changes in the community structure of free-living heterotrophic bacteria in the open tropical Pacific Ocean in response to microalgal lysate-derived dissolved organic matter.

    PubMed

    Tada, Yuya; Suzuki, Koji

    2016-07-01

    Dissolved organic matter derived from phytoplankton (DOMP) can affect the bacterial biomass and community structure in aquatic ecosystems. Here, we examined the community response of free-living heterotrophic bacteria, with respect to cellular nucleic acid levels, to the DOMP lysates derived from three phytoplankton strains in the open tropical Pacific. The free amino acid (FAA) composition of each DOMP lysate differed among the microalgal strains. Terminal restriction fragment-length polymorphism analyses with 16S rRNA genes revealed that the community shifts of high nucleic acid (HNA) and low nucleic acid (LNA) bacteria varied significantly with the different DOMP lysate treatments. Furthermore, the FAA composition in DOMP lysates significantly affected the bacterial community shifts in HNA and LNA. Similarity percentage analysis using 16S rRNA gene deep-sequencing revealed that the DOMP lysates from the pelagophyte Pelagomonas calceolata caused relatively large community shifts with Alcaligenes predominating in the HNA fraction. In contrast, the DOMP lysate from the diatom Thalassiosira oceanica induced a community shift in the LNA fraction with a predominance of uncultured Actinobacteria Thus, the data indicate that the DOMP lysates from different microalgae constitute a primary factor altering the dominant bacterial groups in the open ocean. PMID:27162185

  4. Use of replication restricted recombinant vesicular stomatitis virus vectors for detection of antigen-specific T cells.

    PubMed

    Moseley, Nelson B; Laur, Oskar; Ibegbu, Chris C; Loria, Gilbert D; Ikwuenzunma, Gini; Jayakar, Himangi R; Whitt, Michael A; Altman, John D

    2012-01-31

    Detection of antigen-specific T cells at the single-cell level by ELISpot or flow cytometry techniques employing intracellular cytokine staining (ICS) is now an indispensable tool in many areas of immunology. When precisely mapped, optimal MHC-binding peptide epitopes are unknown, these assays use antigen in a variety of forms, including recombinant proteins, overlapping peptide sets representing one or more target protein sequences, microbial lysates, lysates of microbially-infected cells, or gene delivery vectors such as DNA expression plasmids or recombinant vaccinia or adenoviruses expressing a target protein of interest. Here we introduce replication-restricted, recombinant vesicular stomatitis virus (VSV) vectors as a safe, easy to produce, simple to use, and highly effective vector for genetic antigen delivery for the detection of human antigen-specific helper and cytotoxic T cells. To demonstrate the broad applicability of this approach, we have used these vectors to detect human T cell responses to the immunodominant pp65 antigen of human cytomegalovirus, individual segments of the yellow fever virus polyprotein, and to various influenza proteins. PMID:22004852

  5. Lysate of engineered Escherichia coli supports high-level conversion of glucose to 2,3-butanediol.

    PubMed

    Kay, Jennifer E; Jewett, Michael C

    2015-11-01

    Cell-free metabolic engineering (CFME) is emerging as a powerful approach for the production of target molecules and pathway debugging. Unfortunately, high cofactor costs, limited cofactor and energy regeneration, and low volumetric productivities hamper the widespread use and practical implementation of CFME technology. To address these challenges, we have developed a cell-free system that harnesses ensembles of catalytic proteins prepared from crude lysates, or extracts, of cells to fuel highly active heterologous metabolic conversions. As a model pathway, we selected conversion of glucose to 2,3-butanediol (2,3-BD), a medium level commodity chemical with many industrial applications. Specifically, we engineered a single strain of Escherichia coli to express three pathway enzymes necessary to make meso-2,3-BD (m2,3-BD). We then demonstrated that lysates from this strain, with addition of glucose and catalytic amounts of cofactors NAD+ and ATP, can produce m2,3-BD. Endogenous glycolytic enzymes convert glucose to pyruvate, the starting intermediate for m2,3-BD synthesis. Strikingly, with no strain optimization, we observed a maximal synthesis rate of m2,3-BD of 11.3 ± 0.1 g/L/h with a theoretical yield of 71% (0.36 g m2,3-BD/g glucose) in batch reactions. Titers reached 82 ± 8 g/L m2,3-BD in a 30 h fed-batch reaction. Our results highlight the ability for high-level co-factor regeneration in cell-free lysates. Further, they suggest exciting opportunities to use lysate-based systems to rapidly prototype metabolic pathways and carry out molecular transformations when bioconversion yields (g product/L), productivities (g product/L/h), or cellular toxicity limit commercial feasibility of whole-cell fermentation. PMID:26428449

  6. Relative potencies of four reference endotoxin standards as measured by the Limulus amoebocyte lysate and USP rabbit pyrogen tests.

    PubMed Central

    Weary, M E; Donohue, G; Pearson, F C; Story, K

    1980-01-01

    Four commonly used reference endotoxin standards, Escherichia coli O113:H10:K0, E. coli O55:B5, Salmonella abortusequi, and Shigella dysenteriae were compared by the USP rabbit pyrogen and the Limulus amoebocyte lysate tests. By the rabbit pyrogen test, S. abortus equi was identified as the most potent endotoxin, followed closely by E. coli O113:H10:K0 and E. coli O55:B5. PMID:7006505

  7. Rapid and selective extraction, isolation, preconcentration, and quantitation of small RNAs from cell lysate using on-chip isotachophoresis.

    PubMed

    Schoch, Reto B; Ronaghi, Mostafa; Santiago, Juan G

    2009-08-01

    We present a technique which enables the separation of small RNAs-such as microRNAs (miRNAs), short interfering RNAs (siRNAs), and Piwi-interacting RNAs (piRNAs)-from >or=66 nucleotide RNAs and other biomolecules contained in a cell lysate. In particular, the method achieves separation of small RNAs from precursor miRNAs (pre-miRNAs) in less than 3 min. We use on-chip isotachophoresis (ITP) for the simultaneous extraction, isolation, preconcentration and quantitation of small RNAs (approximately 22 nucleotides) and employ the high-efficiency sieving matrix Pluronic F-127; a thermo-responsive triblock copolymer which allows convenient microchannel loading at low temperature. We present the isolation of small RNAs from the lysate of 293A human kidney cells, and quantitate the number of short RNA molecules per cell to be 2.9x10(7). We estimate this quantity is an aggregate of roughly 500 types of short RNA molecules per 293A cell. Currently, the minimal cell number for small RNA extraction and detection with our method is approximately 900 (from a 5 microL sample volume), and we believe that small RNA analysis from tens of cells is realizable. Techniques for rapid and sensitive extraction and isolation of small RNAs from cell lysate are much-needed to further uncover their full range and functionality, including RNA interference studies. PMID:19606290

  8. Oral application of bacterial lysate in infancy diminishes the prevalence of atopic dermatitis in children at risk for atopy.

    PubMed

    Lau, S

    2014-06-01

    Numerous interventions such as avoidance of food allergens, prolonged breast feeding and supplementation of pro-and/or prebiotics have been tried as primary prevention of atopic dermatitis. Recent data suggest that prevention of infantile eczema is possible in a subgroup of children by feeding bacterial lysates early in life. Bacterial lysates of Escherichia coli and Enterococcus faecalis were found to impair allergic immune responses in rats. An interventional trial in 606 infants at risk for atopy showed a reduction of atopic dermatitis at the end of the treatment phase (month 2 until month 7) of 50% in a subgroup of children with single heredity for atopy. This was even more pronounced in the group of children with paternal heredity for atopy. This effect was still seen at age 1 year. There was no effect on food sensitisation. In conclusion, an immune modulation in terms of prevention of atopic dermatitis in infancy if single atopic family history is present seems to be possible by feeding bacterial lysates early in life. PMID:23886978

  9. An optimized approach for enrichment of glycoproteins from cell culture lysates using native multi-lectin affinity chromatography.

    PubMed

    Lee, Ling Y; Hincapie, Marina; Packer, Nicolle; Baker, Mark S; Hancock, William S; Fanayan, Susan

    2012-09-01

    Lectins are capable of recognizing specific glycan structures and serve as invaluable tools for the separation of glycosylated proteins from nonglycosylated proteins in biological samples. We report on the optimization of native multi-lectin affinity chromatography, combining three lectins, namely, concanavalin A, jacalin, and wheat germ agglutinin for fractionation of cellular glycoproteins from MCF-7 breast cancer lysate. We evaluated several conditions for optimum recovery of total proteins and glycoproteins such as low pH and saccharide elution buffers, and the inclusion of detergents in binding and elution buffers. Optimum recovery was observed with overnight incubation of cell lysate with lectins at 4°C, and inclusion of detergent in binding and saccharide elution buffers. Total protein and bound recoveries were 80 and 9%, respectively. Importantly, we found that high saccharide strength elution buffers were not necessary to release bound glycoproteins. This study demonstrates that multi-lectin affinity chromatography can be extended to total cell lysate to investigate the cellular glycoproteome. PMID:22997032

  10. LPS-Activated Monocytes Are Unresponsive to T4 Phage and T4-Generated Escherichia coli Lysate

    PubMed Central

    Bocian, Katarzyna; Borysowski, Jan; Zarzycki, Michał; Wierzbicki, Piotr; Kłosowska, Danuta; Weber-Dąbrowska, Beata; Korczak-Kowalska, Grażyna; Górski, Andrzej

    2016-01-01

    A growing body of data shows that bacteriophages can interact with different kinds of immune cells. The objective of this study was to investigate whether T4 bacteriophage and T4-generated Escherichia coli lysate affect functions of monocytes, the key population of immune cells involved in antibacterial immunity. To that end, we evaluated how T4 and E. coli lysate influence the expression of main costimulatory molecules including CD40, CD80 and CD86, TLR2, TLR4 on monocytes, as well as the production of IL-6 and IL-12 in cultures of peripheral blood mononuclear cells (PBMCs). Separate experiments were performed on unactivated and LPS-activated PBMCs cultures. Both studied preparations significantly increased the percentage of CD14+CD16-CD40+ and CD14+CD16-CD80+ monocytes in unactivated PBMCs cultures, as well as the concentration of IL-6 and IL-12 in culture supernates. However, neither purified T4 nor E. coli lysate had any significant effect on monocytes in LPS-activated PBMCs cultures. We conclude that LPS-activated monocytes are unresponsive to phages and products of phage-induced lysis of bacteria. This study is highly relevant to phage therapy because it suggests that in patients with infections caused by Gram-negative bacteria the administration of phage preparations to patients and lysis of bacteria by phages are not likely to overly stimulate monocytes.

  11. Efficient ortho-para conversion of H2 on interstellar grain surfaces

    NASA Astrophysics Data System (ADS)

    Bron, Emeric; Le Petit, Franck; Le Bourlot, Jacques

    2016-04-01

    Context. Fast surface conversion between ortho- and para-H2 has been observed in laboratory studies, and it has been proposed that this mechanism plays a role in the control of the ortho-para ratio in the interstellar medium. Observations of rotational lines of H2 in photo-dissociation regions (PDRs) have indeed found significantly lower ortho-para ratios than expected at equilibrium. The mechanisms controlling the balance of the ortho-para ratio in the interstellar medium thus remain incompletely understood, while this ratio can affect the thermodynamical properties of the gas (equation of state, cooling function). Aims: We aim to build an accurate model of ortho-para conversion on dust surfaces based on the most recent experimental and theoretical results, and to validate it by comparison to observations of H2 rotational lines in PDRs. Methods: We propose a statistical model of ortho-para conversion on dust grains with fluctuating dust temperatures. It is based on a master equation approach. This computation is then coupled to full PDR models and compared to PDR observations. Results: We show that the observations of rotational H2 lines indicate a high conversion efficiency on dust grains and that this high efficiency can be accounted for if taking dust temperature fluctuations into account with our statistical model of surface conversion. Simpler models that neglect the dust temperature fluctuations do not reach the high efficiency deduced from the observations. Moreover, this high efficiency induced by dust temperature fluctuations is very insensitive to the values of the model's microphysical parameters. Conclusions: Ortho-para conversion on grains is thus an efficient mechanism in most astrophysical conditions and can play a significant role in controlling the ortho-para ratio.

  12. Engineering Enriched Microenvironments with Gradients of Platelet Lysate in Hydrogel Fibers.

    PubMed

    Santo, Vítor E; Babo, Pedro; Amador, Miguel; Correia, Cláudia; Cunha, Bárbara; Coutinho, Daniela F; Neves, Nuno M; Mano, João F; Reis, Rui L; Gomes, Manuela E

    2016-06-13

    Gradients of physical and chemical cues are characteristic of specific tissue microenvironments and contribute toward morphogenesis and tissue regeneration upon injury. Recent advances on microfluidics and hydrogel manipulation raised the possibility of generating biomimetic biomaterials enriched with bioactive factors and encapsulating cells following designs specifically tailored for a target application. The novelty of this work relies on the combination of methacrylated gellan gum (MeGG) with platelet lysate (PL), aiming to generate novel advanced 3D PL-enriched photo-cross-linkable hydrogels and overcoming the lack of adhesion sites provided by the native MeGG hydrogels. This combination takes advantage of the availability, enriched growth factor composition, and potential autologous application of PL while simultaneously preserving the ability provided by MeGG to tailor mechanical properties, protein release kinetics, and shape of the construct according to the desired goal. Incorporation of PL in the hydrogels significantly improved cellular adhesion and viability in the constructs. The use of microfluidic tools allowed the design of a fiber-like hydrogel incorporating a gradient of PL along the length of the fiber. These spatial protein gradients led to the viability and cell number gradients caused by maintenance of human umbilical vein endothelial cells (HUVECs) survival in the fibers toward the PL-enriched sections in comparison with the nonloaded MeGG sections of the fibers. Altogether, we propose a proof of concept strategy to design a PL gradient biomaterial with potential in tissue engineering approaches and analysis of cell-microenvironment interactions. PMID:27203709

  13. Purification of CD47-streptavidin fusion protein from bacterial lysate using biotin-agarose affinity chromatography.

    PubMed

    Salehi, Nasrin; Peng, Ching-An

    2016-07-01

    CD47 is a widely expressed transmembrane glycoprotein that modulates the activity of a plethora of immune cells via its extracellular domain. Therefore, CD47 plays important roles in the regulation of immune responses and may serve as targets for the development of immunotherapeutic agents. To make sure CD47 functionality is intact under the process of protein conjugation, CD47-streptavidin fusion protein was expressed and purified because it can easily bind to biotin-tagged materials via the unique biotin-streptavidin affinity. In this study, gene sequences of CD47 extracellular domain (CD47ECD) and core streptavidin (coreSA) with a total 834 bp were inserted into pET20b plasmid to construct recombinant plasmid encoding CD47-SA fusion gene. After bacteria transformation, the CD47-SA fusion protein was expressed by isopropyl-β-d-thiogalactopyranoside (IPTG) induction. The collected bacteria lysate was loaded on biotinylated agarose to proceed the purification of CD47-SA fusion protein. Due to the unexpected high affinity between biotin and coreSA, standard washing and elution approaches (e.g., varying pH, using biotin, and applying guanidine hydrochloride) reported for biotin-streptavidin affinity chromatography were not able to separate the target fusion protein. Instead, using low concentration of the non-ionic detergent Triton X-100 followed with alkaline buffer could efficiently weaken the binding between biotin and coreSA, thereby eluting out CD47-SA fusion protein from the biotin agarose column. The purified CD47-SA fusion protein was further characterized by molecular biology methods and its antiphagocytic functionality was confirmed by the phagocytosis assay. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:949-958, 2016. PMID:27110670

  14. Donor Age of Human Platelet Lysate Affects Proliferation and Differentiation of Mesenchymal Stem Cells

    PubMed Central

    Lohmann, Michael; Walenda, Gudrun; Hemeda, Hatim; Joussen, Sylvia; Drescher, Wolf; Jockenhoevel, Stefan; Hutschenreuter, Gabriele; Zenke, Martin; Wagner, Wolfgang

    2012-01-01

    The regenerative potential declines upon aging. This might be due to cell-intrinsic changes in stem and progenitor cells or to influences by the microenvironment. Mesenchymal stem cells (MSC) raise high hopes in regenerative medicine. They are usually culture expanded in media with fetal calf serum (FCS) or other serum supplements such as human platelet lysate (HPL). In this study, we have analyzed the impact of HPL-donor age on culture expansion. 31 single donor derived HPLs (25 to 57 years old) were simultaneously compared for culture of MSC. Proliferation of MSC did not reveal a clear association with platelet counts of HPL donors or growth factors concentrations (PDGF-AB, TGF-β1, bFGF, or IGF-1), but it was significantly higher with HPLs from younger donors (<35 years) as compared to older donors (>45 years). Furthermore, HPLs from older donors increased activity of senescence-associated beta-galactosidase (SA-βgal). HPL-donor age did not affect the fibroblastoid colony-forming unit (CFU-f) frequency, immunophenotype or induction of adipogenic differentiation, whereas osteogenic differentiation was significantly lower with HPLs from older donors. Concentrations of various growth factors (PDGF-AB, TGF-β1, bFGF, IGF-1) or hormones (estradiol, parathormone, leptin, 1,25 vitamin D3) were not associated with HPL-donor age or MSC growth. Taken together, our data support the notion that aging is associated with systemic feedback mechanisms acting on stem and progenitor cells, and this is also relevant for serum supplements in cell culture: HPLs derived from younger donors facilitate enhanced expansion and more pronounced osteogenic differentiation. PMID:22662236

  15. Use of platelet lysate for bone regeneration - are we ready for clinical translation?

    PubMed Central

    Altaie, Ala; Owston, Heather; Jones, Elena

    2016-01-01

    Current techniques to improve bone regeneration following trauma or tumour resection involve the use of autograft bone or its substitutes supplemented with osteoinductive growth factors and/or osteogenic cells such as mesenchymal stem cells (MSCs). Although MSCs are most commonly grown in media containing fetal calf serum, human platelet lysate (PL) offers an effective alternative. Bone marrow - derived MSCs grown in PL-containing media display faster proliferation whilst maintaining good osteogenic differentiation capacity. Limited pre-clinical investigations using PL-expanded MSCs seeded onto osteoconductive scaffolds indicate good potential of such constructs to repair bone in vivo. In an alternative approach, nude PL-coated scaffolds without seeded MSCs have been proposed as novel regenerative medicine devices. Even though methods to coat scaffolds with PL vary, in vitro studies suggest that PL allows for MSC adhesion, migration and differentiation inside these scaffolds. Increased new bone formation and vascularisation in comparison to uncoated scaffolds have also been observed in vivo. This review outlines the state-of-the-art research in the field of PL for ex vivo MSC expansion and in vivo bone regeneration. To minimise inconsistency between the studies, further work is required towards standardisation of PL preparation in terms of the starting material, platelet concentration, leukocyte depletion, and the method of platelet lysis. PL quality control procedures and its “potency” assessment are urgently needed, which could include measurements of key growth and attachment factors important for MSC maintenance and differentiation. Furthermore, different PL formulations could be tailor-made for specific bone repair indications. Such measures would undoubtedly speed up clinical translation of PL-based treatments for bone regeneration. PMID:26981170

  16. Evaluation of human platelet lysate versus fetal bovine serum for culture of mesenchymal stromal cells.

    PubMed

    Hemeda, Hatim; Giebel, Bernd; Wagner, Wolfgang

    2014-02-01

    Culture media for therapeutic cell preparations-such as mesenchymal stromal cells (MSCs)-usually comprise serum additives. Traditionally, fetal bovine serum is supplemented in basic research and in most clinical trials. Within the past years, many laboratories adapted their culture conditions to human platelet lysate (hPL), which further stimulates proliferation and expansion of MSCs. Particularly with regard to clinical application, human alternatives for fetal bovine serum are clearly to be preferred. hPL is generated from human platelet units by disruption of the platelet membrane, which is commonly performed by repeated freeze and thaw cycles. Such culture supplements are notoriously ill-defined, and many parameters contribute to batch-to-batch variation in hPL such as different amounts of plasma, a broad range of growth factors and donor-specific effects. The plasma components of hPL necessitate addition of anticoagulants such as heparins to prevent gelatinization of hPL medium, and their concentration must be standardized. Labels for description of hPL-such as "xenogen-free," "animal-free" and "serum free"-are not used consistently in the literature and may be misleading if not critically assessed. Further analysis of the precise composition of relevant growth factors, attachment factors, microRNAs and exosomes will pave the way for optimized and defined culture conditions. The use of hPL has several advantages and disadvantages: they must be taken into account because the choice of cell culture additive has major impact on cell preparations. PMID:24438898

  17. Synthesis of 9-Fluorenylidenes and 9,10-Phenanthrenes through Palladium-Catalyzed Aryne Annulation by ortho-Halostyrenes and ortho-Halo Allylic Benzenes

    PubMed Central

    Worlikar, Shilpa A.; Larock, Richard C.

    2009-01-01

    A number of functionally substituted 9-fluorenylidenes and 9,10-phenanthrenes have been synthesized from substituted ortho-halostyrenes and ortho-halo allylic benzenes respectively in good yields by the palladium-catalyzed annulation of arynes. The methodology tolerates a variety of functional groups, including cyano, ester, aldehyde and ketone groups, occurs under relatively mild reaction conditions, and involves the generation of two new carbon-carbon bonds, thus providing these important carbocyclic ring systems in a single synthetic step. PMID:19902957

  18. Facile synthesis of acid-labile polymers with pendent ortho esters.

    PubMed

    Cheng, Jing; Ji, Ran; Gao, Shi-Juan; Du, Fu-Sheng; Li, Zi-Chen

    2012-01-01

    This work presents a facile approach for preparation of acid-labile and biocompatible polymers with pendent cyclic ortho esters, which is based on the efficient and mild reactions between cyclic ketene acetal (CKA) and hydroxyl groups. Three CKAs, 2-ethylidene-1,3-dioxane (EDO), 2-ethylidene-1,3-dioxolane (EDL), and 2-ethylidene-4- methyl-1,3-dioxolane (EMD) were prepared from the corresponding cyclic vinyl acetals by catalytic isomerization of the double bond. The reaction of CKAs with different alcohols and diols was examined using trace of p-toluenesulfonic acid as a catalyst. For the monohydroxyl alcohols, cyclic ortho esters were formed by simple addition of the hydroxyl group toward CKAs with ethanol showing a much greater reactivity than iso-propanol. When 1,2- or 1,3-diols were used to react with the CKAs, we observed the isomerized cyclic ortho esters besides the simple addition products. Biocompatible polyols, that is, poly(2-hydroxyethyl acrylate) (PHEA) and poly(vinyl alcohol) (PVA) were then modified with CKAs, and the degree of substitution of the pendent ortho esters can be easily tuned by changing feed ratio. Both the small molecule ortho esters and the CKA-modified polymers demonstrate the pH-dependent hydrolysis profiles, which depend also on the chemical structure of the ortho esters as well as the polymer hydrophobicity. PMID:22176024

  19. Cloning, heterologous expression and antigenicity of a schistosome cercarial protease.

    PubMed

    Price, H P; Doenhoff, M J; Sayers, J R

    1997-05-01

    A gene coding for the 30 kDa Schistosoma mansoni cercarial protease was amplified using the polymerase chain reaction (PCR) from genomic DNA templates. Cloning and sequencing of several independent PCR clones revealed the presence of an intron additional to the one described in the original cloning of the gene. The 3 exons were cloned into expression vectors so that they could be expressed as separate glutathione-S-transferase (GST) translational fusions. Recombinant bacteria carrying these expression plasmids expressed the fusion proteins at high levels. Western blotting of bacterial lysates with sera raised against the native S. mansoni cercarial protease showed that all 3 exons were recognized. Thus we have produced recombinant bacteria capable of providing large amounts of an S. mansoni antigen for immunological studies and evaluation as a candidate vaccine. PMID:9149415

  20. Microbial transformation of bioactive compounds and production of ortho-dihydroxyisoflavones and glycitein from natural fermented soybean paste.

    PubMed

    Roh, Changhyun

    2014-01-01

    Recently, there has been a great deal of remarkable interest in finding bioactive compounds from nutritional foods to replace synthetic compounds. In particular, ortho-dihydroxyisoflavones and glycitein are of growing scientific interest owing to their attractive biological properties. In this study, 7,8-ortho-dihydroxyisoflavone, 6,7-ortho-dihydroxyisoflavone, 3',4'-ortho-dihydroxyisoflavone and 7,4'-dihydroxy-6-methoxyisoflavone were characterized using microorganism screened from soybean Doenjang. Three ortho-dihydroxyisoflavones and glycitein were structurally elucidated by 1H-NMR and GC-MS analysis. Furthermore, bacterial strains from soybean Doenjang with the capacity of biotransformation were screened. The bacterial strain, identified as Bacillus subtilis Roh-1, was shown to convert daidzein into ortho-dihydroxyisoflavones and glycitein. Thus, this study has, for the first time, demonstrated that a bacterial strain had a substrate specificity for multiple modifications of the bioactive compounds. PMID:25513748

  1. Transcutaneous antigen delivery system

    PubMed Central

    Lee, Mi-Young; Shin, Meong-Cheol; Yang, Victor C.

    2013-01-01

    Transcutaneous immunization refers to the topical application of antigens onto the epidermis. Transcutaneous immunization targeting the Langerhans cells of the skin has received much attention due to its safe, needle-free, and noninvasive antigen delivery. The skin has important immunological functions with unique roles for antigen-presenting cells such as epidermal Langerhans cells and dermal dendritic cells. In recent years, novel vaccine delivery strategies have continually been developed; however, transcutaneous immunization has not yet been fully exploited due to the penetration barrier represented by the stratum corneum, which inhibits the transport of antigens and adjuvants. Herein we review recent achievements in transcutaneous immunization, focusing on the various strategies for the enhancement of antigen delivery and vaccination efficacy. [BMB Reports 2013; 46(1): 17-24] PMID:23351379

  2. Measurement of ortho-positronium properties in liquid scintillators

    NASA Astrophysics Data System (ADS)

    Perasso, S.; Consolati, G.; Franco, D.; Jollet, C.; Meregaglia, A.; Tonazzo, A.; Yeh, M.

    2014-03-01

    Pulse shape discrimination is a well-established technique for background rejection in liquid scintillator detectors. It is particularly effective in separating heavy particles from light particles, but not in distinguishing electrons from positrons. This inefficiency can be overtaken by exploiting the formation of ortho-positronium (o-Ps), which alters the time profile of light pulses induced by positrons. We characterized the o-Ps properties in the most commonly used liquid scintillators, i.e. PC, PXE, LAB, OIL and PC + PPO. In addition, we studied the effects of scintillator doping on the o-Ps properties for dopants used in neutrino-less double beta decay experiments (Nd and Te) and in anti-neutrino and neutron detection (Gd and Li respectively). We found that the o-Ps properties are similar in all the tested scintillators, with a lifetime around 3 ns and a formation probability of about 50%. This result indicates that an o-Ps-enhanced pulse shape discrimination can be applied in liquid scintillator detectors for neutrino and anti-neutrino detection and for neutrino-less double beta decay search.

  3. Orthos, an alarm system for the ALICE DAQ operations

    NASA Astrophysics Data System (ADS)

    Chapeland, Sylvain; Carena, Franco; Carena, Wisla; Chibante Barroso, Vasco; Costa, Filippo; Denes, Ervin; Divia, Roberto; Fuchs, Ulrich; Grigore, Alexandru; Simonetti, Giuseppe; Soos, Csaba; Telesca, Adriana; Vande Vyvre, Pierre; von Haller, Barthelemy

    2012-12-01

    ALICE (A Large Ion Collider Experiment) is the heavy-ion detector studying the physics of strongly interacting matter and the quark-gluon plasma at the CERN LHC (Large Hadron Collider). The DAQ (Data Acquisition System) facilities handle the data flow from the detectors electronics up to the mass storage. The DAQ system is based on a large farm of commodity hardware consisting of more than 600 devices (Linux PCs, storage, network switches), and controls hundreds of distributed hardware and software components interacting together. This paper presents Orthos, the alarm system used to detect, log, report, and follow-up abnormal situations on the DAQ machines at the experimental area. The main objective of this package is to integrate alarm detection and notification mechanisms with a full-featured issues tracker, in order to prioritize, assign, and fix system failures optimally. This tool relies on a database repository with a logic engine, SQL interfaces to inject or query metrics, and dynamic web pages for user interaction. We describe the system architecture, the technologies used for the implementation, and the integration with existing monitoring tools.

  4. NIR spectra of vesuvianite a complex ortho-disilicate mineral

    NASA Astrophysics Data System (ADS)

    Paluszkiewicz, C.; Żabiński, W.

    1999-05-01

    Vesuvianite is an ortho-disilicate mineral of a very complex structure. Two structural forms of this mineral were distinguished: high-symmetry vesuvianite with long-range disordered structure and low-symmetry vesuvianite containing ordered domains. These crystallize at different temperatures and under different geological conditions. The infrared spectra of vesuvianites in the OH region were investigated in detail by Groat et al. (L.A. Groat, F.C. Hawthorne, T.S. Ercit, Canad. Miner. 32 (1994) 505). However these authors did not take into account the above mentioned discrimination of two genetic and structural varietes of this mineral. The infrared spectroscopic evidence of ordering in the vesuvianite structure was first reported by the present authors for the MIR region (W. Żabiński, C. Paluszkiewicz, Miner. Polon. 25 (1994) 51; C. Paluszkiewicz, W. Żabiński, Vibr. Spectr. 8 (1995) 315). In this paper particular attention was paid to the NIR region. They confirm well the existence of two structural forms of vesuvianite, differing in the position of overtones and/or combination bands. The differences in chemical composition of two vesuvianite varieties arising from their formation in different geological environments are discussed.

  5. Detection of peptidoglycan and endotoxin in dialysate, using silkworm larvae plasma and limulus amebocyte lysate methods.

    PubMed

    Tsuchida, K; Takemoto, Y; Yamagami, S; Edney, H; Niwa, M; Tsuchiya, M; Kishimoto, T; Shaldon, S

    1997-01-01

    Silkworm larvae plasma (SLP) reagent is activated by peptidoglycan (PG), a fragment of both the gram-positive and gram-negative bacterial cell wall, as well as beta-glucan (BG), a component of fungi. It is possible to measure contamination of gram-positive bacteria quantitatively by combining the conventional limulus amebocyte lysate (LAL) and PG measurement methods. Therefore, a more highly accurate analysis of dialysate can be made using both SLP and LAL methods to detect endotoxin (ET) and/or PG contamination. We studied the effects of contaminated dialysate on human peripheral blood mononuclear cells (PBMC) by producing various cytokines in vitro. Muramyl dipeptide (MDP) was used as the biologically active minimum constituent of PG. A total of 54 dialysate samples were obtained under sterile conditions from 4 sites: (1) reverse osmosis water unit; (2) proportioning unit; (3) multiple dialysate preparation console, and (4) personal dialysate preparation console, at 9 dialysis facilities. To detect bacterial contamination, the samples were measured with LAL(C), LAL(G) and SLP methods. PBMC were collected from 10 healthy controls and from 10 hemodialysis patients and cultured for 24 h with ET, MDP, ET + MDP and contaminated dialysate. IL-1 receptor antagonist (IL-1Ra), IL-1 beta and TNF-alpha in the culture medium supernatants were measured using the ELISA method. PG was not detected in dialysate from sites 1 or 2. However, dialysate from the inlet of the dialyzer at the bedside monitor of the central supply and personal console showed 4.1 +/- 6.1 ng/ml for site 3 (in 7 of 18 samples) and 3.3 +/- 4.6 ng/ml for site 4 (in 3 of 18 samples). Contamination by PG alone and complex contamination by PG and ET were also detected. Furthermore, IL-1Ra, IL-1 beta and TNF-alpha production by PBMC increased in accordance with the concentrations of MDP. Cytokine production was enhanced 5-10 times more where MDP and ET coexisted than where either MDP or ET existed alone, showing

  6. Trypanosoma vivax GM6 Antigen: A Candidate Antigen for Diagnosis of African Animal Trypanosomosis in Cattle

    PubMed Central

    Pillay, Davita; Izotte, Julien; Fikru, Regassa; Büscher, Philipe; Mucache, Hermogenes; Neves, Luis; Boulangé, Alain; Seck, Momar Talla; Bouyer, Jérémy; Napier, Grant B.; Chevtzoff, Cyrille; Coustou, Virginie; Baltz, Théo

    2013-01-01

    Background Diagnosis of African animal trypanosomosis is vital to controlling this severe disease which hampers development across 10 million km2 of Africa endemic to tsetse flies. Diagnosis at the point of treatment is currently dependent on parasite detection which is unreliable, and on clinical signs, which are common to several other prevalent bovine diseases. Methodology/Principle Findings the repeat sequence of the GM6 antigen of Trypanosoma vivax (TvGM6), a flagellar-associated protein, was analysed from several isolates of T. vivax and found to be almost identical despite the fact that T. vivax is known to have high genetic variation. The TvGM6 repeat was recombinantly expressed in E. coli and purified. An indirect ELISA for bovine sera based on this antigen was developed. The TvGM6 indirect ELISA had a sensitivity of 91.4% (95% CI: 91.3 to 91.6) in the period following 10 days post experimental infection with T. vivax, which decreased ten-fold to 9.1% (95% CI: 7.3 to 10.9) one month post treatment. With field sera from cattle infected with T. vivax from two locations in East and West Africa, 91.5% (95% CI: 83.2 to 99.5) sensitivity and 91.3% (95% CI: 78.9 to 93.1) specificity was obtained for the TvGM6 ELISA using the whole trypanosome lysate ELISA as a reference. For heterologous T. congolense field infections, the TvGM6 ELISA had a sensitivity of 85.1% (95% CI: 76.8 to 94.4). Conclusion/Significance this study is the first to analyse the GM6 antigen of T. vivax and the first to test the GM6 antigen on a large collection of sera from experimentally and naturally infected cattle. This study demonstrates that the TvGM6 is an excellent candidate antigen for the development of a point-of-treatment test for diagnosis of T. vivax, and to a lesser extent T. congolense, African animal trypanosomosis in cattle. PMID:24205263

  7. Distinct antigen recognition pattern during zoonotic visceral leishmaniasis in humans and dogs.

    PubMed

    Goto, Yasuyuki; Howard, Randall F; Bhatia, Ajay; Trigo, Joelma; Nakatani, Maria; Netto, Eduardo M; Reed, Steven G

    2009-03-23

    Leishmania infantum is a causative agent of endemic zoonotic visceral leishmaniasis (VL) in regions of South America and the Mediterranean. Dogs are the major reservoirs for L. infantum in these regions, and control of disease in dogs could have a significant impact on human disease. Although dogs share many symptoms of VL with humans as a result of L. infantum infection, they also show some unique clinical manifestations, which are often a combination of visceral and cutaneous leishmaniasis, suggesting different mechanisms of disease development in dogs and humans. Here, we compare antibody responses of dogs and humans with VL to various defined leishmanial antigens. Parasite lysate and K39, the two most commonly used antigens for serodiagnosis of VL, detected the highest levels of antibodies in both humans and dogs with VL, whereas the recognition patterns of these antigens were distinct between the hosts. Among other defined antigens tested, LmSTI1 and CPB detected higher levels of antibodies in dogs and humans, respectively. These results indicate there is a difference between humans and dogs in antigen recognition patterns during VL. We infer that different strategies may need to be used in development of vaccines and diagnostics for humans and for dogs. In addition, we show a correlation between antibody titers to several antigens and severity of clinical symptoms during canine VL. PMID:19059724

  8. [Construction and expression of a eukaryotic vector co-expressing immunodominant antigens of CFP10, ESAT6, Ag85A and Ag85B of Mycobacterium tuberculosis].

    PubMed

    Li, Wu; Deng, Guangcun; Liu, Xiaoming; Wang, Yujiong

    2014-02-01

    CFP10, ESAT6, Antigen 85A (Ag85A) and antigen 85B (Ag85B) are the key immunodominant antigens of Mycobacterium tuberculosis. In order to construct a eukaryotic vector able to co-express the four genes in one vector, we amplified the target gene fragments encoding the CFP10, ESAT6, Ag85A and Ag85B antigens and inserted them into the multicloning site of the shuttle plasmid vector pcDNA3.1 (+), of which the CFP10 and ESAT6 encoding genes were in frame fused with a linker encoding (Gly4Ser)3 residue, before the fused gene was inserted downstream of CMV promoter with a bovine growth hormone poly A(BGH pA) sequence at the 3'-end; Ag85A and Ag85B encoding genes were fused with a separation of internal ribosome entry site (IRES) sequence before the fused gene cassette was inserted downstream of RSV promoter with a BGH pA sequence at the 3'-end. The final plasmid containing all four genes was confirmed by sequence analysis and designated as pcDNA-CFP10-ESAT6-Ag85A-Ag85B (pcDNA-CEAB). In order to verify the ability of this construct to express target proteins, we then transfected the recombinant plasmid into Human embryonic kidney (HEK) 293T cells and harvested the cell lysates, and the cell lysates were then separated by SDS-PAGE and subjected to Western blot analysis 48 h after transfection. All four of the target proteins were detected in the cell lysates against the respective specific antibodies, suggesting that we have successfully constructed a eukaryotic vector co-expressing the four immunodominant antigens of Mycobacterium tuberculosis, which lay a foundation for the further study of the immunogenicity and protective activity of the four antigens. PMID:24941747

  9. Lysates of S. pyogenes serotype M49 induce pancreatic tumor growth delay by specific and unspecific antitumor immune responses.

    PubMed

    Linnebacher, Michael; Maletzki, Claudia; Emmrich, Jörg; Kreikemeyer, Bernd

    2008-10-01

    Treatment of pancreatic cancer by active unspecific bacterial immunotherapy is a promising new strategy. Recently, we showed that a single intratumoral injection of wildtype Streptococcus pyogenes M49 results in complete regression of pancreatic carcinoma in mice mediated both by unspecific cytotoxicity and by specific immune reactions against tumor cells. As for potential clinical use, conditioning and especially inactivation of bacteria would abolish the risk of systemic bacterial infections; we here explored the potential of a streptococcal lysate prepared by bacteriophage lysine to affect pancreatic carcinoma growth in vivo. Application of the lysate into established Panc02 tumors resulted in pronounced growth cessation accompanied by raises in levels of circulating monocytes, granulocytes, and natural killer cells. Detailed analysis of splenocyte subsets revealed lysate-induced transient increases in pre-B cells followed by raised levels of activated T cells. Moreover, blood levels of proinflammatory, T helper-1-type cytokines were significantly elevated. These systemic immunologic effects were accompanied by massive infiltrations of cytotoxic T cells into the tumors. Concomitantly, lymphocytes obtained from treated mice specifically recognized Panc02 tumor cells in IFN-gamma-enzyme-linked immunosorbent spot and in cellular cytotoxicity assays. In rechallenge experiments, these immunologic effector cells were found to delay, but not completely prevent growth of secondary tumors. However, when considering the notoriously depressed immune status of individuals suffering from pancreatic carcinoma, the orchestrated antitumoral immune responses we analyzed here in detail significantly strengthen the potential usefulness of microbial compounds as active unspecific immunotherapeutic agent for treatment of pancreatic carcinoma. PMID:18779749

  10. Quantitative, real-time analysis of base excision repair activity in cell lysates utilizing lesion-specific molecular beacons.

    PubMed

    Svilar, David; Vens, Conchita; Sobol, Robert W

    2012-01-01

    We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base excision repair (BER) molecular beacons. The substrate (beacon) is comprised of a deoxyoligonucleotide containing a single base lesion with a 6-Carboxyfluorescein (6-FAM) moiety conjugated to the 5'end and a Dabcyl moiety conjugated to the 3' end of the oligonucleotide. The BER molecular beacon is 43 bases in length and the sequence is designed to promote the formation of a stem-loop structure with 13 nucleotides in the loop and 15 base pairs in the stem. When folded in this configuration the 6-FAM moiety is quenched by Dabcyl in a non-fluorescent manner via Förster Resonance Energy Transfer (FRET). The lesion is positioned such that following base lesion removal and strand scission the remaining 5 base oligonucleotide containing the 6-FAM moiety is released from the stem. Release and detachment from the quencher (Dabcyl) results in an increase of fluorescence that is proportionate to the level of DNA repair. By collecting multiple reads of the fluorescence values, real-time assessment of BER activity is possible. The use of standard quantitative real-time PCR instruments allows the simultaneous analysis of numerous samples. The design of these BER molecular beacons, with a single base lesion, is amenable to kinetic analyses, BER quantification and inhibitor validation and is adaptable for quantification of DNA Repair activity in tissue and tumor cell lysates or with purified proteins. The analysis of BER activity in tumor lysates or tissue aspirates using these molecular beacons may be applicable to functional biomarker measurements. Further, the analysis of BER activity with purified proteins using this quantitative assay provides a rapid, high-throughput method for the discovery and validation of BER inhibitors. PMID:22895410

  11. Studies on the adsorption of cell impurities from plasmid-containing lysates to phenyl boronic acid chromatographic beads.

    PubMed

    Gomes, A Gabriela; Azevedo, Ana M; Aires-Barros, M Raquel; Prazeres, D Miguel F

    2011-12-01

    Plasmid DNA (pDNA) is purified directly from alkaline lysis-derived Escherichia coli (E. coli) lysates by phenyl boronate (PB) chromatography. The method explores the ability of PB ligands to bind covalently, but reversibly, to cis-diol-containing impurities like RNA and lipopolysaccharides (LPS), leaving pDNA in solution. In spite of this specificity, cis-diol free species like proteins and genomic DNA (gDNA) are also removed. This is a major advantage since the process is designed to keep the target pDNA from binding. The focus of this paper is on the study of the secondary interactions between the impurities (RNA, gDNA, proteins, LPS) in a pDNA-containing lysate and 3-amino PB controlled pore glass (CPG) matrices. Runs were designed to evaluate the role of adsorption buffer composition, feed type (pH, salt content), CPG matrix and sample pretreatment (RNase A, isopropanol precipitation). Water was chosen as the adsorption buffer over MgCl(2) solutions since it maximised pDNA yield (96.2±4.9%) and protein removal (61.3±3.0%), while providing for a substantial removal of RNA (65.5±3.5%) and gDNA (44.7±14.1%). Although the use of pH 3.5 maximised removal of impurities (~75%), the best compromise between plasmid yield (~96%) and RNA clearance (~60-70%) was obtained for a pH of 5.2. Plasmid yield was maximal (>96%) when the concentration of acetate and potassium ions in the incoming lysate feed were 1.7 M and 1.0 M, respectively. The pre-treatment of lysates with RNase A deteriorated the performance since the resulting oligoribonucleotides lack the cis-diol group at their 3' termini. Overall, the results support the idea that charge transfer interactions between the boron atom at acidic pH and electron donor groups in the aromatic bases of nucleic acids and side residues of proteins are responsible for the non-specific removal of gDNA, RNA and proteins. PMID:22024344

  12. Data from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with Withaferin A (WA).

    PubMed

    Narayan, Malathi; Seeley, Kent W; Jinwal, Umesh K

    2016-06-01

    Mass spectrometry data collected in a study analyzing the effect of withaferin A (WA) on a mouse microglial (N9) cell line is presented in this article. Data was collected from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with either WA or DMSO vehicle control. This article reports all the proteins that were identified in this analysis. The data presented here is related to the published research article on the effect of WA on the differential regulation of proteins in mouse microglial cells [1]. Mass spectrometry data has also been deposited in the ProteomeXchange with the identifier PXD003032. PMID:27054189

  13. Data from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with Withaferin A (WA)

    PubMed Central

    Narayan, Malathi; Seeley, Kent W.; Jinwal, Umesh K.

    2016-01-01

    Mass spectrometry data collected in a study analyzing the effect of withaferin A (WA) on a mouse microglial (N9) cell line is presented in this article. Data was collected from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with either WA or DMSO vehicle control. This article reports all the proteins that were identified in this analysis. The data presented here is related to the published research article on the effect of WA on the differential regulation of proteins in mouse microglial cells [1]. Mass spectrometry data has also been deposited in the ProteomeXchange with the identifier PXD003032. PMID:27054189

  14. Hydrogen-atom attack on phenol and toluene is ortho-directed.

    PubMed

    Krechkivska, Olha; Wilcox, Callan M; Troy, Tyler P; Nauta, Klaas; Chan, Bun; Jacob, Rebecca; Reid, Scott A; Radom, Leo; Schmidt, Timothy W; Kable, Scott H

    2016-03-28

    The reaction of H + phenol and H/D + toluene has been studied in a supersonic expansion after electric discharge. The (1 + 1') resonance-enhanced multiphoton ionization (REMPI) spectra of the reaction products, at m/z = parent + 1, or parent + 2 amu, were measured by scanning the first (resonance) laser. The resulting spectra are highly structured. Ionization energies were measured by scanning the second (ionization) laser, while the first laser was tuned to a specific transition. Theoretical calculations, benchmarked to the well-studied H + benzene → cyclohexadienyl radical reaction, were performed. The spectrum arising from the reaction of H + phenol is attributed solely to the ortho-hydroxy-cyclohexadienyl radical, which was found in two conformers (syn and anti). Similarly, the reaction of H/D + toluene formed solely the ortho isomer. The preference for the ortho isomer at 100-200 K in the molecular beam is attributed to kinetic, not thermodynamic effects, caused by an entrance channel barrier that is ∼5 kJ mol(-1) lower for ortho than for other isomers. Based on these results, we predict that the reaction of H + phenol and H + toluene should still favour the ortho isomer under elevated temperature conditions in the early stages of combustion (200-400 °C). PMID:26948897

  15. On the Ortho:Para Ratio of H+ 3 in Diffuse Molecular Clouds

    NASA Astrophysics Data System (ADS)

    Crabtree, Kyle N.; Indriolo, Nick; Kreckel, Holger; Tom, Brian A.; McCall, Benjamin J.

    2011-03-01

    The excitation temperature T 01 derived from the relative intensities of the J = 0 (para) and J = 1 (ortho) rotational levels of H2 has been assumed to be an accurate measure of the kinetic temperature in interstellar environments. In diffuse molecular clouds, the average value of T 01 is ~70 K. However, the excitation temperature T(H+ 3) derived from the (J, K) = (1, 1) (para) and (1, 0) (ortho) rotational levels of H+ 3 has been observed to be ~30 K in the same types of environments. In this work, we present observations of H+ 3 in three additional diffuse cloud sight lines for which H2 measurements are available, showing that in four of five cases T 01 and T(H+ 3) are discrepant. We then examine the thermalization mechanisms for the ortho:para ratios of H+ 3 and H2, concluding that indeed T 01 is an accurate measure of the cloud kinetic temperature, while the ortho:para ratio of H+ 3 need not be thermal. By constructing a steady-state chemical model taking into account the nuclear spin dependence of reactions involving H+ 3, we show that the ortho:para ratio of H+ 3 in diffuse molecular clouds is likely governed by a competition between dissociative recombination with electrons and thermalization via reactive collisions with H2. Based in part on observations made with ESO Telescopes at the La Silla or Paranal Observatories under program ID 384.C-0618.

  16. Ortho-para conversion of endohedral water in the fullerene C60 at cryogenic temperatures

    NASA Astrophysics Data System (ADS)

    Shugai, Anna; Nagel, U.; Rõõm, T.; Mamone, S.; Concistrè, M.; Meier, B.; Krachmalnicoff, A.; Whitby, R. J.; Levitt, M. H.; Lei, Xuegong; Li, Yongjun; Turro, N. J.

    2015-03-01

    Water displays the phenomenon of spin isomerism in which the two proton spins either couple to form a triplet (ortho water, I = 1) or a singlet nuclear spin state (para water, I = 0). Here we study the interconversion of para and ortho water. The exact mechanism of this process is still not fully understood. In order to minimize interactions between molecules we use a sample where a single H2O is trapped in the C60 molecular cage (H2O@C60)andH2O@C60iscrystallized.H2O@C60 has long-lived ortho state and ortho-para conversion kinetics is non-exponential at LHeT. We studied mixtures of H2O@C60, D2O@C60 and C60 using IR absorption, NMR and dielectric measurements. We saw the speeding up of the interconversion with the growth of H2O@C60 concentration in C60 or when D2O@C60 was added. At some temperatures the kinetics is exponential. Models are discussed in order to explain the T and concentration dependence of ortho-para interconversion kinetics. This work was supported by institutional research funding IUT23-3 of the Estonian Ministry of Education and Research.

  17. The formaldehyde ortho/para ratio as a probe of dark cloud chemistry and evolution

    NASA Technical Reports Server (NTRS)

    Dickens, J. E.; Irvine, W. M.

    1999-01-01

    We present measurements of the H2CO ortho/para ratio toward four star-forming cores, L723, L1228, L1527, and L43, and one quiescent core, L1498. Combining these data with earlier results by Minh et al., three quiescent cores are found to have ortho/para ratios near 3, the ratio of statistical weights expected for gas-phase formation processes. In contrast, ortho/para ratios are 1.5-2.1 in five star-forming cores, suggesting thermalization at a kinetic temperature of 10 K. We attribute modification of the ortho/para ratio in the latter cores to formation and/or equilibration of H2CO on grains with sub-sequent release back into the gas phase due to the increased energy inputs from the forming star and outflow. We see accompanying enhancements in the H2CO abundance relative to H, to support this idea. The results suggest that the formaldehyde ortho/para ratio can differentiate between quiescent cores and those in which low-mass star formation has occurred.

  18. Ortho Image and DTM Generation with Intelligent Methods

    NASA Astrophysics Data System (ADS)

    Bagheri, H.; Sadeghian, S.

    2013-10-01

    Nowadays the artificial intelligent algorithms has considered in GIS and remote sensing. Genetic algorithm and artificial neural network are two intelligent methods that are used for optimizing of image processing programs such as edge extraction and etc. these algorithms are very useful for solving of complex program. In this paper, the ability and application of genetic algorithm and artificial neural network in geospatial production process like geometric modelling of satellite images for ortho photo generation and height interpolation in raster Digital Terrain Model production process is discussed. In first, the geometric potential of Ikonos-2 and Worldview-2 with rational functions, 2D & 3D polynomials were tested. Also comprehensive experiments have been carried out to evaluate the viability of the genetic algorithm for optimization of rational function, 2D & 3D polynomials. Considering the quality of Ground Control Points, the accuracy (RMSE) with genetic algorithm and 3D polynomials method for Ikonos-2 Geo image was 0.508 pixel sizes and the accuracy (RMSE) with GA algorithm and rational function method for Worldview-2 image was 0.930 pixel sizes. For more another optimization artificial intelligent methods, neural networks were used. With the use of perceptron network in Worldview-2 image, a result of 0.84 pixel sizes with 4 neurons in middle layer was gained. The final conclusion was that with artificial intelligent algorithms it is possible to optimize the existing models and have better results than usual ones. Finally the artificial intelligence methods, like genetic algorithms as well as neural networks, were examined on sample data for optimizing interpolation and for generating Digital Terrain Models. The results then were compared with existing conventional methods and it appeared that these methods have a high capacity in heights interpolation and that using these networks for interpolating and optimizing the weighting methods based on inverse

  19. Evaluation of a combined lysate/recombinant antigen anti-HTLV-I/II ELISA in high and low endemic areas of HTLV-I/II infection.

    PubMed

    Vrielink, H; Sisay, Y; Reesink, H W; Woerdeman, M; Winkel, C; de Leeuw, S J; Lelie, P N; van der Poel, C L

    1995-06-01

    The Wellcozyme HTLV-I/II ELISA (Murex Diagnostics) was evaluated in 7800 samples of various serum panels. Repeat activity was found by Wellcozyme in (A) 1/2181 (0.05%) Dutch blood donors, (B) 44/3036 (1.4%) Curaçao (Caribbean area) blood donors, (C) 46/2533 (1.8%) individuals of different Ethiopian population subsets, (D) 30/30 (100%) confirmed anti-HTLV-I positive samples and (E) 20/20 (100%) HTLV-II PCR-positive samples. All 91 Wellcozyme-positive samples were tested for confirmation by Western blot (WB, Diagnostic Biotechnology). Among Wellcozyme HTLV-I/II ELISA-positive individuals, HTLV-I/II WB positivity was found in 0/1 Dutch blood donors, 40/44 (88.9%) Curaçao blood donors and 20/46 (43.5%) Ethiopian individuals. HTLV-I positivity was found in 40 (1.3%) WB-positive Curaçao blood donors and in 9 (0.35%) Ethiopian individuals. HTLV-II positivity was found in 11 (0.43%) WB-positive Ethiopian individuals. The Wellcozyme HTLV-I/II ELISA had a specificity of 99.95% in Dutch blood donors and a sensitivity of 100% on confirmed HTLV-I- and HTLV-II-positive samples. In Ethiopia 55% of the HTLV-I/II WB-positive individuals were exclusively HTLV-II positive, whereas in Curaçao no HTLV-II infections were found. PMID:7655577

  20. Cross-presentation of viral antigens in dribbles leads to efficient activation of virus-specific human memory t cells

    PubMed Central

    2014-01-01

    Background Autophagy regulates innate and adaptive immune responses to pathogens and tumors. We have reported that autophagosomes derived from tumor cells after proteasome inhibition, DRibbles (Defective ribosomal products in blebs), were excellent sources of antigens for efficient cross priming of tumor-specific CD8+ T cells, which mediated regression of established tumors in mice. But the activity of DRibbles in human has not been reported. Methods DRibbles or cell lysates derived from HEK293T or UbiLT3 cell lines expressing cytomegalovirus (CMV) pp65 protein or transfected with a plasmid encoding dominant HLA-A2 restricted CMV, Epstein-Barr virus (EBV), and Influenza (Flu) epitopes (CEF) were loaded onto human monocytes or PBMCs and the response of human CMV pp65 or CEF antigen-specific CD4+ and CD8+ memory T cells was detected by intracellular staining. The effect of cytokines (GM-CSF, IL-4, IL-12, TNF-α, IFN-α and IFN-γ) TLR agonists (Lipopolysaccharide, Polyinosinic-polycytidylic acid (poly(I:C), M52-CpG, R848, TLR2 ligand) and CD40 ligand on the cross-presentation of antigens contained in DRibbles or cell lysates was explored. Results In this study we showed that purified monocytes, or human PBMCs, loaded with DRibbles isolated from cells expressing CMV or CEF epitopes, could activate CMV- or CEF-specific memory T cells. DRibbles were significantly more efficient at stimulating CD8+ memory T cells compared to cell lysates expressing the same antigenic epitopes. We optimized the conditions for T-cell activation and IFN-γ production following direct loading of DRibbles onto PBMCs. We found that the addition of Poly(I:C), CD40 ligand, and GM-CSF to the PBMCs together with DRibbles significantly increased the level of CD8+ T cell responses. Conclusions DRibbles containing specific viral antigens are an efficient ex vivo activator of human antigen-specific memory T cells specific for those antigens. This function could be enhanced by combining with Poly

  1. The ortho-substituent effect on the Ag-catalysed decarboxylation of benzoic acids.

    PubMed

    Grainger, Rachel; Cornella, Josep; Blakemore, David C; Larrosa, Igor; Campanera, Josep M

    2014-12-01

    A combined experimental and computational investigation on the Ag-catalysed decarboxylation of benzoic acids is reported herein. The present study demonstrates that a substituent at the ortho position exerts dual effects in the decarboxylation event. On one hand, ortho-substituted benzoic acids are inherently destabilised starting materials compared to their meta- and para-substituted counterparts. On the other hand, the presence of an ortho-electron-withdrawing group results in an additional stabilisation of the transition state. The combination of both effects results in an overall reduction of the activation energy barrier associated with the decarboxylation event. Furthermore, the Fujita-Nishioka linear free energy relationship model indicates that steric bulk of the substituent can also exert a negative effect by destabilising the transition state of decarboxylation. PMID:25336158

  2. Accuracy Comparison of Vhr Systematic-Ortho Satellite Imageries against Vhr Orthorectified Imageries Using Gcp

    NASA Astrophysics Data System (ADS)

    Widyaningrum, E.; Fajari, M.; Octariady, J.

    2016-06-01

    The Very High Resolution (VHR) satellite imageries such us Pleiades, WorldView-2, GeoEye-1 used for precise mapping purpose must be corrected from any distortion to achieve the expected accuracy. Orthorectification is performed to eliminate geometric errors of the VHR satellite imageries. Orthorectification requires main input data such as Digital Elevation Model (DEM) and Ground Control Point (GCP). The VHR systematic-ortho imageries were generated using SRTM 30m DEM without using any GCP data. The accuracy value differences of VHR systematic-ortho imageries and VHR orthorectified imageries using GCP currently is not exactly defined. This study aimed to identified the accuracy comparison of VHR systematic-ortho imageries against orthorectified imageries using GCP. Orthorectified imageries using GCP created by using Rigorous model. Accuracy evaluation is calculated by using several independent check points.

  3. Terahertz absorption spectrum of para and ortho water vapors at different humidities at room temperature

    NASA Astrophysics Data System (ADS)

    Xin, X.; Altan, H.; Saint, A.; Matten, D.; Alfano, R. R.

    2006-11-01

    Terahertz time-domain spectroscopy has been used to measure the absorption of water vapor in 0.2-2.4THz range from low to high humidity at room temperature. The observed absorption lines are due to the water molecular rotations in the ground vibrational state. We find that the absorption strength of para transitions increases as humidity increases, while the absorption strength of ortho transitions increases and then decreases in intensity with increasing humidity. We explain this difference based on the nuclear spin statistics based ratio of ortho to para water monomer populations at room temperature. The preferential adsorption on the solid surfaces of para water leads to an ortho dominated vapor cloud whose monomer rotational absorption intensity decreases due to the effects of dimerization, molecular collisions, clustering, and interactions with liquid droplets at high concentrations.

  4. Electrical detection of ortho-para conversion in fullerene-encapsulated water

    NASA Astrophysics Data System (ADS)

    Meier, Benno; Mamone, Salvatore; Concistrè, Maria; Alonso-Valdesueiro, Javier; Krachmalnicoff, Andrea; Whitby, Richard J.; Levitt, Malcolm H.

    2015-08-01

    Water exists in two spin isomers, ortho and para, that have different nuclear spin states. In bulk water, rapid proton exchange and hindered molecular rotation obscure the direct observation of two spin isomers. The supramolecular endofullerene H2O@C60 provides freely rotating, isolated water molecules even at cryogenic temperatures. Here we show that the bulk dielectric constant of this substance depends on the ortho/para ratio, and changes slowly in time after a sudden temperature jump, due to nuclear spin conversion. The attribution of the effect to ortho-para conversion is validated by comparison with nuclear magnetic resonance and quantum theory. The change in dielectric constant is consistent with an electric dipole moment of 0.51+/-0.05 Debye for an encapsulated water molecule, indicating the partial shielding of the water dipole by the encapsulating cage. The dependence of bulk dielectric constant on nuclear spin isomer composition appears to be a previously unreported physical phenomenon.

  5. Prevention of respiratory tract infections with bacterial lysate OM-85 bronchomunal in children and adults: a state of the art

    PubMed Central

    2013-01-01

    Respiratory tract infections (RTIs) are a leading cause of morbidity and also represent a cause of death in some parts of the world. The treatment of RTIs implies a continuous search for stronger therapies and represents an economical burden for health services and society. In this context the prevention of infections is absolutely required. The use of bacterial lysates as immuno-modulators to boost immunological response is widely debated. Aim of this review is to summarize the main clinical studies on the effect of the bacterial lysate OM-85 in treating RTIs in susceptible subjects - namely children and chronic obstructive pulmonary disease (COPD)-affected adults. Results from clinical trials and recent systematic reviews are reported. The results show that mean number of RTIs decreases upon treatment with OM-85, as measured by frequency of exacerbations or number of antibiotic courses. Data from systematic reviews indicated that OM-85 is particularly beneficial in children at high risk of RTIs. In COPD-affected adults, clinical studies showed that treatment with OM-85 reduced exacerbations, although systematic reviews did not legitimate the protective effect of OM-85 toward COPD as significant. The use of OM-85 could be efficacious in reducing exacerbation frequency of RTIs in children and adults at risk. However further high-quality studies are needed to better explain the mechanism of action and confirm the beneficial results of OM85. PMID:23692890

  6. Effectiveness of Polyvalent Bacterial Lysate and Autovaccines Against Upper Respiratory Tract Bacterial Colonization by Potential Pathogens: A Randomized Study

    PubMed Central

    Zagólski, Olaf; Stręk, Paweł; Kasprowicz, Andrzej; Białecka, Anna

    2015-01-01

    Background Polyvalent bacterial lysate (PBL) is an oral immunostimulating vaccine consisting of bacterial standardized lysates obtained by lysis of different strains of bacteria. Autovaccines are individually prepared based on the results of smears obtained from the patient. Both types of vaccine can be used to treat an ongoing chronic infection. This study sought to determine which method is more effective against nasal colonization by potential respiratory tract pathogens. Material/Methods We enrolled 150 patients with aerobic Gram stain culture and count results indicating bacterial colonization of the nose and/or throat by potential pathogens. The participants were randomly assigned to each of the following groups: 1. administration of PBL, 2. administration of autovaccine, and 3. no intervention (controls). Results Reduction of the bacterial count in Streptococcus pneumoniae-colonized participants was significant after the autovaccine (p<0.001) and PBL (p<0.01). Reduction of the bacterial count of other β-hemolytic streptococcal strains after treatment with the autovaccine was significant (p<0.01) and was non-significant after PBL. In Haemophilus influenzae colonization, significant reduction in the bacterial count was noted in the PBL group (p<0.01). Methicillin-resistant Staphylococcus aureus colonization did not respond to either treatment. Conclusions The autovaccine is more effective than PBL for reducing bacterial count of Streptococcus pneumoniae and β-hemolytic streptococci, while PBL was more effective against Haemophilus influenzae colonization. PMID:26434686

  7. Prevention of respiratory tract infections with bacterial lysate OM-85 bronchomunal in children and adults: a state of the art.

    PubMed

    De Benedetto, Fernando; Sevieri, Gianfranco

    2013-01-01

    Respiratory tract infections (RTIs) are a leading cause of morbidity and also represent a cause of death in some parts of the world. The treatment of RTIs implies a continuous search for stronger therapies and represents an economical burden for health services and society. In this context the prevention of infections is absolutely required. The use of bacterial lysates as immuno-modulators to boost immunological response is widely debated. Aim of this review is to summarize the main clinical studies on the effect of the bacterial lysate OM-85 in treating RTIs in susceptible subjects - namely children and chronic obstructive pulmonary disease (COPD)-affected adults. Results from clinical trials and recent systematic reviews are reported.The results show that mean number of RTIs decreases upon treatment with OM-85, as measured by frequency of exacerbations or number of antibiotic courses. Data from systematic reviews indicated that OM-85 is particularly beneficial in children at high risk of RTIs. In COPD-affected adults, clinical studies showed that treatment with OM-85 reduced exacerbations, although systematic reviews did not legitimate the protective effect of OM-85 toward COPD as significant.The use of OM-85 could be efficacious in reducing exacerbation frequency of RTIs in children and adults at risk. However further high-quality studies are needed to better explain the mechanism of action and confirm the beneficial results of OM85. PMID:23692890

  8. The virion N protein of infectious bronchitis virus is more phosphorylated than the N protein from infected cell lysates

    SciTech Connect

    Jayaram, Jyothi; Youn, Soonjeon; Collisson, Ellen W. . E-mail: ecollisson@cvm.tamu.edu

    2005-08-15

    Because phosphorylation of the infectious bronchitis virus (IBV) nucleocapsid protein (N) may regulate its multiple roles in viral replication, the dynamics of N phosphorylation were examined. {sup 32}P-orthophosphate labeling and Western blot analyses confirmed that N was the only viral protein that was phosphorylated. Pulse labeling with {sup 32}P-orthophosphate indicated that the IBV N protein was phosphorylated in the virion, as well as at all times during infection in either chicken embryo kidney cells or Vero cells. Pulse-chase analyses followed by immunoprecipitation of IBV N proteins using rabbit anti-IBV N polyclonal antibody demonstrated that the phosphate on the N protein was stable for at least 1 h. Simultaneous labeling with {sup 32}P-orthophosphate and {sup 3}H-leucine identified a 3.5-fold increase in the {sup 32}P:{sup 3}H counts per minute (cpm) ratio of N in the virion as compared to the {sup 32}P:{sup 3}H cpm ratio of N in the cell lysates from chicken embryo kidney cells, whereas in Vero cells the {sup 32}P:{sup 3}H cpm ratio of N from the virion was 10.5-fold greater than the {sup 32}P:{sup 3}H cpm ratio of N from the cell lysates. These studies are consistent with the phosphorylation of the IBV N playing a role in assembly or maturation of the viral particle.

  9. OrthoVenn: a web server for genome wide comparison and annotation of orthologous clusters across multiple species.

    PubMed

    Wang, Yi; Coleman-Derr, Devin; Chen, Guoping; Gu, Yong Q

    2015-07-01

    Genome wide analysis of orthologous clusters is an important component of comparative genomics studies. Identifying the overlap among orthologous clusters can enable us to elucidate the function and evolution of proteins across multiple species. Here, we report a web platform named OrthoVenn that is useful for genome wide comparisons and visualization of orthologous clusters. OrthoVenn provides coverage of vertebrates, metazoa, protists, fungi, plants and bacteria for the comparison of orthologous clusters and also supports uploading of customized protein sequences from user-defined species. An interactive Venn diagram, summary counts, and functional summaries of the disjunction and intersection of clusters shared between species are displayed as part of the OrthoVenn result. OrthoVenn also includes in-depth views of the clusters using various sequence analysis tools. Furthermore, OrthoVenn identifies orthologous clusters of single copy genes and allows for a customized search of clusters of specific genes through key words or BLAST. OrthoVenn is an efficient and user-friendly web server freely accessible at http://probes.pw.usda.gov/OrthoVenn or http://aegilops.wheat.ucdavis.edu/OrthoVenn. PMID:25964301

  10. Environmental occurrence and potential toxicity of planar, mono-, and di-ortho polychlorinated biphenyls in the biota

    SciTech Connect

    Hong, C.S.; Xiao, J.; Bush, B.

    1995-12-31

    Coplanar PCBs without ortho-chlorine substituents and semicoplanar PCBs with one ortho-chlorination are stereochemically similar to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and, because of this similarity, exert biochemical activity and toxicity to biota comparable to that of TCDD. Four non-ortho-, eight mono-ortho-, and two di-ortho-chlorinated congeners have been determined in insect larvae, fresh water and salt water mussels, fish, mallard duck, seals, and in human milk and adipose tissue. The PCB congeners are separated from the remainder of PCBs by activated carbon chromatography or HPLC on porous graphitic carbon followed by gas chromatography with electron capture detection. PCB toxic equivalency factors (TEFs) (1) recommended by WHO for 3 non-ortho, 8 mono-ortho and 2 di-ortho PCBs and a TEF for congener 81 (3,4,4{prime},5-tetrachlorobiphenyl) suggested by Harris et al. from AHH induction in rainbow trout (2) were used for calculation of the contribution to dioxin-like toxicity to each life form. In all the biota examined, PCB congener number 126 was the major contributor to PCB toxic equivalents (TEQs), followed by congener numbers 118, 114, 156, and 105. The ability to separate out planar PCBs from the majority of PCBs has allowed the use of TCDD toxicity equivalence to compare the relative dioxin-like potency of PCB residues in various species from 12 different locations.

  11. Merging Photoredox with Palladium Catalysis: Decarboxylative ortho-Acylation of Acetanilides with α-Oxocarboxylic Acids under Mild Reaction Conditions.

    PubMed

    Zhou, Chao; Li, Pinhua; Zhu, Xianjin; Wang, Lei

    2015-12-18

    A room temperature decarboxylative ortho-acylation of acetanilides with α-oxocarboxylic acids has been developed via a novel Eosin Y with Pd dual catalytic system. This dual catalytic reaction shows a broad substrate scope and good functional group tolerance, and an array of ortho-acylacetanilides can be afforded in high yields under mild conditions. PMID:26646667

  12. Discovery of Native Autoantigens via Antigen Surrogate Technology: Application to Type 1 Diabetes

    PubMed Central

    2014-01-01

    A fundamental goal in understanding the mechanisms of autoimmune disease is the characterization of autoantigens that are targeted by autoreactive antibodies and T cells. Unfortunately, the identification of autoantigens is a difficult problem. We have begun to explore a novel route to the discovery of autoantibody/autoantigen pairs that involves comparative screening of combinatorial libraries of unnatural, synthetic molecules for compounds that bind antibodies present at much higher levels in the serum of individuals with a given autoimmune disease than in the serum of control individuals. We have shown that this approach can yield “antigen surrogates” capable of capturing disease-specific autoantibodies from serum. In this report, we demonstrate that the synthetic antigen surrogates can be used to affinity purify the autoantibodies from serum and that these antibodies can then be used to identify their cognate autoantigen in an appropriate tissue lysate. Specifically, we report the discovery of a peptoid able to bind autoantibodies present in about one-third of nonobese diabetic (NOD) mice. The peptoid-binding autoantibodies were highly enriched through peptoid affinity chromatography and employed to probe mouse pancreatic and brain lysates. This resulted in identification of murine GAD65 as the native autoantigen. GAD65 is a known humoral autoantigen in human type 1 diabetes mellitus (T1DM), but its existence in mice had been controversial. This study demonstrates the potential of this chemical approach for the unbiased identification of autoantigen/autoantibody complexes. PMID:25474415

  13. Diagnostic value of carbohydrate antigens in supernatants and sediments of pleural effusions.

    PubMed

    Terracciano, Daniela; Mazzarella, Claudia; Cicalese, Marcellino; Galzerano, Sonia; Apostolico, Gianfranco; DI Carlo, Angelina; Mariano, Angela; Cecere, Ciriaco; Macchia, Vincenzo

    2010-05-01

    A panel of tumour markers including carcinoembryonic antigen (CEA), carbohydrate antigen (Ca)15-3, Ca125 and Ca19-9 were measured in the lysate of sediments and in the supernatants of pleural effusions of patients with benign and malignant disease. The tumour markers were also measured in the serum of the same patients. Of these patients, 32 had benign diseases (12 trasudative effusions associated with cirrhosis and 20 with non-malignant exudates: 12 pleuritis and 8 other inflammations) and 103 had malignant effusions (37 breast cancers, 29 lung cancers, 10 ovary cancers, 6 kidney cancers, 11 mesotheliomas and 10 lymphomas). We showed the highest level of CEA in pleural effusions of lung cancer followed by that in pleural effusions of breast cancer; whereas Ca15-3 was very high in the pleural effusions of breast and lung cancer. Concerning the lysate of sediment, CEA was high in the pleural effusions of patients with lung cancer and Ca15-3 in those of patients with breast cancer. The other markers are much less useful. For the remaining tumours, none of the markers tested appear to aid in the diagnosis of disease. In conclusion, our data suggest that the combined determination of tumour markers on supernatants and sediments of pleural effusion may provide additional information on the nature of pleural effusion, especially for cases with negative cytology. PMID:22966327

  14. Diagnostic value of carbohydrate antigens in supernatants and sediments of pleural effusions

    PubMed Central

    TERRACCIANO, DANIELA; MAZZARELLA, CLAUDIA; CICALESE, MARCELLINO; GALZERANO, SONIA; APOSTOLICO, GIANFRANCO; DI CARLO, ANGELINA; MARIANO, ANGELA; CECERE, CIRIACO; MACCHIA, VINCENZO

    2010-01-01

    A panel of tumour markers including carcinoembryonic antigen (CEA), carbohydrate antigen (Ca)15-3, Ca125 and Ca19-9 were measured in the lysate of sediments and in the supernatants of pleural effusions of patients with benign and malignant disease. The tumour markers were also measured in the serum of the same patients. Of these patients, 32 had benign diseases (12 trasudative effusions associated with cirrhosis and 20 with non-malignant exudates: 12 pleuritis and 8 other inflammations) and 103 had malignant effusions (37 breast cancers, 29 lung cancers, 10 ovary cancers, 6 kidney cancers, 11 mesotheliomas and 10 lymphomas). We showed the highest level of CEA in pleural effusions of lung cancer followed by that in pleural effusions of breast cancer; whereas Ca15-3 was very high in the pleural effusions of breast and lung cancer. Concerning the lysate of sediment, CEA was high in the pleural effusions of patients with lung cancer and Ca15-3 in those of patients with breast cancer. The other markers are much less useful. For the remaining tumours, none of the markers tested appear to aid in the diagnosis of disease. In conclusion, our data suggest that the combined determination of tumour markers on supernatants and sediments of pleural effusion may provide additional information on the nature of pleural effusion, especially for cases with negative cytology. PMID:22966327

  15. T-cell-specific membrane antigens in the Mexican axolotl (urodele amphibian).

    PubMed

    Kerfourn, F; Guillet, F; Charlemagne, J; Tournefier, A

    1992-01-01

    Comparative analysis of SDS-PAGE patterns of axolotl spleen cells membrane detergent lysates showed important discrepancies between control and thymectomized animals. Among these, a 38-kD protein band, which appeared as a major protein in controls, was not or poorly expressed after thymectomy. A rabbit antiserum (L12) raised against the 38-kD eluted band labeled in indirect immunofluorescence 80-86% of thymocytes and 40-46% of mIg- lymphoid cells in the spleen. The anti-38-kD antibodies stained in Western blotting two antigenically related polypeptides of 38- and 36-kD on splenocyte membrane lysates. Two-dimensional NEPHGE-PAGE analysis indicated that the anti-38-kD antibodies reacted in the spleen with several gathered spots in the 7.8-8.2 pI range, corresponding to 38-36-kD microheterogeneous polypeptides. Most of these spots are not further expressed in thymectomized animals. These results support evidence that the 38-kD surface antigens can be considered as specific surface markers of the axolotl thymus-derived lymphocytes. PMID:1627952

  16. Microencapsulation of Streptococcus equi antigens in biodegradable microspheres and preliminary immunisation studies.

    PubMed

    Azevedo, Ana F; Galhardas, Jorge; Cunha, António; Cruz, Patrícia; Gonçalves, Lídia M D; Almeida, António J

    2006-10-01

    Streptococcus equi subspecies equi is the causative agent of strangles, a bacterial infection of the respiratory tract of equidae. Current strategies to prevent strangles rely on antimicrobial therapy or immunisation with inactivated bacteria, S. equi bacterin, or M-like protein (SeM) extract. The aim of this work was to investigate whether immunisation with whole killed S. equi or a bacterial lysate entrapped in poly(lactide-co-glycolide) (PLGA) microspheres might induce protective immunity to mice. Animals were treated with a dose of antigen equivalent to 25 microg of SeM. For intranasal route animals were primed on days 1, 2 and 3 and were boosted on day 29. For intramuscular route, primary immunisation was carried out with a single injection on day 1 and animals were boosted on day 29. On day 43 animals were submitted to a challenge with a virulent strain of S. equi. Vaccination with antigen-containing microspheres induced higher serum antibody levels in mice treated by the intranasal route, whereas intramuscular immunisation did not reveal any difference between control and treatment groups. Microencapsulated antigens achieved to fully protect mice against experimental infection irrespective of the route of administration used. Following intranasal or intramuscular administration soluble antigen failed to protect mice against challenge. These studies indicate that PLGA microspheres are a potential carrier system for the delivery of S. equi antigens. PMID:16846728

  17. Presentation of hepatocellular antigens

    PubMed Central

    Grakoui, Arash; Crispe, Ian Nicholas

    2016-01-01

    The liver is an organ in which antigen-specific T-cell responses manifest a bias toward immune tolerance. This is clearly seen in the rejection of allogeneic liver transplants, and multiple other phenomena suggest that this effect is more general. These include tolerance toward antigens introduced via the portal vein, immune failure to several hepatotropic viruses, the lack of natural liver-stage immunity to malaria parasites, and the frequent metastasis of cancers to the liver. Here we review the mechanisms by which T cells engage with hepatocellular antigens, the context in which such encounters occur, and the mechanisms that act to suppress a full T-cell response. While many mechanisms play a role, we will argue that two important processes are the constraints on the cross-presentation of hepatocellular antigens, and the induction of negative feedback inhibition driven by interferons. The constant exposure of the liver to microbial products from the intestine may drive innate immunity, rendering the local environment unfavorable for specific T-cell responses through this mechanism. Nevertheless, tolerance toward hepatocellular antigens is not monolithic and under specific circumstances allows both effective immunity and immunopathology. PMID:26924525

  18. Critical evaluation of polychlorinated biphenyl toxicity in terrestrial and marine mammals: increasing impact of non-ortho and mono-ortho coplanar polychlorinated biphenyls from land to ocean.

    PubMed

    Kannan, N; Tanabe, S; Ono, M; Tatsukawa, R

    1989-11-01

    Residues of potentially toxic non-ortho chlorine substituted coplanar 3,3',4,4'-tetra-,3,3',4,4',5-penta-, 3,3',4,4',5,5'-hexachlorobiphenyl and their mono- and di-ortho analogs 2,3',4,4',5-penta, 2,3,3',4,4'-penta-, 2,3,3',4,4',5-hexa- and 2,2',3,3',4,4'-hexa-, 2,2',3,4,4',5-hexachlorobiphenyl) were determined in humans, dogs, cats (terrestrial), a finless porpoise (Neophocoena phocoenoides--coastal), Dall's porpoises (Phocoenoides dalli, dalli), Baird's beaked whales (Berardius bairdii) and killer whales (Orcinus orca--open ocean). Among the coplanar polychlorinated biphenyl (PCB) congeners, the concentration of the di-ortho congeners was the highest and the non-ortho congeners was the lowest. However, all three coplanar PCBs occurred at significantly higher levels than toxic polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). The relative bioconcentration and metabolic capacity of terrestrial and marine mammals to these chemicals, suggest that the toxic threat of coplanar PCBs increases from land to ocean, but the reverse is true for PCDDs and PCDFs. The toxic threat of coplanar PCBs to higher aquatic predators such as cetaceans was principally assessed by 2,3,7,8-T4CDD Toxic Equivalent Analysis which is based on the induction of arylhydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD). Analysis indicates, in particular, that the bioaccumulation of toxic 3,3',4,4',5-penta- and 2,3,3',4,4'-pentachlorobiphenyls in carnivorous marine mammals is a cause for considerable concern. PMID:2515809

  19. Characterization of the O-antigen Polymerase (Wzy) of Francisella tularensis*

    PubMed Central

    Kim, Tae-Hyun; Sebastian, Shite; Pinkham, Jessica T.; Ross, Robin A.; Blalock, LeeAnn T.; Kasper, Dennis L.

    2010-01-01

    The O-antigen polymerase of Gram-negative bacteria has been difficult to characterize. Herein we report the biochemical and functional characterization of the protein product (Wzy) of the gene annotated as the putative O-antigen polymerase, which is located in the O-antigen biosynthetic locus of Francisella tularensis. In silico analysis (homology searching, hydropathy plotting, and codon usage assessment) strongly suggested that Wzy is an O-antigen polymerase whose function is to catalyze the addition of newly synthesized O-antigen repeating units to a glycolipid consisting of lipid A, inner core polysaccharide, and one repeating unit of the O-polysaccharide (O-PS). To characterize the function of the Wzy protein, a non-polar deletion mutant of wzy was generated by allelic replacement, and the banding pattern of O-PS was observed by immunoblot analysis of whole-cell lysates obtained by SDS-PAGE and stained with an O-PS-specific monoclonal antibody. These immunoblot analyses showed that O-PS of the wzy mutant expresses only one repeating unit of O-antigen. Further biochemical characterization of the subcellular fractions of the wzy mutant demonstrated that (as is characteristic of O-antigen polymerase mutants) the low molecular weight O-antigen accumulates in the periplasm of the mutant. Site-directed mutagenesis based on protein homology and topology, which was carried out to locate a catalytic residue of the protein, showed that modification of specific residues (Gly176, Asp177, Gly323, and Tyr324) leads to a loss of O-PS polymerization. Topology models indicate that these amino acids most likely lie in close proximity on the bacterial surface. PMID:20605777

  20. Expression Cloning of Camelid Nanobodies Specific for Xenopus Embryonic Antigens

    PubMed Central

    Itoh, Keiji; Sokol, Sergei Y.

    2014-01-01

    Developmental biology relies heavily on the use of conventional antibodies, but their production and maintenance involves significant effort. Here we use an expression cloning approach to identify variable regions of llama single domain antibodies (known as nanobodies), which recognize specific embryonic antigens. A nanobody cDNA library was prepared from lymphocytes of a llama immunized with Xenopus embryo lysates. Pools of bacterially expressed cDNAs were sib-selected for the ability to produce specific staining patterns in gastrula embryos. Three different nanobodies were isolated: NbP1 and NbP3 stained yolk granules, while the reactivity of NbP7 was predominantly restricted to the cytoplasm and the cortex. The isolated nanobodies recognized specific protein bands in immunoblot analysis. A reverse proteomic approach identified NbP1 target antigen as EP45/Seryp, a serine protease inhibitor. Given the unique stability of nanobodies and the ease of their expression in diverse systems, we propose that nanobody cDNA libraries represent a promising resource for molecular markers for developmental biology. PMID:25285446

  1. Pathways of Antigen Processing

    PubMed Central

    Blum, Janice S.; Wearsch, Pamela A.; Cresswell, Peter

    2014-01-01

    T cell recognition of antigen presenting cells depends on their expression of a spectrum of peptides bound to Major Histocompatibility Complex class I (MHC-I) and class II (MHC-II) molecules. Conversion of antigens from pathogens or transformed cells into MHC-I and MHC-II-bound peptides is critical for mounting protective T cell responses, and similar processing of self proteins is necessary to establish and maintain tolerance. Cells use a variety of mechanisms to acquire protein antigens, from translation in the cytosol to variations on the theme of endocytosis, and to degrade them once acquired. In this review we highlight the aspects of MHC-I and MHC-II biosynthesis and assembly that have evolved to intersect these pathways and sample the peptides that are produced. PMID:23298205

  2. Evaluation of ToxA and Vibrio parahaemolyticus lysate on humoral immune response and immune-related genes in Pacific red snapper.

    PubMed

    Reyes-Becerril, Martha; Maldonado-García, Minerva; Guluarte, Crystal; León-Gallo, Amalia; Rosales-Mendoza, Sergio; Ascencio, Felipe; Hirono, Ikuo; Angulo, Carlos

    2016-09-01

    Immunogenicity of ToxA and Vibrio parahaemolyticus lysate was evaluated in a double immunostimulation scheme in Pacific red snapper after V. parahaemolyticus infection. Three groups of Pacific red snapper were intraperitonealy (i.p.) injected with phosphate-buffered saline (PBS group), ToxA of V. parahaemolyticus (ToxA-Vp group) or V. parahaemolyticus lysate (lysate-Vp group) (first injection, day 1; second injection, day 7). Fish were subsequently infected with live V. parahaemolyticus. Humoral immune parameters in skin mucus and serum were evaluated on days 1, 7, 8 and 14 days post-immunostimulation and 7 days post-infection. Moreover expression of immune-related genes was quantified by real time PCR in head-kidney leukocytes, spleen, liver, and intestine. The ToxA-Vp-treated group showed a higher anti-protease and catalase activity in skin mucus when compared with the PBS group. Measurements of SOD and CAT activities showed an increment in both activities a day after the second boost with ToxA-Vp or lysate-Vp. Interestingly, IgM levels in mucus and transcripts were enhanced followed the ToxA-Vp treatment even after challenge. Furthermore, IL-1β was strongly expressed in all analyzed cell or tissues followed ToxA-Vp or Vp-lysate treatments. Finally, SOD and CAT gene expression was up-regulated in fish immunostimulated with either treatment ToxA-Vp or lysate-Vp, mainly after infection in head-kidney leukocytes and intestine. This is the first study where the effects of ToxA from V. parahaemolyticus in the immune system of Pacific red snapper was evaluated. These results suggest that ToxA-Vp would positively affect humoral immune response and up-regulate expression of genes involved in the immune system function; and could help in the control of V. parahaemolyticus infection in Pacific red snapper Lutjanus peru, an economic important fish in Mexico. PMID:27417232

  3. Lipid antigens in immunity

    PubMed Central

    Dowds, C. Marie; Kornell, Sabin-Christin

    2014-01-01

    Lipids are not only a central part of human metabolism but also play diverse and critical roles in the immune system. As such, they can act as ligands of lipid-activated nuclear receptors, control inflammatory signaling through bioactive lipids such as prostaglandins, leukotrienes, lipoxins, resolvins, and protectins, and modulate immunity as intracellular phospholipid- or sphingolipid-derived signaling mediators. In addition, lipids can serve as antigens and regulate immunity through the activation of lipid-reactive T cells, which is the topic of this review. We will provide an overview of the mechanisms of lipid antigen presentation, the biology of lipid-reactive T cells, and their contribution to immunity. PMID:23999493

  4. Ortho-Para Mixing Interaction in the Vinyl Radical Detected by Millimeter-Wave Spectroscopy and Prediction of Fast Ortho-Para Conversion Rate

    NASA Astrophysics Data System (ADS)

    Tanaka, Keiichi; Hayashi, Masato; Ohtsuki, Mitsuhiko; Harada, Kensuke; Tanaka, Takehiko

    2010-06-01

    Ortho-para mixing interaction due to the coupling of nuclear and electron spins was detected for the first time by millimeter-wave spectroscopy of deuterated vinyl radicals, H_2CCD and D_2CCD, of which the ground states are split by the tunneling motion of the α deuteron into two components 0^+ and 0^-, whose separations have been determined to be Δ E_0 = 1186.644(16) and 771.978(18) MHz, respectively. The observed tunneling-rotation spectra are significantly perturbed by the othro-para mixing interaction expressed by < 0^± |H'|0^mprangle = (δ aF(β)/2) S \\cdot(I_β 1-I_β 2), where I_β 1 and I_β 2 are spins of the two hydrogen nuclei in the β position and S is the electron spin, which connects rotational levels in the 0^+ and 0^- states, one being an ortho level and the other a para level. The δ aF(β) constants for H_2CCD and D_2CCD have been determined to be 68.06(53) and 10.63(94) MHz, respectively, consistent each other within the isotopic mass relation. The othro and para states are mixed by about 0.097% and 0.0123% due to this interaction. The δ aF constant for H_2CCH should be similar to that for H_2CCD because of the same probability density of the unpaired electron at the β protons, but could not be determined independently in our previous study. It is because the mixing of para- and ortho-levels of about 0.00044% is much smaller than that for H_2CCD due to the large tunneling splitting of Δ E_0=16271.8429(59) MHz. The rate constant of para to ortho (I_β = 0 → 1) conversion is predicted as 1.2× 10^5 s-1 torr-1 for H_2CCD, suggesting extremely rapid mutual conversion between ortho and para nuclear spin isomers of H_2CCD, which is more than 106 times faster compared with that in closed shell molecules such as H_2CO and H_2CCH_2. K. Tanaka, M. Hayashi, M. Ohtsuki, K. Harada, T. Tanaka, J. Chem. Phys., 131, 111101 (2009). K. Tanaka, M. Toshimitsu, K. Harada, T. Tanaka, J. Chem. Phys., 120, 3604 (2004).

  5. Derivatization of (quinolin-8-yl)phosphinimidic amides via ortho-lithiation revisited.

    PubMed

    Fernández Sáez, Nerea; García López, Jesús; Iglesias, María José; López Ortiz, Fernando

    2015-07-01

    5The direct ortho-lithiation of N-H containing (quinolin-8-yl)phosphinimidic amides by reaction with 1 equiv. of n-BuLi described by Wang and co-workers has been re-examined. The multinuclear magnetic resonance ((1)H, (2)H, (7)Li, (13)C, (15)N and (31)P) study of the species formed in the monolithiation of N-(tert-butyl)-P,P-diphenyl-N'-(quinolin-8-yl)phosphinimidic amide 5 with n-BuLi in THF showed that proton abstraction occurred exclusively and quantitatively at the NH. The combination of the NMR results with a DFT study made it possible to describe the structure of the N-lithiated species 9 as a dimer consisting of an eight-membered ring showing two lithium ions triply coordinated to nitrogen atoms corresponding to the deprotonated amine and aminoquinoline moieties of different monomers. The formation of a polymer featuring the same coordination mode couldn't be excluded. In addition, optimized conditions for the efficient derivatization of 5 via ortho-lithiation were realised. The reaction of 5 with 2.4 equiv. of t-BuLi in THF in the temperature range of -80 °C to 25 °C for 3 h afforded a N,C(ortho)-dilithiated species that was trapped with a series of electrophiles leading to new functionalized ortho derivatives of 5 in good yields. PMID:26030882

  6. ortho-Fluoroazobenzenes: visible light switches with very long-Lived Z isomers.

    PubMed

    Knie, Christopher; Utecht, Manuel; Zhao, Fangli; Kulla, Hannes; Kovalenko, Sergey; Brouwer, Albert M; Saalfrank, Peter; Hecht, Stefan; Bléger, David

    2014-12-01

    Improving the photochemical properties of molecular photoswitches is crucial for the development of light-responsive systems in materials and life sciences. ortho-Fluoroazobenzenes are a new class of rationally designed photochromic azo compounds with optimized properties, such as the ability to isomerize with visible light only, high photoconversions, and unprecedented robust bistable character. Introducing σ-electron-withdrawing F atoms ortho to the NN unit leads to both an effective separation of the n→π* bands of the E and Z isomers, thus offering the possibility of using these two transitions for selectively inducing E/Z isomerizations, and greatly enhanced thermal stability of the Z isomers. Additional para-electron-withdrawing groups (EWGs) work in concert with ortho-F atoms, giving rise to enhanced separation of the n→π* transitions. A comprehensive study of the effect of substitution on the key photochemical properties of ortho-fluoroazobenzenes is reported herein. In particular, the position, number, and nature of the EWGs have been varied, and the visible light photoconversions, quantum yields of isomerization, and thermal stabilities have been measured and rationalized by DFT calculations. PMID:25352421

  7. Single C-F Bond Cleavage of Trifluoromethylarenes with an ortho-Silyl Group.

    PubMed

    Yoshida, Suguru; Shimomori, Ken; Kim, Youngchan; Hosoya, Takamitsu

    2016-08-22

    The transformation of a single C-F bond of trifluoromethylarenes bearing a hydrosilyl group at the ortho position was achieved. The activation of the hydrosilyl group with a trityl cation in the presence of nucleophiles allowed for selective C-F bond functionalization, for example, by allylation, carboxylation, or chlorination. Further derivatization of the resulting fluorosilylarenes afforded various aromatic difluoromethylene compounds. PMID:27312982

  8. OrthoEvidence™: A Clinical Resource for Evidence-Based Orthopedics

    PubMed Central

    Sprague, Sheila; Smith, Chris; Bhandari, Mohit

    2015-01-01

    The prevalence of musculoskeletal issues in clinical practice, and the limited focus placed upon musculoskeletal conditions by current electronic summary resources, highlights the need for a resource that provides access to simple and concise summaries of top-quality orthopedic literature for orthopedic surgeons and allied healthcare professionals. OrthoEvidence™ is an online clinical resource that addresses the paucity of adequate evidence-based summary tools in the field of orthopedic surgery. OrthoEvidence™ uses a rigorous, transparent, and unique process to review, evaluate, and summarize high quality research studies and their implications for orthopedic clinical practice. Randomized controlled trials and meta-analyses are identified and reviewed by an expert medical writing team, who prepare Advanced Clinical Evidence (ACETM) reports: one or two detailed pages including critical appraisals and synopses of key research. These timely and targeted reports provide a clear understanding about the quality of evidence associated with each summarized study, and can be organized by users to identify trending information. OrthoEvidence™ allows members to use their time efficiently and to stay current by having access to a breadth of timely, high-quality research output. OrthoEvidence™ is easily accessible through the internet and is available at the point-of-care, which allows treating orthopedic surgeons and allied health professionals to easily practice the principles of evidence-based medicine within their clinical practices.. PMID:26330990

  9. Reaction of arynes with vinylogous amides: nucleophilic addition to the ortho-quinodimethide intermediate.

    PubMed

    Li, Ran; Wang, Xuemei; Wei, Zhibin; Wu, Chunrui; Shi, Feng

    2013-09-01

    The reaction of arynes with vinylogous amides containing no free N-H bonds proceeds in a [2 + 2] cycloaddition fashion at ambient temperature. The electronic properties of the vinylogous amides allow for the cycloadducts undergoing a facile ring-opening process, leading to electronically biased ortho-quinodimethide intermediates. Subsequent nucleophilic addition with alcohols affords 2-substituted benzaldehydes or ketones. PMID:23957502

  10. POLYCHLORINATED BIPHENYLS AS INDUCERS OF HEPATIC MICROSOMAL ENZYMES: EFFECTS OF DI-ORTHO SUBSTITUTION

    EPA Science Inventory

    All of the 13 possible polychlorinated biphenyl (PCB) isomers and congeners substituted at both para positions, at least two meta positions (but not necessarily on the same ring) and at two ortho positions have been synthesized and tested as rat hepatic microsomal enzyme inducers...

  11. Rotational excitation of formaldehyde by hydrogen molecules: ortho-H_2CO at low temperature

    NASA Astrophysics Data System (ADS)

    Troscompt, N.; Faure, A.; Wiesenfeld, L.; Ceccarelli, C.; Valiron, P.

    2009-01-01

    Aims: Rate coefficients for the rotational excitation of the ten lowest levels of ortho-H_2CO by collisions with H2 molecules are computed for kinetic temperatures in the range 5-100 K. Methods: Cross sections are obtained from extensive, fully converged, quantum-mechanical scattering calculations using a highly accurate potential energy surface computed at the CCSD(T) level with a basis set extrapolation procedure. Scattering calculations are carried out for H2 molecules in both para and ortho rotational levels. Results: The present rates are shown to differ significantly from those available in the literature. Moreover, the strength of propensity rules is found to depend on the para/ortho form of H2. Radiative transfer modeling also shows that the new rates have a significant impact on H_2CO emission line fluxes and that they should be adopted in any detailed radiative transfer model of ortho-H_2CO in cold environments (T ⪉ 30 K). This paper is dedicated to the memory of our friend and colleague, Pierre Valiron, who died on 31 August 2008. Table of rate coefficients is available in electronic form at the CDS via anonymous ftp to cdsarc.u-strasbg.fr (130.79.128.5) or via http://cdsweb.u-strasbg.fr/cgi-bin/qcat?J/A+A/493/687

  12. Copper-mediated ortho C-H sulfonylation of benzoic acid derivatives with sodium sulfinates.

    PubMed

    Liu, Jidan; Yu, Lin; Zhuang, Shaobo; Gui, Qingwen; Chen, Xiang; Wang, Wenduo; Tan, Ze

    2015-04-14

    Copper-mediated direct ortho C-H bond sulfonylation of benzoic acid derivatives with sodium sulfinates was achieved by employing an 8-aminoquinoline moiety as the bidentate directing group. Various aryl sulfones were synthesized in good yields with excellent regioselectivity. PMID:25766975

  13. The Ortho-Syllable as a Processing Unit in Handwriting: The Mute E Effect

    ERIC Educational Resources Information Center

    Lambert, Eric; Sausset, Solen; Rigalleau, François

    2015-01-01

    Some research on written production has focused on the role of the syllable as a processing unit. However, the precise nature of this syllable unit has yet to be elucidated. The present study examined whether the nature of this processing unit is orthographic (i.e., an ortho-syllable) or phonological. We asked French adults to copy three-syllable…

  14. Palladium-Catalyzed Ortho-Arylation of Carbamate-Protected Estrogens.

    PubMed

    Bedford, Robin B; Brenner, Peter B; Durrant, Steven J; Gallagher, Timothy; Méndez-Gálvez, Carolina; Montgomery, Michelle

    2016-05-01

    The palladium-catalyzed ortho-arylation of diethyl carbamate-protected estrone and estriol with aryl iodides gives the 2-arylated analogues. Subsequent removal of the carbamate directing group furnishes 2-arylated estrone, estradiol, or estriol depending on the method used. PMID:27057762

  15. Regioselective desymmetrization of diaryltetrahydrofurans via directed ortho-lithiation: an unexpected help from green chemistry.

    PubMed

    Mallardo, Valentina; Rizzi, Ruggiero; Sassone, Francesca C; Mansueto, Rosmara; Perna, Filippo M; Salomone, Antonio; Capriati, Vito

    2014-08-14

    An efficient functionalization of diaryltetrahydrofurans via a regioselective THF-directed ortho-lithiation is first described. This reaction can be successfully carried out in cyclopentyl methyl ether as a "greener" alternative to Et2O, with better results in terms of yield and selectivity and, surprisingly, also in protic eutectic mixtures competitively with protonolysis. PMID:24968025

  16. USE OF THE 'ORTHO EFFECT' FOR CHLORINATED BIPHENYL AND BROMINATED BIPHENYL ISOMER IDENTIFICATION

    EPA Science Inventory

    The ortho effect observed for chlorinated biphenyls (PCBs) and brominated biphenyls (PBBs) having 2,2; 2,2', 6 or 2,2', 6,6' halogens, can be combined with GC retention index for isomer specific identifications by gas chromatography/mass spectrometry (GC/MS). This technique relia...

  17. Gut-targeted immunonutrition boosting natural killer cell activity using Saccharomyces boulardii lysates in immuno-compromised healthy elderly subjects.

    PubMed

    Naito, Yasuhiro; Marotta, Francesco; Kantah, Makoto K; Zerbinati, Nicola; Kushugulova, Almagul; Zhumadilov, Zhaxybay; Illuzzi, Nicola; Sapienza, Chiara; Takadanohara, Hiroshi; Kobayashi, Riyichi; Catanzaro, Roberto

    2014-04-01

    The aim of this study was to assess the immunomodulatory effect of KC-1317 (a symbiotic mixture containing Saccharomyces boulardii lysate in a cranberry, colostrum-derived lactoferrin, fragaria, and lactose mixture) supplementation in immune-compromised but otherwise healthy elderly subjects. A liquid formulation of KC-1317 was administered in a randomized controlled trial (RCT) fashion to healthy volunteers (65-79 years) previously selected for low natural killer (NK) cell activity, and this parameter was checked at the completion of the study. A significant improvement in NK cell activity of KC-1317 consumers was observed as compared to placebo at the end of 2 months. Although preliminary, these beneficial immune-modulatory effects of KC-1317 in aged individuals might indicate its employment within a wider age-management strategy. PMID:24059806

  18. Tobacco BY-2 cell-free lysate: an alternative and highly-productive plant-based in vitro translation system

    PubMed Central

    2014-01-01

    Background Cell-free protein synthesis is a rapid and efficient method for the production of recombinant proteins. Usage of prokaryotic cell-free extracts often leads to non-functional proteins. Eukaryotic counterparts such as wheat germ extract (WGE) and rabbit reticulocyte lysate (RLL) may improve solubility and promote the correct folding of eukaryotic multi-domain proteins that are difficult to express in bacteria. However, the preparation of WGEs is complex and time-consuming, whereas RLLs suffer from low yields. Here we report the development of a novel cell-free system based on tobacco Bright Yellow 2 (BY-2) cells harvested in the exponential growth phase. Results The highly-productive BY-2 lysate (BYL) can be prepared quickly within 4–5 h, compared to 4–5 d for WGE. The efficiency of the BYL was tested using three model proteins: enhanced yellow fluorescent protein (eYFP) and two versions of luciferase. The added mRNA was optimized by testing different 5’ and 3’ untranslated regions (UTRs). The protein yield in batch and dialysis reactions using BYL was much higher than that of a commercial Promega WGE preparation, achieving a maximum yield of 80 μg/mL of eYFP and 100 μg/mL of luciferase, compared to only 45 μg/mL of eYFP and 35 μg/mL of luciferase in WGEs. In dialysis reactions, the BYL yielded about 400 μg/mL eYFP, representing up to 50% more of the target protein than the Promega WGE, and equivalent to the amount using 5Prime WGE system. Conclusions Due to the high yield and the short preparation time the BYL represents a remarkable improvement over current eukaryotic cell-free systems. PMID:24886601

  19. Antigen detection systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infectious agents or their constituent parts (antigens or nucleic acids) can be detected in fresh, frozen, or fixed tissue using a variety of direct or indirect assays. The assays can be modified to yield the greatest sensitivity and specificity but in most cases a particular methodology is chosen ...

  20. Antigen smuggling in tuberculosis.

    PubMed

    Hudrisier, Denis; Neyrolles, Olivier

    2014-06-11

    The importance of CD4 T lymphocytes in immunity to M. tuberculosis is well established; however, how dendritic cells activate T cells in vivo remains obscure. In this issue of Cell Host & Microbe, Srivastava and Ernst (2014) report a mechanism of antigen transfer for efficient activation of antimycobacterial T cells. PMID:24922567

  1. Antigen detection systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infectious agents or their constituent parts (antigens or nucleic acids) can be detected in fresh, frozen, or fixed tissues or other specimens, using a variety of direct or indirect assays. The assays can be modified to yield the greatest sensitivity and specificity but in most cases a particular m...

  2. Aspergillus antigen skin test (image)

    MedlinePlus

    The aspergillus antigen skin test determines whether or not a person has been exposed to the mold aspergillus. It is performed by injecting an aspergillus antigen under the skin with a needle. After 48 ...

  3. Microwave-Based Reaction Screening: Tandem Retro-Diels-Alder/Diels-Alder Cycloadditions of ortho-Quinol Dimers

    PubMed Central

    Dong, Suwei; Cahill, Kath arine J.; Kang, Moon -Il; Colburn, Nancy H.; Henrich, Curtis J.; Wilson, Jennifer A.; Beutler, John A.; Johnson, Richard P.; Porco, John A.

    2011-01-01

    We have accomplished a parallel screen of cycloaddition partners for ortho-quinols utilizing a plate-based microwave system. Microwave irradiation improves the efficiency of retro-Diels-Alder/Diels-Alder cascades of ortho-quinol dimers which generally proceed in a diastereoselective fashion. Computational studies indicate that asynchronous transition states are favored in Diels-Alder cycloadditions of ortho-quinols. Subsequent biological evaluation of a collection of cycloadducts has identified an inhibitor of activator protein-1 (AP-1), an oncogenic transcription factor. PMID:21942286

  4. Antigenic characterization of Helicobacter pylori strains from different parts of the world.

    PubMed Central

    Höök-Nikanne, J; Perez-Perez, G I; Blaser, M J

    1997-01-01

    Although Helicobacter pylori is considered to be relatively homogeneous at the phenotypic level, our aim was to describe its antigenic heterogeneity and to examine differences in host response. Whole-cell lysates of H. pylori strains originally isolated from persons from Africa, the People's Republic of China, Japan, Peru, Thailand, or the United States or from monkeys were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblots were performed by using sera from H. pylori-infected persons from different areas of the world and rabbit immune sera against H. pylori antigens. Specific H. pylori antibody responses in persons from the United States and the People's Republic of China were analyzed by enzyme-linked immunosorbent assay with antigens prepared from U.S. or Chinese strains. Despite diverse origins, the strains showed conserved major bands of 84, 60, 56, 31, and 25 kDa. Although there were clear differences in minor bands, there was no obvious geographic pattern. The anti-CagA serum recognized 120- to 140-kDa bands in cagA+ strains from around the world. Although antigenic preparations from individual U.S. or Chinese strains were not optimally sensitive for serologic detection of infection in the heterologous country, use of pools of strains largely overcame this phenomenon. We conclude that conserved H. pylori antigens exist and are recognized by sera from persons from many parts of the world. The heterogeneity of H. pylori antigens and the serological responses of infected hosts is not fully explained by geographic differences. Use of pools may allow development of antigens for serologic testing in any country. PMID:9302211

  5. Vaccination with autologous dendritic cells loaded with autologous tumor lysate or homogenate combined with immunomodulating radiotherapy and/or preleukapheresis IFN-α in patients with metastatic melanoma: a randomised “proof-of-principle” phase II study

    PubMed Central

    2014-01-01

    Background Vaccination with dendritic cells (DC) loaded with tumor antigens elicits tumor-specific immune responses capable of killing cancer cells without inducing meaningful side-effects. Patients with advanced melanoma enrolled onto our phase II clinical studies have been treated with autologous DC loaded with autologous tumor lysate/homogenate matured with a cytokine cocktail, showing a clinical benefit (PR + SD) in 55.5% of evaluable cases to date. The beneficial effects of the vaccine were mainly restricted to patients who developed vaccine-specific immune response after treatment. However, immunological responses were only induced in about two-thirds of patients, and treatments aimed at improving immunological responsiveness to the vaccine are needed. Methods/Design This is a phase II, “proof-of-principle”, randomized, open-label trial of vaccination with autologous DC loaded with tumor lysate or homogenate in metastatic melanoma patients combined with immunomodulating RT and/or preleukapheresis IFN-α. All patients will receive four bi-weekly doses of the vaccine during the induction phase and monthly doses thereafter for up to a maximum of 14 vaccinations or until confirmed progression. Patients will be randomized to receive: (1.) three daily doses of 8 Gy up to 12 Gy radiotherapy delivered to one non-index metastatic field between vaccine doses 1 and 2 and, optionally, between doses 7 and 8, using IMRT-IMAT techniques; (2.) daily 3 MU subcutaneous IFN-α for 7 days before leukapheresis; (3.) both 1 and 2; (4.) neither 1 nor 2. At least six patients eligible for treatment will be enrolled per arm. Daily 3 MU IL-2 will be administered subcutaneously for 5 days starting from the second day after each vaccine dose. Serial DTH testing and blood sampling to evaluate treatment-induced immune response will be performed. Objective response will be evaluated according to immune-related response criteria (irRC). Discussion Based upon the emerging role of

  6. Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins

    PubMed Central

    Zhong, Nan; Loppnau, Peter; Seitova, Alma; Ravichandran, Mani; Fenner, Maria; Jain, Harshika; Bhattacharya, Anandi; Hutchinson, Ashley; Paduch, Marcin; Lu, Vincent; Olszewski, Michal; Kossiakoff, Anthony A.; Dowdell, Evan; Koide, Akiko; Koide, Shohei; Huang, Haiming; Nadeem, Vincent; Sidhu, Sachdev S.; Greenblatt, Jack F.; Marcon, Edyta; Arrowsmith, Cheryl H.; Edwards, Aled M.; Gräslund, Susanne

    2015-01-01

    We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols. PMID:26437229

  7. Cancer testis antigen and immunotherapy

    PubMed Central

    Krishnadas, Deepa Kolaseri; Bai, Fanqi; Lucas, Kenneth G

    2013-01-01

    The identification of cancer testis (CT) antigens has been an important advance in determining potential targets for cancer immunotherapy. Multiple previous studies have shown that CT antigen vaccines, using both peptides and dendritic cell vaccines, can elicit clinical and immunologic responses in several different tumors. This review details the expression of melanoma antigen family A, 1 (MAGE-A1), melanoma antigen family A, 3 (MAGE-A3), and New York esophageal squamous cell carcinoma-1 (NY-ESO-1) in various malignancies, and presents our current understanding of CT antigen based immunotherapy.

  8. The Metabolic Fate of ortho-Quinones Derived from Catecholamine Metabolites

    PubMed Central

    Ito, Shosuke; Yamanaka, Yuta; Ojika, Makoto; Wakamatsu, Kazumasa

    2016-01-01

    ortho-Quinones are produced in vivo through the oxidation of catecholic substrates by enzymes such as tyrosinase or by transition metal ions. Neuromelanin, a dark pigment present in the substantia nigra and locus coeruleus of the brain, is produced from dopamine (DA) and norepinephrine (NE) via an interaction with cysteine, but it also incorporates their alcoholic and acidic metabolites. In this study we examined the metabolic fate of ortho-quinones derived from the catecholamine metabolites, 3,4-dihydroxyphenylethanol (DOPE), 3,4-dihydroxyphenylethylene glycol (DOPEG), 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylmandelic acid (DOMA). The oxidation of catecholic substrates by mushroom tyrosinase was followed by UV-visible spectrophotometry. HPLC analysis after reduction with NaBH4 or ascorbic acid enabled measurement of the half-lives of ortho-quinones and the identification of their reaction products. Spectrophotometric examination showed that the ortho-quinones initially formed underwent extensive degradation at pH 6.8. HPLC analysis showed that DOPE-quinone and DOPEG-quinone degraded with half-lives of 15 and 30 min at pH 6.8, respectively, and >100 min at pH 5.3. The major product from DOPE-quinone was DOPEG which was produced through the addition of a water molecule to the quinone methide intermediate. DOPEG-quinone yielded a ketone, 2-oxo-DOPE, through the quinone methide intermediate. DOPAC-quinone and DOMA-quinone degraded immediately with decarboxylation of the ortho-quinone intermediates to form 3,4-dihydroxybenzylalcohol (DHBAlc) and 3,4-dihydroxybenzaldehyde (DHBAld), respectively. DHBAlc-quinone was converted to DHBAld with a half-life of 9 min, while DHBAld-quinone degraded rapidly with a half-life of 3 min. This study confirmed the fact that ortho-quinones from DOPE, DOPEG, DOPAC and DOMA are converted to quinone methide tautomers as common intermediates, through proton rearrangement or decarboxylation. The unstable quinone methides

  9. The Metabolic Fate of ortho-Quinones Derived from Catecholamine Metabolites.

    PubMed

    Ito, Shosuke; Yamanaka, Yuta; Ojika, Makoto; Wakamatsu, Kazumasa

    2016-01-01

    ortho-Quinones are produced in vivo through the oxidation of catecholic substrates by enzymes such as tyrosinase or by transition metal ions. Neuromelanin, a dark pigment present in the substantia nigra and locus coeruleus of the brain, is produced from dopamine (DA) and norepinephrine (NE) via an interaction with cysteine, but it also incorporates their alcoholic and acidic metabolites. In this study we examined the metabolic fate of ortho-quinones derived from the catecholamine metabolites, 3,4-dihydroxyphenylethanol (DOPE), 3,4-dihydroxyphenylethylene glycol (DOPEG), 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylmandelic acid (DOMA). The oxidation of catecholic substrates by mushroom tyrosinase was followed by UV-visible spectrophotometry. HPLC analysis after reduction with NaBH₄ or ascorbic acid enabled measurement of the half-lives of ortho-quinones and the identification of their reaction products. Spectrophotometric examination showed that the ortho-quinones initially formed underwent extensive degradation at pH 6.8. HPLC analysis showed that DOPE-quinone and DOPEG-quinone degraded with half-lives of 15 and 30 min at pH 6.8, respectively, and >100 min at pH 5.3. The major product from DOPE-quinone was DOPEG which was produced through the addition of a water molecule to the quinone methide intermediate. DOPEG-quinone yielded a ketone, 2-oxo-DOPE, through the quinone methide intermediate. DOPAC-quinone and DOMA-quinone degraded immediately with decarboxylation of the ortho-quinone intermediates to form 3,4-dihydroxybenzylalcohol (DHBAlc) and 3,4-dihydroxybenzaldehyde (DHBAld), respectively. DHBAlc-quinone was converted to DHBAld with a half-life of 9 min, while DHBAld-quinone degraded rapidly with a half-life of 3 min. This study confirmed the fact that ortho-quinones from DOPE, DOPEG, DOPAC and DOMA are converted to quinone methide tautomers as common intermediates, through proton rearrangement or decarboxylation. The unstable quinone

  10. Hydrogen adsorption on two catalysts for the ortho- to parahydrogen conversion: Cr-doped silica and ferric oxide gel.

    PubMed

    Hartl, Monika; Gillis, Robert Chad; Daemen, Luke; Olds, Daniel P; Page, Katherine; Carlson, Stefan; Cheng, Yongqiang; Hügle, Thomas; Iverson, Erik B; Ramirez-Cuesta, A J; Lee, Yongjoong; Muhrer, Günter

    2016-06-29

    Molecular hydrogen exists in two spin-rotation coupled states: parahydrogen and orthohydrogen. Due to the variation of energy with rotational level, the occupation of ortho- and parahydrogen states is temperature dependent, with parahydrogen being the dominant species at low temperatures. The equilibrium at 20 K (99.8% parahydrogen) can be reached by natural conversion only after a lengthy process. With the use of a suitable catalyst, this process can be shortened significantly. Two types of commercial catalysts currently being used for ortho- to parahydrogen conversion are: iron(iii) oxide (Fe2O3, IONEX®), and chromium(ii) oxide doped silica catalyst (CrO·SiO2, OXISORB®). We investigate the interaction of ortho- and parahydrogen with the surfaces of these ortho-para conversion catalysts using neutron vibrational spectroscopy. The catalytic surfaces have been characterized using X-ray absorption fine structure (XAFS) and X-ray/neutron pair distribution function measurements. PMID:27149564

  11. A Catalyst-Controlled Aerobic Coupling of ortho-Quinones and Phenols Applied to the Synthesis of Aryl Ethers.

    PubMed

    Huang, Zheng; Lumb, Jean-Philip

    2016-09-12

    ortho-Quinones are underutilized six-carbon-atom building blocks. We herein describe an approach for controlling their reactivity with copper that gives rise to a catalytic aerobic cross-coupling with phenols. The resulting aryl ethers are generated in high yield across a broad substrate scope under mild conditions. This method represents a unique example where the covalent modification of an ortho-quinone is catalyzed by a transition metal, creating new opportunities for their utilization in synthesis. PMID:27513295

  12. Double-antigen sandwich ELISA for the detection of anti-hepatitis C virus antibodies.

    PubMed

    He, Jing; Xiu, Bingshui; Wang, Guohua; Chen, Kun; Feng, Xiaoyan; Song, Xiaoguo; Zhu, Cuixia; Ling, Shigan; Zhang, Heqiu

    2011-01-01

    A double-antigen sandwich ELISA was developed a detection of HCV antibodies by a recombinant multi-epitope HCV antigen and a biotin-streptavidin amplification system. Three plasma specimens from 1708 individuals who were suspected previously to be HCV-positive using an HCV antibody diagnostic kit (Chuangxin, Xiamen, China) displayed negative results when using the ELISA. These results were validated by a recombinant immunoblotting assay (two were negative, and one was indeterminate). Among 889 blood specimens donated for clinical evaluation, 246 were positive and 630 were negative using the ELISA. The sensitivity and specificity of the ELISA were 98.7% and 100%, respectively. In 43 donors and 14 patients with chronic hepatitis C, the detectable rates for HCV IgM by both ELISA and the HCV anti-IgM detection reagents (Huimin, Shenyang, China) were 100%, and the detectable rate for HCV IgG using an indirect HCV-antibody detection kit (GWK, Beijing, China) was 98.3%. Thus, the double-antigen sandwich ELISA exhibits strong specificity and sensitivity and has been approved by the China State Food and Drug Administration (SFDA). The performance of the double-antigen sandwich ELISA was similar to the Ortho ELISA 3.0. It did not give false-negative results otherwise IgM was undetectable using an indirect HCV-antibody detection kit. This ELISA provides another method for the detection of HCV antibodies. PMID:21029749

  13. An antibacterial ortho-quinone diterpenoid and its derivatives from Caryopteris mongolica.

    PubMed

    Saruul, Erdenebileg; Murata, Toshihiro; Selenge, Erdenechimeg; Sasaki, Kenroh; Yoshizaki, Fumihiko; Batkhuu, Javzan

    2015-06-15

    To identify antibacterial components in traditional Mongolian medicinal plant Caryopteris mongolica, an ortho-quinone abietane caryopteron A (1) and three its derivatives caryopteron B-D (2-4) were isolated from the roots of the plant together with three known abietanes demethylcryptojaponol (5), 6α-hydroxydemethyl cryptojaponol (6), and 14-deoxycoleon U (7). The chemical structures of these abietane derivatives were elucidated on the basis of spectroscopic data. Compounds 1-4 had C-13 methylcyclopropane substructures, and 2-4 had a hexanedioic anhydride ring C instead of ortho-quinone in 1. The stereochemistry of these compound was assumed from NOE spectra and ECD Cotton effects. Compounds 1 and 5-7 showed antibacterial activities against the Gram-positive bacteria Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Micrococcus luteus, being 1 the more potent. PMID:25958242

  14. Luminescence properties of Sm3+-doped alkaline earth ortho-stannates

    NASA Astrophysics Data System (ADS)

    Stanulis, Andrius; Katelnikovas, Artūras; Enseling, David; Dutczak, Danuta; Šakirzanovas, Simas; Bael, Marlies Van; Hardy, An; Kareiva, Aivaras; Jüstel, Thomas

    2014-05-01

    A series of Sm3+ doped M2SnO4 (M = Ca, Sr and Ba) samples were prepared by a conventional high temperature solid-state reaction route. All samples were characterized by powder X-ray diffraction (XRD) analysis, photoluminescence (PL), photoluminescence thermal quenching (TQ) and fluorescence lifetime (FL) measurements. The morphology of synthesized phosphor powders was examined by scanning electron microscopy (SEM). Moreover, luminous efficacies (LE) and color points of the CIE 1931 color space diagram were calculated and discussed. Synthesized powders showed bright orange-red emission under UV excitation. Based on the results obtained we demonstrate that Sm3+ ions occupy Ca and Sr sites in the Ca2SnO4 and Sr2SnO4 ortho-stannate structures, respectively. In contrast, Sm3+ substitutes Sn in the barium ortho-stannate Ba2SnO4 structure.

  15. A DFT study of permanganate oxidation of toluene and its ortho-nitroderivatives.

    PubMed

    Adamczyk, Paweł; Wijker, Reto S; Hofstetter, Thomas B; Paneth, Piotr

    2014-02-01

    Calculations of alternative oxidation pathways of toluene and its ortho-substituted nitro derivatives by permanganate anion have been performed. The competition between methyl group and ring oxidation has been addressed. Acceptable results have been obtained using IEFPCM/B3LYP/6-31+G(d,p) calculations with zero-point (ZPC) and thermal corrections, as validated by comparison with the experimental data. It has been shown that ring oxidation reactions proceed via relatively early transition states that become quite unsymmetrical for reactions involving ortho-nitrosubstituted derivatives. Transition states for the hydrogen atom abstraction reactions, on the other hand, are late. All favored reactions are characterized by the Gibbs free energy of activation, ΔG(≠), of about 25 kcal mol(-1). Methyl group oxidations are exothermic by about 20 kcal mol(-1) while ring oxidations are around thermoneutrality. PMID:24526379

  16. Interplay of ortho- with spiro-cyclisation during iminyl radical closures onto arenes and heteroarenes

    PubMed Central

    2013-01-01

    Summary Sensitised photolyses of ethoxycarbonyl oximes of aromatic and heteroaromatic ketones yielded iminyl radicals, which were characterised by EPR spectroscopy. Iminyls with suitably placed arene or heteroarene acceptors underwent cyclisations yielding phenanthridine-type products from ortho-additions. For benzofuran and benzothiophene acceptors, spiro-cyclisation predominated at low temperatures, but thermodynamic control ensured ortho-products, benzofuro- or benzothieno-isoquinolines, formed at higher temperatures. Estimates by steady-state kinetic EPR established that iminyl radical cyclisations onto aromatics took place about an order of magnitude more slowly than prototypical C-centred radicals. The cyclisation energetics were investigated by DFT computations, which gave insights into factors influencing the two cyclisation modes. PMID:23766822

  17. orthoFind Facilitates the Discovery of Homologous and Orthologous Proteins.

    PubMed

    Mier, Pablo; Andrade-Navarro, Miguel A; Pérez-Pulido, Antonio J

    2015-01-01

    Finding homologous and orthologous protein sequences is often the first step in evolutionary studies, annotation projects, and experiments of functional complementation. Despite all currently available computational tools, there is a requirement for easy-to-use tools that provide functional information. Here, a new web application called orthoFind is presented, which allows a quick search for homologous and orthologous proteins given one or more query sequences, allowing a recurrent and exhaustive search against reference proteomes, and being able to include user databases. It addresses the protein multidomain problem, searching for homologs with the same domain architecture, and gives a simple functional analysis of the results to help in the annotation process. orthoFind is easy to use and has been proven to provide accurate results with different datasets. Availability: http://www.bioinfocabd.upo.es/orthofind/. PMID:26624019

  18. Geometry and B(1s) core excitons of ortho-carborane

    SciTech Connect

    Green, T.A. )

    1991-07-01

    The optimized C{sub 2v} geometry of ortho-carborane, 1,2-C{sub 2}B{sub 10}H{sub 12}, is determined from Hartree-Fock calculations. For this geometry, a carbon atom is substituted for a boron atom at one of the 4 inequivalent boron sites and the ground-state unrestricted Hartree-Fock eigenvalues and molecular orbitals are found. One thus obtains the valence structure of the B(1s) core-excited molecule according to the Z+1 approximation. The eigenvalue of the highest occupied molecular orbital is then subtracted from the experimental B(1s) ionization energy of the same site in ortho-carborane. This determines the excitation energy of the most tightly bound exciton for that site. Three of the sites yield nearly identical excitation energies of 191.9 eV; the fourth site yields an excitation energy of 190.9 eV.

  19. 1,2-Azaborine: The Boron-Nitrogen Derivative of ortho-Benzyne.

    PubMed

    Edel, Klara; Brough, Sarah A; Lamm, Ashley N; Liu, Shih-Yuan; Bettinger, Holger F

    2015-06-26

    The BN analogue of ortho-benzyne, 1,2-azaborine, is generated by flash vacuum pyrolysis, trapped under cryogenic conditions, and studied by direct spectroscopic techniques. The parent BN aryne spontaneously binds N2 and CO2, thus demonstrating its highly reactive nature. The interaction with N2 is photochemically reversible. The CO2 adduct of 1,2-azaborine is a cyclic carbamate which undergoes photocleavage, thus resulting in overall CO2 splitting. PMID:26095444

  20. Constraining the ortho-to-para ratio of H2 with anomalous H_2CO absorption

    NASA Astrophysics Data System (ADS)

    Troscompt, N.; Faure, A.; Maret, S.; Ceccarelli, C.; Hily-Blant, P.; Wiesenfeld, L.

    2009-11-01

    Context: The ortho-to-para ratio (OPR) of molecular hydrogen is a fundamental parameter in understanding the physics and chemistry of molecular clouds. In dark and cold regions, however, H2 is not directly observable and the OPR of H2 in these sources has so far remained elusive. Aims: We show that the 6 cm absorption line of ortho-formaldehyde (H2CO) can be employed to constrain both the density and the OPR of H2 in dark clouds. Methods: Green Bank Telescope (GBT) observations of ortho-H2CO toward the molecular cloud Barnard 68 (B68) are reported. Non-LTE radiative transfer calculations combined with the well-constrained structure of B68 are then employed to derive the physical conditions in the absorption region. Results: We provide the first firm confirmation of the Townes & Cheung mechanism: propensity rules for the collisions of H2CO with H2 molecules are responsible for the sub-2.7 K cooling of the 6 cm doublet. Non-LTE calculations show that in the absorption region of B68, the kinetic temperature is ˜ 10 K, the ortho-H2CO column density amounts to ˜ 2.2× 1013 cm-2, the H2 density is in the range 1.4{-}2.4× 10 4 cm-3, and the OPR of H2 is close to zero. Our observations thus provide fresh evidence that H2 is mostly in its para form in the cold gas, as expected from theoretical considerations. Our results also suggest that formaldehyde absorption originates in the edge of B68, at visual extinctions A_V⪉ 0.5 mag. This work has been inspired by our colleague and friend Pierre Valiron, who passed away in August 2008. This paper is dedicated to his memory.

  1. Enantioselective Addition of Boronates to Ortho-Quinone Methides Catalyzed by Chiral Biphenols

    PubMed Central

    Luan, Yi; Schaus, Scott E.

    2014-01-01

    Chiral biphenols were found to catalyze the enantioselective asymmetric addition of aryl- or alkenyl-boronates to ortho-quinone methides. Substituted 2-styryl phenols were obtained in good yields (up to 95%) and high enantiomeric ratios (up to 98:2) in presence of 10 mol % of 3,3′-Br2-BINOL. A two step synthesis of (S)-4-Methoxy-dalbergione is achieved in good yield and selectivity. PMID:23206197

  2. Branch-Selective Alkene Hydroarylation by Cooperative Destabilization: Iridium-Catalyzed ortho-Alkylation of Acetanilides

    PubMed Central

    Crisenza, Giacomo E M; Sokolova, Olga O; Bower, John F

    2015-01-01

    An iridium(I) catalyst system, modified with the wide-bite-angle and electron-deficient bisphosphine dFppb (1,4-bis(di(pentafluorophenyl)phosphino)butane) promotes highly branch-selective hydroarylation reactions between diverse acetanilides and aryl- or alkyl-substituted alkenes. This provides direct and ortho-selective access to synthetically challenging anilines, and addresses long-standing issues associated with related Friedel–Crafts alkylations. PMID:26490739

  3. Palladium-Catalyzed Heteroarylation and Concomitant ortho-Alkylation of Aryl Iodides.

    PubMed

    Lei, Chuanhu; Jin, Xiaojia; Zhou, Jianrong Steve

    2015-11-01

    Three-component couplings were achieved from common aryl halides, alkyl halides, and heteroarenes under palladium and norbornene co-catalysis. The reaction forges hindered aryl-heteroaryl bonds and introduces ortho-alkyl groups to aryl rings. Various heterocycles such as oxazoles, thiazoles and thiophenes underwent efficient coupling. The heteroarenes were deprotonated in situ by bases without the assistance of palladium catalysts. PMID:26358935

  4. Copper-catalyzed divergent oxidative pathways of 2-naphthol derivatives: ortho-naphthoquinones versus 2-BINOLs.

    PubMed

    Kim, H Y; Takizawa, S; Oh, K

    2016-07-26

    Catalyst-dependent divergent pathways of 2-naphthol derivatives have been investigated. A readily available CuCl2-DMAP catalyst system promotes the aerobic oxidation of 2-naphthol derivatives to ortho-naphthoquinones whereas switching the catalyst system to Cu(OAc)2-DBN under an argon atmosphere allows the oxidative coupling of 2-naphthols to 1,1'-bi-2-naphthols (BINOLs) in good to excellent yields. PMID:27404292

  5. Cranioplasty Results after Application of Anti-adhesive Films (OrthoWrap™) in Traumatic Decompressive Craniectomy

    PubMed Central

    Khalili, Hosseinali; Omidvar, Aydin; Ghaffarpasand, Fariborz; Yadollahikhales, Golnaz

    2016-01-01

    Objective: To determine the effects of application of anti-adhesive films (OrthoWrap™) in traumatic decompressive craniectomy on prevention of adhesion formation and facilitation of subsequent cranioplasty. Methods: This was a retrospective cohort study being performed in ShahidRajaei hospital (Shiraz Level I trauma center) during a 12-month period (from March 2012 to April 2013) including 93 patients undergoing traumatic decompressivecraniectomy.Patients who received OrthoWrap™ during the initial craniectomy (n=44) were compared to those who did not (n=49). Two study groups were matched regarding the baseline characteristics. The perioperative indices including the surgical time, amount of bleeding, transfusion and 6-month Glasgow Outcome Scale (GOS) were compared between two study groups. Results: There was no significant difference between two study groups regarding the baseline characteristics. We found that the cranioplasty duration (113.3±33.2 vs. 146.9±34.9 minutes; p<0.001) and amount of intraoperative bleeding (182.1±98.3 vs. 270.6±77.6 mL; p=0.043) was significantly lower in those who had OrthoWrap™ compared to control group. The final GCS (p=0.052) as well as GOSE (p=0.653) was comparable between groups. The infection rate was comparable between two study groups (p=0.263). Conclusion: Application of OrthoWrap™ during decompressive craniectomy in those with severe traumatic brain injury is associated with shorter duration of operation and less intraoperative bleeding in subsequent cranioplasty. Infection rate and neurologic outcome was comparable between study groups. PMID:27162923

  6. The ortho:para-H_2 ratio in C- and J-type shocks

    NASA Astrophysics Data System (ADS)

    Wilgenbus, D.; Cabrit, S.; Pineau des Forêts, G.; Flower, D. R.

    2000-04-01

    We have computed extensive grids of models of both C- and J-type planar shock waves, propagating in dark, cold molecular clouds, in order to study systematically the behaviour of the ortho:para-H_2 ratio. Careful attention was paid to both macroscopic (dynamical) and microscopic (chemical reactions and collisional population transfer in H_2) aspects. We relate the predictions of the models to observational determinations of the ortho:para-H_2 ratio using both pure rotational lines and rovibrational lines. As an illustration, we consider ISO and ground-based H_2 observations of HH 54. Neither planar C-type nor planar J-type shocks appear able to account fully for these observations. Given the additional constraints provided by the observed ortho:para H_2 ratios, a C-type bowshock, or a C-type precursor followed by a J-type shock, remain as plausible models. Tables~2a-f and 4a-f are only available in electronic form at the CDS via anonymous ftp to cdsarc.u-strasbg.fr (130.79.128.5) or via http://cdsweb.u-strasbg.fr/Abstract.html

  7. Theoretical study of the design of a catalyst for para to ortho hydrogen conversion

    NASA Technical Reports Server (NTRS)

    Coffman, Robert E.

    1992-01-01

    The theory of Petzinger and Scalapino (1973) was thoroughly reviewed, and all of the basic equations for paramagnetic para to ortho hydrogen catalysis re-derived. There are only a few minor phase errors and errors of omission in the description of the theory. Three models (described by Petzinger and Scalapino) for the rate of para to ortho H2 catalysis were worked out, and uniform agreement obtained to within a constant factor of 2 pi. The analytical methods developed in the course of this study were then extended to two new models, which more adequately describe the process of surface catalysis including transfer of hydrogen molecules onto and off of the surface. All five equations for the para to ortho catalytic rate of conversion are described. The two new equations describe the catalytic rate for these models: H2 on the surface is a 2-D gas with lifetime tau; and H2 on the surface is a 2-D liquid undergoing Brownian motion (diffusion) with surface lifetime tau.

  8. The ortho:para ratio of H{sub 3}{sup +} in laboratory and astrophysical plasmas

    SciTech Connect

    Crabtree, Kyle N.; Indriolo, Nick; Kreckel, Holger; McCall, Benjamin J.

    2015-01-22

    The discovery of H{sub 3}{sup +} in the diffuse interstellar medium has dramatically changed our view of the cosmic-ray ionization rate in diffuse molecular clouds. However, another surprise has been that the ortho:para ratio of H{sub 3}{sup +} in these clouds is inconsistent with the temperature derived from the excitation of H{sub 2}, the dominant species in these clouds. In an effort to understand this discrepancy, we have embarked on an experimental program to measure the nuclear spin dependence of the dissociative electron recombination rate of H{sub 3}{sup +} using the CRYRING and TSR ion storage rings. We have also performed the first measurements of the reaction H{sub 3}{sup +}+H{sub 2}→H{sub 2}+H{sub 3}{sup +} below room temperature. This reaction is likely the most common bimolecular reaction in the universe, and plays an important role in interconverting ortho- and para-H{sub 3}{sup +}. Finally, we have constructed a steady-state chemical model for diffuse clouds, which takes into account the spin-dependence of the formation of H{sub 3}{sup +}, its electron recombination, and its reaction with H{sub 2}. We find that the ortho:para ratio of H{sub 3}{sup +} in diffuse clouds is likely governed by a competition between dissociative recombination and thermalization by reactive collisions.

  9. Electrochemistry and spectroscopy of ortho-metalated complexes of Ir(III) and Rh(III)

    SciTech Connect

    Ohsawa, Y.; Sprouse, S.; King, K.A.; DeArmond, M.K.; Hanck, K.W.; Watts, R.J.

    1987-02-26

    The electrochemical and UV-visible spectroscopic properties of Rh(III) and Ir(III) complexes of the ortho-metalating (NC) ligands, 2-phenylpyridine (ppy) and benzo(h)quinone (bzq), have been studied. Cyclic voltammetric studies of several of the dimeric species, (M(NC)/sub 2/Cl)/sub 2/, indicate metal-centered oxidation occurs at moderate potentials. Cationic monomers of the type M(NC)/sub 2/(NN)/sup +/ where (NN) = 2,2'-bipyridine or 1,10-phenanthroline have been prepared by reaction of the chelating ligands with the parent dimers. Cyclic voltammetric studies of these monomers indicate that several reversible ligand-centered reductions are generally observed and that the chelating ligand is more easily reduced than is the ortho-metalating ligand. Spectroscopic studies of the mixed ligand monomers indicate that dual emissions from MLCT states associated with the ortho-metalating and chelating ligands occur in the Ir(III) complexes whereas a single emission from a ligand-localized excited state is observed in the Rh(III) complexes. These results are discussed in terms of electronic and nuclear coupling factors analogous to those encountered in descriptions of bimolecular energy and electron-transfer processes.

  10. Synthesis, structure, and spectroscopic properties of ortho-metalated platinum(II) complexes

    SciTech Connect

    Mdleleni, M.M.; Bridgewater, J.S.; Watts, R.J.; Ford, P.C.

    1995-04-26

    The ortho-metalated Pt(II) complexes Pt(ppy)(CO)Cl (1), Pt(ptpy)(CO)Cl (2), and Pt(ppy)(Hppy)Cl (3) (where ppy and ptpy are respectively the ortho-C-deprotonated forms of 2-phenylpyridine and 2-p-tolylpyridine and Hppy is 2-phenylpyridine) have been prepared. The CO ligand is coordinated trans to the nitrogen atom of the ortho-metalated ligand and exerts a strong trans effect resulting in a relatively long Pt-N bond [2.114(19) {angstrom}]. This structure shows both the bidentate ppy ligand and the monodentate Hppy with the nitrogens of these ligands trans to each other. The UV/vis electronic absorption spectra of 1-3 have intense bands in the near-UV region ({approximately}375 nm) which have been assigned as metal to ligand charge transfer (MLCT) transitions, and higher energy bands were assigned as ligand-centered transitions. Each complex exhibits relatively long-lived structured emissions in the solid state at ambient temperature and at 77 K and 77 K glassy toluene solutions. These emissions are proposed to originate from triplet MLCT states. Notably, in solution both the lifetime and spectrum of 2 proved to be a function of the concentration, a phenomenon interpreted in terms of the propensity of square planar d{sup 8} complexes to oligomerize. In contrast, the more sterically hindered complex 3 displayed no such tendency toward oligomerization.