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Sample records for affinity maturation process

  1. Strategies to guide the antibody affinity maturation process.

    PubMed

    Doria-Rose, Nicole A; Joyce, M Gordon

    2015-04-01

    Antibodies with protective activity are critical for vaccine efficacy. Affinity maturation increases antibody activity through multiple rounds of somatic hypermutation and selection in the germinal center. Identification of HIV-1 specific and influenza-specific antibody developmental pathways, as well as characterization of B cell and virus co-evolution in patients, has informed our understanding of antibody development. In order to counteract HIV-1 and influenza viral diversity, broadly neutralizing antibodies precisely target specific sites of vulnerability and require high levels of affinity maturation. We present immunization strategies that attempt to recapitulate these natural processes and guide the affinity maturation process. PMID:25913818

  2. Strategies to guide the antibody affinity maturation process

    PubMed Central

    Doria-Rose, Nicole A.; Joyce, M. Gordon

    2015-01-01

    Antibodies with protective activity are critical for vaccine efficacy. Affinity maturation increases antibody activity through multiple rounds of somatic hypermutation and selection in the germinal center. Identification of HIV-1 specific and influenza-specific antibody developmental pathways, as well as characterization of B cell and virus co-evolution in patients, has informed our understanding of antibody development. In order to counteract HIV-1 and Influenza viral diversity, broadly neutralizing antibodies precisely target specific sites of vulnerability and require high levels of affinity maturation. We present immunization strategies that attempt to recapitulate these natural processes and guide the affinity maturation process. PMID:25913818

  3. Visualizing antibody affinity maturation in germinal centers.

    PubMed

    Tas, Jeroen M J; Mesin, Luka; Pasqual, Giulia; Targ, Sasha; Jacobsen, Johanne T; Mano, Yasuko M; Chen, Casie S; Weill, Jean-Claude; Reynaud, Claude-Agnès; Browne, Edward P; Meyer-Hermann, Michael; Victora, Gabriel D

    2016-03-01

    Antibodies somatically mutate to attain high affinity in germinal centers (GCs). There, competition between B cell clones and among somatic mutants of each clone drives an increase in average affinity across the population. The extent to which higher-affinity cells eliminating competitors restricts clonal diversity is unknown. By combining multiphoton microscopy and sequencing, we show that tens to hundreds of distinct B cell clones seed each GC and that GCs lose clonal diversity at widely disparate rates. Furthermore, efficient affinity maturation can occur in the absence of homogenizing selection, ensuring that many clones can mature in parallel within the same GC. Our findings have implications for development of vaccines in which antibodies with nonimmunodominant specificities must be elicited, as is the case for HIV-1 and influenza. PMID:26912368

  4. Antibody Affinity Maturation in Fishes—Our Current Understanding

    PubMed Central

    Magor, Brad G.

    2015-01-01

    It has long been believed that fish lack antibody affinity maturation, in part because they were thought to lack germinal centers. Recent research done on sharks and bony fishes indicates that these early vertebrates are able to affinity mature their antibodies. This article reviews the functionality of the fish homologue of the immunoglobulin (Ig) mutator enzyme activation-induced cytidine deaminase (AID). We also consider the protein and molecular evidence for Ig somatic hypermutation and antibody affinity maturation. In the context of recent evidence for a putative proto-germinal center in fishes we propose some possible reasons that observed affinity maturation in fishes often seems lacking and propose future work that might shed further light on this process in fishes. PMID:26264036

  5. Co-administration of CpG oligonucleotides enhances the late affinity maturation process of human anti-hepatitis B vaccine response.

    PubMed

    Siegrist, Claire-Anne; Pihlgren, Maria; Tougne, Chantal; Efler, Sue M; Morris, Mary Lou; AlAdhami, Mohammed J; Cameron, D William; Cooper, Curtis L; Heathcote, Jenny; Davis, Heather L; Lambert, Paul-Henri

    2004-12-16

    We assessed the avidity maturation process elicited by human immunization with alum-adsorbed HBsAg alone or with a novel adjuvant containing CpG motifs (CpG 7909). Mean avidity indexes and distribution of low- and high-avidity anti-HBs indicated that avidity maturation essentially takes place late after priming. CpG 7909 markedly enhanced this affinity maturation process, increasing the pool of high-avidity antibodies. The influence of CpG 7909 was antigen-specific, isotype-specific and distinct from the influence on anti-HBs production, as avidity did not correlate with anti-HBs IgG titers. This is the first demonstration that a novel human adjuvant may induce antibodies with higher antigen-binding affinity. PMID:15542181

  6. Aptamer Affinity Maturation by Resampling and Microarray Selection.

    PubMed

    Kinghorn, Andrew B; Dirkzwager, Roderick M; Liang, Shaolin; Cheung, Yee-Wai; Fraser, Lewis A; Shiu, Simon Chi-Chin; Tang, Marco S L; Tanner, Julian A

    2016-07-19

    Aptamers have significant potential as affinity reagents, but better approaches are critically needed to discover higher affinity nucleic acids to widen the scope for their diagnostic, therapeutic, and proteomic application. Here, we report aptamer affinity maturation, a novel aptamer enhancement technique, which combines bioinformatic resampling of aptamer sequence data and microarray selection to navigate the combinatorial chemistry binding landscape. Aptamer affinity maturation is shown to improve aptamer affinity by an order of magnitude in a single round. The novel aptamers exhibited significant adaptation, the complexity of which precludes discovery by other microarray based methods. Honing aptamer sequences using aptamer affinity maturation could help optimize a next generation of nucleic acid affinity reagents. PMID:27346322

  7. Streamlining the Pipeline for Generation of Recombinant Affinity Reagents by Integrating the Affinity Maturation Step.

    PubMed

    Huang, Renhua; Gorman, Kevin T; Vinci, Chris R; Dobrovetsky, Elena; Gräslund, Susanne; Kay, Brian K

    2015-01-01

    Often when generating recombinant affinity reagents to a target, one singles out an individual binder, constructs a secondary library of variants, and affinity selects a tighter or more specific binder. To enhance the throughput of this general approach, we have developed a more integrated strategy where the "affinity maturation" step is part of the phage-display pipeline, rather than a follow-on process. In our new schema, we perform two rounds of affinity selection, followed by error-prone PCR on the pools of recovered clones, generation of secondary libraries, and three additional rounds of affinity selection, under conditions of off-rate competition. We demonstrate the utility of this approach by generating low nanomolar fibronectin type III (FN3) monobodies to five human proteins: ubiquitin-conjugating enzyme E2 R1 (CDC34), COP9 signalosome complex subunit 5 (COPS5), mitogen-activated protein kinase kinase 5 (MAP2K5), Splicing factor 3A subunit 1 (SF3A1) and ubiquitin carboxyl-terminal hydrolase 11 (USP11). The affinities of the resulting monobodies are typically in the single-digit nanomolar range. We demonstrate the utility of two binders by pulling down the targets from a spiked lysate of HeLa cells. This integrated approach should be applicable to directed evolution of any phage-displayed affinity reagent scaffold. PMID:26437402

  8. In vitro affinity maturation of a natural human antibody overcomes a barrier to in vivo affinity maturation

    PubMed Central

    Li, Bing; Fouts, Ashley E; Stengel, Katharina; Luan, Peng; Dillon, Michael; Liang, Wei-Ching; Feierbach, Becket; Kelley, Robert F; Hötzel, Isidro

    2014-01-01

    Antibodies isolated from human donors are increasingly being developed for anti-infective therapeutics. These antibodies undergo affinity maturation in vivo, minimizing the need for engineering of therapeutic leads for affinity. However, the affinities required for some therapeutic applications may be higher than the affinities of the leads obtained, requiring further affinity maturation in vitro. To improve the neutralization potency of natural human antibody MSL-109 targeting human cytomegalovirus (CMV), we affinity matured the antibody against the gH/gL glycoprotein complex. A phage display library where most of the six complementary-determining regions (CDRs) were allowed to vary in only one amino acid residue at a time was used to scan for mutations that improve binding affinity. A T55R mutation and multiple mutations in position 53 of the heavy chain were identified that, when present individually or in combination, resulted in higher apparent affinities to gH/gL and improved CMV neutralization potency of Fab fragments expressed in bacterial cells. Three of these mutations in position 53 introduced glycosylation sites in heavy chain CDR 2 (CDR H2) that impaired binding of antibodies expressed in mammalian cells. One high affinity (KD < 10 pM) variant was identified that combined the D53N and T55R mutations while avoiding glycosylation of CDR H2. However, all the amino acid substitutions identified by phage display that improved binding affinity without introducing glycosylation sites required between two and four simultaneous nucleotide mutations to avoid glycosylation. These results indicate that the natural human antibody MSL-109 is close to a local affinity optimum. We show that affinity maturation by phage display can be used to identify and bypass barriers to in vivo affinity maturation of antibodies imposed by glycosylation and codon usage. These constraints may be relatively prevalent in human antibodies due to the codon usage and the amino acid

  9. Manipulating the selection forces during affinity maturation to generate cross-reactive HIV antibodies

    PubMed Central

    Wang, Shenshen; Mata-Fink, Jordi; Kriegsman, Barry; Hanson, Melissa; Irvine, Darrell J.; Eisen, Herman N.; Burton, Dennis R.; Wittrup, K. Dane; Kardar, Mehran; Chakraborty, Arup K.

    2015-01-01

    Summary Generation of potent antibodies by a mutation-selection process called affinity maturation is a key component of effective immune responses. Antibodies that protect against highly mutable pathogens must neutralize diverse strains. Developing effective immunization strategies to drive their evolution requires understanding how affinity maturation happens in an enviroment where variants of the same antigen are present. We present an in silico model of affinity maturation driven by antigen variants which reveals that induction of cross-reactive antibodies often occurs with low probability because conflicting selection forces, imposed by different antigen variants, can frustrate affinity maturation. We describe how variables such as temporal pattern of antigen administration influence the outcome of this frustrated evolutionary process. Our calculations predict, and experiments in mice with variant gp120 constructs of the HIV envelope protein confirm, that sequential immunization with antigen variants is preferred over a cocktail for induction of cross-reactive antibodies focused on the shared CD4 binding site epitope. PMID:25662010

  10. Streamlining the Pipeline for Generation of Recombinant Affinity Reagents by Integrating the Affinity Maturation Step

    PubMed Central

    Huang, Renhua; Gorman, Kevin T.; Vinci, Chris R.; Dobrovetsky, Elena; Gräslund, Susanne; Kay, Brian K.

    2015-01-01

    Often when generating recombinant affinity reagents to a target, one singles out an individual binder, constructs a secondary library of variants, and affinity selects a tighter or more specific binder. To enhance the throughput of this general approach, we have developed a more integrated strategy where the “affinity maturation” step is part of the phage-display pipeline, rather than a follow-on process. In our new schema, we perform two rounds of affinity selection, followed by error-prone PCR on the pools of recovered clones, generation of secondary libraries, and three additional rounds of affinity selection, under conditions of off-rate competition. We demonstrate the utility of this approach by generating low nanomolar fibronectin type III (FN3) monobodies to five human proteins: ubiquitin-conjugating enzyme E2 R1 (CDC34), COP9 signalosome complex subunit 5 (COPS5), mitogen-activated protein kinase kinase 5 (MAP2K5), Splicing factor 3A subunit 1 (SF3A1) and ubiquitin carboxyl-terminal hydrolase 11 (USP11). The affinities of the resulting monobodies are typically in the single-digit nanomolar range. We demonstrate the utility of two binders by pulling down the targets from a spiked lysate of HeLa cells. This integrated approach should be applicable to directed evolution of any phage-displayed affinity reagent scaffold. PMID:26437402

  11. Reconstructing a B-Cell Clonal Lineage. II. Mutation, Selection, and Affinity Maturation

    PubMed Central

    Kepler, Thomas B.; Munshaw, Supriya; Wiehe, Kevin; Zhang, Ruijun; Yu, Jae-Sung; Woods, Christopher W.; Denny, Thomas N.; Tomaras, Georgia D.; Alam, S. Munir; Moody, M. Anthony; Kelsoe, Garnett; Liao, Hua-Xin; Haynes, Barton F.

    2014-01-01

    Affinity maturation of the antibody response is a fundamental process in adaptive immunity during which B-cells activated by infection or vaccination undergo rapid proliferation accompanied by the acquisition of point mutations in their rearranged immunoglobulin (Ig) genes and selection for increased affinity for the eliciting antigen. The rate of somatic hypermutation at any position within an Ig gene is known to depend strongly on the local DNA sequence, and Ig genes have region-specific codon biases that influence the local mutation rate within the gene resulting in increased differential mutability in the regions that encode the antigen-binding domains. We have isolated a set of clonally related natural Ig heavy chain–light chain pairs from an experimentally infected influenza patient, inferred the unmutated ancestral rearrangements and the maturation intermediates, and synthesized all the antibodies using recombinant methods. The lineage exhibits a remarkably uniform rate of improvement of the effective affinity to influenza hemagglutinin (HA) over evolutionary time, increasing 1000-fold overall from the unmutated ancestor to the best of the observed antibodies. Furthermore, analysis of selection reveals that selection and mutation bias were concordant even at the level of maturation to a single antigen. Substantial improvement in affinity to HA occurred along mutationally preferred paths in sequence space and was thus strongly facilitated by the underlying local codon biases. PMID:24795717

  12. Tailored Immunogens Direct Affinity Maturation toward HIV Neutralizing Antibodies.

    PubMed

    Briney, Bryan; Sok, Devin; Jardine, Joseph G; Kulp, Daniel W; Skog, Patrick; Menis, Sergey; Jacak, Ronald; Kalyuzhniy, Oleksandr; de Val, Natalia; Sesterhenn, Fabian; Le, Khoa M; Ramos, Alejandra; Jones, Meaghan; Saye-Francisco, Karen L; Blane, Tanya R; Spencer, Skye; Georgeson, Erik; Hu, Xiaozhen; Ozorowski, Gabriel; Adachi, Yumiko; Kubitz, Michael; Sarkar, Anita; Wilson, Ian A; Ward, Andrew B; Nemazee, David; Burton, Dennis R; Schief, William R

    2016-09-01

    Induction of broadly neutralizing antibodies (bnAbs) is a primary goal of HIV vaccine development. VRC01-class bnAbs are important vaccine leads because their precursor B cells targeted by an engineered priming immunogen are relatively common among humans. This priming immunogen has demonstrated the ability to initiate a bnAb response in animal models, but recall and maturation toward bnAb development has not been shown. Here, we report the development of boosting immunogens designed to guide the genetic and functional maturation of previously primed VRC01-class precursors. Boosting a transgenic mouse model expressing germline VRC01 heavy chains produced broad neutralization of near-native isolates (N276A) and weak neutralization of fully native HIV. Functional and genetic characteristics indicate that the boosted mAbs are consistent with partially mature VRC01-class antibodies and place them on a maturation trajectory that leads toward mature VRC01-class bnAbs. The results show how reductionist sequential immunization can guide maturation of HIV bnAb responses. PMID:27610570

  13. Affinity Maturation to Improve Human Monoclonal Antibody Neutralization Potency and Breadth against Hepatitis C Virus*

    PubMed Central

    Wang, Yong; Keck, Zhen-yong; Saha, Anasuya; Xia, Jinming; Conrad, Fraser; Lou, Jianlong; Eckart, Michael; Marks, James D.; Foung, Steven K. H.

    2011-01-01

    A potent neutralizing antibody to a conserved hepatitis C virus (HCV) epitope might overcome its extreme variability, allowing immunotherapy. The human monoclonal antibody HC-1 recognizes a conformational epitope on the HCV E2 glycoprotein. Previous studies showed that HC-1 neutralizes most HCV genotypes but has modest potency. To improve neutralization, we affinity-matured HC-1 by constructing a library of yeast-displayed HC-1 single chain Fv (scFv) mutants, using for selection an E2 antigen from one of the poorly neutralized HCVpp. We developed an approach by parallel mutagenesis of the heavy chain variable (VH) and κ-chain variable (Vk) genes separately, then combining the optimized VH and Vk mutants. This resulted in the generation of HC-1-related scFv variants exhibiting improved affinities. The best scFv variant had a 92-fold improved affinity. After conversion to IgG1, some of the antibodies exhibited a 30-fold improvement in neutralization activity. Both surface plasmon resonance and solution kinetic exclusion analysis showed that the increase in affinity was largely due to a lowering of the dissociation rate constant, Koff. Neutralization against a panel of HCV pseudoparticles and infectious 2a HCV virus improved with the affinity-matured IgG1 antibodies. Interestingly, some of these antibodies neutralized a viral isolate that was not neutralized by wild-type HC-1. Moreover, propagating 2a HCVcc under the selective pressure of WT HC-1 or affinity-matured HC-1 antibodies yielded no viral escape mutants and, with the affinity-matured IgG1, needed 100-fold less antibody to achieve complete virus elimination. Taken together, these findings suggest that affinity-matured HC-1 antibodies are excellent candidates for therapeutic development. PMID:22002064

  14. Shark Attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation.

    PubMed

    Zielonka, Stefan; Weber, Niklas; Becker, Stefan; Doerner, Achim; Christmann, Andreas; Christmann, Christine; Uth, Christina; Fritz, Janine; Schäfer, Elena; Steinmann, Björn; Empting, Martin; Ockelmann, Pia; Lierz, Michael; Kolmar, Harald

    2014-12-10

    A novel method for stepwise in vitro affinity maturation of antigen-specific shark vNAR domains is described that exclusively relies on semi-synthetic repertoires derived from non-immunized sharks. Target-specific molecules were selected from a CDR3-randomized bamboo shark (Chiloscyllium plagiosum) vNAR library using yeast surface display as platform technology. Various antigen-binding vNAR domains were easily isolated by screening against several therapeutically relevant antigens, including the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Affinity maturation was demonstrated for EpCAM and HTRA1 by diversifying CDR1 of target-enriched populations which allowed for the rapid selection of nanomolar binders. EpCAM-specific vNAR molecules were produced as soluble proteins and more extensively characterized via thermal shift assays and biolayer interferometry. Essentially, we demonstrate that high-affinity binders can be generated in vitro without largely compromising the desirable high thermostability of the vNAR scaffold. PMID:24862193

  15. Quantifying evolutionary constraints on B-cell affinity maturation

    PubMed Central

    McCoy, Connor O.; Bedford, Trevor; Minin, Vladimir N.; Bradley, Philip; Robins, Harlan; Matsen, Frederick A.

    2015-01-01

    The antibody repertoire of each individual is continuously updated by the evolutionary process of B-cell receptor (BCR) mutation and selection. It has recently become possible to gain detailed information concerning this process through high-throughput sequencing. Here, we develop modern statistical molecular evolution methods for the analysis of B-cell sequence data, and then apply them to a very deep short-read dataset of BCRs. We find that the substitution process is conserved across individuals but varies significantly across gene segments. We investigate selection on BCRs using a novel method that side-steps the difficulties encountered by previous work in differentiating between selection and motif-driven mutation; this is done through stochastic mapping and empirical Bayes estimators that compare the evolution of in-frame and out-of-frame rearrangements. We use this new method to derive a per-residue map of selection, which provides a more nuanced view of the constraints on framework and variable regions. PMID:26194758

  16. Quantifying evolutionary constraints on B-cell affinity maturation.

    PubMed

    McCoy, Connor O; Bedford, Trevor; Minin, Vladimir N; Bradley, Philip; Robins, Harlan; Matsen, Frederick A

    2015-09-01

    The antibody repertoire of each individual is continuously updated by the evolutionary process of B-cell receptor (BCR) mutation and selection. It has recently become possible to gain detailed information concerning this process through high-throughput sequencing. Here, we develop modern statistical molecular evolution methods for the analysis of B-cell sequence data, and then apply them to a very deep short-read dataset of BCRs. We find that the substitution process is conserved across individuals but varies significantly across gene segments. We investigate selection on BCRs using a novel method that side-steps the difficulties encountered by previous work in differentiating between selection and motif-driven mutation; this is done through stochastic mapping and empirical Bayes estimators that compare the evolution of in-frame and out-of-frame rearrangements. We use this new method to derive a per-residue map of selection, which provides a more nuanced view of the constraints on framework and variable regions. PMID:26194758

  17. Affinity maturation of anti-(4-hydroxy-3-nitrophenyl)acetyl antibodies accompanies a modulation of antigen specificity.

    PubMed

    Oda, Masayuki; Azuma, Takachika

    2016-02-01

    Anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies bearing λ1 chains are known to possess fine specificity, referred to as heterocliticity, which causes these antibodies to bind to hapten analogues such as (4-hydroxy-3-iodo-5-nitrophenyl)acetyl (NIP) and (4-hydroxy-3,5-dinitrophenyl)acetyl (NNP) with higher affinity than to the autologous hapten, NP. They also show preferential binding to the phenolate form of hapten than to the phenolic form. We address here the question of whether affinity maturation accompanies in the fine specificity of these antibodies by analyzing the interaction between NP1-, NIP1-, or NNP1-hen egg lysozyme and anti-NP antibodies that possess different association constants to NP using a surface plasmon resonance biosensor. We measured interactions at various pH values and found that heterocliticity as well as preferential binding to the phenolate form of hapten were most prominent in a germline antibody having immature affinity and that fine specificity becomes less evident, i.e., anti-NP antibodies become more specific to the immunizing antigen, NP during the process of affinity maturation. PMID:26688069

  18. Affinity maturation generates greatly improved xyloglucan-specific carbohydrate binding modules

    PubMed Central

    2009-01-01

    Background Molecular evolution of carbohydrate binding modules (CBM) is a new approach for the generation of glycan-specific molecular probes. To date, the possibility of performing affinity maturation on CBM has not been investigated. In this study we show that binding characteristics such as affinity can be improved for CBM generated from the CBM4-2 scaffold by using random mutagenesis in combination with phage display technology. Results Two modified proteins with greatly improved affinity for xyloglucan, a key polysaccharide abundant in the plant kingdom crucial for providing plant support, were generated. Both improved modules differ from other existing xyloglucan probes by binding to galactose-decorated subunits of xyloglucan. The usefulness of the evolved binders was verified by staining of plant sections, where they performed better than the xyloglucan-binding module from which they had been derived. They discriminated non-fucosylated from fucosylated xyloglucan as shown by their ability to stain only the endosperm, rich in non-fucosylated xyloglucan, but not the integument rich in fucosylated xyloglucan, on tamarind seed sections. Conclusion We conclude that affinity maturation of CBM selected from molecular libraries based on the CBM4-2 scaffold is possible and has the potential to generate new analytical tools for detection of plant carbohydrates. PMID:19878581

  19. Salmonella Infection Drives Promiscuous B Cell Activation Followed by Extrafollicular Affinity Maturation.

    PubMed

    Di Niro, Roberto; Lee, Seung-Joo; Vander Heiden, Jason A; Elsner, Rebecca A; Trivedi, Nikita; Bannock, Jason M; Gupta, Namita T; Kleinstein, Steven H; Vigneault, Francois; Gilbert, Tamara J; Meffre, Eric; McSorley, Stephen J; Shlomchik, Mark J

    2015-07-21

    The B cell response to Salmonella typhimurium (STm) occurs massively at extrafollicular sites, without notable germinal centers (GCs). Little is known in terms of its specificity. To expand the knowledge of antigen targets, we screened plasmablast (PB)-derived monoclonal antibodies (mAbs) for Salmonella specificity, using ELISA, flow cytometry, and antigen microarray. Only a small fraction (0.5%-2%) of the response appeared to be Salmonella-specific. Yet, infection of mice with limited B cell receptor (BCR) repertoires impaired the response, suggesting that BCR specificity was important. We showed, using laser microdissection, that somatic hypermutation (SHM) occurred efficiently at extrafollicular sites leading to affinity maturation that in turn led to detectable STm Ag-binding. These results suggest a revised vision of how clonal selection and affinity maturation operate in response to Salmonella. Clonal selection initially is promiscuous, activating cells with virtually undetectable affinity, yet SHM and selection occur during the extrafollicular response yielding higher affinity, detectable antibodies. PMID:26187411

  20. First molecular and biochemical analysis of in vivo affinity maturation in an ectothermic vertebrate

    PubMed Central

    Dooley, Helen; Stanfield, Robyn L.; Brady, Rebecca A.; Flajnik, Martin F.

    2006-01-01

    The cartilaginous fish are the oldest phylogenetic group in which Igs have been found. Sharks produce a unique Ig isotype, IgNAR, a heavy-chain homodimer that does not associate with light chains. Instead, the variable (V) regions of IgNAR bind antigen as soluble single domains. Our group has shown that IgNAR plays an integral part in the humoral response of nurse sharks (Ginglymostoma cirratum) upon antigen challenge. Here, we generated phage-displayed libraries of IgNAR V regions from an immunized animal and found a family of clones derived from the same rearrangement event but differentially mutated during expansion. Because of the cluster organization of shark Ig genes and the paucicopy nature of IgNAR, we were able to construct the putative ancestor of this family. By studying mutations in the context of clone affinities, we found evidence that affinity maturation occurs for this isotype. Subsequently, we were able to identify mutations important in the affinity improvement of this family. Because the family clones were all obtained after immunization, they provide insight into the in vivo maturation mechanisms, in general, and for single-domain antibody fragments. PMID:16446445

  1. CD4+ T Cells Promote Antibody Production but Not Sustained Affinity Maturation during Borrelia burgdorferi Infection

    PubMed Central

    Elsner, Rebecca A.; Hastey, Christine J.

    2014-01-01

    CD4 T cells are crucial for enhancing B cell-mediated immunity, supporting the induction of high-affinity, class-switched antibody responses, long-lived plasma cells, and memory B cells. Previous studies showed that the immune response to Borrelia burgdorferi appears to lack robust T-dependent B cell responses, as neither long-lived plasma cells nor memory B cells form for months after infection, and nonswitched IgM antibodies are produced continuously during this chronic disease. These data prompted us to evaluate the induction and functionality of B. burgdorferi infection-induced CD4 TFH cells. We report that CD4 T cells were effectively primed and TFH cells induced after B. burgdorferi infection. These CD4 T cells contributed to the control of B. burgdorferi burden and supported the induction of B. burgdorferi-specific IgG responses. However, while affinity maturation of antibodies against a prototypic T-dependent B. burgdorferi protein, Arthritis-related protein (Arp), were initiated, these increases were reversed later, coinciding with the previously observed involution of germinal centers. The cessation of affinity maturation was not due to the appearance of inhibitory or exhausted CD4 T cells or a strong induction of regulatory T cells. In vitro T-B cocultures demonstrated that T cells isolated from B. burgdorferi-infected but not B. burgdorferi-immunized mice supported the rapid differentiation of B cells into antibody-secreting plasma cells rather than continued proliferation, mirroring the induction of rapid short-lived instead of long-lived T-dependent antibody responses in vivo. The data further suggest that B. burgdorferi infection drives the humoral response away from protective, high-affinity, and long-lived antibody responses and toward the rapid induction of strongly induced, short-lived antibodies of limited efficacy. PMID:25312948

  2. Antibody response and antibody affinity maturation in cats with experimental proliferative immune complex glomerulonephritis.

    PubMed

    Bishop, S A; Bailey, M; Lucke, V M; Stokes, C R

    1992-07-01

    An experimental model of proliferative glomerulonephritis (GN) in the cat, which closely resembles human proliferative forms of GN, has been used to study the role of antibody and antibody affinity in the development of immune complex-mediated renal disease. The serum IgG and IgM antibody response to antigen, average antibody affinity (avidity) and affinity heterogeneity of the IgG and IgM populations was assessed at varying times after commencement of chronic immunization with the antigen, human serum albumin (HSA), by enzyme immunoassay. Cats could be classified according to whether they were "low", "intermediate" or "high" IgG responders, by quantification of serum IgG values. Cats with the lowest serum IgG values failed to develop glomerulonephritis. However, there was no relationship between actual IgG values and the severity of the induced disease. In contrast to IgG, there was no division of cats into low or high IgM anti-HSA responders. Again, cats with the lowest IgM values failed to develop GN, but, more interestingly, a late, marked increase in serum IgM anti-HSA occurred only in cats that developed clinical signs of GN (anterior uveitis and nephrotic syndrome). Maturation of average, functional IgG affinity (avidity) for HSA following chronic immunization was clearly demonstrated for all cats. At the end of the experiment, all cats had IgG of high affinity for HSA and the average affinity heterogeneity of the IgG populations was less than in measurements taken earlier. Values of IgG affinity at the end of the experiment were very similar both in cats which developed GN and in those which remained clinically, biochemically and pathologically normal. In contrast to IgG antibody, some cats developed IgM of increased affinity, whilst others produced antibody of reduced affinity, following chronic immunization. There was no correlation between the development of disease and the production of either low or high affinity IgM antibody. Data indicated that an

  3. Guiding the evolution to catch the virus: An in silico study of affinity maturation against rapidly mutating antigen

    NASA Astrophysics Data System (ADS)

    Wang, Shenshen; Burton, Dennis; Kardar, Mehran; Chakraborty, Arup

    2014-03-01

    The immune system comprises an intricate and evolving collection of cells and molecules that enables a defense against pathogenic agents. Its workings present a rich source of physical problems that impact human health. One intriguing example is the process of affinity maturation (AM) through which an antibody (Ab)--a component of the host immune system--evolves to more efficiently bind an antigen (Ag)--a unique part of a foreign pathogen such as a virus. Sufficiently strong binding to the Ag enables recognition and neutralization. A major challenge is to contain a diversifying mixture of Ag variants, that arise in natural infection, from evading Ab neutralization. This entails a thorough understanding of AM against multiple Ag species and mutating Ag. During AM, Ab-encoding cells undergo cycles of mutation and selection, a process reminiscent of Darwinian evolution yet occurring in real time. We first cast affinity-dependent selection into an extreme value problem and show how the binding characteristics scale with Ag diversity. We then develop an agent-based residue-resolved computational model of AM which allows us to track the evolutionary trajectories of individual cells. This dynamic model not only reveals significant stochastic effects associated with the relatively small and highly dynamic population size, it also uncovers the markedly distinct maturation outcomes if designed Ag variants are presented in different temporal procedures. Insights thus obtained would guide rational design of vaccination protocols.

  4. Structure-based affinity maturation of a chimeric anti-ricin antibody C4C13.

    PubMed

    Luo, Longlong; Luo, Qun; Guo, Leiming; Lv, Ming; Lin, Zhou; Geng, Jing; Li, Xinying; Li, Yan; Shen, Beifen; Qiao, Chunxia; Feng, Jiannan

    2014-01-01

    Ricin is a highly lethal toxin. Anti-ricin chimeric monoclonal antibody (mAb) C4C13 was prepared in our lab; however, its binding affinity was much weaker than that of the parent antibody 4C13. In this study, based on the computer-guided homology modeling and conformational optimization methods, the 3-D structure of C4C13 variable regions Fv was constructed and optimized. Using molecular docking and dynamics simulation methods, the 3-D complex structure of ricin and C4C13 Fv was obtained. Considering the orientation property, surface electrostatic distribution, residues chemical and physical character and intermolecular hydrogen bond, the binding mode and key residues were predicted. According to C4C13 Fv fragment and ricin complementary binding surface, electrostatic attraction periphery and van der Waals interaction interface, three mutants (i.e., M1 (N(H102)F, W(H103)Y); M2 (W(H103)Y) and M3 (R(L90)G)) were designed, in which M1 and M2 were predicted to possess higher antigen-binding activity than C4C13, while M3 was weaker. The relative affinity assays by ELISA showed that M1 and M2 mutations had higher affinity (9.6 and 18.3 nmol/L) than C4C13 (130 nmol/L) and M3 had weaker affinity (234.5 nmol/L) than C4C13. The results showed that the modeling complex structure of the antigen (ricin) and antibody (C4C13) is reasonable. Our work offered affinity maturated antibodies by site mutations, which were beneficial for valuable anti-ricin antibody design and preparation in future. PMID:23527922

  5. Maturation processing and characterization of streptopain.

    PubMed

    Chen, Chiu-Yueh; Luo, Shih-Chi; Kuo, Chih-Feng; Lin, Yee-Shin; Wu, Jiunn-Jong; Lin, Ming T; Liu, Ching-Chuan; Jeng, Wen-Yih; Chuang, Woei-Jer

    2003-05-01

    Streptopain is a cysteine protease expressed by Streptococcus pyogenes. To study the maturation mechanism of streptopain, wild-type and Q186N, C192S, H340R, N356D and W357A mutant proteins were expressed in Escherichia coli and purified to homogeneity. Proteolytic analyses showed that the maturation of prostreptococcal pyrogenic exotoxin B zymogen (pro-SPE B) involves eight intermediates with a combination of cis- and trans-processing. Based on the sequences of these intermediates, the substrate specificity of streptopain favors a hydrophobic residue at the P2 site. The relative autocatalytic rates of these mutants exhibited the order Q186N > W357A > N356D, C192S, H340R. Interestingly, the N356D mutant containing protease activity could not be converted into the 28-kDa form by autoprocessing. This observation suggested that Asn(356) might involve the cis-processing of the propeptide. In addition, the maturation rates of pro-SPE B with trypsin and plasmin were 10- and 60-fold slower than that with active mature streptopain. These findings indicate that active mature streptopain likely plays the most important role in the maturation of pro-SPE B under physiological conditions. PMID:12621045

  6. Metal-affinity separations: A new dimension in protein processing

    SciTech Connect

    Arnold, F.H. )

    1991-02-01

    Rapid growth in the preparative and high-resolution analytical applications of metal-affinity chromatography demonstrate the appeal of metal recognition as a basis for protein separations. Stable, inexpensive chelated metals effectively mimic biospecific interactions, providing selective ligands for protein binding. This article reviews recent progress in understanding the mechanisms of metal-protein recognition that underlie metal-affinity separations. Also discussed are schemes for integrating metal-affinity purifications into the expression and bioprocessing of recombinant proteins. Promising future developments include new metal-affinity processes for analytical and preparative-scale separations and a range of techniques for enhancing the selectivity of metal-affinity separations.

  7. Affinity maturation in an HIV broadly neutralizing B-cell lineage through reorientation of variable domains.

    PubMed

    Fera, Daniela; Schmidt, Aaron G; Haynes, Barton F; Gao, Feng; Liao, Hua-Xin; Kepler, Thomas B; Harrison, Stephen C

    2014-07-15

    Rapidly evolving pathogens, such as human immunodeficiency and influenza viruses, escape immune defenses provided by most vaccine-induced antibodies. Proposed strategies to elicit broadly neutralizing antibodies require a deeper understanding of antibody affinity maturation and evolution of the immune response to vaccination or infection. In HIV-infected individuals, viruses and B cells evolve together, creating a virus-antibody "arms race." Analysis of samples from an individual designated CH505 has illustrated the interplay between an antibody lineage, CH103, and autologous viruses at various time points. The CH103 antibodies, relatively broad in their neutralization spectrum, interact with the CD4 binding site of gp120, with a contact dominated by CDRH3. We show by analyzing structures of progenitor and intermediate antibodies and by correlating them with measurements of binding to various gp120s that there was a shift in the relative orientation of the light- and heavy-chain variable domains during evolution of the CH103 lineage. We further show that mutations leading to this conformational shift probably occurred in response to insertions in variable loop 5 (V5) of the HIV envelope. The shift displaced the tips of the light chain away from contact with V5, making room for the inserted residues, which had allowed escape from neutralization by the progenitor antibody. These results, which document the selective mechanism underlying this example of a virus-antibody arms race, illustrate the functional significance of affinity maturation by mutation outside the complementarity determining region surface of the antibody molecule. PMID:24982157

  8. Affinity maturation in an HIV broadly neutralizing B-cell lineage through reorientation of variable domains

    PubMed Central

    Fera, Daniela; Schmidt, Aaron G.; Haynes, Barton F.; Gao, Feng; Liao, Hua-Xin; Kepler, Thomas B.; Harrison, Stephen C.

    2014-01-01

    Rapidly evolving pathogens, such as human immunodeficiency and influenza viruses, escape immune defenses provided by most vaccine-induced antibodies. Proposed strategies to elicit broadly neutralizing antibodies require a deeper understanding of antibody affinity maturation and evolution of the immune response to vaccination or infection. In HIV-infected individuals, viruses and B cells evolve together, creating a virus−antibody “arms race.” Analysis of samples from an individual designated CH505 has illustrated the interplay between an antibody lineage, CH103, and autologous viruses at various time points. The CH103 antibodies, relatively broad in their neutralization spectrum, interact with the CD4 binding site of gp120, with a contact dominated by CDRH3. We show by analyzing structures of progenitor and intermediate antibodies and by correlating them with measurements of binding to various gp120s that there was a shift in the relative orientation of the light- and heavy-chain variable domains during evolution of the CH103 lineage. We further show that mutations leading to this conformational shift probably occurred in response to insertions in variable loop 5 (V5) of the HIV envelope. The shift displaced the tips of the light chain away from contact with V5, making room for the inserted residues, which had allowed escape from neutralization by the progenitor antibody. These results, which document the selective mechanism underlying this example of a virus−antibody arms race, illustrate the functional significance of affinity maturation by mutation outside the complementarity determining region surface of the antibody molecule. PMID:24982157

  9. Engineering protein therapeutics: predictive performances of a structure-based virtual affinity maturation protocol.

    PubMed

    Oberlin, Michael; Kroemer, Romano; Mikol, Vincent; Minoux, Hervé; Tastan, Erdogan; Baurin, Nicolas

    2012-08-27

    The implementation of a structure based virtual affinity maturation protocol and evaluation of its predictivity are presented. The in silico protocol is based on conformational sampling of the interface residues (using the Dead End Elimination/A* algorithm), followed by the estimation of the change of free energy of binding due to a point mutation, applying MM/PBSA calculations. Several implementations of the protocol have been evaluated for 173 mutations in 7 different protein complexes for which experimental data were available: the use of the Boltzamnn averaged predictor based on the free energy of binding (ΔΔG(*)) combined with the one based on its polar component only (ΔΔE(pol*)) led to the proposal of a subset of mutations out of which 45% would have successfully enhanced the binding. When focusing on those mutations that are less likely to be introduced by natural in vivo maturation methods (99 mutations with at least two base changes in the codon), the success rate is increased to 63%. In another evaluation, focusing on 56 alanine scanning mutations, the in silico protocol was able to detect 89% of the hot-spots. PMID:22788756

  10. Affinity Maturation of a Potent Family of HIV Antibodies Is Primarily Focused on Accommodating or Avoiding Glycans.

    PubMed

    Garces, Fernando; Lee, Jeong Hyun; de Val, Natalia; de la Pena, Alba Torrents; Kong, Leopold; Puchades, Cristina; Hua, Yuanzi; Stanfield, Robyn L; Burton, Dennis R; Moore, John P; Sanders, Rogier W; Ward, Andrew B; Wilson, Ian A

    2015-12-15

    The high-mannose patch on the HIV-1 envelope (Env) glycoprotein is the epicenter for binding of the potent broadly neutralizing PGT121 family of antibodies, but strategies for generating such antibodies by vaccination have not been defined. We generated structures of inferred antibody intermediates by X-ray crystallography and electron microscopy to elucidate the molecular events that occurred during evolution of this family. Binding analyses revealed that affinity maturation was primarily focused on avoiding, accommodating, or binding the N137 glycan. The overall antibody approach angle to Env was defined very early in the maturation process, yet some variation evolved in the PGT121 family branches that led to differences in glycan specificities in their respective epitopes. Furthermore, we determined a crystal structure of the recombinant BG505 SOSIP.664 HIV-1 trimer with a PGT121 family member at 3.0 Å that, in concert with these antibody intermediate structures, provides insights to advance design of HIV vaccine candidates. PMID:26682982

  11. A Simple Flow-Cytometric Method Measuring B Cell Surface Immunoglobulin Avidity Enables Characterization of Affinity Maturation to Influenza A Virus

    PubMed Central

    Frank, Gregory M.; Angeletti, Davide; Ince, William L.; Gibbs, James S.; Khurana, Surender; Wheatley, Adam K.; Max, Edward E.; McDermott, Adrian B.; Golding, Hana; Stevens, James; Bennink, Jack R.

    2015-01-01

    ABSTRACT Antibody (Ab) affinity maturation enables an individual to maintain immunity to an increasing number of pathogens within the limits of a total Ig production threshold. A better understanding of this process is critical for designing vaccines that generate optimal Ab responses to pathogens. Our study describes a simple flow-cytometric method that enumerates virus-specific germinal center (GC) B cells as well as their AC50, a measure of Ab avidity, defined as the antigen concentration required to detect 50% of specific B cells. Using a model of mouse Ab responses to the influenza A virus hemagglutinin (IAV HA), we obtained data indicating that AC50 decreases with time postinfection in an affinity maturation-dependent process. As proof of principle of the utility of the method, our data clearly show that relative to intranasal IAV infection, intramuscular immunization against inactivated IAV in adjuvant results in a diminished GC HA B cell response, with increased AC50 correlating with an increased serum Ab off-rate. Enabling simultaneous interrogation of both GC HA B cell quantity and quality, this technique should facilitate study of affinity maturation and rational vaccine design. PMID:26242629

  12. Dual-display of small molecules enables the discovery of ligand pairs and facilitates affinity maturation

    NASA Astrophysics Data System (ADS)

    Wichert, Moreno; Krall, Nikolaus; Decurtins, Willy; Franzini, Raphael M.; Pretto, Francesca; Schneider, Petra; Neri, Dario; Scheuermann, Jörg

    2015-03-01

    In contrast to standard fragment-based drug discovery approaches, dual-display DNA-encoded chemical libraries have the potential to identify fragment pairs that bind simultaneously and benefit from the chelate effect. However, the technology has been limited by the difficulty in unambiguously decoding the ligand pairs from large combinatorial libraries. Here we report a strategy that overcomes this limitation and enables the efficient identification of ligand pairs that bind to a target protein. Small organic molecules were conjugated to the 5‧ and 3‧ ends of complementary DNA strands that contain a unique identifying code. DNA hybridization followed by an inter-strand code-transfer created a stable dual-display DNA-encoded chemical library of 111,100 members. Using this approach we report the discovery of a low micromolar binder to alpha-1-acid glycoprotein and the affinity maturation of a ligand to carbonic anhydrase IX, an established marker of renal cell carcinoma. The newly discovered subnanomolar carbonic anhydrase IX binder dramatically improved tumour targeting performance in vivo.

  13. Combining somatic mutations present in different in vivo affinity-matured antibodies isolated from immunized Lama glama yields ultra-potent antibody therapeutics.

    PubMed

    Klarenbeek, Alex; Blanchetot, Christophe; Schragel, Georg; Sadi, Ava S; Ongenae, Nico; Hemrika, Wieger; Wijdenes, John; Spinelli, Silvia; Desmyter, Aline; Cambillau, Christian; Hultberg, Anna; Kretz-Rommel, Anke; Dreier, Torsten; De Haard, Hans J W; Roovers, Rob C

    2016-04-01

    Highly potent human antibodies are required to therapeutically neutralize cytokines such as interleukin-6 (IL-6) that is involved in many inflammatory diseases and malignancies. Although a number of mutagenesis approaches exist to perform antibody affinity maturation, these may cause antibody instability and production issues. Thus, a robust and easy antibody affinity maturation strategy to increase antibody potency remains highly desirable. By immunizing llama, cloning the 'immune' antibody repertoire and using phage display, we selected a diverse set of IL-6 antagonistic Fabs. Heavy chain shuffling was performed on the Fab with lowest off-rate, resulting in a panel of variants with even lower off-rate. Structural analysis of the Fab:IL-6 complex suggests that the increased affinity was partly due to a serine to tyrosine switch in HCDR2. This translated into neutralizing capacity in an in vivo model of IL-6 induced SAA production. Finally, a novel Fab library was designed, encoding all variations found in the natural repertoire of VH genes identified after heavy chain shuffling. High stringency selections resulted in identification of a Fab with 250-fold increased potency when re-formatted into IgG1. Compared with a heavily engineered anti-IL-6 monoclonal antibody currently in clinical development, this IgG was at least equally potent, showing the engineering process to have had led to a highly potent anti-IL-6 antibody. PMID:26945588

  14. Combining different design strategies for rational affinity maturation of the MICA-NKG2D interface

    PubMed Central

    Henager, Samuel H; Hale, Melissa A; Maurice, Nicholas J; Dunnington, Erin C; Swanson, Carter J; Peterson, Megan J; Ban, Joseph J; Culpepper, David J; Davies, Luke D; Sanders, Lisa K; McFarland, Benjamin J

    2012-01-01

    We redesigned residues on the surface of MICA, a protein that binds the homodimeric immunoreceptor NKG2D, to increase binding affinity with a series of rational, incremental changes. A fixed-backbone RosettaDesign protocol scored a set of initial mutations, which we tested by surface plasmon resonance for thermodynamics and kinetics of NKG2D binding, both singly and in combination. We combined the best four mutations at the surface with three affinity-enhancing mutations below the binding interface found with a previous design strategy. After curating design scores with three cross-validated tests, we found a linear relationship between free energy of binding and design score, and to a lesser extent, enthalpy and design score. Multiple mutants bound with substantial subadditivity, but in at least one case full additivity was observed when combining distant mutations. Altogether, combining the best mutations from the two strategies into a septuple mutant enhanced affinity by 50-fold, to 50 nM, demonstrating a simple, effective protocol for affinity enhancement. PMID:22761154

  15. What limits affinity maturation of antibodies in Xenopus--the rate of somatic mutation or the ability to select mutants?

    PubMed Central

    Wilson, M; Hsu, E; Marcuz, A; Courtet, M; Du Pasquier, L; Steinberg, C

    1992-01-01

    Although the Xenopus immunoglobulin heavy chain locus is structurally and functionally similar to mammalian IgH loci, Xenopus antibodies are limited in heterogeneity, and they mature only slightly in affinity during immune responses. During the antibody response of isogenic frogs to DNP-KLH, mu and upsilon cDNA sequences using elements of the VH1 family were cloned, sequenced and compared with germline counterparts. There were zero to four mutations per sequence, mostly single base substitutions, in the framework and CDRs 1 and 2 of VH. No mutations were found in JH. Since the point mutation rate was only 4- to 7-fold lower than that calculated for mice, affinity maturation does not seem to be limited by mutant availability. Because of a relatively low ratio of replacement to silent mutations in the CDRs and a very high ratio of GC to AT base pairs altered by mutation, it is suggested that the problem results from the absence of an effective mechanism for selecting mutants, which in turn might be related to the absence of germinal centers in Xenopus. Images PMID:1425571

  16. Antibody affinity maturation through combining display of two-chain paired antibody and precision flow cytometric sorting.

    PubMed

    Sun, Shuang; Yang, Xiao; Wang, Haifeng; Zhao, Yun; Lin, Yan; Ye, Chen; Fang, Xiangdong; Hang, Haiying

    2016-07-01

    Recombination of antibody light and heavy chain libraries greatly increases the size of a two-chain paired antibody library, thus easing the construction of large antibody libraries. Here, light and heavy chain variable domains paired by a coiled coil were applied to a bacterial inner membrane display system. However, the probability of the correct pairing of light and heavy chains through random recombination after each round of flow cytometric sorting and cloning was very low in the presence of mostly unmatched light and heavy chain genes, resulting in inefficient enrichment; a target antibody clone in the ratio of 1:100,000 negative control spheroplasts was unable to be enriched by six rounds of sorting and cloning by a conventional sorting strategy (sorting the top 1 %). By just sorting the top 0.000025 % of spheroplasts, we succeeded in enriching the target antibody clone mixed with negative control spheroplasts in a ratio of 1:10(8) by just one round of sorting and cloning. Furthermore, using this gating strategy, we efficiently enriched for an antibody clone with an affinity slightly better than the parent antibody clone from mixed spheroplasts which were present in the ratio of 1 better affinity clone to 10 parent clones to 10(6) negative control clones after just two rounds of sorting and cloning, suggesting that this gating strategy is highly sensitive in distinguishing between clones with a small difference in affinity and also enriching for clones with a higher affinity. Taken together, the combination of the display of a two-chain paired antibody library and the use of stringent gating has significantly increased the efficiency of the antibody maturation system. PMID:27142297

  17. Affinity maturation of T-cell receptor-like antibodies for Wilms tumor 1 peptide greatly enhances therapeutic potential

    PubMed Central

    Zhao, Qi; Ahmed, Mahiuddin; Tassev, Dimiter V.; Hasan, Aisha; Kuo, Tzu-Yun; Guo, Hong-fen; O’Reilly, Richard J.; Cheung, Nai-Kong V.

    2016-01-01

    WT1126 (RMFPNAPYL) is a human leukocyte antigen-A2 (HLA-A2) restricted peptide derived from Wilms tumor protein (WT1), which is widely expressed in a broad spectrum of leukemias, lymphomas and solid tumors. A novel T-cell-receptor (TCR)-like single chain variable fragment (scFv) antibody specific for the T cell epitope consisting of the WT1/HLA-A2 complex was isolated from a human scFv phage library. This scFv was affinity-matured by mutagenesis combined with yeast display, and structurally analyzed using a homology model. This monovalent scFv showed a 100-fold affinity improvement (dissociation constant [KD]= 3nM) and exquisite specificity towards its targeted epitope or HLA-A2+/WT1+ tumor cells. Bivalent scFv-huIgG1-Fc fusion protein demonstrated an even higher avidity (KD = 2pM) binding to the T cell epitope and to tumor targets, and was capable of mediating antibody-dependent cell-mediated cytotoxicity or tumor lysis by chimeric antigen receptor (CAR)-expressing human T or NK-92-MI transfected cells. This antibody demonstrated specific and potent cytotoxicity in vivo towards WT1-positive leukemia xenograft that was HLA-A2 restricted. In summary, T cell epitopes can provide novel targets for antibody-based therapeutics. By combining phage and yeast displays and scFv-Fc fusion platforms, a strategy for developing high affinity TCR-like antibodies could be rapidly explored for potential clinical development. PMID:25987253

  18. Maturation of Cognitive Processes From Late Childhood to Adulthood

    ERIC Educational Resources Information Center

    Luna,Beatriz; Garver,Krista E.; Urban,Trinity A.; Lazar,Nicole A.; Sweeney,John A.

    2004-01-01

    To characterize cognitive maturation through adolescence, processing speed, voluntary response suppression, and spatial working memory were measured in 8- to 30-year-old (N=245) healthy participants using oculomotor tasks. Development progressed with a steep initial improvement in performance followed by stabilization in adolescence. Adult-level…

  19. Expression of the transferrin receptor gene during the process of mononuclear phagocyte maturation

    SciTech Connect

    Hirata, T.; Bitterman, P.B.; Mornex, J.; Crystal, R.G.

    1986-02-15

    The expression of transferrin receptors by blood monocytes, human alveolar macrophages, and in vitro matured macrophages was evaluated by immunofluorescence, radioligand binding, and Northern analysis, using the monoclonal anti-human transferrin receptor antibody OKT9, (/sup 125/I)-labeled human transferrin and a (/sup 32/P)-labeled human transferrin receptor cDNA probe, respectively. By immunofluorescence, the majority of alveolar macrophages expressed transferrin receptors (86 +/- 3%). The radioligand binding assay demonstrated the affinity constant (K/sub a/) of the alveolar macrophage transferrin receptor was 4.4 +/- 0.7 x 10/sup 8/ M/sup -1/, and the number of receptors per cell was 4.4 +/- 1.2 x 10/sup 4/. In marked contrast, transferrin receptors were not present on the surface or in the cytoplasm of blood monocytes, the precursors of the alveolar macrophages. However, when monocytes were cultured in vitro and allowed to mature, > 80% expressed transferrin receptors by day 6, and the receptors could be detected by day 3. Consistent with these observations, a transferrin receptor mRNA with a molecular size of 4.9 kb was demonstrated in alveolar macrophages and in vitro matured macrophages but not in blood monocytes. Thus, although blood monocytes do not express the transferrin receptor gene, it is expressed by mature macrophages, an event that probably occurs relatively early in the process of monocyte differentiation to macrophages.

  20. Structural Basis of HIV-1 Neutralization by Affinity Matured Fabs Directed against the Internal Trimeric Coiled-Coil of gp41

    SciTech Connect

    Gustchina, Elena; Li, Mi; Louis, John M.; Anderson, D.Eric; Lloyd, John; Frisch, Christian; Bewley, Carole A.; Gustchina, Alla; Wlodawer, Alexander; Clore, G.Marius

    2010-12-03

    The conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 is transiently exposed during the fusion process by forming a pre-hairpin intermediate, thus representing an attractive target for the design of fusion inhibitors and neutralizing antibodies. In previous studies we reported a series of broadly neutralizing mini-antibodies derived from a synthetic naive human combinatorial antibody library by panning against a mimetic of the trimeric N-HR coiled coil, followed by affinity maturation using targeted diversification of the CDR-H2 loop. Here we report crystal structures of the N-HR mimetic 5-Helix with two Fabs that represent the extremes of this series: Fab 8066 is broadly neutralizing across a wide panel of B and C type HIV-1 viruses, whereas Fab 8062 is non-neutralizing. The crystal structures reveal important differences in the conformations of the CDR-H2 loops in the complexes that propagate into other regions of the antigen-antibody interface, and suggest that both neutralization properties and affinity for the target can be attributed, at least in part, to the differences in the interactions of the CDR-H2 loops with the antigen. Furthermore, modeling of the complex of an N-HR trimer with three Fabs suggests that the CDR-H2 loop may be involved in close intermolecular contacts between neighboring antibody molecules, and that such contacts may hinder the formation of complexes between the N-HR trimer and more than one antibody molecule depending on the conformation of the bound CDR-H2 loop which is defined by its interactions with antigen. Comparison with the crystal structure of the complex of 5-Helix with another neutralizing monoclonal antibody known as D5, derived using an entirely different antibody library and panning procedure, reveals remarkable convergence in the optimal sequence and conformation of the CDR-H2 loop.

  1. The maturation of incentive processing and cognitive control

    PubMed Central

    Geier, Charles; Luna, Beatriz

    2009-01-01

    Understanding how immaturities in the reward system affect decision-making can inform us on adolescent vulnerabilities to risk-taking, which is a primary contributor to mortality and substance abuse in this age group. In this paper, we review the literature characterizing the neurodevelopment of reward and cognitive control and propose a model for adolescent reward processing. While the functional neuroanatomy of the mature reward system has been well-delineated, adolescent reward processing is just beginning to be understood. Results indicate that adolescents relative to adults demonstrate decreased anticipatory processing and assessment of risk, but an increased consummatory response. Such differences could result in suboptimal representations of reward valence and value and bias adolescent decision-making. These functional differences in reward processing occur in parallel with on-going structural and pharmacological maturation in the adolescent brain. In addition to limitations in incentive processing, basic cognitive control abilities, including working memory and inhibitory control, continue to mature during adolescence. Consequently, adolescents may be limited, relative to adults, in their abilities to inhibit impulsive behaviors and reliably hold ‘on-line’ comparisons of potential rewards/punishments during decision-making. PMID:19593842

  2. Process maturity progress at Motorola Cellular Systems Division

    NASA Technical Reports Server (NTRS)

    Borgstahl, Ron; Criscione, Mark; Dobson, Kim; Willey, Allan

    1994-01-01

    We believe that the key success elements are related to our recognition that Software Process Improvement (SPI) can and should be organized, planned, managed, and measured as if it were a project to develop a new process, analogous to a software product. We believe that our process improvements have come as the result of these key elements: use of a rigorous, detailed requirements set (Capability Maturity Model, CMM); use of a robust, yet flexible architecture (IEEE 1074); use of a SPI project, resourced and managed like other work, to produce the specifications and implement them; and development of both internal and external goals, with metrics to support them.

  3. High-Resolution Longitudinal Study of HIV-1 Env Vaccine-Elicited B Cell Responses to the Virus Primary Receptor Binding Site Reveals Affinity Maturation and Clonal Persistence.

    PubMed

    Wang, Yimeng; Sundling, Christopher; Wilson, Richard; O'Dell, Sijy; Chen, Yajing; Dai, Kaifan; Phad, Ganesh E; Zhu, Jiang; Xiao, Yongli; Mascola, John R; Karlsson Hedestam, Gunilla B; Wyatt, Richard T; Li, Yuxing

    2016-05-01

    Because of the genetic variability of the HIV-1 envelope glycoproteins (Env), the elicitation of neutralizing Abs to conserved neutralization determinants including the primary receptor binding site, CD4 binding site (CD4bs), is a major focus of vaccine development. To gain insight into the evolution of Env-elicited Ab responses, we used single B cell analysis to interrogate the memory B cell Ig repertoires from two rhesus macaques after five serial immunizations with Env/adjuvant. We observed that the CD4bs-specific repertoire displayed unique features in the third CDR of Ig H chains with minor alterations along the immunization course. Progressive affinity maturation occurred as evidenced by elevated levels of somatic hypermutation (SHM) in Ab sequences isolated at the late immunization time point compared with the early time point. Abs with higher SHM were associated with increased binding affinity and virus neutralization capacity. Moreover, a notable portion of the CD4bs-specific repertoire was maintained between early and late immunization time points, suggesting that persistent clonal lineages were induced by Env vaccination. Furthermore, we found that the predominant persistent CD4bs-specific clonal lineages had larger population sizes and higher affinities than that from the rest of the repertoires, underscoring the critical role of Ag affinity selection in Ab maturation and clonal expansion. Genetic and functional analyses revealed that the accumulation of SHM in both framework regions and CDRs contributed to the clonal affinity and antigenicity evolution. Our longitudinal study provides high-resolution understanding of the dynamically evolving CD4bs-specific B cell response after Env immunization in primates. PMID:27001953

  4. Technology maturation process: the NASA Strategic Astrophysics Technology (SAT) program

    NASA Astrophysics Data System (ADS)

    Perez, Mario R.; Pham, Bruce T.; Lawson, Peter R.

    2014-08-01

    In 2009 the Astrophysics Division at NASA Headquarters established the Strategic Astrophysics Technology (SAT) solicitation as a new technology maturation program to fill the needed gap for mid-Technology Readiness Level (TRL) levels (3≤ TRL <6). In three full proposal selection cycles since the inception of this program, more than 40 investigations have been selected, many meritorious milestones have been met and advances have been achieved. In this paper, we review the process of establishing technology priorities, the management of technology advancements and milestones, and the incipient success of some of these investigations in light of the need of future space missions.

  5. Maturation processes and structures of small secreted peptides in plants

    PubMed Central

    Tabata, Ryo; Sawa, Shinichiro

    2014-01-01

    In the past decade, small secreted peptides have proven to be essential for various aspects of plant growth and development, including the maintenance of certain stem cell populations. Most small secreted peptides identified in plants to date are recognized by membrane-localized receptor kinases, the largest family of receptor proteins in the plant genome. This peptide-receptor interaction is essential for initiating intracellular signaling cascades. Small secreted peptides often undergo post-translational modifications and proteolytic processing to generate the mature peptides. Recent studies suggest that, in contrast to the situation in mammals, the proteolytic processing of plant peptides involves a number of complex steps. Furthermore, NMR-based structural analysis demonstrated that post-translational modifications induce the conformational changes needed for full activity. In this mini review, we summarize recent advances in our understanding of how small secreted peptides are modified and processed into biologically active peptides and describe the mature structures of small secreted peptides in plants. PMID:25071794

  6. Affinity maturation of single-chain variable fragment specific for aflatoxin B(1) using yeast surface display.

    PubMed

    Min, Won-Ki; Kim, Sung-Gun; Seo, Jin-Ho

    2015-12-01

    As aflatoxin B1 is one of the most toxic mycotoxins, it is important to detect and to quantify aflatoxin B1 accurately by immunological methods. To enhance aflatoxin B1-binding affinity of the single-chain variable fragment, yeast surface display technique combined with fluorescence-activated cell sorting was applied. A randomly mutated scFv library was subjected to 4 rounds of fluorescence-activated cell sorting, resulting in isolation of 5 scFv variants showing an affinity improvement compared to the parental wild type scFv. The best scFv with a 9-fold improvement in affinity for aflatoxin B1 exhibited similar specificity to the monoclonal antibody. Most of the mutations in scFv-M37 were located outside of the canonical antigen-contact loops, suggesting that its affinity improvement might be driven by an allosteric effect inducing scFv-M37 to form a more favorable binding pocket for aflatoxin B1 than the wild type scFv. PMID:26041237

  7. Electrophysiological evidence for late maturation of strategic episodic retrieval processes.

    PubMed

    Sprondel, Volker; Kipp, Kerstin H; Mecklinger, Axel

    2012-05-01

    Improvement in source memory performance throughout development is thought to be mediated by strategic processes that facilitate the retrieval of task-relevant information. Using event-related potentials (ERPs), we examined developmental changes in these processes during adolescence. Adolescents (13-14 years) and adults (19-29 years) completed a memory exclusion task which required the discrimination between words studied in one color ('targets') and words studied in the alternative color ('non-targets') under two conditions that put different demands on strategic control. Memory accuracy improved with age and also increased with decreasing control demands in both age groups. The parietal old/new effect, an ERP correlate of recollection, was reliable for targets across conditions in both age groups. By contrast, ERP correlates of non-target recollection were present in adolescents across conditions but not in adults. This suggests that adults implemented a strategy to prioritize recollection of target information with greater success than adolescents regardless of control demands, presumably reflecting maturational differences in cognitive control. In support of this view, the ERP amplitude difference between targets and non-targets was positively correlated with a measure of working memory capacity (WMC) in adults but not in adolescents. A further age-related difference was that ERP correlates of post-retrieval processing, including late right-frontal old/new effects and late posterior negativities, were observed in adults only. Together, our data suggest protracted maturation in the strategic processes that underlie selective recollection and post-retrieval control. PMID:22490174

  8. XGFR*, a novel affinity-matured bispecific antibody targeting IGF-1R and EGFR with combined signaling inhibition and enhanced immune activation for the treatment of pancreatic cancer.

    PubMed

    Schanzer, Juergen M; Wartha, Katharina; Moessner, Ekkehard; Hosse, Ralf J; Moser, Samuel; Croasdale, Rebecca; Trochanowska, Halina; Shao, Cuiying; Wang, Peng; Shi, Lei; Weinzierl, Tina; Rieder, Natascha; Bacac, Marina; Ries, Carola H; Kettenberger, Hubert; Schlothauer, Tilman; Friess, Thomas; Umana, Pablo; Klein, Christian

    2016-01-01

    The epidermal growth factor receptor (EGFR) and the insulin-like growth factor-1 receptor (IGF-1R) play critical roles in tumor growth, providing a strong rationale for the combined inhibition of IGF-1R and EGFR signaling in cancer therapy. We describe the design, affinity maturation, in vitro and in vivo characterization of the bispecific anti-IGF-1R/EGFR antibody XGFR*. XGFR* is based on the bispecific IgG antibody XGFR, which enabled heterodimerization of an IGF-1R binding scFab heavy chain with an EGFR-binding light and heavy chain by the "knobs-into-holes" technology. XGFR* is optimized for monovalent binding of human EGFR and IGF-1R with increased binding affinity for IGF-1R due to affinity maturation and highly improved protein stability to oxidative and thermal stress. It bears an afucosylated Fc-portion for optimal induction of antibody-dependent cell-mediated cytotoxicity (ADCC). Stable Chinese hamster ovary cell clones with production yields of 2-3 g/L were generated, allowing for large scale production of the bispecific antibody. XGFR* potently inhibits EGFR- and IGF-1R-dependent receptor phosphorylation, reduces tumor cell proliferation in cells with heterogeneous levels of IGF-1R and EGFR receptor expression and induces strong ADCC in vitro. A comparison of pancreatic and colorectal cancer lines demonstrated superior responsiveness to XGFR*-mediated signaling and tumor growth inhibition in pancreatic cancers that frequently show a high degree of IGF-1R/EGFR co-expression. XGFR* showed potent anti-tumoral efficacy in the orthotopic MiaPaCa-2 pancreatic xenograft model, resulting in nearly complete tumor growth inhibition with significant number of tumor remissions. In summary, the bispecific anti-IGF-1R/EGFR antibody XGFR* combines potent signaling and tumor growth inhibition with enhanced ADCC induction and represents a clinical development candidate for the treatment of pancreatic cancer. PMID:26984378

  9. XGFR*, a novel affinity-matured bispecific antibody targeting IGF-1R and EGFR with combined signaling inhibition and enhanced immune activation for the treatment of pancreatic cancer

    PubMed Central

    Schanzer, Juergen M.; Wartha, Katharina; Moessner, Ekkehard; Hosse, Ralf J.; Moser, Samuel; Croasdale, Rebecca; Trochanowska, Halina; Shao, Cuiying; Wang, Peng; Shi, Lei; Weinzierl, Tina; Rieder, Natascha; Bacac, Marina; Ries, Carola H.; Kettenberger, Hubert; Schlothauer, Tilman; Friess, Thomas; Umana, Pablo; Klein, Christian

    2016-01-01

    ABSTRACT The epidermal growth factor receptor (EGFR) and the insulin-like growth factor-1 receptor (IGF-1R) play critical roles in tumor growth, providing a strong rationale for the combined inhibition of IGF-1R and EGFR signaling in cancer therapy. We describe the design, affinity maturation, in vitro and in vivo characterization of the bispecific anti-IGF-1R/EGFR antibody XGFR*. XGFR* is based on the bispecific IgG antibody XGFR, which enabled heterodimerization of an IGF-1R binding scFab heavy chain with an EGFR-binding light and heavy chain by the “knobs-into-holes” technology. XGFR* is optimized for monovalent binding of human EGFR and IGF-1R with increased binding affinity for IGF-1R due to affinity maturation and highly improved protein stability to oxidative and thermal stress. It bears an afucosylated Fc-portion for optimal induction of antibody-dependent cell-mediated cytotoxicity (ADCC). Stable Chinese hamster ovary cell clones with production yields of 2–3 g/L were generated, allowing for large scale production of the bispecific antibody. XGFR* potently inhibits EGFR- and IGF-1R-dependent receptor phosphorylation, reduces tumor cell proliferation in cells with heterogeneous levels of IGF-1R and EGFR receptor expression and induces strong ADCC in vitro. A comparison of pancreatic and colorectal cancer lines demonstrated superior responsiveness to XGFR*-mediated signaling and tumor growth inhibition in pancreatic cancers that frequently show a high degree of IGF-1R/EGFR co-expression. XGFR* showed potent anti-tumoral efficacy in the orthotopic MiaPaCa-2 pancreatic xenograft model, resulting in nearly complete tumor growth inhibition with significant number of tumor remissions. In summary, the bispecific anti-IGF-1R/EGFR antibody XGFR* combines potent signaling and tumor growth inhibition with enhanced ADCC induction and represents a clinical development candidate for the treatment of pancreatic cancer. PMID:26984378

  10. Recombinant pro-regions from papain and papaya proteinase IV-are selective high affinity inhibitors of the mature papaya enzymes.

    PubMed

    Taylor, M A; Baker, K C; Briggs, G S; Connerton, I F; Cummings, N J; Pratt, K A; Revell, D F; Freedman, R B; Goodenough, P W

    1995-01-01

    Proteolytic enzymes require the presence of their pro-regions for correct folding. Of the four proteolytic enzymes from Carica papaya, papain and papaya proteinase IV (PPIV) have 68% sequence identity. We find that their pro-regions are even more similar, exhibiting 73.6% identity. cDNAs encoding the pro-regions of these two proteinases have been expressed in Escherichia coli independently from their mature enzymes. The recombinant pro-regions of papain and PPIV have been shown to be high affinity inhibitors of all four of the mature native papaya cysteine proteinases. Their inhibition constants are in the range 10(-6) - 10(-9) M. PPIV was inhibited two to three orders of magnitude less effectively than papain, chymopapain and caricain. The pro-region of PPIV, however, inhibited its own mature enzyme more effectively than did the pro-region of papain. Alignment of the sequences of the four papaya enzymes shows that there is a highly variable section towards the C-terminal of the pro-region. This region may therefore confer selectivity to the pro-regions for the individual proteolytic enzymes. PMID:7770454

  11. Process for recovering metals from solution utilizing metalloprotein affinity chromatography

    SciTech Connect

    Spears, D.R.; Vincent, J.B.

    1993-11-29

    The invention relates generally to a process for recovering metals from an aqueous metal-bearing solution and, more particularly, to a process which utilizes metalloproteins immobilized on an insoluble support to remove metal ions such as the main group, transition, lanthanide, and actinide ions from the aqueous metal-ion bearing solution.

  12. Delta Inulin Adjuvant Enhances Plasmablast Generation, Expression of Activation-Induced Cytidine Deaminase and B-Cell Affinity Maturation in Human Subjects Receiving Seasonal Influenza Vaccine

    PubMed Central

    Li, Lei; Honda-Okubo, Yoshikazu; Li, Connie; Sajkov, Dimitar; Petrovsky, Nikolai

    2015-01-01

    There is a major need for new adjuvants to improve the efficacy of seasonal and pandemic influenza vaccines. Advax is a novel polysaccharide adjuvant based on delta inulin that has been shown to enhance the immunogenicity of influenza vaccine in animal models and human clinical trials. To better understand the mechanism for this enhancement, we sought to assess its effect on the plasmablast response in human subjects. This pilot study utilised cryopreserved 7 day post-vaccination (7dpv) peripheral blood mononuclear cell samples obtained from a subset of 25 adult subjects from the FLU006-12 trial who had been immunized intramuscularly with a standard dose of 2012 trivalent inactivated influenza vaccine (TIV) alone (n=9 subjects) or combined with 5mg (n=8) or 10mg (n=8) of Advax adjuvant. Subjects receiving Advax adjuvant had increased 7dpv plasmablasts, which in turn exhibited a 2-3 fold higher rate of non-silent mutations in the B-cell receptor CDR3 region associated with higher expression of activation-induced cytidine deaminase (AID), the major enzyme controlling BCR affinity maturation. Together, these data suggest that Advax adjuvant enhances influenza immunity in immunized subjects via multiple mechanisms including increased plasmablast generation, AID expression and CDR3 mutagenesis resulting in enhanced BCR affinity maturation and increased production of high avidity antibody. How Advax adjuvant achieves these beneficial effects on plasmablasts remains the subject of ongoing investigation. Trial Registration Australia New Zealand Clinical Trials Register ACTRN12612000709842 https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=362709 PMID:26177480

  13. Affinity-matured recombinant immunotoxin targeting gangliosides 3′-isoLM1 and 3′,6'-isoLD1 on malignant gliomas

    PubMed Central

    Piao, Hailan; Kuan, Chien-Tsun; Chandramohan, Vidya; Keir, Stephen T; Pegram, Charles N; Bao, Xuhui; Månsson, Jan-Eric; Pastan, Ira H; Bigner, Darell D

    2013-01-01

    About 60 percent of glioblastomas highly express the gangliosides 3′-isoLM1 and 3′,6′-isoLD1 on the cell surface, providing ideal targets for brain tumor immunotherapy. A novel recombinant immunotoxin, DmAb14m-(scFv)-PE38KDEL (DmAb14m-IT), specific for the gangliosides 3′-isoLM1 and 3′,6′-isoLD1, was constructed with improved affinity and increased cytotoxicity for immunotherapeutic targeting of glioblastoma. We isolated an scFv parental clone from a previously established murine hybridoma, DmAb14, that is specific to both 3′-isoLM1 and 3′,6′-isoLD1. We then performed in vitro affinity maturation by CDR hotspot random mutagenesis. The binding affinity and specificity of affinity-matured DmAb14m-IT were measured by surface-plasmon resonance, flow cytometry, and immunohistochemical analysis. In vitro cytotoxicity of DmAb14m-IT was measured by protein synthesis inhibition and cell death assays in human cell lines expressing gangliosides 3′-isoLM1 and 3′,6′-isoLD1 (D54MG and D336MG) and xenograft-derived cells (D2224MG). As a result, the KD of DmAb14m-IT for gangliosides 3′-isoLM1 and 3′,6′-isoLD1 was 2.6 × 10−9M. Also, DmAb14m-IT showed a significantly higher internalization rate in cells expressing 3′-isoLM1 and 3′,6′-isoLD1. The DmAb14m-IT IC50 was 80 ng/mL (1194 pM) on the D54MG cell line, 5 ng/ml (75 pM) on the D336MG cell line, and 0.5 ng/ml (7.5 pM) on the D2224MG xenograft-derived cells. There was no cytotoxicity on ganglioside-negative HEK293 cells. Immunohistochemical analysis confirmed the specific apparent affinity of DmAb14m-IT with 3′-isoLM1 and 3′,6′-isoLD1. In conclusion, DmAb14m-IT showed specific binding affinity, a significantly high internalization rate, and selective cytotoxicity on glioma cell lines and xenograft-derived cells expressing 3′-isoLM1 and 3′,6′-isoLD1, thereby displaying robust therapeutic potential for testing the antitumor efficacy of DmAb14m-IT at the preclinical level and

  14. Development of a specific affinity-matured exosite inhibitor to MT1-MMP that efficiently inhibits tumor cell invasion in vitro and metastasis in vivo

    PubMed Central

    Botkjaer, Kenneth A.; Kwok, Hang Fai; Terp, Mikkel G.; Karatt-Vellatt, Aneesh; Santamaria, Salvatore; McCafferty, John; Andreasen, Peter A.; Itoh, Yoshifumi; Ditzel, Henrik J.; Murphy, Gillian

    2016-01-01

    The membrane-associated matrix metalloproteinase-14, MT1-MMP, has been implicated in pericellular proteolysis with an important role in cellular invasion of collagenous tissues. It is substantially upregulated in various cancers and rheumatoid arthritis, and has been considered as a potential therapeutic target. Here, we report the identification of antibody fragments to MT1-MMP that potently and specifically inhibit its cell surface functions. Lead antibody clones displayed inhibitory activity towards pro-MMP-2 activation, collagen-film degradation and gelatin-film degradation, and were shown to bind to the MT1-MMP catalytic domain outside the active site cleft, inhibiting binding to triple helical collagen. Affinity maturation using CDR3 randomization created a second generation of antibody fragments with dissociation constants down to 0.11 nM, corresponding to an improved affinity of 332-fold with the ability to interfere with cell-surface MT1-MMP functions, displaying IC50 values down to 5 nM. Importantly, the new inhibitors were able to inhibit collagen invasion by tumor-cells in vitro and in vivo primary tumor growth and metastasis of MDA-MB-231 cells in a mouse orthotopic xenograft model. Herein is the first demonstration that an inhibitory antibody targeting sites outside the catalytic cleft of MT1-MMP can effectively abrogate its in vivo activity during tumorigenesis and metastasis. PMID:26934448

  15. Affinity maturation of anti-TNF-alpha scFv with somatic hypermutation in non-B cells.

    PubMed

    Chen, Shaopeng; Qiu, Junkang; Chen, Chuan; Liu, Chunchun; Liu, Yuheng; An, Lili; Jia, Junying; Tang, Jie; Wu, Lijun; Hang, Haiying

    2012-06-01

    Activation-induced cytidine deaminase (AID) is required for the generation of antibody diversity through initiating both somatic hypermutation (SHM) and class switch recombination. A few research groups have successfully used the feature of AID for generating mutant libraries in directed evolution of target proteins in B cells in vitro. B cells, cultured in suspension, are not convenient for transfection and cloning. In this study, we established an AID-based mutant accumulation and sorting system in adherent human cells. Mouse AID gene was first transfected into the human non-small cell lung carcinoma H1299 cells, and a stable cell clone (H1299-AID) was selected. Afterwards, anti-hTNF-α scFv (ATscFv) was transfected into H1299-AID cells and ATscFv was displayed on the surface of H1299-AID cells. By 4-round amplification/flow cytometric sorting for cells with the highest affinities to hTNF-alpha, two ATscFv mutant gene clones were isolated. Compared with the wild type ATscFv, the two mutants were much more efficient in neutralizing cytotoxicity of hTNF-alpha. The results indicate that directed evolution by somatic hypermutation can be carried out in adherent non-B cells, which makes directed evolution in mammalian cells easier and more efficient. PMID:22467272

  16. Twin-column CaptureSMB: a novel cyclic process for protein A affinity chromatography.

    PubMed

    Angarita, Monica; Müller-Späth, Thomas; Baur, Daniel; Lievrouw, Roel; Lissens, Geert; Morbidelli, Massimo

    2015-04-10

    A twin-column counter-current chromatography processes, CaptureSMB, was used for the protein A affinity capture of a monoclonal antibody (mAb). By means of sequential loading, the process improves the utilization of the stationary phase by achieving loadings much closer to the static binding capacity of the resin in comparison to batch chromatography. Using a mAb capture case study with protein A affinity chromatography, the performance and product quality obtained from CaptureSMB and batch processes were compared. The effect of the flow rate, column length and titer concentration on the process performance and product quality were evaluated. CaptureSMB showed superior performance compared to batch chromatography with respect to productivity, capacity utilization, product concentration and buffer consumption. A simplified economic evaluation showed that CaptureSMB could decrease resin costs of 10-30% depending on the manufacturing scenario. PMID:25748537

  17. Initial differences in lipid processing leading to pig-and beef-derived mature adipocyte differentiation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clonal cultures of pig-derived mature adipocytes are capable of dedifferentiating and forming proliferative-competent progeny cells in vitro. Initial lipid processing, is different to that observed in cultures of beef-derived adipocytes. Mature pig adipocytes extrude lipid before proliferation, wher...

  18. Corn silage management: effects of hybrid, maturity, inoculation, and mechanical processing on fermentation characteristics.

    PubMed

    Johnson, L M; Harrison, J H; Davidson, D; Mahanna, W C; Shinners, K

    2003-01-01

    Two experiments were conducted to evaluate the effects of hybrid, maturity, mechanical processing, and inoculation of corn silage on fermentation characteristics. In experiment 1, Pioneer hybrid 3845 corn silage was harvested at three maturities (hard dough, one-third milkline, two-thirds milkline). In experiment 2, Pioneer hybrids 3845 and Quanta were harvested at three maturities (one-third milkline, two-thirds milkline, and blackline). In both experiments, corn silage was harvested at each maturity with and without mechanical processing and with and without inoculation. In experiments 1 and 2, corn silage was harvested at a theoretical length-of-cut of 6.4 and 12.7 mm, respectively. Maturity at harvest tended to have a greater impact on silage fermentation characteristics of corn silage than mechanical processing and inoculation. In experiments 1 and 2, corn silage harvested at the earliest maturity tended to have decreased dry matter content and increased water-soluble carbohydrate concentrations during the ensiling process than corn silage harvested at advanced maturities. In experiment 2, pH levels were lower for corn silage harvested at the early maturity (one-third milkline) compared with advanced maturities (two-thirds milkline and blackline) by 57 d after ensiling. The difference in pH can be explained by the greater concentration of water-soluble carbohydrates at the early maturity (one-third ML) soon after ensiling (2, 3, 6 and 10 d after ensiling) compared with advanced maturities (two-thirds ML and BL). The increased water-soluble carbohydrate concentrations in the less mature corn silage provided nutrients for bacteria to grow and produce primarily lactic acid (6, 10, and 57 d after ensiling) and some acetic acid (2, 3, 6, and 10 d after ensiling) which reduced the pH of corn silage more than at the advanced maturities. There was a slight change in silage fermentation characteristics when corn silage was inoculated with Pioneer 1132 inoculant in

  19. Crystal Structure of an Affinity-matured Prolactin Complexed to Its Dimerized Receptor Reveals the Topology of Hormone Binding Site 2*

    PubMed Central

    Broutin, Isabelle; Jomain, Jean-Baptiste; Tallet, Estelle; van Agthoven, Jan; Raynal, Bertrand; Hoos, Sylviane; Kragelund, Birthe B.; Kelly, Paul A.; Ducruix, Arnaud; England, Patrick; Goffin, Vincent

    2010-01-01

    We report the first crystal structure of a 1:2 hormone·receptor complex that involves prolactin (PRL) as the ligand, at 3.8-Å resolution. Stable ternary complexes were obtained by generating affinity-matured PRL variants harboring an N-terminal tail from ovine placental lactogen, a closely related PRL receptor (PRLR) ligand. This structure allows one to draw up an exhaustive inventory of the residues involved at the PRL·PRLR site 2 interface, consistent with all previously reported site-directed mutagenesis data. We propose, with this description, an interaction model involving three structural components of PRL site 2 (“three-pin plug”): the conserved glycine 129 of helix α3, the hydrogen bond network involving surrounding residues (glycine cavity), and the N terminus. The model provides a molecular basis for the properties of the different PRL analogs designed to date, including PRLR antagonists. Finally, comparison of our 1:2 PRL·PRLR2 structure with those of free PRL and its 1:1 complex indicates that the structure of PRL undergoes significant changes when binding the first, but not the second receptor. This suggests that the second PRLR moiety adapts to the 1:1 complex rather than the opposite. In conclusion, this structure will be a useful guiding tool for further investigations of the molecular mechanisms involved in PRLR dimerization and activation, as well as for the optimization of PRLR antagonists, an emerging class of compounds with high therapeutic potential against breast and prostate cancer. PMID:20053995

  20. Electrophysiological Evidence for Late Maturation of Strategic Episodic Retrieval Processes

    ERIC Educational Resources Information Center

    Sprondel, Volker; Kipp, Kerstin H.; Mecklinger, Axel

    2012-01-01

    Improvement in source memory performance throughout development is thought to be mediated by strategic processes that facilitate the retrieval of task-relevant information. Using event-related potentials (ERPs), we examined developmental changes in these processes during adolescence. Adolescents (13-14 years) and adults (19-29 years) completed a…

  1. Using Dual Process Models to Examine Impulsivity Throughout Neural Maturation.

    PubMed

    Leshem, Rotem

    2016-01-01

    The multivariate construct of impulsivity is examined through neural systems and connections that comprise the executive functioning system. It is proposed that cognitive and behavioral components of impulsivity can be divided into two distinct groups, mediated by (1) the cognitive control system: deficits in top-down cognitive control processes referred to as action/cognitive impulsivity and (2) the socioemotional system: related to bottom-up affective/motivational processes referred to as affective impulsivity. Examination of impulsivity from a developmental viewpoint can guide future research, potentially enabling the selection of more effective interventions for impulsive individuals, based on the cognitive components requiring improvement. PMID:27186976

  2. A conceptual framework and classification of capability areas for business process maturity

    NASA Astrophysics Data System (ADS)

    Van Looy, Amy; De Backer, Manu; Poels, Geert

    2014-03-01

    The article elaborates on business process maturity, which indicates how well an organisation can perform based on its business processes, i.e. on its way of working. This topic is of paramount importance for managers who try to excel in today's competitive world. Hence, business process maturity is an emerging research field. However, no consensus exists on the capability areas (or skills) needed to excel. Moreover, their theoretical foundation and synergies with other fields are frequently neglected. To overcome this gap, our study presents a conceptual framework with six main capability areas and 17 sub areas. It draws on theories regarding the traditional business process lifecycle, which are supplemented by recognised organisation management theories. The comprehensiveness of this framework is validated by mapping 69 business process maturity models (BPMMs) to the identified capability areas, based on content analysis. Nonetheless, as a consensus neither exists among the collected BPMMs, a classification of different maturity types is proposed, based on cluster analysis and discriminant analysis. Consequently, the findings contribute to the grounding of business process literature. Possible future avenues are evaluating existing BPMMs, directing new BPMMs or investigating which combinations of capability areas (i.e. maturity types) contribute more to performance than others.

  3. Maturation of Fibrinolytic Bacillopeptidase F Involves both Hetero- and Autocatalytic Processes.

    PubMed

    Meng, Dongheng; Dai, Meihong; Xu, Bi-Lin; Zhao, Zhong-Shu; Liang, Xiaoliang; Wang, Mingqiu; Tang, Xiao-Feng; Tang, Bing

    2016-01-01

    Bacillopeptidase F (Bpr) is a fibrinolytic serine protease produced by Bacillus subtilis. Its precursor is composed of a signal peptide, an N-terminal propeptide, a catalytic domain, and a long C-terminal extension (CTE). Several active forms of Bpr have been previously reported, but little is known about the maturation of this enzyme. Here, a gene encoding a Bpr (BprL) was cloned from B. subtilis LZW and expressed in B. subtilis WB700, and three fibrinolytic mature forms with apparent molecular masses of 45, 75, and 85 kDa were identified in the culture supernatant. After treatment with urea, the 75-kDa mature form had the same molecular mass as the 85-kDa mature form, from which we infer that they adopt different conformations. Mutational analysis revealed that while the 85-kDa mature form is generated via heterocatalytic processing of a BprL proform by an unidentified protease of B. subtilis, the production of the 75- and 45-kDa mature forms involves both hetero- and autocatalytic events. From in vitro analysis of BprL and its sequential C-terminal truncation variants, it appears that partial removal of the CTE is required for the initiation of autoprocessing of the N-terminal propeptide, which is composed of a core domain (N*) and a 15-residue linker peptide, thereby yielding the 45-kDa mature form. These data suggest that the differential processing of BprL, either heterocatalytically or autocatalytically, leads to the formation of multiple mature forms with different molecular masses or conformations. PMID:26497454

  4. Maturation of Fibrinolytic Bacillopeptidase F Involves both Hetero- and Autocatalytic Processes

    PubMed Central

    Meng, Dongheng; Dai, Meihong; Xu, Bi-Lin; Zhao, Zhong-Shu; Liang, Xiaoliang; Wang, Mingqiu; Tang, Xiao-Feng

    2015-01-01

    Bacillopeptidase F (Bpr) is a fibrinolytic serine protease produced by Bacillus subtilis. Its precursor is composed of a signal peptide, an N-terminal propeptide, a catalytic domain, and a long C-terminal extension (CTE). Several active forms of Bpr have been previously reported, but little is known about the maturation of this enzyme. Here, a gene encoding a Bpr (BprL) was cloned from B. subtilis LZW and expressed in B. subtilis WB700, and three fibrinolytic mature forms with apparent molecular masses of 45, 75, and 85 kDa were identified in the culture supernatant. After treatment with urea, the 75-kDa mature form had the same molecular mass as the 85-kDa mature form, from which we infer that they adopt different conformations. Mutational analysis revealed that while the 85-kDa mature form is generated via heterocatalytic processing of a BprL proform by an unidentified protease of B. subtilis, the production of the 75- and 45-kDa mature forms involves both hetero- and autocatalytic events. From in vitro analysis of BprL and its sequential C-terminal truncation variants, it appears that partial removal of the CTE is required for the initiation of autoprocessing of the N-terminal propeptide, which is composed of a core domain (N*) and a 15-residue linker peptide, thereby yielding the 45-kDa mature form. These data suggest that the differential processing of BprL, either heterocatalytically or autocatalytically, leads to the formation of multiple mature forms with different molecular masses or conformations. PMID:26497454

  5. Neural classifier in the estimation process of maturity of selected varieties of apples

    NASA Astrophysics Data System (ADS)

    Boniecki, P.; Piekarska-Boniecka, H.; Koszela, K.; Zaborowicz, M.; Przybył, K.; Wojcieszak, D.; Zbytek, Z.; Ludwiczak, A.; Przybylak, A.; Lewicki, A.

    2015-07-01

    This paper seeks to present methods of neural image analysis aimed at estimating the maturity state of selected varieties of apples which are popular in Poland. An identification of the degree of maturity of selected varieties of apples has been conducted on the basis of information encoded in graphical form, presented in the digital photos. The above process involves the application of the BBCH scale, used to determine the maturity of apples. The aforementioned scale is widely used in the EU and has been developed for many species of monocotyledonous plants and dicotyledonous plants. It is also worth noticing that the given scale enables detailed determinations of development stage of a given plant. The purpose of this work is to identify maturity level of selected varieties of apples, which is supported by the use of image analysis methods and classification techniques represented by artificial neural networks. The analysis of graphical representative features based on image analysis method enabled the assessment of the maturity of apples. For the utilitarian purpose the "JabVis 1.1" neural IT system was created, in accordance with requirements of the software engineering dedicated to support the decision-making processes occurring in broadly understood production process and processing of apples.

  6. N-terminal processing of affinity-tagged recombinant proteins purified by IMAC procedures.

    PubMed

    Mooney, Jane T; Fredericks, Dale P; Christensen, Thorkild; Bruun Schiødt, Christine; Hearn, Milton T W

    2015-07-01

    The ability of a new class of metal binding tags to facilitate the purification of recombinant proteins, exemplified by the tagged glutathione S-transferase and human growth hormone, from Escherichia coli fermentation broths and lysates has been further investigated. These histidine-containing tags exhibit high affinity for borderline metal ions chelated to the immobilised ligand, 1,4,7-triazacyclononane (tacn). The use of this tag-tacn immobilised metal ion affinity chromatography (IMAC) system engenders high selectivity with regard to host cell protein removal and permits facile tag removal from the E. coli-expressed recombinant protein. In particular, these tags were specifically designed to enable their efficient removal by the dipeptidyl aminopeptidase 1 (DAP-1), thus capturing the advantages of high substrate specificity and rates of cleavage. MALDI-TOF MS analysis of the cleaved products from the DAP-1 digestion of the recombinant N-terminally tagged proteins confirmed the complete removal of the tag within 4-12 h under mild experimental conditions. Overall, this study demonstrates that the use of tags specifically designed to target tacn-based IMAC resins offers a comprehensive and flexible approach for the purification of E. coli-expressed recombinant proteins, where complete removal of the tag is an essential prerequisite for subsequent application of the purified native proteins in studies aimed at delineating the molecular and cellular basis of specific biological processes. PMID:25727088

  7. Maturational Constraints on the Recruitment of Early Processes for Syntactic Processing

    PubMed Central

    Pakulak, Eric; Neville, Helen J.

    2011-01-01

    An enduring question in the study of second language acquisition concerns the relative contributions of age of acquisition (AOA) and ultimate linguistic proficiency to neural organization for second language processing. Several event-related potential (ERP) and neuroimaging studies of second language learners have found that neural organization for syntactic processing is sensitive to delays in second language acquisition. However, such delays in second language acquisition are typically associated with lower language proficiency, rendering it difficult to assess whether differences in AOA or proficiency lead to these effects. Here we examined the effects of delayed second language acquisition while controlling for proficiency differences by examining participants who differ in AOA but who were matched for proficiency in the same language. We compared the ERP response to auditory English phrase structure violations in a group of late learners of English matched for grammatical proficiency with a group of English native speakers. In the native speaker group violations elicited a bilateral and prolonged anterior negativity, with onset at 100 ms, followed by a posterior positivity (P600). In contrast, in the non-native speaker group violations did not elicit the early anterior negativity, but did elicit a P600 which was more widespread spatially and temporally than that of the native speaker group. These results suggest that neural organization for syntactic processing is sensitive to delays in language acquisition independently of proficiency level. More specifically they suggest that both early and later syntactic processes are sensitive to maturational constraints. These results also suggest that late learners who reach a high level of second language proficiency rely on different neural mechanisms than native speakers of that language. PMID:20964590

  8. CEval: All-in-one software for data processing and statistical evaluations in affinity capillary electrophoresis.

    PubMed

    Dubský, Pavel; Ördögová, Magda; Malý, Michal; Riesová, Martina

    2016-05-01

    We introduce CEval software (downloadable for free at echmet.natur.cuni.cz) that was developed for quicker and easier electrophoregram evaluation and further data processing in (affinity) capillary electrophoresis. This software allows for automatic peak detection and evaluation of common peak parameters, such as its migration time, area, width etc. Additionally, the software includes a nonlinear regression engine that performs peak fitting with the Haarhoff-van der Linde (HVL) function, including automated initial guess of the HVL function parameters. HVL is a fundamental peak-shape function in electrophoresis, based on which the correct effective mobility of the analyte represented by the peak is evaluated. Effective mobilities of an analyte at various concentrations of a selector can be further stored and plotted in an affinity CE mode. Consequently, the mobility of the free analyte, μA, mobility of the analyte-selector complex, μAS, and the apparent complexation constant, K('), are first guessed automatically from the linearized data plots and subsequently estimated by the means of nonlinear regression. An option that allows two complexation dependencies to be fitted at once is especially convenient for enantioseparations. Statistical processing of these data is also included, which allowed us to: i) express the 95% confidence intervals for the μA, μAS and K(') least-squares estimates, ii) do hypothesis testing on the estimated parameters for the first time. We demonstrate the benefits of the CEval software by inspecting complexation of tryptophan methyl ester with two cyclodextrins, neutral heptakis(2,6-di-O-methyl)-β-CD and charged heptakis(6-O-sulfo)-β-CD. PMID:27062723

  9. Coupled diffusion processes and 2D affinities of adhesion molecules at synthetic membrane junctions

    NASA Astrophysics Data System (ADS)

    Peel, Christopher; Choudhuri, Kaushik; Schmid, Eva M.; Bakalar, Matthew H.; Ann, Hyoung Sook; Fletcher, Daniel A.; Journot, Celine; Turberfield, Andrew; Wallace, Mark; Dustin, Michael

    A more complete understanding of the physically intrinsic mechanisms underlying protein mobility at cellular interfaces will provide additional insights into processes driving adhesion and organization in signalling junctions such as the immunological synapse. We observed diffusional slowing of structurally diverse binding proteins at synthetic interfaces formed by giant unilamellar vesicles (GUVs) on supported lipid bilayers (SLBs) that shows size dependence not accounted for by existing models. To model the effects of size and intermembrane spacing on interfacial reaction-diffusion processes, we describe a multistate diffusion model incorporating entropic effects of constrained binding. This can be merged with hydrodynamic theories of receptor-ligand diffusion and coupling to thermal membrane roughness. A novel synthetic membrane adhesion assay based on reversible and irreversible DNA-mediated interactions between GUVs and SLBs is used to precisely vary length, affinity, and flexibility, and also provides a platform to examine these effects on the dynamics of processes such as size-based segregation of binding and non-binding species.

  10. Spectral response variation of a negative-electron-affinity photocathode in the preparation process

    SciTech Connect

    Liu Lei; Du Yujie; Chang Benkang; Yunsheng Qian

    2006-08-20

    In order to research the spectral response variation of a negative electron affinity (NEA) photocathode in the preparation process, we have done two experiments on a transmission-type GaAs photocathode.First, an automatic spectral response recording system is described, which is used to take spectral response curves during the activation procedure of the photocathode. By this system, the spectral response curves of a GaAs:Cs-Ophotocathode measured in situ are presented. Then, after the cathode is sealed with a microchannel plate and a fluorescence screen into the image tube, we measure the spectral response of the tube by another measurement instrument. By way of comparing and analyzing these curves, we can find the typical variation in spectral-responses.The reasons for the variation are discussed. Based on these curves, spectral matching factors of a GaAs cathode for green vegetation and rough concrete are calculated. The visual ranges of night-vision goggles under specific circumstances are estimated. The results show that the spectral response of the NEA photocathode degraded in the sealing process, especially at long wavelengths. The variation has also influenced the whole performance of the intensifier tube.

  11. Spectral response variation of a negative-electron-affinity photocathode in the preparation process.

    PubMed

    Liu, Lei; Du, Yujie; Chang, Benkang; Yunsheng, Qian

    2006-08-20

    In order to research the spectral response variation of a negative electron affinity (NEA) photocathode in the preparation process, we have done two experiments on a transmission-type GaAs photocathode. First, an automatic spectral response recording system is described, which is used to take spectral response curves during the activation procedure of the photocathode. By this system, the spectral response curves of a GaAs:Cs-O photocathode measured in situ are presented. Then, after the cathode is sealed with a microchannel plate and a fluorescence screen into the image tube, we measure the spectral response of the tube by another measurement instrument. By way of comparing and analyzing these curves, we can find the typical variation in spectral-responses. The reasons for the variation are discussed. Based on these curves, spectral matching factors of a GaAs cathode for green vegetation and rough concrete are calculated. The visual ranges of night-vision goggles under specific circumstances are estimated. The results show that the spectral response of the NEA photocathode degraded in the sealing process, especially at long wavelengths. The variation has also influenced the whole performance of the intensifier tube. PMID:16892108

  12. Coconut water of different maturity stages ameliorates inflammatory processes in model of inflammation

    PubMed Central

    Rao, Sadia Saleem; Najam, Rahila

    2016-01-01

    Aim: Coconut water is a natural beverage that is a part of daily diet of many people. This study was designed to explore the anti-inflammatory activity of coconut water of different maturation stages (young and mature) with rat paw edema model of inflammation using plethysmometer. Methodology: For this study, albino rats were selected and divided into four equal groups (10 rats in each group). Group 1 was set as control and administered distilled water 1 ml orally; Groups 2 and 3 were treated with young and mature coconut water, respectively, at 4 ml/100 g dose orally. Group 4 was treated with the standard drug (ibuprofen) at 400 mg/70 kg. 0.1 ml of 1% w/v acetic acid was administered in the subplantar tissue of rat paw 30 min after oral treatments of groups. Plethysmometer was used to measure rat paw edema. Results: Results revealed that both coconut water possess significant anti-inflammatory activity (P < 0.001). In comparison to control, percent inhibition by young coconut water was 20.22%, 35.13%, 42.52%, and 36% at 1, 2, 3, and 4 h of acetic acid administration, respectively. However, maximum percent inhibition (42.52%) was observed in the second phase of the inflammatory process. On the other hand, percent inhibition by mature coconut water was 18.80%, 25.94%, 24.13%, and 18.66% at 1, 2, 3, and 4 h of acetic acid administration, respectively. However, maximum percent inhibition (25.94%) was observed in the first phase of the inflammatory process. Conclusions: This study strongly suggests the use of young coconut water for potent anti-inflammatory effect and mature coconut water for moderate anti-inflammatory effect. PMID:27366350

  13. V3 interneuron subpopulations in the mouse spinal cord undergo distinctive postnatal maturation processes.

    PubMed

    Borowska, J; Jones, C T; Deska-Gauthier, D; Zhang, Y

    2015-06-01

    Mice develop weight-bearing locomotion within the first 2-3 weeks of birth, a period during which motoneurons (MNs) and interneurons (INs) that control locomotor activities undergo rapid maturation. In this study, we investigate the maturation of two subpopulations of V3 INs in the mouse spinal cord during this period. To do this, we conducted whole-cell patch-clamp recordings of tdTomato fluorescent protein-expressing spinal V3 INs from Sim1(Cre/+);tdTom mice at post-natal day (P) 0, P4, P9 and P14 and compared their properties to those at P21. Combining electrophysiology with computational analyses, we show that dorsal and ventral V3 subpopulations are physiologically distinct at birth, but the electrophysiological properties of V3 INs change significantly during the first three post-natal weeks. We further reveal that there are multiple developmental phases of both V3 subpopulations during the maturation process. The different developmental trajectories of physiological properties also coincide with changes in an animal's locomotor behavior. These properties likely reflect the differential functions of V3 subpopulations in maturing spinal locomotor circuits. PMID:25800308

  14. Phagosome maturation during endosome interaction revealed by partial rhodopsin processing in retinal pigment epithelium

    PubMed Central

    Wavre-Shapton, Silène T.; Meschede, Ingrid P.; Seabra, Miguel C.; Futter, Clare E.

    2014-01-01

    ABSTRACT Defects in phagocytosis and degradation of photoreceptor outer segments (POS) by the retinal pigment epithelium (RPE) are associated with aging and retinal disease. The daily burst of rod outer segment (ROS) phagocytosis by the RPE provides a unique opportunity to analyse phagosome processing in vivo. In mouse retinae, phagosomes containing stacked rhodopsin-rich discs were identified by immuno-electron microscopy. Early apical phagosomes stained with antibodies against both cytoplasmic and intradiscal domains of rhodopsin. During phagosome maturation, a remarkably synchronised loss of the cytoplasmic epitope coincided with movement to the cell body and preceded phagosome–lysosome fusion and disc degradation. Loss of the intradiscal rhodopsin epitope and disc digestion occurred upon fusion with cathepsin-D-positive lysosomes. The same sequential stages of phagosome maturation were identified in cultured RPE and macrophages challenged with isolated POS. Loss of the cytoplasmic rhodopsin epitope was insensitive to pH but sensitive to protease inhibition and coincided with the interaction of phagosomes with endosomes. Thus, during pre-lysosomal maturation of ROS-containing phagosomes, limited rhodopsin processing occurs upon interaction with endosomes. This potentially provides a sensitive readout of phagosome–endosome interactions that is applicable to multiple phagocytes. PMID:25074813

  15. Different visuomotor processes maturation rates in children support dual visuomotor learning systems.

    PubMed

    Gómez-Moya, Rosinna; Díaz, Rosalinda; Fernandez-Ruiz, Juan

    2016-04-01

    Different processes are involved during visuomotor learning, including an error-based procedural and a strategy based cognitive mechanism. Our objective was to analyze if the changes in the adaptation or the aftereffect components of visuomotor learning measured across development, reflected different maturation rates of the aforementioned mechanisms. Ninety-five healthy children aged 4-12years and a group of young adults participated in a wedge prism and a dove prism throwing task, which laterally displace or horizontally reverse the visual field respectively. The results show that despite the age-related differences in motor control, all children groups adapted in the error-based wedge prisms condition. However, when removing the prism, small children showed a slower aftereffects extinction rate. On the strategy-based visual reversing task only the older children group reached adult-like levels. These results are consistent with the idea of different mechanisms with asynchronous maturation rates participating during visuomotor learning. PMID:26802974

  16. A late-acting quality control process for mature eukaryotic rRNAs.

    PubMed

    LaRiviere, Frederick J; Cole, Sarah E; Ferullo, Daniel J; Moore, Melissa J

    2006-11-17

    Ribosome biogenesis is a multifaceted process involving a host of trans-acting factors mediating numerous chemical reactions, RNA conformational changes, and RNA-protein associations. Given this high degree of complexity, tight quality control is likely crucial to ensure structural and functional integrity of the end products. We demonstrate that ribosomal RNAs (rRNAs) containing individual point mutations, in either the 25S peptidyl transferase center or 18S decoding site, that adversely affect ribosome function are strongly downregulated in Saccharomyces cerevisiae. This downregulation occurs via decreased stability of the mature rRNA contained in fully assembled ribosomes and ribosomal subunits. Thus, eukaryotes possess a quality-control mechanism, nonfunctional rRNA decay (NRD), capable of detecting and eliminating the rRNA component of mature ribosomes. PMID:17188037

  17. Sustaining a Mature Risk Management Process: Ensuring the International Space Station for a Vibrant Future

    NASA Technical Reports Server (NTRS)

    Raftery, Michael; Carter-Journet, Katrina

    2013-01-01

    The International Space Station (ISS) risk management methodology is an example of a mature and sustainable process. Risk management is a systematic approach used to proactively identify, analyze, plan, track, control, communicate, and document risks to help management make risk-informed decisions that increase the likelihood of achieving program objectives. The ISS has been operating in space for over 14 years and permanently crewed for over 12 years. It is the longest surviving habitable vehicle in low Earth orbit history. Without a mature and proven risk management plan, it would be increasingly difficult to achieve mission success throughout the life of the ISS Program. A successful risk management process must be able to adapt to a dynamic program. As ISS program-level decision processes have evolved, so too has the ISS risk management process continued to innovate, improve, and adapt. Constant adaptation of risk management tools and an ever-improving process is essential to the continued success of the ISS Program. Above all, sustained support from program management is vital to risk management continued effectiveness. Risk management is valued and stressed as an important process by the ISS Program.

  18. Effects of drying process on the physicochemical properties of nopal cladodes at different maturity stages.

    PubMed

    Contreras-Padilla, Margarita; Gutiérrez-Cortez, Elsa; Valderrama-Bravo, María Del Carmen; Rojas-Molina, Isela; Espinosa-Arbeláez, Diego Germán; Suárez-Vargas, Raúl; Rodríguez-García, Mario Enrique

    2012-03-01

    Chemical proximate analysis was done in order to determine the changes of nutritional characteristics of nopal powders from three different maturity stages 50, 100, and 150 days and obtained by three different drying processes: freeze dried, forced air oven, and tunnel. Results indicate that nopal powder obtained by the process of freeze dried retains higher contents of protein, soluble fiber, and fat than the other two processes. Also, freeze dried process had less effect on color hue variable. No changes were observed in insoluble fiber content, chroma and lightness with the three different drying processes. Furthermore, the soluble fibers decreased with the age of nopal while insoluble fibers and ash content shows an opposite trend. In addition, the luminosity and hue values did not show differences among the maturity stages studied. The high content of dietary fibers of nopal pad powder could to be an interesting source of these important components for human diets and also could be used in food, cosmetics and pharmaceutical industry. PMID:22183868

  19. Implementing NASA's Capability-Driven Approach: Insight into NASA's Processes for Maturing Exploration Systems

    NASA Technical Reports Server (NTRS)

    Williams-Byrd, Julie; Arney, Dale; Rodgers, Erica; Antol, Jeff; Simon, Matthew; Hay, Jason; Larman, Kevin

    2015-01-01

    NASA is engaged in transforming human spaceflight. The Agency is shifting from an exploration-based program with human activities focused on low Earth orbit (LEO) and targeted robotic missions in deep space to a more sustainable and integrated pioneering approach. Through pioneering, NASA seeks to address national goals to develop the capacity for people to work, learn, operate, live, and thrive safely beyond the Earth for extended periods of time. However, pioneering space involves more than the daunting technical challenges of transportation, maintaining health, and enabling crew productivity for long durations in remote, hostile, and alien environments. This shift also requires a change in operating processes for NASA. The Agency can no longer afford to engineer systems for specific missions and destinations and instead must focus on common capabilities that enable a range of destinations and missions. NASA has codified a capability driven approach, which provides flexible guidance for the development and maturation of common capabilities necessary for human pioneers beyond LEO. This approach has been included in NASA policy and is captured in the Agency's strategic goals. It is currently being implemented across NASA's centers and programs. Throughout 2014, NASA engaged in an Agency-wide process to define and refine exploration-related capabilities and associated gaps, focusing only on those that are critical for human exploration beyond LEO. NASA identified 12 common capabilities ranging from Environmental Control and Life Support Systems to Robotics, and established Agency-wide teams or working groups comprised of subject matter experts that are responsible for the maturation of these exploration capabilities. These teams, called the System Maturation Teams (SMTs) help formulate, guide and resolve performance gaps associated with the identified exploration capabilities. The SMTs are defining performance parameters and goals for each of the 12 capabilities

  20. Physical, chemical and nutritional characteristics of premature-processed and matured green legumes.

    PubMed

    Bhattacharya, Sila; Malleshi, N G

    2012-08-01

    Premature green legumes are good sources of nutraceuticals and antioxidants and are consumed as snacks as well as vegetables. They are seasonal and have limited shelf-life. Efforts are provided to prepare shelf-stable green legumes to extend their availability throughout the year. Green legumes from chick pea or Bengal gram (Cicer arietinum) and field bean (Dolichos lablab) have been processed to enhance their shelf-life, and determined their nutritional, physico-chemical and nutraceutical qualities. The shelf stable green legumes (SSGL) show higher water absorption capacity compared to matured dry legumes (MDL). The total colour change in the processed/dried SSGL and MDL samples increased significantly (p ≤ 0.05) compared to the freshly harvested green samples. The carotenoid content of Bengal gram and field bean SSGLs are 8.0 and 3.2 mg/100 g, and chlorophyll contents are 12.5 and 0.5 mg/100 g, respectively, which are in negligible quantities in matured legumes; the corresponding polyphenol contents are 197.8 and 153.1 mg/100 g. These results indicate that SSGLs possess potential antioxidant activity. PMID:23904654

  1. Affinity Inequality among Serum Antibodies That Originate in Lymphoid Germinal Centers

    PubMed Central

    Eisen, Ellen A.; Chakraborty, Arup K.

    2015-01-01

    Upon natural infection with pathogens or vaccination, antibodies are produced by a process called affinity maturation. As affinity maturation ensues, average affinity values between an antibody and ligand increase with time. Purified antibodies isolated from serum are invariably heterogeneous with respect to their affinity for the ligands they bind, whether macromolecular antigens or haptens (low molecular weight approximations of epitopes on antigens). However, less is known about how the extent of this heterogeneity evolves with time during affinity maturation. To shed light on this issue, we have taken advantage of previously published data from Eisen and Siskind (1964). Using the ratio of the strongest to the weakest binding subsets as a metric of heterogeneity (or affinity inequality), we analyzed antibodies isolated from individual serum samples. The ratios were initially as high as 50-fold, and decreased over a few weeks after a single injection of small antigen doses to around unity. This decrease in the effective heterogeneity of antibody affinities with time is consistent with Darwinian evolution in the strong selection limit. By contrast, neither the average affinity nor the heterogeneity evolves much with time for high doses of antigen, as competition between clones of the same affinity is minimal. PMID:26444899

  2. Maturational Constraints on the Recruitment of Early Processes for Syntactic Processing

    ERIC Educational Resources Information Center

    Pakulak, Eric; Neville, Helen J.

    2011-01-01

    An enduring question in the study of second-language acquisition concerns the relative contributions of age of acquisition (AOA) and ultimate linguistic proficiency to neural organization for second-language processing. Several ERP and neuroimaging studies of second-language learners have found that neural organization for syntactic processing is…

  3. The Maturation Process of pVP2 Requires Assembly of Infectious Bursal Disease Virus Capsids

    PubMed Central

    Chevalier, Christophe; Lepault, Jean; Erk, Inge; Da Costa, Bruno; Delmas, Bernard

    2002-01-01

    Infectious bursal disease virus (IBDV) is a nonenveloped avian virus with a two-segment double-stranded RNA genome. Its T=13 icosahedral capsid is most probably assembled with 780 subunits of VP2 and 600 copies of VP3 and has a diameter of about 60 nm. VP1, the RNA-dependent RNA polymerase, resides inside the viral particle. Using a baculovirus expression system, we first observed that expression of the pVP2-VP4-VP3 polyprotein encoded by the genomic segment IBDA results mainly in the formation of tubules with a diameter of about 50 nm and composed of pVP2, the precursor of VP2. Very few virus-like particles (VLPs) and VP4 tubules with a diameter of about 25 nm were also identified. The inefficiency of VLP assembly was further investigated by expression of additional IBDA-derived constructs. Expression of pVP2 without any other polyprotein components results in the formation of isometric particles with a diameter of about 30 nm. VLPs were observed mainly when a large exogeneous polypeptide sequence (the green fluorescent protein sequence) was fused to the VP3 C-terminal domain. Large numbers of VLPs were visualized by electron microscopy, and single particles were shown to be fluorescent by standard and confocal microscopy analysis. Moreover, the final maturation process converting pVP2 into the VP2 mature form was observed on generated VLPs. We therefore conclude that the correct scaffolding of the VP3 can be artificially induced to promote the formation of VLPs and that the final processing of pVP2 to VP2 is controlled by this particular assembly. To our knowledge, this is the first report of the engineering of a morphogenesis switch to control a particular type of capsid protein assembly. PMID:11836416

  4. Analyzing a Mature Software Inspection Process Using Statistical Process Control (SPC)

    NASA Technical Reports Server (NTRS)

    Barnard, Julie; Carleton, Anita; Stamper, Darrell E. (Technical Monitor)

    1999-01-01

    This paper presents a cooperative effort where the Software Engineering Institute and the Space Shuttle Onboard Software Project could experiment applying Statistical Process Control (SPC) analysis to inspection activities. The topics include: 1) SPC Collaboration Overview; 2) SPC Collaboration Approach and Results; and 3) Lessons Learned.

  5. Affinity Chromatography.

    ERIC Educational Resources Information Center

    Gray, Gary R.

    1980-01-01

    Presents selected recent advances in immobilization chemistry which have important connections to affinity chromatography. Discusses ligand immobilization and support modification. Cites 51 references. (CS)

  6. EFFECT OF CORN SILAGE MATURITY AND MECHANICAL PROCESSING ON NUTRIENT DIGESTIBILITY BY LACTATING DAIRY COWS OF DIFFERENT LACTATION STAGES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Starch digestibility (StarchD) can decrease when corn silage is harvested at advanced stages of maturity. Processing corn silage through rollers during chopping typically increases StarchD. We hypothesized that corn silage processing increases StarchD in lactating dairy cows to a greater extent when...

  7. Bioelectrochemically-assisted anaerobic composting process enhancing compost maturity of dewatered sludge with synchronous electricity generation.

    PubMed

    Yu, Hang; Jiang, Junqiu; Zhao, Qingliang; Wang, Kun; Zhang, Yunshu; Zheng, Zhen; Hao, Xiaodi

    2015-10-01

    Bioelectrochemically-assisted anaerobic composting process (AnCBE) with dewatered sludge as the anode fuel was constructed to accelerate composting of dewatered sludge, which could increase the quality of the compost and harvest electric energy in comparison with the traditional anaerobic composting (AnC). Results revealed that the AnCBE yielded a voltage of 0.60 ± 0.02 V, and total COD (TCOD) removal reached 19.8 ± 0.2% at the end of 35 d. The maximum power density was 5.6 W/m(3). At the end of composting, organic matter content (OM) reduction rate increased to 19.5 ± 0.2% in AnCBE and to 12.9 ± 0.1% in AnC. The fuzzy comprehensive assessment (FCA) result indicated that the membership degree of class I of AnCBE compost (0.64) was higher than that of AnC compost (0.44). It was demonstrated that electrogenesis in the AnCBE could improve the sludge stabilization degree, accelerate anaerobic composting process and enhance composting maturity with bioelectricity generation. PMID:26115526

  8. Natural antisense RNA promotes 3′ end processing and maturation of MALAT1 lncRNA

    PubMed Central

    Zong, Xinying; Nakagawa, Shinichi; Freier, Susan M.; Fei, Jingyi; Ha, Taekjip; Prasanth, Supriya G.; Prasanth, Kannanganattu V.

    2016-01-01

    The RNase P-mediated endonucleolytic cleavage plays a crucial role in the 3′ end processing and cellular accumulation of MALAT1, a nuclear-retained long noncoding RNA that promotes malignancy. The regulation of this cleavage event is largely undetermined. Here we characterize a broadly expressed natural antisense transcript at the MALAT1 locus, designated as TALAM1, that positively regulates MALAT1 levels by promoting the 3′ end cleavage and maturation of MALAT1 RNA. TALAM1 RNA preferentially localizes at the site of transcription, and also interacts with MALAT1 RNA. Depletion of TALAM1 leads to defects in the 3′ end cleavage reaction and compromises cellular accumulation of MALAT1. Conversely, overexpression of TALAM1 facilitates the cleavage reaction in trans. Interestingly, TALAM1 is also positively regulated by MALAT1 at the level of both transcription and RNA stability. Together, our data demonstrate a novel feed-forward positive regulatory loop that is established to maintain the high cellular levels of MALAT1, and also unravel the existence of sense-antisense mediated regulatory mechanism for cellular lncRNAs that display RNase P-mediated 3′ end processing. PMID:26826711

  9. Maturation of the Central Auditory Nervous System in Children with Auditory Processing Disorder.

    PubMed

    Tomlin, Dani; Rance, Gary

    2016-02-01

    Neurodevelopmental delay has been proposed as the underlying cause of the majority of cases of auditory processing disorder (APD). The current study employs the cortical auditory evoked potential (CAEP) to assess if maturational differences of the central auditory nervous system (CANS) can be identified between children who do and do not meet the diagnostic criterion for APD. The P1-N1 complex of the CAEP has previously been used for tracking development of the CANS in children with hearing impairment. Twenty-seven children (7 to 12 years old) who failed an APD behavioral test battery were age-matched (within 3 months) to children who had passed the same battery. CAEP responses to 500-Hz tone burst stimuli were recorded and analyzed for latency and amplitude measures. The P1-N1 complex of the CAEP, which has previously been used for tracking development of the CANS in children with hearing impairment, showed significant group differences. The children diagnosed with APD showed significantly increased latency (∼10 milliseconds) and significantly reduced amplitude (∼10 μV) of the early components of the CAEP compared with children with normal auditory processing. No significant differences were seen in the later P2 wave. The normal developmental course is for a decrease in latency and increase in amplitude as a function of age. The results of this study are, therefore, consistent with an immaturity of the CANS as an underlying cause of APD in children. PMID:27587924

  10. Microscopic dynamics research on the "mature" process of dye-sensitized solar cells after injection of highly concentrated electrolyte.

    PubMed

    Liang, Zhongguan; Liu, Weiqing; Chen, Jun; Hu, Linhua; Dai, Songyuan

    2015-01-21

    After injection of electrolyte, the internal three-dimensional solid-liquid penetration system of dye-sensitized solar cells (DSCs) can take a period of time to reach "mature" state. This paper studies the changes of microscopic processes of DSCs including TiO2 energy-level movement, localized state distribution, charge accumulation, electron transport, and recombination dynamics, from the beginning of electrolyte injection to the time of reached mature state. The results show that the microscopic dynamics process of DSCs exhibited a time-dependent behavior and achieved maturity ∼12 h after injecting the electrolyte into DSCs. Within 0-12 h, several results were observed: (1) the conduction band edge of TiO2 moved slightly toward negative potential direction; (2) the localized states in the band gap of TiO2 was reduced according to the same distribution law; (3) the transport resistance in TiO2 film increased, and electron transport time was prolonged as the time of maturity went on, which indicated that the electron transport process is impeded gradually; (4) the recombination resistance at the TiO2/electrolyte (EL) interface increases, and electron lifetime gradually extends, therefore, the recombination process is continuously suppressed. Furthermore, results suggest that the parameters of EL/Pt-transparent conductive oxide (TCO) interface including the interfacial capacitance, electron-transfer resistance, and transfer time constant would change with time of maturity, indicating that the EL/Pt-TCO interface is a potential factor affecting the mature process of DSCs. PMID:25517177

  11. Process characterization for metal-affinity chromatography of an Fc fusion protein: a design-of-experiments approach.

    PubMed

    Shukla, A A; Sorge, L; Boldman, J; Waugh, S

    2001-10-01

    The utility of a design-of-experiments approach was investigated for process characterization of a metal-affinity chromatographic purification process for an Fc fusion protein. This approach gave a better understanding of some of the key process variables as well as robustness for this step in the purification process. Single-variable experiments were employed to screen some of the potentially important variables in this step. Ranges for these variables were set based on prior experience in clinical manufacturing with similar processes. Following these experiments, a fractional factorial study was employed to further investigate the most important variables and their interactions. Key operational variables that had an impact on step yield and eluate purity were identified. In addition, the study helped identify a worst-case scenario for the step purity and helped assure that the rest of the process would successfully purify the product. This paper demonstrates the utility of a design-of-experiments approach for the characterization and validation of process chromatography steps in downstream processing. In addition, this study emphasizes the utility of robustness studies early in process development and establishes a strategy for future robustness studies. PMID:11592911

  12. Whey protein processing influences formula-induced gut maturation in preterm pigs.

    PubMed

    Li, Yanqi; Østergaard, Mette V; Jiang, Pingping; Chatterton, Dereck E W; Thymann, Thomas; Kvistgaard, Anne S; Sangild, Per T

    2013-12-01

    Immaturity of the gut predisposes preterm infants to nutritional challenges potentially leading to clinical complications such as necrotizing enterocolitis. Feeding milk formulas is associated with greater risk than fresh colostrum or milk, probably due to loss of bioactive proteins (e.g., immunoglobulins, lactoferrin, insulin-like growth factor, transforming growth factor-β) during industrial processing (e.g., pasteurization, filtration, spray-drying). We hypothesized that the processing method for whey protein concentrate (WPC) would affect gut maturation in formula-fed preterm pigs used as a model for preterm infants. Fifty-five caesarean-delivered preterm pigs were distributed into 4 groups given 1 of 4 isoenergetic diets: formula containing conventional WPC (filtration, multi-pasteurization, standard spray-drying) (CF); formula containing gently treated WPC (reduced filtration and pasteurization, gentle spray-drying) (GF); formula containing minimally treated WPC (rennet precipitation, reduced filtration, heat treatment <40°C, freeze-drying) (MF); and bovine colostrum (used as a positive reference group) (BC). Relative to CF, GF, and MF pigs, BC pigs had greater villus heights, lactose digestion, and absorption and lower gut permeability (P < 0.05). MF and BC pigs had greater plasma citrulline concentrations than CF and GF pigs and intestinal interleukin-8 was lower in BC pigs than in the other groups (P < 0.05). MF pigs had lower concentrations of intestinal claudin-4, cleaved caspase-3, and phosphorylated c-Jun than CF pigs (P < 0.05). The conventional and gently treated WPCs had similar efficacy in stimulating proliferation of porcine intestinal epithelial cells. We conclude that processing of WPC affects intestinal structure, function, and integrity when included in formulas for preterm pigs. Optimization of WPC processing technology may be important to preserve the bioactivity and nutritional value of formulas for sensitive newborns. PMID:24047702

  13. The Native Form and Maturation Process of Hepatitis C Virus Core Protein

    PubMed Central

    Yasui, Kohichiroh; Wakita, Takaji; Tsukiyama-Kohara, Kyoko; Funahashi, Shin-Ichi; Ichikawa, Masumi; Kajita, Tadahiro; Moradpour, Darius; Wands, Jack R.; Kohara, Michinori

    1998-01-01

    The maturation and subcellular localization of hepatitis C virus (HCV) core protein were investigated with both a vaccinia virus expression system and CHO cell lines stably transformed with HCV cDNA. Two HCV core proteins, with molecular sizes of 21 kDa (p21) and 23 kDa (p23), were identified. The C-terminal end of p23 is amino acid 191 of the HCV polyprotein, and p21 is produced as a result of processing between amino acids 174 and 191. The subcellular localization of the HCV core protein was examined by confocal laser scanning microscopy. Although HCV core protein resided predominantly in the cytoplasm, it was also found in the nucleus and had the same molecular size as p21 in both locations, as determined by subcellular fractionation. The HCV core proteins had different immunoreactivities to a panel of monoclonal antibodies. Antibody 5E3 stained core protein in both the cytoplasm and the nucleus, C7-50 stained core protein only in the cytoplasm, and 499S stained core protein only in the nucleus. These results clearly indicate that the p23 form of HCV core protein is processed to p21 in the cytoplasm and that the core protein in the nucleus has a higher-order structure different from that of p21 in the cytoplasm. HCV core protein in sera of patients with HCV infection was analyzed in order to determine the molecular size of genuinely processed HCV core protein. HCV core protein in sera was found to have exactly the same molecular weight as the p21 protein. These results suggest that p21 core protein is a component of native viral particles. PMID:9621068

  14. Dynamic laser speckle applied to the analysis of maturation process of irradiated fresh fruits

    NASA Astrophysics Data System (ADS)

    Vincitorio, F. M.; Budini, N.; Freyre, C.; Mulone, C.; Fiorucci, M. P.; López, A. J.; Ramil, A.

    2012-10-01

    The treatment of fresh fruits with different doses of ionizing radiation has been found effective for delaying ripening and, in this way, to extend shelf life. This preservation method is likely to produce some functional or constitutive changes in the cellular structure of the fruit. In this work, a test of the effectiveness of fruit irradiation with relatively low doses was performed by using dynamic speckle imaging. Bananas from a same lot were chosen, being a first series of them irradiated with different doses of 0.2, 0.4 and 0.6 kGy (Gy = J/kg) and a second series with doses of 0.2, 0.4, 0.6 and 1 kGy. Non irradiated bananas (0 kGy) were considered as the lot reference for contrast. Irradiation was carried out at the Semi-Industrial Cobalt 60 facility of the Ezeiza Atomic Center, with an activity of 6 × 105 Curie and a dose rate of 28.5 Gy/min. The objective of this work is to analyze differences in the maturation process between irradiated and nonirradiated fruits by means of dynamic speckle pattern evaluation.

  15. Pre-adolescent alcohol expectancies: critical shifts and associated maturational processes.

    PubMed

    Bekman, Nicole M; Goldman, Mark S; Worley, Matthew J; Anderson, Kristen G

    2011-12-01

    Children's alcohol expectancies shift in late childhood/early adolescence in ways thought to lead to increased risk for adolescent alcohol use. The precise nature of this shift and the maturational processes that may influence it remain to be clarified. To these ends, we compared expectancy endorsement by grade across four expectancy domains: positive, negative, arousal, and sedation, in a cross-sectional sample of 3rd-6th grade children attending afterschool programs (n = 299). Structural equation modeling (SEM) was then used to describe the relationships between expectancies and differences in (a) cognitive ability and concept formation, (b) risk-taking personality traits, and (c) social exposure or values regarding alcohol-related information. Results showed those children in higher grades endorsed significantly more positive, negative, and sedating expectancies for alcohol than their younger peers. Concept formation partially and fully mediated the relationships between grade and both positive and sedating expectancies, respectively, but not the relationship between grade and negative expectancies. Sensation seeking did not increase across grades in this sample, and the relationship between sensation seeking and positive expectancies was fully mediated by reported alcohol exposure and values. This study provides a basis for future exploration of developmental influences on alcohol expectancies, an understanding of which may be helpful in the design of prevention efforts targeting high-risk youth before adolescence. PMID:21942260

  16. Individual heterogeneity in growth and age at sexual maturity: a gamma process analysis of capture–mark–recapture data

    USGS Publications Warehouse

    Link, William; Hesed, Kyle Miller

    2015-01-01

    Knowledge of organisms’ growth rates and ages at sexual maturity is important for conservation efforts and a wide variety of studies in ecology and evolutionary biology. However, these life history parameters may be difficult to obtain from natural populations: individuals encountered may be of unknown age, information on age at sexual maturity may be uncertain and interval-censored, and growth data may include both individual heterogeneity and measurement errors. We analyzed mark–recapture data for Red-backed Salamanders (Plethodon cinereus) to compare sex-specific growth rates and ages at sexual maturity. Aging of individuals was made possible by the use of a von Bertalanffy model of growth, complemented with models for interval-censored and imperfect observations at sexual maturation. Individual heterogeneity in growth was modeled through the use of Gamma processes. Our analysis indicates that female P. cinereus mature earlier and grow more quickly than males, growing to nearly identical asymptotic size distributions as males.

  17. From the affinity constant to the half-saturation index: understanding conventional modeling concepts in novel wastewater treatment processes.

    PubMed

    Arnaldos, Marina; Amerlinck, Youri; Rehman, Usman; Maere, Thomas; Van Hoey, Stijn; Naessens, Wouter; Nopens, Ingmar

    2015-03-01

    The "affinity constant" (KS) concept is applied in wastewater treatment models to incorporate the effect of substrate limitation on process performance. As an increasing number of wastewater treatment processes rely on low substrate concentrations, a proper understanding of these so-called constants is critical in order to soundly model and evaluate emerging treatment systems. In this paper, an in-depth analysis of the KS concept has been carried out, focusing on the different physical and biological phenomena that affect its observed value. By structuring the factors influencing half-saturation indices (newly proposed nomenclature) into advectional, diffusional and biological, light has been shed onto some of the apparent inconsistencies present in the literature. Particularly, the importance of non-ideal mixing as a source of variability between observed KS values in different systems has been illustrated. Additionally, discussion on the differences existent between substrates that affect half-saturation indices has been carried out; it has been shown that the observed KS for some substrates will reflect transport or biological limitations more than others. Finally, potential modeling strategies that could alleviate the shortcomings of the KS concept have been provided. These could be of special importance when considering the evaluation and design of emerging wastewater treatment processes. PMID:25576693

  18. Exploring the Decision Process of "School Leavers" and "Mature Students" in University Choice.

    ERIC Educational Resources Information Center

    Harker, Debra; Slade, Peter; Harker, Michael

    2001-01-01

    Examined potential differences in how Australian mature entrants and those who have just left school undertake the decision to attend a new university. Found differences between the two groups in terms of their need for public transportation and scheduling convenience, emphasis on program quality, and college search strategies. (EV)

  19. Piloting a Process Maturity Model as an e-Learning Benchmarking Method

    ERIC Educational Resources Information Center

    Petch, Jim; Calverley, Gayle; Dexter, Hilary; Cappelli, Tim

    2007-01-01

    As part of a national e-learning benchmarking initiative of the UK Higher Education Academy, the University of Manchester is carrying out a pilot study of a method to benchmark e-learning in an institution. The pilot was designed to evaluate the operational viability of a method based on the e-Learning Maturity Model developed at the University of…

  20. [Pharmaceutical Assistance in the Family Healthcare Program: points of affinity and discord in the organization process].

    PubMed

    Silva Oliveira, Tatiana de Alencar; Maria, Tatiane de Oliveira Silva; Alves do Nascimento, Angela Maria; do Nascimento, Angela Alves

    2011-09-01

    The scope of this study was to discuss the organization of the pharmaceutical assistance service in the family healthcare program. Qualitative research from a critical/analytical perspective was conducted in family healthcare units in a municipality of the state of Bahia, Brazil. Data was collected on the basis of systematic observation, semi-structured interviews and documents analysis from a dialectic standpoint. The organization of Pharmaceutical Assistance consisted of selection, planning, acquisition, storage and dispensing activities. The process was studied in the implementation phase, which was occurring in a centralized and uncoordinated fashion, without the proposed team work. An excess of activity was observed among the healthcare workers and there was an absence of a continued education policy for the workers. For the transformation of this situation and to ensure the organization of pharmaceutical assistance with quality and in an integrated manner, a reworking of the manner of thinking and action of the players concerned (managers, health workers and users), who participate directly in the organization, is necessary. Furthermore, mechanical, bureaucratic and impersonal work practices need to be abandoned. PMID:21987338

  1. Maturation of auditory neural processes in autism spectrum disorder — A longitudinal MEG study

    PubMed Central

    Port, Russell G.; Edgar, J. Christopher; Ku, Matthew; Bloy, Luke; Murray, Rebecca; Blaskey, Lisa; Levy, Susan E.; Roberts, Timothy P.L.

    2016-01-01

    Background Individuals with autism spectrum disorder (ASD) show atypical brain activity, perhaps due to delayed maturation. Previous studies examining the maturation of auditory electrophysiological activity have been limited due to their use of cross-sectional designs. The present study took a first step in examining magnetoencephalography (MEG) evidence of abnormal auditory response maturation in ASD via the use of a longitudinal design. Methods Initially recruited for a previous study, 27 children with ASD and nine typically developing (TD) children, aged 6- to 11-years-old, were re-recruited two to five years later. At both timepoints, MEG data were obtained while participants passively listened to sinusoidal pure-tones. Bilateral primary/secondary auditory cortex time domain (100 ms evoked response latency (M100)) and spectrotemporal measures (gamma-band power and inter-trial coherence (ITC)) were examined. MEG measures were also qualitatively examined for five children who exhibited “optimal outcome”, participants who were initially on spectrum, but no longer met diagnostic criteria at follow-up. Results M100 latencies were delayed in ASD versus TD at the initial exam (~ 19 ms) and at follow-up (~ 18 ms). At both exams, M100 latencies were associated with clinical ASD severity. In addition, gamma-band evoked power and ITC were reduced in ASD versus TD. M100 latency and gamma-band maturation rates did not differ between ASD and TD. Of note, the cohort of five children that demonstrated “optimal outcome” additionally exhibited M100 latency and gamma-band activity mean values in-between TD and ASD at both timepoints. Though justifying only qualitative interpretation, these “optimal outcome” related data are presented here to motivate future studies. Conclusions Children with ASD showed perturbed auditory cortex neural activity, as evidenced by M100 latency delays as well as reduced transient gamma-band activity. Despite evidence for maturation

  2. Mitochondrial Polyadenylation Is a One-Step Process Required for mRNA Integrity and tRNA Maturation

    PubMed Central

    Calvo-Garrido, Javier; Maffezzini, Camilla; Felser, Andrea; Wibom, Rolf; Wedell, Anna; Wredenberg, Anna

    2016-01-01

    Polyadenylation has well characterised roles in RNA turnover and translation in a variety of biological systems. While polyadenylation on mitochondrial transcripts has been suggested to be a two-step process required to complete translational stop codons, its involvement in mitochondrial RNA turnover is less well understood. We studied knockdown and knockout models of the mitochondrial poly(A) polymerase (MTPAP) in Drosophila melanogaster and demonstrate that polyadenylation of mitochondrial mRNAs is exclusively performed by MTPAP. Further, our results show that mitochondrial polyadenylation does not regulate mRNA stability but protects the 3' terminal integrity, and that despite a lack of functioning 3' ends, these trimmed transcripts are translated, suggesting that polyadenylation is not required for mitochondrial translation. Additionally, loss of MTPAP leads to reduced steady-state levels and disturbed maturation of tRNACys, indicating that polyadenylation in mitochondria might be important for the stability and maturation of specific tRNAs. PMID:27176048

  3. Opportunistic proteolytic processing of carbonic anhydrase 1 from Chlamydomonas in Arabidopsis reveals a novel route for protein maturation.

    PubMed

    Juvale, Parijat S; Wagner, Ryan L; Spalding, Martin H

    2016-04-01

    Proteolytic processing of secretory proteins to yield an active form generally involves specific proteolytic cleavage of a pre-protein. Multiple specific proteases have been identified that target specific pre-protein processing sites in animals. However, characterization of site-specific proteolysis of plant pre-proteins is still evolving. In this study, we characterized proteolytic processing of Chlamydomonas periplasmic carbonic anhydrase 1 (CAH1) in Arabidopsis. CAH1 pre-protein undergoes extensive post-translational modification in the endomembrane system, including glycosylation, disulfide bond formation and proteolytic removal of a peptide 'spacer' region, resulting in a mature, heterotetrameric enzyme with two large and two small subunits. We generated a series of small-scale and large-scale modifications to the spacer and flanking regions to identify potential protease target motifs. Surprisingly, we found that the endoproteolytic removal of the spacer from the CAH1 pre-protein proceeded via an opportunistic process apparently followed by further maturation via amino and carboxy peptidases. We also discovered that the spacer itself is not required for processing, which appears to be dependent only on the number of amino acids separating two key disulfide-bond-forming cysteines. Our data suggest a novel, opportunistic route for pre-protein processing of CAH1. PMID:26917556

  4. Opportunistic proteolytic processing of carbonic anhydrase 1 from Chlamydomonas in Arabidopsis reveals a novel route for protein maturation

    PubMed Central

    Juvale, Parijat S.; Wagner, Ryan L.; Spalding, Martin H.

    2016-01-01

    Proteolytic processing of secretory proteins to yield an active form generally involves specific proteolytic cleavage of a pre-protein. Multiple specific proteases have been identified that target specific pre-protein processing sites in animals. However, characterization of site-specific proteolysis of plant pre-proteins is still evolving. In this study, we characterized proteolytic processing of Chlamydomonas periplasmic carbonic anhydrase 1 (CAH1) in Arabidopsis. CAH1 pre-protein undergoes extensive post-translational modification in the endomembrane system, including glycosylation, disulfide bond formation and proteolytic removal of a peptide ‘spacer’ region, resulting in a mature, heterotetrameric enzyme with two large and two small subunits. We generated a series of small-scale and large-scale modifications to the spacer and flanking regions to identify potential protease target motifs. Surprisingly, we found that the endoproteolytic removal of the spacer from the CAH1 pre-protein proceeded via an opportunistic process apparently followed by further maturation via amino and carboxy peptidases. We also discovered that the spacer itself is not required for processing, which appears to be dependent only on the number of amino acids separating two key disulfide-bond-forming cysteines. Our data suggest a novel, opportunistic route for pre-protein processing of CAH1. PMID:26917556

  5. Epithelial SCAP/INSIG/SREBP Signaling Regulates Multiple Biological Processes during Perinatal Lung Maturation

    PubMed Central

    Bridges, James P.; Schehr, Angelica; Wang, Yanhua; Huo, Liya; Besnard, Valérie; Ikegami, Machiko; Whitsett, Jeffrey A.; Xu, Yan

    2014-01-01

    Pulmonary surfactant is required for lung function at birth and throughout postnatal life. Defects in the surfactant system are associated with common pulmonary disorders including neonatal respiratory distress syndrome and acute respiratory distress syndrome in children and adults. Lipogenesis is essential for the synthesis of pulmonary surfactant by type II epithelial cells lining the alveoli. This study sought to identify the role of pulmonary epithelial SREBP, a transcriptional regulator of cellular lipid homeostasis, during a critical time period of perinatal lung maturation in the mouse. Genome wide mRNA expression profiling of lung tissue from transgenic mice with epithelial-specific deletions of Scap (ScapΔ/Δ, resulting in inactivation of SREBP signaling) or Insig1 and Insig2 (Insig1/2Δ/Δ, resulting in activation of SREBP signaling) was assessed. Differentially expressed genes responding to SREBP perturbations were identified and subjected to functional enrichment analysis, pathway mapping and literature mining to predict upstream regulators and transcriptional networks regulating surfactant lipid homeostasis. Through comprehensive data analysis and integration, time dependent effects of epithelial SCAP/INSIG/SREBP deletion and defined SCAP/INSIG/SREBP-associated genes, bioprocesses and downstream pathways were identified. SREBP signaling influences epithelial development, cell death and cell proliferation at E17.5, while primarily influencing surfactant physiology, lipid/sterol synthesis, and phospholipid transport after birth. SREBP signaling integrated with the Wnt/β-catenin and glucocorticoid receptor signaling pathways during perinatal lung maturation. SREBP regulates perinatal lung lipogenesis and maturation through multiple mechanisms by interactions with distinct sets of regulatory partners. PMID:24806461

  6. Identification of new participants in the rainbow trout (Oncorhynchus mykiss) oocyte maturation and ovulation processes using cDNA microarrays

    PubMed Central

    Bobe, Julien; Montfort, Jerôme; Nguyen, Thaovi; Fostier, Alexis

    2006-01-01

    Background The hormonal control of oocyte maturation and ovulation as well as the molecular mechanisms of nuclear maturation have been thoroughly studied in fish. In contrast, the other molecular events occurring in the ovary during post-vitellogenesis have received far less attention. Methods Nylon microarrays displaying 9152 rainbow trout cDNAs were hybridized using RNA samples originating from ovarian tissue collected during late vitellogenesis, post-vitellogenesis and oocyte maturation. Differentially expressed genes were identified using a statistical analysis. A supervised clustering analysis was performed using only differentially expressed genes in order to identify gene clusters exhibiting similar expression profiles. In addition, specific genes were selected and their preovulatory ovarian expression was analyzed using real-time PCR. Results From the statistical analysis, 310 differentially expressed genes were identified. Among those genes, 90 were up-regulated at the time of oocyte maturation while 220 exhibited an opposite pattern. After clustering analysis, 90 clones belonging to 3 gene clusters exhibiting the most remarkable expression patterns were kept for further analysis. Using real-time PCR analysis, we observed a strong up-regulation of ion and water transport genes such as aquaporin 4 (aqp4) and pendrin (slc26). In addition, a dramatic up-regulation of vasotocin (avt) gene was observed. Furthermore, angiotensin-converting-enzyme 2 (ace2), coagulation factor V (cf5), adam 22, and the chemokine cxcl14 genes exhibited a sharp up-regulation at the time of oocyte maturation. Finally, ovarian aromatase (cyp19a1) exhibited a dramatic down-regulation over the post-vitellogenic period while a down-regulation of Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (cmah) was observed at the time of oocyte maturation. Conclusion We showed the over or under expression of more that 300 genes, most of them being previously unstudied or unknown in the

  7. Changes in the processing of beta-endorphin in the hypothalamus and pituitary gland of female rats during sexual maturation.

    PubMed

    Martensz, N D

    1985-11-01

    Puberty in the female rat is accompanied by a marked attenuation of the opioid inhibition of luteinizing hormone secretion. One factor which may contribute to this altered role is a change in the metabolism of opioid peptides during sexual maturation. beta-Endorphin undergoes a considerable degree of metabolism through both C-terminal proteolysis and N-acetylation, and these metabolites do not possess opioid activity. The processing of beta-endorphin in the hypothalamus and in the anterior and neurointermediate lobes of the pituitary gland in prepubertal and adult female rats was studied using gel filtration and high performance liquid chromatography coupled with radioimmunoassay. In the anterior lobe, high molecular weight precursors of beta-endorphin (pro-opiomelanocortin and beta-lipotropin) were present in prepubertal (28 days old) rats, but little authentic beta-endorphin was detected. In contrast, only beta-lipotropin and beta-endorphin were present in mature (70 days old) animals. Only beta-endorphin-sized peptides were present in the neurointermediate lobes of both prepubertal and adult rats. However, the proportion of N-acetylated metabolites was higher in sexually mature animals. In the hypothalamus, only beta-endorphin-sized peptides were present in both juvenile and adult animals. However, C-terminal proteolysis increased with age (no acetylated metabolites were detectable in this tissue). The proportion of the total beta-endorphin-like immunoreactivity attributable to beta-endorphin was lower in young adult (first dioestrus after vaginal opening) (55%) and mature (dioestrus, 61-64 days old) rats (56%) compared to prepubertal (30 days old) animals (75%) and the proportions of non-acetylated metabolites [beta-endorphin-(1-27) in young adults and beta-endorphin-(1-26) in adults] were increased concomitantly. These changes were correlated with a reduced luteinizing hormone response to the opiate antagonist naloxone in adult compared to prepubertal rats

  8. Changes in esculeoside A content in different regions of the tomato fruit during maturation and heat processing.

    PubMed

    Katsumata, Akiko; Kimura, Mizuki; Saigo, Hiromi; Aburaya, Kei; Nakano, Masako; Ikeda, Tsuyoshi; Fujiwara, Yukio; Nagai, Ryoji

    2011-04-27

    We previously demonstrated that esculeogenin A, a new aglycone of the tomato sapogenol esculeoside A, inhibits both acyl coenzyme A:cholesterol acyl-transferase (ACAT)-1 and -2 and ameliorates the pathogenesis of atherosclerosis in apoE deficient mice. Although we believe that daily intake of esculeoside A from tomato products can play a beneficial role in preventing the pathogenesis of atherosclerosis, the compound is not being used for preventive medicine due to the lack of information on methods for quantitative analysis and the content and stability of the compound in tomato products. In the present study, we report the development of a high-performance liquid chromatography (HPLC) method using an instrument equipped with a refractive index (RI) detector for esculeoside A quantification. We used this method to measure the changes in esculeoside A content during maturation, its distribution in the fruit body, and its stability during the heating process. The contents of esculeoside A in cherry tomatoes and Momotaro tomatoes were 21- and 9-fold, respectively, higher than that of lycopene, which is the most well-known compound in tomatoes. Furthermore, the esculeoside A content in pericarp wall was higher than in the whole tomato fruit and increased in a time-dependent manner during maturation. Although the melting point of purified esculeoside A was 225 °C, the esculeoside A in crude tomato extract decreased in a temperature-dependent manner. Degradation due to the heating process was inhibited under a pH of 9. These results demonstrated that the esculeoside A content differs in the various types of tomatoes, during maturation, and during the heating process used for preservation. PMID:21395308

  9. Large scale affinity calculations of cyclodextrin host-guest complexes: Understanding the role of reorganization in the molecular recognition process

    PubMed Central

    Wickstrom, Lauren; He, Peng; Gallicchio, Emilio; Levy, Ronald M.

    2013-01-01

    Host-guest inclusion complexes are useful models for understanding the structural and energetic aspects of molecular recognition. Due to their small size relative to much larger protein-ligand complexes, converged results can be obtained rapidly for these systems thus offering the opportunity to more reliably study fundamental aspects of the thermodynamics of binding. In this work, we have performed a large scale binding affinity survey of 57 β-cyclodextrin (CD) host guest systems using the binding energy distribution analysis method (BEDAM) with implicit solvation (OPLS-AA/AGBNP2). Converged estimates of the standard binding free energies are obtained for these systems by employing techniques such as parallel Hamitionian replica exchange molecular dynamics, conformational reservoirs and multistate free energy estimators. Good agreement with experimental measurements is obtained in terms of both numerical accuracy and affinity rankings. Overall, average effective binding energies reproduce affinity rank ordering better than the calculated binding affinities, even though calculated binding free energies, which account for effects such as conformational strain and entropy loss upon binding, provide lower root mean square errors when compared to measurements. Interestingly, we find that binding free energies are superior rank order predictors for a large subset containing the most flexible guests. The results indicate that, while challenging, accurate modeling of reorganization effects can lead to ligand design models of superior predictive power for rank ordering relative to models based only on ligand-receptor interaction energies. PMID:25147485

  10. Maturation of Visual and Auditory Temporal Processing in School-Aged Children

    ERIC Educational Resources Information Center

    Dawes, Piers; Bishop, Dorothy V. M.

    2008-01-01

    Purpose: To examine development of sensitivity to auditory and visual temporal processes in children and the association with standardized measures of auditory processing and communication. Methods: Normative data on tests of visual and auditory processing were collected on 18 adults and 98 children aged 6-10 years of age. Auditory processes…

  11. Molecular and physiological study of the artificial maturation process in European eel males: from brain to testis.

    PubMed

    Peñaranda, David S; Pérez, Luz; Gallego, Víctor; Jover, Miguel; Tveiten, Helge; Baloche, Sylvie; Dufour, Sylvie; Asturiano, Juan F

    2010-03-01

    European eel males can be artificially matured (1.5IU hCG/g fish), but the regulatory mechanisms of their reproductive development are practically unknown. Spermatogenic stages (S1-S6), biometric characters [eye index (EI), gonadosomatic index (GSI), hepatosomatic index (HSI)] and sperm quality parameters (motility, viability and head spermatozoa morphometry) were analysed. Moreover, the present study evaluated the expression of GnRHs (mammal and chicken II Gonadotropin Release Hormone I) and gonadotrophins (FSHbeta and LHbeta) during hormonal treatment, as well as 11-ketotestosterone (11-KT) and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) plasma levels. One week was enough to observe the S2 of gonad development, but it was necessary to reach the 7th week of treatment to obtain animals that presented the most advanced stage of development (S6). Differential regulation of the two GnRH expressions was found, supporting the main role of mGnRH in the control of gonadotrophin release. One hCG injection was enough to dramatically decrease the FSHbeta expression, being close to zero during the rest of the treatment. LHbeta expression and 17,20beta-P registered a significant increase in the same stage of development, S3/4, confirming the role of this gonadotrophin in the last steps of maturation and 17,20beta-P in the spermatozoa maturation. The 11-KT increased with GSI, and the highest 11-KT values coincided with the advanced steps of spermatogenesis prior to spermiation. Being consistent with the known role of the steroid in these processes. Furthermore, this study supports a role for 11-KT in stimulating eye growth, presenting high values when EI increased. Sperm production was obtained from the 4th week of treatment, but it was in the 8th week when a significant increase was observed in sperm quality [viability, high motility (>75%)]. PMID:19699741

  12. Maturation of Rapid Auditory Temporal Processing and Subsequent Nonword Repetition Performance in Children

    ERIC Educational Resources Information Center

    Fox, Allison M.; Reid, Corinne L.; Anderson, Mike; Richardson, Cassandra; Bishop, Dorothy V. M.

    2012-01-01

    According to the rapid auditory processing theory, the ability to parse incoming auditory information underpins learning of oral and written language. There is wide variation in this low-level perceptual ability, which appears to follow a protracted developmental course. We studied the development of rapid auditory processing using event-related…

  13. Can bovine in vitro-matured oocytes selectively process X- or Y-sorted sperm differentially?

    PubMed

    Bermejo-Alvarez, P; Rizos, D; Rath, D; Lonergan, P; Gutiérrez-Adán, A

    2008-10-01

    It has been reported that the mammalian female could have a preconceptual influence on the sex of her offspring, and it has been hypothesized that this influence could go some way toward accounting for the reported lower fertility following insemination with sex-sorted sperm. To test whether in vitro matured oocytes are able to select X- or Y-bearing spermatozoa following in vitro fertilization (IVF), we fertilized in vitro 1788 oocytes with X-sorted semen, Y-sorted semen, a mix of X- and Y-sorted semen, and unsorted semen from the same bull, and cultured until Day 9. Fertility was assessed by recording cleavage rate at 48 h postinsemination (hpi) and blastocyst development until Day 9. Embryos were sexed at the two- to four-cell stage and the blastocyst stage. The proportion of zygotes cleaving at 48 hpi was not different between X- and Y-sorted groups and the mix of X- and Y-sorted semen group; however, all were significantly lower than the unsorted group (P < 0.001). Blastocyst yield on Day 6 was significantly higher (P < or = 0.01) in the control group compared with the rest of the groups. Cumulative blastocyst yields on Days 7, 8, and 9 were also significantly higher (P < or = 0.01) in the unsorted group compared with the sorted groups. The proportion of female and male two- to four-cell embryos obtained following IVF with X- and Y-sorted sperm was 88% and 89%, respectively and the sex ratio at the two- to four-cell stage was not different following IVF with unsorted or sorted/recombined sperm (56.9% males vs. 57% males, respectively). At the blastocyst stage, similar percentages were obtained. In conclusion, the differences in cleavage and blastocyst development using sorted versus unsorted sperm are not due to the oocyte preferentially selecting sperm of one sex over another, but are more likely due to spermatic damage caused by the sorting procedure. PMID:18579751

  14. Theory of morphological transformation of viral capsid shell during the maturation process in the HK97 bacteriophage and similar viruses.

    PubMed

    Konevtsova, O V; Lorman, V L; Rochal, S B

    2016-05-01

    We consider the symmetry and physical origin of collective displacement modes playing a crucial role in the morphological transformation during the maturation of the HK97 bacteriophage and similar viruses. It is shown that the experimentally observed hexamer deformation and pentamer twist in the HK97 procapsid correspond to the simplest irreducible shear strain mode of a spherical shell. We also show that the icosahedral faceting of the bacteriophage capsid shell is driven by the simplest irreducible radial displacement field. The shear field has the rotational icosahedral symmetry group I while the radial field has the full icosahedral symmetry I_{h}. This difference makes their actions independent. The radial field sign discriminates between the icosahedral and the dodecahedral shapes of the faceted capsid shell, thus making the approach relevant not only for the HK97-like viruses but also for the parvovirus family. In the frame of the Landau-Ginzburg formalism we propose a simple phenomenological model valid for the first reversible step of the HK97 maturation process. The calculated phase diagram illustrates the discontinuous character of the virus shape transformation. The characteristics of the virus shell faceting and expansion obtained in the in vitro and in vivo experiments are related to the decrease in the capsid shell thickness and to the increase of the internal capsid pressure. PMID:27300929

  15. Theory of morphological transformation of viral capsid shell during the maturation process in the HK97 bacteriophage and similar viruses

    NASA Astrophysics Data System (ADS)

    Konevtsova, O. V.; Lorman, V. L.; Rochal, S. B.

    2016-05-01

    We consider the symmetry and physical origin of collective displacement modes playing a crucial role in the morphological transformation during the maturation of the HK97 bacteriophage and similar viruses. It is shown that the experimentally observed hexamer deformation and pentamer twist in the HK97 procapsid correspond to the simplest irreducible shear strain mode of a spherical shell. We also show that the icosahedral faceting of the bacteriophage capsid shell is driven by the simplest irreducible radial displacement field. The shear field has the rotational icosahedral symmetry group I while the radial field has the full icosahedral symmetry Ih. This difference makes their actions independent. The radial field sign discriminates between the icosahedral and the dodecahedral shapes of the faceted capsid shell, thus making the approach relevant not only for the HK97-like viruses but also for the parvovirus family. In the frame of the Landau-Ginzburg formalism we propose a simple phenomenological model valid for the first reversible step of the HK97 maturation process. The calculated phase diagram illustrates the discontinuous character of the virus shape transformation. The characteristics of the virus shell faceting and expansion obtained in the in vitro and in vivo experiments are related to the decrease in the capsid shell thickness and to the increase of the internal capsid pressure.

  16. Market maturity

    SciTech Connect

    Meade, B.; Bowden, S.; Ellis, M

    1995-02-01

    The power sector in the Philipines provides one of the most mature independent power markets in Asia. Over the past five years, National Power Corp. (NPC), the government owned utility, has actively invited the power sector into power generation. Distribution has remained in the hands of private and rural cooperative utilities. Private utilities have been operating as full requirements customers of NPC while the growth in capacity additions by independent power producers (IPPs) has outpaced NPC`s for the second year in a row. With a recovering economy and regulatory reform proceeding, the outlook for independent power remains strong through the end of the decade. The Philipine Congress is now reviewing draft legislation that will decentralize NPC and begin the process of privatization and market-based reforms throughout the country`s power sector.

  17. Acute stress alters transcript expression pattern and reduces processing of proBDNF to mature BDNF in Dicentrarchus labrax

    PubMed Central

    2010-01-01

    Background Stress involves alterations of brain functioning that may precipitate to mood disorders. The neurotrophin Brain Derived Neurotrophic Factor (BDNF) has recently been involved in stress-induced adaptation. BDNF is a key regulator of neuronal plasticity and adaptive processes. Regulation of BDNF is complex and may reflect not only stress-specific mechanisms but also hormonal and emotional responses. For this reason we used, as an animal model of stress, a fish whose brain organization is very similar to that of higher vertebrates, but is generally considered free of emotional reactions. Results We provide a comprehensive characterization of BDNF gene in the Dicentrarchus labrax and its transcriptional, translational and post-translational regulation following acute stress. While total BDNF mRNA levels are unchanged, BDNF transcripts 1c and 1d resulted down regulated after acute stress. Acute stress induces also a significant increase in proBDNF levels and reduction in mature BDNF suggesting altered regulation of proBDNF proteolytic processing. Notably, we provide here the first evidence that fishes possess a simplified proteolytic regulation of BDNF since the pro28Kda form, generated by the SKI-1 protease in mammals, is absent in fishes because the cleavage site has first emerged in reptilians. Finally, we show that the proBDNF/totBDNF ratio is a highly predictive novel quantitative biomarker to detect stress in fishes with sensitivity = 100%, specificity = 87%, and Negative Predictive Value = 100%. Conclusion The high predictivity of proBDNF/totBDNF ratio for stress in lower vertebrates indicates that processing of BDNF is a central mechanism in adaptation to stress and predicts that a similar regulation of pro/mature BDNF has likely been conserved throughout evolution of vertebrates from fish to man. PMID:20074340

  18. Identification of tubulin drug binding sites and prediction of relative differences in binding affinities to tubulin isotypes using digital signal processing.

    PubMed

    Chen, Ke; Huzil, J Torin; Freedman, Holly; Ramachandran, Parameswaran; Antoniou, Andreas; Tuszynski, Jack A; Kurgan, Lukasz

    2008-11-01

    Microtubules are involved in numerous cellular processes including chromosome segregation during mitosis and, as a result, their constituent protein, tubulin, has become a successful target of several chemotherapeutic drugs. In general, these drugs bind indiscriminately to tubulin within both cancerous and healthy cells, resulting in unwanted side effects. However, differences between beta-tubulin isotypes expressed in a wide range of cell types may aid in the development of anti-tubulin drugs having increased specificity for only certain types of cells. Here, we describe a digital signal processing (DSP) method that is capable of predicting hot spots for the tubulin family of proteins as well as determining relative differences in binding affinities to these hot spots based only on the primary sequence of 10 human tubulin isotypes. Due to the fact that several drug binding sites have already been characterized within beta-tubulin, we are able to correlate hot spots with the binding sites for known chemotherapy drugs. We have also verified the accuracy of this method using the correlation between the binding affinities of characterized drugs and the tubulin isotypes. Additionally, the DSP method enables the rapid estimation of relative differences in binding affinities within the binding sites of tubulin isotypes that are yet to be experimentally determined. PMID:18951052

  19. Long-term performance and microbial ecology of a two-stage PN-ANAMMOX process treating mature landfill leachate.

    PubMed

    Li, Huosheng; Zhou, Shaoqi; Ma, Weihao; Huang, Pengfei; Huang, Guotao; Qin, Yujie; Xu, Bin; Ouyang, Hai

    2014-05-01

    Long-term performance of a two-stage partial nitritation (PN)-anaerobic ammonium oxidation (ANAMMOX) process treating mature landfill leachate was investigated. Stable partial nitritation performance was achieved in a sequencing batch reactor (SBR) using endpoint pH control, providing an effluent with a ratio of NO2(-)-N/NH4(+)-N at 1.23 ± 0.23. High rate nitrogen removal over 4 kg N/m(3)/d was observed in the ANAMMOX reactor in the first three months. However, during long-term operation, the ANAMMOX reactor can only stably operate under nitrogen load of 1 kg N/m(3)/d, with 85 ± 1% of nitrogen removal. The ammonium oxidizing bacteria (AOB) in the PN-SBR were mainly affiliated to Nitrosomonas sp. IWT514, Nitrosomonas eutropha and Nitrosomonas eutropha, the anaerobic ammonium oxidizing bacteria (AnAOB) in the ANAMMOX reactor were mainly affiliated to Kuenenia stuttgartiensis. PMID:24681301

  20. An aspartyl cathepsin, CTH3, is essential for proprotein processing during secretory granule maturation in Tetrahymena thermophila

    PubMed Central

    Kumar, Santosh; Briguglio, Joseph S.; Turkewitz, Aaron P.

    2014-01-01

    In Tetrahymena thermophila, peptides secreted via dense-core granules, called mucocysts, are generated by proprotein processing. We used expression profiling to identify candidate processing enzymes, which localized as cyan fluorescent protein fusions to mucocysts. Of note, the aspartyl cathepsin Cth3p plays a key role in mucocyst-based secretion, since knockdown of this gene blocked proteolytic maturation of the entire set of mucocyst proproteins and dramatically reduced mucocyst accumulation. The activity of Cth3p was eliminated by mutation of two predicted active-site mutations, and overexpression of the wild-type gene, but not the catalytic-site mutant, partially rescued a Mendelian mutant defective in mucocyst proprotein processing. Our results provide the first direct evidence for the role of proprotein processing in this system. Of interest, both localization and the CTH3 disruption phenotype suggest that the enzyme provides non–mucocyst-related functions. Phylogenetic analysis of the T. thermophila cathepsins, combined with prior work on the role of sortilin receptors in mucocyst biogenesis, suggests that repurposing of lysosomal enzymes was an important step in the evolution of secretory granules in ciliates. PMID:24943840

  1. Mature landfill leachate treatment by coagulation/flocculation combined with Fenton and solar photo-Fenton processes.

    PubMed

    Amor, Carlos; De Torres-Socías, Estefanía; Peres, José A; Maldonado, Manuel I; Oller, Isabel; Malato, Sixto; Lucas, Marco S

    2015-04-01

    This work reports the treatment of a mature landfill leachate through the application of chemical-based treatment processes in order to achieve the discharge legal limits into natural water courses. Firstly, the effect of coagulation/flocculation with different chemicals was studied, evaluating the role of different initial pH and chemicals concentration. Afterwards, the efficiency of two different advanced oxidation processes for leachate remediation was assessed. Fenton and solar photo-Fenton processes were applied alone and in combination with a coagulation/flocculation pre-treatment. This physicochemical conditioning step, with 2 g L(-1) of FeCl3 · 6H2O at pH 5, allowed removing 63% of COD, 80% of turbidity and 74% of total polyphenols. Combining the coagulation/flocculation pre-treatment with Fenton reagent, it was possible to reach 89% of COD removal in 96 h. Moreover, coagulation/flocculation combined with solar photo-Fenton revealed higher DOC (75%) reductions than single solar photo-Fenton (54%). In the combined treatment (coagulation/flocculation and solar photo-Fenton), it was reached a DOC reduction of 50% after the chemical oxidation, with 110 kJ L(-1) of accumulated UV energy and a H2O2 consumption of 116 mM. Toxicity and biodegradability assays were performed to evaluate possible variations along the oxidation processes. After the combined treatment, the leachate under study presented non-toxicity but biodegradability increased. PMID:25590819

  2. MicroRNA-155 controls affinity-based selection by protecting c-MYC+ B cells from apoptosis

    PubMed Central

    Nakagawa, Rinako; Leyland, Rebecca; Meyer-Hermann, Michael; Lu, Dong; Turner, Martin; Arbore, Giuseppina; Phan, Tri Giang; Brink, Robert; Vigorito, Elena

    2015-01-01

    The production of high-affinity antibodies by B cells is essential for pathogen clearance. Antibody affinity for antigen is increased through the affinity maturation in germinal centers (GCs). This is an iterative process in which B cells cycle between proliferation coupled with the acquisition of mutations and antigen-based positive selection, resulting in retention of the highest-affinity B cell clones. The posttranscriptional regulator microRNA-155 (miR-155) is critical for efficient affinity maturation and the maintenance of the GCs; however, the cellular and molecular mechanism by which miR-155 regulates GC responses is not well understood. Here, we utilized a miR-155 reporter mouse strain and showed that miR-155 is coexpressed with the proto-oncogene encoding c-MYC in positively selected B cells. Functionally, miR-155 protected positively selected c-MYC+ B cells from apoptosis, allowing clonal expansion of this population, providing an explanation as to why Mir155 deletion impairs affinity maturation and promotes the premature collapse of GCs. We determined that miR-155 directly inhibits the Jumonji family member JARID2, which enhances B cell apoptosis when overexpressed, and thereby promotes GC B cell survival. Our findings also suggest that there is cooperation between c-MYC and miR-155 during the normal GC response, a cooperation that may explain how c-MYC and miR-155 can collaboratively function as oncogenes. PMID:26657861

  3. Role of Endoproteolytic Dibasic Proprotein Processing in Maturation of Secretory Proteins in Trichoderma reesei

    PubMed Central

    Goller, Sabine P.; Schoisswohl, Doris; Baron, Michel; Parriche, Martine; Kubicek, Christian P.

    1998-01-01

    Cell extracts of Trichoderma reesei exhibited dibasic endopeptidase activity toward the carboxylic side of KR, RR, and PR sequences. This activity was stimulated by the presence of Ca2+ ions and localized in vesicles of low bouyant density; it therefore exhibited some similarity to yeast Kex2. Analytical chromatofocusing revealed a single peak of activity. The dibasic endopeptidase activity was strongly and irreversibly inhibited in vitro as well as in vivo by 1 mM p-amidinophenylmethylsulfonyl fluoride (pAPMSF) but not by PMSF at concentrations up to 5 mM. We therefore used pAPMSF to study the role of the dibasic endopeptidase in the secretion of protein by T. reesei. Secretion of xylanase I (proprotein processing sequence -R-R-↓-R-↓-A-) and xylanase II (-K-R-↓-Q-) was strongly inhibited by 1 mM pAPMSF, and a larger, unprocessed enzyme form was detected intracellularly under these conditions. Secretion of cellobiohydrolase II (CBH II; -E-R-↓-Q-) was only slightly inhibited by pAPMSF, and no accumulation of unprocessed precursors was detected. In contrast, secretion of CBH I (-R-A-↓-Q-) was stimulated by pAPMSF addition, and a simultaneous decrease in the concentration of intracellular CBH I was detected. Similar experiments were also carried out with a single heterologous protein, ShBLE, the phleomycin-binding protein from Streptoalloteichus hindustanus, fused to a series of model proprotein-processing sequences downstream of the expression signals of the Aspergillus nidulans gpdA promoter. Consistent with the results obtained with homologous proteins, pAPMSF inhibited the secretion of ShBLE with fusions containing dibasic (RK and KR) target sequences, but it even stimulated secretion in fusions to LR, NHA, and EHA target sequences. Addition of 5 mM PMSF, a nonspecific inhibitor of serine protease, nonspecifically inhibited the secretion of heterologous proteins from fusions bearing the NHA and LR targets. These data point to the existence of different

  4. 3[prime] end maturation of the Chlamydomonas reinhardtii chloroplast atpB mRNA is a two-step process

    SciTech Connect

    Stern, D.B.; Kindle, K.L. )

    1993-04-01

    The research studied the 3[prime] end maturation of green algae chloroplast atpB mRNA. Most data on transcription termination and 3[prime] end maturation in chloroplasts have been based on in vitro experiments. Newly developed chloroplast transformation techniques have allowed the use of a green algae, Chlamydomonas reinhardtii, to examine chloroplast mRNA 3[prime] end stability determinants and mRNA processing both in vitro and in vivo. The results of this research showed that Chlamydomonas chloroplast protein extracts contain an endonuclease activity that cleaves a synthetic precursor of atpB mRNA 10 nucleotides downstream on the mature 3[prime] end in vitro. Rapid cleavage by this endonuclease is followed by exonucleolytic removal of 10 nucleotides to yield the mature 3[prime] end.

  5. Report: Affinity Chromatography.

    ERIC Educational Resources Information Center

    Walters, Rodney R.

    1985-01-01

    Supports, affinity ligands, immobilization, elution methods, and a number of applications are among the topics considered in this discussion of affinity chromatography. An outline of the basic principles of affinity chromatography is included. (JN)

  6. Process Maturity and Organizational Structure as a Framework for Performance Improvements

    NASA Astrophysics Data System (ADS)

    Škrinjar, Rok; Dimovski, Vlado; Škerlavaj, Miha; Indihar Šternberger, Mojca

    Business and technological change threatens organizational sustainability, which depends on a balance between change and order. Few organizations can control the forces that affect them, but they can control the way in which they deal with those forces. Organizations today face increasing pressures to integrate their processes across disparate divisions and functional units, in order to remove inefficiencies as well as to enhance manageability. Although many enterprises acknowledge the need to be quick and responsive in a global economy and market that is constantly changing, many are not adequately structured to do so. The functional/divisional organizational structure, the legacy form industrial age and one of the major agility inhibitors, is still predominant.

  7. Oral Priming with Replicating Adenovirus Serotype 4 Followed by Subunit H5N1 Vaccine Boost Promotes Antibody Affinity Maturation and Expands H5N1 Cross-Clade Neutralization

    PubMed Central

    Khurana, Surender; Coyle, Elizabeth M.; Manischewitz, Jody; King, Lisa R.; Ishioka, Glenn; Alexander, Jeff; Smith, Jon; Gurwith, Marc; Golding, Hana

    2015-01-01

    A Phase I trial conducted in 2009–2010 demonstrated that oral vaccination with a replication competent Ad4-H5 (A/Vietnam) vector with dosages ranging from 107-1011 viral particles was well tolerated. HA-specific T-cell responses were efficiently induced, but very limited hemagglutination-inhibiting (HI) humoral responses were measured. However, a single boost of Ad4-H5-Vtn vaccinated individuals with a unadjuvanted licensed H5N1 (A/Vietnam) subunit vaccine resulted in superior HI titers compared with unprimed subjects. In the current study, the impact of Ad4-H5 priming on the quality of the polyclonal humoral immune response was evaluated using a real-time kinetics assay by surface plasmon resonance (SPR). Total binding of serum polyclonal antibodies from the Ad4-H5-Vtn primed groups against both homologous H5N1-A/Vietnam/1194/2004 (clade 1) and heterologous A/Indonesia-5/2005 (clade 2.1) HA1 head domain was significantly higher compared with sera from individuals that received subunit H5N1 vaccination alone. SPR measurements also demonstrated that the antigen-antibody complex dissociation rates (a surrogate for antibody affinity) of serum antibodies against the HA1 of H5N1-A/Vietnam were significantly higher in the Ad4-H5 primed groups compared with those from the unprimed group. Furthermore, strong correlations were observed between the antibody affinities for HA1 (but not HA2) and the virus neutralization titers against the homologous strain and a panel of heterologous clade 2 H5N1 strains. These findings support the concept of oral prime-boost vaccine approaches against pandemic influenza to elicit long-term memory B cells with high affinity capable of rapid response to variant pandemic viruses likely to emerge and adapt to human transmissions. PMID:25629161

  8. Oral priming with replicating adenovirus serotype 4 followed by subunit H5N1 vaccine boost promotes antibody affinity maturation and expands H5N1 cross-clade neutralization.

    PubMed

    Khurana, Surender; Coyle, Elizabeth M; Manischewitz, Jody; King, Lisa R; Ishioka, Glenn; Alexander, Jeff; Smith, Jon; Gurwith, Marc; Golding, Hana

    2015-01-01

    A Phase I trial conducted in 2009-2010 demonstrated that oral vaccination with a replication competent Ad4-H5 (A/Vietnam) vector with dosages ranging from 107-1011 viral particles was well tolerated. HA-specific T-cell responses were efficiently induced, but very limited hemagglutination-inhibiting (HI) humoral responses were measured. However, a single boost of Ad4-H5-Vtn vaccinated individuals with a unadjuvanted licensed H5N1 (A/Vietnam) subunit vaccine resulted in superior HI titers compared with unprimed subjects. In the current study, the impact of Ad4-H5 priming on the quality of the polyclonal humoral immune response was evaluated using a real-time kinetics assay by surface plasmon resonance (SPR). Total binding of serum polyclonal antibodies from the Ad4-H5-Vtn primed groups against both homologous H5N1-A/Vietnam/1194/2004 (clade 1) and heterologous A/Indonesia-5/2005 (clade 2.1) HA1 head domain was significantly higher compared with sera from individuals that received subunit H5N1 vaccination alone. SPR measurements also demonstrated that the antigen-antibody complex dissociation rates (a surrogate for antibody affinity) of serum antibodies against the HA1 of H5N1-A/Vietnam were significantly higher in the Ad4-H5 primed groups compared with those from the unprimed group. Furthermore, strong correlations were observed between the antibody affinities for HA1 (but not HA2) and the virus neutralization titers against the homologous strain and a panel of heterologous clade 2 H5N1 strains. These findings support the concept of oral prime-boost vaccine approaches against pandemic influenza to elicit long-term memory B cells with high affinity capable of rapid response to variant pandemic viruses likely to emerge and adapt to human transmissions. PMID:25629161

  9. Residual effects of low oxygen storage of mature green fruit on ripening processes and ester biosynthesis during ripening in bananas

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mature green banana (Musa sapientum L. cv. Cavendish) fruit were stored in 0.5%, 2 %, or 21% O2 for 7 days at 20 °C before ripening was initiated by ethylene. Residual effects of low O2 storage in mature green fruit on ripening and ester biosynthesis in fruit were investigated during ripening period...

  10. On the Meaning of Affinity Limits in B-Cell Epitope Prediction for Antipeptide Antibody-Mediated Immunity

    PubMed Central

    Caoili, Salvador Eugenio C.

    2012-01-01

    B-cell epitope prediction aims to aid the design of peptide-based immunogens (e.g., vaccines) for eliciting antipeptide antibodies that protect against disease, but such antibodies fail to confer protection and even promote disease if they bind with low affinity. Hence, the Immune Epitope Database (IEDB) was searched to obtain published thermodynamic and kinetic data on binding interactions of antipeptide antibodies. The data suggest that the affinity of the antibodies for their immunizing peptides appears to be limited in a manner consistent with previously proposed kinetic constraints on affinity maturation in vivo and that cross-reaction of the antibodies with proteins tends to occur with lower affinity than the corresponding reaction of the antibodies with their immunizing peptides. These observations better inform B-cell epitope prediction to avoid overestimating the affinity for both active and passive immunization; whereas active immunization is subject to limitations of affinity maturation in vivo and of the capacity to accumulate endogenous antibodies, passive immunization may transcend such limitations, possibly with the aid of artificial affinity-selection processes and of protein engineering. Additionally, protein disorder warrants further investigation as a possible supplementary criterion for B-cell epitope prediction, where such disorder obviates thermodynamically unfavorable protein structural adjustments in cross-reactions between antipeptide antibodies and proteins. PMID:23209458

  11. Folate content in faba beans (Vicia faba L.)-effects of cultivar, maturity stage, industrial processing, and bioprocessing.

    PubMed

    Hefni, Mohammed E; Shalaby, Mohamed T; Witthöft, Cornelia M

    2015-01-01

    Faba beans are an important source of folate and commonly consumed in Egypt. This study examined the effects of Egyptian industrial food processing (e.g., canning and freezing), germination, cultivar, and maturity stages on folate content, with the aim to develop a candidate functional canned faba bean food with increased folate content. The folate content in four cultivars of green faba beans ranged from 110 to 130 μg 100 g(-1) fresh weight (535-620 μg 100 g(-1) dry matter [DM]), which was four- to sixfold higher than in dried seeds. Industrial canning of dried seeds resulted in significant folate losses of ∼20% (P = 0.004), while industrial freezing had no effect. Germination of faba beans increased the folate content by >40% (P < 0.0001). A novel industrial canning process involving pregermination of dried faba beans resulted in a net folate content of 194 μg 100 g(-1) DM, which is 52% more than in conventional canned beans. The consumption of green faba beans should be recommended, providing ∼120 μg dietary folate equivalents per 100 g/portion. PMID:25650294

  12. Folate content in faba beans (Vicia faba L.)—effects of cultivar, maturity stage, industrial processing, and bioprocessing

    PubMed Central

    Hefni, Mohammed E; Shalaby, Mohamed T; Witthöft, Cornelia M

    2015-01-01

    Faba beans are an important source of folate and commonly consumed in Egypt. This study examined the effects of Egyptian industrial food processing (e.g., canning and freezing), germination, cultivar, and maturity stages on folate content, with the aim to develop a candidate functional canned faba bean food with increased folate content. The folate content in four cultivars of green faba beans ranged from 110 to 130 μg 100 g−1 fresh weight (535–620 μg 100 g−1 dry matter [DM]), which was four- to sixfold higher than in dried seeds. Industrial canning of dried seeds resulted in significant folate losses of ∼20% (P = 0.004), while industrial freezing had no effect. Germination of faba beans increased the folate content by >40% (P < 0.0001). A novel industrial canning process involving pregermination of dried faba beans resulted in a net folate content of 194 μg 100 g−1 DM, which is 52% more than in conventional canned beans. The consumption of green faba beans should be recommended, providing ∼120 μg dietary folate equivalents per 100 g/portion. PMID:25650294

  13. Efficient fabrication of high-capacity immobilized metal ion affinity chromatographic media: The role of the dextran-grafting process and its manipulation.

    PubMed

    Zhao, Lan; Zhang, Jingfei; Huang, Yongdong; Li, Qiang; Zhang, Rongyue; Zhu, Kai; Suo, Jia; Su, Zhiguo; Zhang, Zhigang; Ma, Guanghui

    2016-03-01

    Novel high-capacity Ni(2+) immobilized metal ion affinity chromatographic media were prepared through the dextran-grafting process. Dextran was grafted to an allyl-activated agarose-based matrix followed by functionalization for the immobilized metal ion affinity chromatographic media. With elaborate regulation of the allylation degree, dextran was completely or partly grafted to agarose microspheres, namely, completely dextran-grafted agarose microspheres and partly dextran-grafted ones, respectively. Confocal laser scanning microscope results demonstrated that a good adjustment of dextran-grafting degree was achieved, and dextran was distributed uniformly in whole completely dextran-grafted microspheres, while just distributed around the outside of the partly dextran-grafted ones. Flow hydrodynamic properties were improved greatly after the dextran-grafting process, and the flow velocity increased by about 30% compared with that of a commercial chromatographic medium (Ni Sepharose FF). A significant improvement of protein binding performance was also achieved by the dextran-grafting process, and partly dextran-grafted Ni(2+) chelating medium had a maximum binding capacity for His-tagged lactate dehydrogenase about 2.5 times higher than that of Ni Sepharose FF. The results indicated that this novel chromatographic medium is promising for applications in high-efficiency and large-scale protein purification. PMID:26786724

  14. The SPS100 gene of Saccharomyces cerevisiae is activated late in the sporulation process and contributes to spore wall maturation.

    PubMed Central

    Law, D T; Segall, J

    1988-01-01

    We previously described the use of a differential hybridization screen of a genomic DNA library of Saccharomyces cerevisiae to identify sporulation-specific (SPS) genes (A. Percival-Smith and J. Segall, Mol. Cell. Biol. 4:142-150, 1984). This initial screen identified 14 SPS genes that are first expressed 6 to 8 h after transfer of cells to sporulation medium. Accumulation of transcripts corresponding to these genes becomes maximal at 8 to 12 h of sporulation, the time at which meiotic events are nearing completion, and by 15 h of sporulation, transcript levels are beginning to decrease. In the present study two additional SPS genes, first expressed at 12 h of sporulation, were isolated. The steady-state level of transcripts corresponding to these two genes, termed SPS100 and SPS101, remains unchanged from 15 to 35 h, a time coincident with spore wall maturation. The nature of the putative 34.2-kilodalton protein encoded by the SPS100 gene is consistent with its being a component of the glycoprotein matrix of the spore wall; the protein contains a potential signal sequence and cleavage site and numerous sites for potential glycosylation. A MATa sps100/MAT alpha sps100 strain was found to be indistinguishable from the wild-type strain when assessed for efficiency of ascus formation and spore viability. However, a more detailed analysis of the mutant strain revealed that the SPS100 gene product serves a protective role during the early stages of spore wall formation. The time at which resistance to ether could first be detected in developing spores was delayed by 5 h in the mutant strain relative to the wild-type strain. This phenotype is presumably a reflection of a defect in spore wall maturation. This study has confirmed that temporally distinct classes of sporulation-specific genes are sequentially activated during the process of meiosis and spore formation and has shown that the SPS100 gene, identified on the basis of its developmental-specific expression

  15. Nutrient Limitation on Ecosystem Productivity and Processes of Mature and Old-Growth Subtropical Forests in China

    PubMed Central

    Hou, Enqing; Chen, Chengrong; McGroddy, Megan E.; Wen, Dazhi

    2012-01-01

    Nitrogen (N) is considered the dominant limiting nutrient in temperate regions, while phosphorus (P) limitation frequently occurs in tropical regions, but in subtropical regions nutrient limitation is poorly understood. In this study, we investigated N and P contents and N:P ratios of foliage, forest floors, fine roots and mineral soils, and their relationships with community biomass, litterfall C, N and P productions, forest floor turnover rate, and microbial processes in eight mature and old-growth subtropical forests (stand age >80 yr) at Dinghushan Biosphere Reserve, China. Average N:P ratios (mass based) in foliage, litter (L) layer and mixture of fermentation and humus (F/H) layer, and fine roots were 28.3, 42.3, 32.0 and 32.7, respectively. These values are higher than the critical N:P ratios for P limitation proposed (16–20 for foliage, ca. 25 for forest floors). The markedly high N:P ratios were mainly attributed to the high N concentrations of these plant materials. Community biomass, litterfall C, N and P productions, forest floor turnover rate and microbial properties were more strongly related to measures of P than N and frequently negatively related to the N:P ratios, suggesting a significant role of P availability in determining ecosystem production and productivity and nutrient cycling at all the study sites except for one prescribed disturbed site where N availability may also be important. We propose that N enrichment is probably a significant driver of the potential P limitation in the study area. Low P parent material may also contribute to the potential P limitation. In general, our results provided strong evidence supporting a significant role for P availability, rather than N availability, in determining ecosystem primary productivity and ecosystem processes in subtropical forests of China. PMID:23284873

  16. The Origin of Voluminous Dacite (vs. Andesite) at Mature, Thick Continental Arcs: A Reflection of Processes in the Deep Crust

    NASA Astrophysics Data System (ADS)

    Lange, R. A.

    2013-12-01

    arc crust, but at melt fractions too low (≤15%) to permit efficient transport to the upper crust (Vigneresse and Tikoff, 1999). It is further proposed that the reason why dacite is so abundant at mature thick continental arcs (e.g., Altiplano-Puno complex) may be because mantle-derived basalts are primarily emplaced at similar depths (~30-40 km) in continental arc crustal columns. If so, in the central Andean arc, a depth of 30-40 km is within the middle dioritic crust (Graeber and Asch, 1999). Partial melts of hornblende diorite (vs. hornblende gabbro) are predicted to be dacitic (vs. andesitic) at melt fractions of 20-25%, which permits transport to the upper crust. It is therefore proposed that it is deep crustal processes that determine whether andesite or dacite is the most voluminous magma type emplaced into the upper crust and erupted at arcs.

  17. Neutrophilic Cathepsin C Is Maturated by a Multistep Proteolytic Process and Secreted by Activated Cells during Inflammatory Lung Diseases.

    PubMed

    Hamon, Yveline; Legowska, Monika; Hervé, Virginie; Dallet-Choisy, Sandrine; Marchand-Adam, Sylvain; Vanderlynden, Lise; Demonte, Michèle; Williams, Rich; Scott, Christopher J; Si-Tahar, Mustapha; Heuzé-Vourc'h, Nathalie; Lalmanach, Gilles; Jenne, Dieter E; Lesner, Adam; Gauthier, Francis; Korkmaz, Brice

    2016-04-15

    The cysteine protease cathepsin C (CatC) activates granule-associated proinflammatory serine proteases in hematopoietic precursor cells. Its early inhibition in the bone marrow is regarded as a new therapeutic strategy for treating proteolysis-driven chronic inflammatory diseases, but its complete inhibition is elusive in vivo Controlling the activity of CatC may be achieved by directly inhibiting its activity with a specific inhibitor or/and by preventing its maturation. We have investigated immunochemically and kinetically the occurrence of CatC and its proform in human hematopoietic precursor cells and in differentiated mature immune cells in lung secretions. The maturation of proCatC obeys a multistep mechanism that can be entirely managed by CatS in neutrophilic precursor cells. CatS inhibition by a cell-permeable inhibitor abrogated the release of the heavy and light chains from proCatC and blocked ∼80% of CatC activity. Under these conditions the activity of neutrophil serine proteases, however, was not abolished in precursor cell cultures. In patients with neutrophilic lung inflammation, mature CatC is found in large amounts in sputa. It is secreted by activated neutrophils as confirmed through lipopolysaccharide administration in a nonhuman primate model. CatS inhibitors currently in clinical trials are expected to decrease the activity of neutrophilic CatC without affecting those of elastase-like serine proteases. PMID:26884336

  18. AN INVESTIGATION OF THE IMPACT OF TELEVISION UPON THE MATURING PROCESS OF THE ADULT (PH.D. THESIS).

    ERIC Educational Resources Information Center

    TADROS, SAMY S.

    A SURVEY MADE OF EMPIRICAL RESEARCH RELATED TO TELEVISION CONTENT AND EFFECTS ON ADULTS TURNED UP 39 PERTINENT STUDIES WHICH WERE ANALYZED USING FIVE CRITERIA OF ADULT MATURITY, GLEANED FROM THE LITERATURE. OPINIONS OF 44 AMERICAN UNIVERSITY ADULT EDUCATORS REGARDING PROBLEM AREAS NEEDING RESEARCH WERE SOLICITED. IT WAS CONCLUDED THAT TELEVISION…

  19. Exploring Enantiospecific Ligand-Protein Interactions Using Cellular Membrane Affinity Chromatography: Chiral Recognition as a Dynamic Process

    PubMed Central

    Jozwiak, Krzysztof; Moaddel, Ruin; Ravichandran, Sarangan; Plazinska, Anita; Kozak, Joanna; Patel, Sharvil; Yamaguchi, Rika; Wainer, Irving

    2008-01-01

    The chiral recognition mechanisms responsible for the enantioselective binding on the α3β4 nicotinic acetyl choline receptor (α3β4 nAChR) and human organic cation transporter 1 (hOCT1) have been reviewed. The results indicate that chiral recognition on the α3β4 nAChR is a process involving initial tethering of dextromethorphan and levomethorphan at hydrophobic pockets within the central lumen followed by hydrogen bonding interactions favoring dextromethorphan. The second step is the defining enantioselective step. Studies with the hOCT1 indentified four binding sites within the transporter that participated in chiral recognition. Each of the enantiomers of the compounds used in the study interacted with three of these sites, while (R)-verapamil interacted with all four. Chiral recognition arose from the conformational adjustments required to produce optimum interactions. With respect to the prevailing interaction-based models, the data suggest that chiral recognition is a dynamic process and that the static point-based models should be amended to reflect this. PMID:18723411

  20. Prepubertal increases in gonadotropin-releasing hormone mRNA, gonadotropin-releasing hormone precursor, and subsequent maturation of precursor processing in male rats.

    PubMed Central

    Dutlow, C M; Rachman, J; Jacobs, T W; Millar, R P

    1992-01-01

    Changes in gonadotropins and gonadal steroids during sexual maturation in rats and humans are well documented but little is known about hypothalamic gonadotropin-releasing hormone (GnRH) gene expression in relation to these events. This study measured hypothalamic proGnRH mRNA, GnRH precursor, and fully processed GnRH from postnatal day 8 until day 62 in male rats. GnRH precursor increased on day 22, reached a peak on day 24, declined on day 25 and returned to infantile levels by day 28. A secondary rise in precursor occurred at about day 40 when testosterone levels increased. GnRH mRNA increased on day 22 and remained elevated over the study period to day 26. GnRH increased on day 24 and remained at this level until a secondary rise occurred coincident with the testosterone rise at about day 40. The ratio of GnRH precursor to GnRH was high until day 24 and was low from day 26 onwards, reflecting a maturation of the processing enzyme system between these 2 d. Thus, an abrupt increase in GnRH gene transcription (mRNA) occurs early in juvenile male rats (day 22), well before the onset of puberty. An increase in GnRH precursor accompanies these early changes and this is followed by the maturation of processing as evidenced by the rapid decline of precursor and increase in GnRH from day 24 onward. Images PMID:1469100

  1. Dual Roles for Ste24p in Yeast a-Factor Maturation: NH2-terminal Proteolysis and COOH-terminal CAAX Processing

    PubMed Central

    Tam, Amy; Nouvet, Franklin J.; Fujimura-Kamada, Konomi; Slunt, Hilda; Sisodia, Sangram S.; Michaelis, Susan

    1998-01-01

    Maturation of the Saccharomyces cerevisiae a-factor precursor involves COOH-terminal CAAX processing (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) followed by cleavage of an NH2-terminal extension (two sequential proteolytic processing steps). The aim of this study is to clarify the precise role of Ste24p, a membrane-spanning zinc metalloprotease, in the proteolytic processing of the a-factor precursor. We demonstrated previously that Ste24p is necessary for the first NH2-terminal processing step by analysis of radiolabeled a-factor intermediates in vivo (Fujimura-Kamada, K., F.J. Nouvet, and S. Michaelis. 1997. J. Cell Biol. 136:271–285). In contrast, using an in vitro protease assay, others showed that Ste24p (Afc1p) and another gene product, Rce1p, share partial overlapping function as COOH-terminal CAAX proteases (Boyartchuk, V.L., M.N. Ashby, and J. Rine. 1997. Science. 275:1796–1800). Here we resolve these apparently conflicting results and provide compelling in vivo evidence that Ste24p indeed functions at two steps of a-factor maturation using two methods. First, direct analysis of a-factor biosynthetic intermediates in the double mutant (ste24Δ rce1Δ) reveals a previously undetected species (P0*) that fails to be COOH terminally processed, consistent with redundant roles for Ste24p and Rce1p in COOH-terminal CAAX processing. Whereas a-factor maturation appears relatively normal in the rce1Δ single mutant, the ste24Δ single mutant accumulates an intermediate that is correctly COOH terminally processed but is defective in cleavage of the NH2-terminal extension, demonstrating that Ste24p is also involved in NH2-terminal processing. Together, these data indicate dual roles for Ste24p and a single role for Rce1p in a-factor processing. Second, by using a novel set of ubiquitin–a-factor fusions to separate the NH2- and COOH-terminal processing events of a-factor maturation, we provide independent evidence for the dual roles of Ste

  2. Solar Ion Processing of Major Element Surface Compositions of Mature Mare Soils: Insights from Combined XPS and Analytical TEM Observations

    NASA Technical Reports Server (NTRS)

    Christoffersen, R.; Dukes, C.; Keller, L. P.; Baragiola, R.

    2012-01-01

    Solar wind ions are capable of altering the sur-face chemistry of the lunar regolith by a number of mechanisms including preferential sputtering, radiation-enhanced diffusion and sputter erosion of space weathered surfaces containing pre-existing compositional profiles. We have previously reported in-situ ion irradiation experiments supported by X-ray photoelectron spectroscopy (XPS) and analytical TEM that show how solar ions potentially drive Fe and Ti reduction at the monolayer scale as well as the 10-100 nm depth scale in lunar soils [1]. Here we report experimental data on the effect of ion irradiation on the major element surface composition in a mature mare soil.

  3. Current challenges for pre-earthquake electromagnetic emissions: shedding light from micro-scale plastic flow, granular packings, phase transitions and self-affinity notion of fracture process

    NASA Astrophysics Data System (ADS)

    Eftaxias, K.; Potirakis, S. M.

    2013-10-01

    Are there credible electromagnetic (EM) potential earthquake (EQ) precursors? This a question debated in the scientific community and there may be legitimate reasons for the critical views. The negative view concerning the existence of EM potential precursors is enhanced by features that accompany their observation which are considered as paradox ones, namely, these signals: (i) are not observed at the time of EQs occurrence and during the aftershock period, (ii) are not accompanied by large precursory strain changes, (iii) are not accompanied by simultaneous geodetic or seismological precursors and (iv) their traceability is considered problematic. In this work, the detected candidate EM potential precursors are studied through a shift in thinking towards the basic science findings relative to granular packings, micron-scale plastic flow, interface depinning, fracture size effects, concepts drawn from phase transitions, self-affine notion of fracture and faulting process, universal features of fracture surfaces, recent high quality laboratory studies, theoretical models and numerical simulations. We try to contribute to the establishment of strict criteria for the definition of an emerged EM anomaly as a possibly EQ-related one, and to the explanation of potential precursory EM features which have been considered as paradoxes. A three-stage model for EQ generation by means of pre-EQ fracture-induced EM emissions is proposed. The claim that the observed EM potential precursors may permit a real-time and step-by-step monitoring of the EQ generation is tested.

  4. Determinism and stochasticity during maturation of the zebrafish antibody repertoire

    PubMed Central

    Jiang, Ning; Weinstein, Joshua A.; Penland, Lolita; White, Richard A.; Fisher, Daniel S.; Quake, Stephen R.

    2011-01-01

    It is thought that the adaptive immune system of immature organisms follows a more deterministic program of antibody creation than is found in adults. We used high-throughput sequencing to characterize the diversifying antibody repertoire in zebrafish over five developmental time points. We found that the immune system begins in a highly stereotyped state with preferential use of a small number of V (variable) D (diverse) J (joining) gene segment combinations, but that this stereotypy decreases dramatically as the zebrafish mature, with many of the top VDJ combinations observed in 2-wk-old zebrafish virtually disappearing by 1 mo. However, we discovered that, in the primary repertoire, there are strong correlations in VDJ use that increase with zebrafish maturity, suggesting that VDJ recombination involves a level of deterministic programming that is unexpected. This stereotypy is masked by the complex diversification processes of antibody maturation; the variation and lack of correlation in full repertoires between individuals appears to be derived from randomness in clonal expansion during the affinity maturation process. These data provide a window into the mechanisms of VDJ recombination and diversity creation and allow us to better understand how the adaptive immune system achieves diversity. PMID:21393572

  5. E-selectin ligand–1 regulates growth plate homeostasis in mice by inhibiting the intracellular processing and secretion of mature TGF-β

    PubMed Central

    Yang, Tao; Mendoza-Londono, Roberto; Lu, Huifang; Tao, Jianning; Li, Kaiyi; Keller, Bettina; Jiang, Ming Ming; Shah, Rina; Chen, Yuqing; Bertin, Terry K.; Engin, Feyza; Dabovic, Branka; Rifkin, Daniel B.; Hicks, John; Jamrich, Milan; Beaudet, Arthur L.; Lee, Brendan

    2010-01-01

    The majority of human skeletal dysplasias are caused by dysregulation of growth plate homeostasis. As TGF-β signaling is a critical determinant of growth plate homeostasis, skeletal dysplasias are often associated with dysregulation of this pathway. The context-dependent action of TFG-β signaling is tightly controlled by numerous mechanisms at the extracellular level and downstream of ligand-receptor interactions. However, TGF-β is synthesized as an inactive precursor that is cleaved to become mature in the Golgi apparatus, and the regulation of this posttranslational intracellular processing and trafficking is much less defined. Here, we report that a cysteine-rich protein, E-selectin ligand–1 (ESL-1), acts as a negative regulator of TGF-β production by binding TGF-β precursors in the Golgi apparatus in a cell-autonomous fashion, inhibiting their maturation. Furthermore, ESL-1 inhibited the processing of proTGF-β by a furin-like protease, leading to reduced secretion of mature TGF-β by primary mouse chondrocytes and HEK293 cells. In vivo loss of Esl1 in mice led to increased TGF-β/SMAD signaling in the growth plate that was associated with reduced chondrocyte proliferation and delayed terminal differentiation. Gain-of-function and rescue studies of the Xenopus ESL-1 ortholog in the context of early embryogenesis showed that this regulation of TGF-β/Nodal signaling was evolutionarily conserved. This study identifies what we believe to be a novel intracellular mechanism for regulating TGF-β during skeletal development and homeostasis. PMID:20530870

  6. Partial nitrification and denitrification of mature landfill leachate using a pilot-scale continuous activated sludge process at low dissolved oxygen.

    PubMed

    Chen, Zhenguo; Wang, Xiaojun; Yang, YongYuan; Mirino, Markus W; Yuan, Yanlei

    2016-10-01

    Controlling of low dissolved oxygen (DO) levels (0.1-0.5mg/L), a cost-effective strategy, was applied to a pilot-scale anoxic-oxic-oxic-anoxic process for partial nitrification and denitrification of mature landfill leachate. High ammonium removal efficiency, stable nitrite accumulation rate and total nitrogen removal efficiency was higher than 95.0%, 90.0% and 66.4%, respectively, implying potential application of this process for nitrogen removal of mature landfill leachate. Efficient nitrite accumulation in the first oxic reactor depended on low DO conditions and sufficient alkalinity. However, operational limit was mainly decided by actual hydraulic retention time (AHRT) of the first oxic reactor and appeared with AHRT less than 13.9h under DO of 0.3-0.5mg/L. High-throughput sequencing analysis demonstrated significant change of bacterial diversity in the first oxic reactor after a long-term operation and dominant bacteria genus Nitrosomonas was shown to be responsible for NH4(+)-N removal and nitrite accumulation under low DO levels. PMID:27403860

  7. Special Report: Affinity Chromatography.

    ERIC Educational Resources Information Center

    Parikh, Indu; Cuatrecasas, Pedro

    1985-01-01

    Describes the nature of affinity chromatography and its use in purifying enzymes, studying cell interactions, exploring hormone receptors, and other areas. The potential the technique may have in treating disease is also considered. (JN)

  8. Resveratrol Ameliorates the Maturation Process of β-Cell-Like Cells Obtained from an Optimized Differentiation Protocol of Human Embryonic Stem Cells

    PubMed Central

    Pezzolla, Daniela; López-Beas, Javier; Lachaud, Christian C.; Domínguez-Rodríguez, Alejandro; Smani, Tarik; Hmadcha, Abdelkrim; Soria, Bernat

    2015-01-01

    Human embryonic stem cells (hESCs) retain the extraordinary capacity to differentiate into different cell types of an adult organism, including pancreatic β-cells. For this particular lineage, although a lot of effort has been made in the last ten years to achieve an efficient and reproducible differentiation protocol, it was not until recently that this aim was roughly accomplished. Besides, several studies evidenced the impact of resveratrol (RSV) on insulin secretion, even though the mechanism by which this polyphenol potentiates glucose-stimulated insulin secretion (GSIS) is still not clear. The aim of this study was to optimize an efficient differentiation protocol that mimics in vivo pancreatic organogenesis and to investigate whether RSV may improve the final maturation step to obtain functional insulin-secreting cells. Our results indicate that treatment of hESCs (HS-181) with activin-A induced definitive endoderm differentiation as detected by the expression of SOX17 and FOXA2. Addition of retinoic acid (RA), Noggin and Cyclopamine promoted pancreatic differentiation as indicated by the expression of the early pancreatic progenitor markers ISL1, NGN3 and PDX1. Moreover, during maturation in suspension culture, differentiating cells assembled in islet-like clusters, which expressed specific endocrine markers such as PDX1, SST, GCG and INS. Similar results were confirmed with the human induced Pluripotent Stem Cell (hiPSC) line MSUH-001. Finally, differentiation protocols incorporating RSV treatment yielded numerous insulin-positive cells, induced significantly higher PDX1 expression and were able to transiently normalize glycaemia when transplanted in streptozotocin (STZ) induced diabetic mice thus promoting its survival. In conclusion, our strategy allows the efficient differentiation of hESCs into pancreatic endoderm capable of generating β-cell-like cells and demonstrates that RSV improves the maturation process. PMID:25774684

  9. Resveratrol ameliorates the maturation process of β-cell-like cells obtained from an optimized differentiation protocol of human embryonic stem cells.

    PubMed

    Pezzolla, Daniela; López-Beas, Javier; Lachaud, Christian C; Domínguez-Rodríguez, Alejandro; Smani, Tarik; Hmadcha, Abdelkrim; Soria, Bernat

    2015-01-01

    Human embryonic stem cells (hESCs) retain the extraordinary capacity to differentiate into different cell types of an adult organism, including pancreatic β-cells. For this particular lineage, although a lot of effort has been made in the last ten years to achieve an efficient and reproducible differentiation protocol, it was not until recently that this aim was roughly accomplished. Besides, several studies evidenced the impact of resveratrol (RSV) on insulin secretion, even though the mechanism by which this polyphenol potentiates glucose-stimulated insulin secretion (GSIS) is still not clear. The aim of this study was to optimize an efficient differentiation protocol that mimics in vivo pancreatic organogenesis and to investigate whether RSV may improve the final maturation step to obtain functional insulin-secreting cells. Our results indicate that treatment of hESCs (HS-181) with activin-A induced definitive endoderm differentiation as detected by the expression of SOX17 and FOXA2. Addition of retinoic acid (RA), Noggin and Cyclopamine promoted pancreatic differentiation as indicated by the expression of the early pancreatic progenitor markers ISL1, NGN3 and PDX1. Moreover, during maturation in suspension culture, differentiating cells assembled in islet-like clusters, which expressed specific endocrine markers such as PDX1, SST, GCG and INS. Similar results were confirmed with the human induced Pluripotent Stem Cell (hiPSC) line MSUH-001. Finally, differentiation protocols incorporating RSV treatment yielded numerous insulin-positive cells, induced significantly higher PDX1 expression and were able to transiently normalize glycaemia when transplanted in streptozotocin (STZ) induced diabetic mice thus promoting its survival. In conclusion, our strategy allows the efficient differentiation of hESCs into pancreatic endoderm capable of generating β-cell-like cells and demonstrates that RSV improves the maturation process. PMID:25774684

  10. Rapid changes in diatom silica surface charge density, silanol abundance, and oxygen isotope values elucidate silica maturation processes in biogenic silica

    NASA Astrophysics Data System (ADS)

    Wiedenheft, W.; Dodd, J. P.; Sunderlin, L.

    2014-12-01

    Oxygen isotope values of biogenic silica are increasingly used as proxies of paleoenvironmental conditions. Numerous studies have demonstrated a strong relationship between the diatom silica and the temperature/oxygen isotope value of the formation water; however, some studies have indicated that early diagenesis of biogenic silica may alter the oxygen isotope values by several permil. Quantification of the maturation process has proven difficult since the mechanisms that drive post-mortem changes in the silica oxygen isotope values have not been well characterized. New silica maturation data from marine diatoms, Stephanopyxis turris, cultured in a controlled laboratory experiment demonstrate rapid post-mortem decline in silica reactivity. A decrease in relative abundance of surface silanol groups coincides with a decrease in the surface charge density (excess proton concentration) of freshly harvested frustules. Over a maturation period of 20 days at 85ºC, S. turris samples in a 0.7 M NaCl solution at a pH of 8.0 demonstrate a rapid decrease in the surface charge density from -380 μmoles/g to -16 μmoles/g (Figure 1). FTIR analyses reveal a decrease in the abundance of silanol groups (Si-OH) in the diatom frustules occurs over the same time period. It is important to note that the surface charge density and silanol relative abundance appear to have an asymptotic change through time, indicating that further alteration/reactivity is greatly reduced. Preliminary data indicate that post-mortem increases in the oxygen isotope values of diatom silica observed here and in other studies are coincident with a reduction in the surface charge density and silanol abundance. These experiments demonstrate that rapid post-mortem alteration of biogenic silica is occurring and provide a possible mechanism for alteration of oxygen isotope values in biogenic silica.

  11. Synthesis of boronate-silica hybrid affinity monolith via a one-pot process for specific capture of glycoproteins at neutral conditions.

    PubMed

    Yang, F; Mao, J; He, X W; Chen, L X; Zhang, Y K

    2013-08-01

    In this study, a boronate-silica hybrid affinity monolith was prepared for specific capture of glycoproteins at neutral pH condition. The monolith was synthesized via a facile one-pot procedure in a stainless steel column by concurrently mixing hydrolyzed alkoxysilanes tetramethoxysilane and vinyltrimethoxysilane, organic monomer 3-acrylamidophenylboronic acid and initiator 2,2'-azobisisobutyronitrile together. The polycondensation of alkoxysilanes and copolymerization of organic monomer and vinyl-silica monolith were carried out successively by reacting at different temperatures. After optimizing the preparation conditions, the resulting hybrid affinity monolith was systematically characterized and exhibited excellent affinity to both cis-diol-containing small molecules and glycoproteins at neutral and physiological pH, including adenosine, horseradish peroxidase, transferrin and ovalbumin. The binding capacity of ovalbumin on monolith was measured to be 2.5 mg g(-1) at pH 7.0. Furthermore, the hybrid affinity monolith was applied to the separation of transferrin from bovine serum sample at a physiological condition. Good repeatability was obtained and the relative standard deviations of retention time were 1.15 and 4.77 % (n = 5) for run-to-run and column-to-column, respectively. PMID:23807307

  12. Identity, Affinity, Reality: Making the Case for Affinity Groups in Elementary School

    ERIC Educational Resources Information Center

    Parsons, Julie; Ridley, Kimberly

    2012-01-01

    Affinity groups are places where students build connections and process "ouch" moments from their classes. Children talk about the isolation they sometimes feel. The relationships students gain through race-based affinity groups enable them to feel less alone with their emotions and help them build a stronger sense of self. At the same time,…

  13. Processing and maturation of the pilin of the type IV secretion system encoded within the gonococcal genetic island.

    PubMed

    Jain, Samta; Kahnt, Jörg; van der Does, Chris

    2011-12-23

    The type IV secretion system (T4SS) encoded within the gonococcal genetic island (GGI) of Neisseria gonorrhoeae has homology to the T4SS encoded on the F plasmid. The GGI encodes the putative pilin protein TraA and a serine protease TrbI, which is homologous to the TraF protein of the RP4 plasmid involved in circularization of pilin subunits of P-type pili. TraA was processed to a 68-amino acid long circular peptide by leader peptidase and TrbI. Processing occurred after co-translational membrane insertion and was independent of other proteins. Circularization occurred after removal of three C-terminal amino acids. Mutational analysis of TraA revealed limited flexibility at the cleavage and joining sites. Mutagenesis of TrbI showed that the conserved Lys-93 and Asp-155 are essential, whereas mutagenesis of Ser-52, the putative catalytic serine did not influence circularization. Further mutagenesis of other serine residues did not identify a catalytic serine, indicating that TrbI either contains redundant catalytic serine residues or does not function via a serine-lysine dyad mechanism. In vitro studies revealed that circularization occurs via a covalent intermediate between the C terminus of TraA and TrbI. The intermediate is processed to the circular form after cleavage of the N-terminal signal sequence. This is the first demonstration of a covalent intermediate in the circularization mechanism of conjugative pili. PMID:22006923

  14. Effect of canola oil emulsion injection on processing characteristics and consumer acceptability of three muscles from mature beef.

    PubMed

    Pietrasik, Z; Wang, H; Janz, J A M

    2013-02-01

    The study was undertaken to investigate the impact of the combined effect of blade tenderization and canola oil emulsion injection on processing yield and eating quality-related parameters of selected loin and hip muscles (longissimus lumborum, LL, biceps femoris, BF and semimembranosus, SM) from over thirty month (OTM) cattle. Canola oil emulsion injection significantly reduced shear force, increased sensory scores for juiciness and tenderness, and made connective tissue less perceptible. Targeted levels of omega-3 fatty acids can be achieved by the inclusion of canola oil containing marinades/emulsions at levels sufficient to retain omega-3 fatty acids in cooked product. All consumer acceptability attributes of OTM muscles were improved with the use of canola oil emulsion injection treatments without compromising colour although slightly decreasing oxidative stability of BF muscle. Injection of omega-3 oil emulsions in combination with blade tenderization can be effectively utilized to enrich injected products in essential fatty acids and enhance eating quality of OTM beef. PMID:23089241

  15. Detection of radiation-induced hydrocarbons in Camembert irradiated before and after the maturing process-comparison of florisil column chromatography and on-line coupled liquid chromatography-gas chromatography

    SciTech Connect

    Schulzki, G.; Spiegelberg, A.; Schreiber, G.A.

    1995-02-01

    The influence of the maturing process on the detection of radiation-induced volatile hydrocarbons in the fat of Camembert has been investigated. Two analytical methods for separation of the hydrocarbon fraction from the lipid were applied: Florisil column chromatography with subsequent gas chromatographic-mass spectrometric (GC-MS) determination as well as on-line coupled liquid chromatography-GC-MS. The maturing process had no influence on the detection of radiation-induced volatiles. Comparable results were achieved with both analytical methods. However, preference is given to the more effective on-line coupled LC-GC method. 17 refs., 5 figs., 2 tabs.

  16. The SMC-5/6 Complex and the HIM-6 (BLM) Helicase Synergistically Promote Meiotic Recombination Intermediate Processing and Chromosome Maturation during Caenorhabditis elegans Meiosis.

    PubMed

    Hong, Ye; Sonneville, Remi; Agostinho, Ana; Meier, Bettina; Wang, Bin; Blow, J Julian; Gartner, Anton

    2016-03-01

    Meiotic recombination is essential for the repair of programmed double strand breaks (DSBs) to generate crossovers (COs) during meiosis. The efficient processing of meiotic recombination intermediates not only needs various resolvases but also requires proper meiotic chromosome structure. The Smc5/6 complex belongs to the structural maintenance of chromosome (SMC) family and is closely related to cohesin and condensin. Although the Smc5/6 complex has been implicated in the processing of recombination intermediates during meiosis, it is not known how Smc5/6 controls meiotic DSB repair. Here, using Caenorhabditis elegans we show that the SMC-5/6 complex acts synergistically with HIM-6, an ortholog of the human Bloom syndrome helicase (BLM) during meiotic recombination. The concerted action of the SMC-5/6 complex and HIM-6 is important for processing recombination intermediates, CO regulation and bivalent maturation. Careful examination of meiotic chromosomal morphology reveals an accumulation of inter-chromosomal bridges in smc-5; him-6 double mutants, leading to compromised chromosome segregation during meiotic cell divisions. Interestingly, we found that the lethality of smc-5; him-6 can be rescued by loss of the conserved BRCA1 ortholog BRC-1. Furthermore, the combined deletion of smc-5 and him-6 leads to an irregular distribution of condensin and to chromosome decondensation defects reminiscent of condensin depletion. Lethality conferred by condensin depletion can also be rescued by BRC-1 depletion. Our results suggest that SMC-5/6 and HIM-6 can synergistically regulate recombination intermediate metabolism and suppress ectopic recombination by controlling chromosome architecture during meiosis. PMID:27010650

  17. The SMC-5/6 Complex and the HIM-6 (BLM) Helicase Synergistically Promote Meiotic Recombination Intermediate Processing and Chromosome Maturation during Caenorhabditis elegans Meiosis

    PubMed Central

    Hong, Ye; Sonneville, Remi; Agostinho, Ana; Meier, Bettina; Wang, Bin; Blow, J. Julian; Gartner, Anton

    2016-01-01

    Meiotic recombination is essential for the repair of programmed double strand breaks (DSBs) to generate crossovers (COs) during meiosis. The efficient processing of meiotic recombination intermediates not only needs various resolvases but also requires proper meiotic chromosome structure. The Smc5/6 complex belongs to the structural maintenance of chromosome (SMC) family and is closely related to cohesin and condensin. Although the Smc5/6 complex has been implicated in the processing of recombination intermediates during meiosis, it is not known how Smc5/6 controls meiotic DSB repair. Here, using Caenorhabditis elegans we show that the SMC-5/6 complex acts synergistically with HIM-6, an ortholog of the human Bloom syndrome helicase (BLM) during meiotic recombination. The concerted action of the SMC-5/6 complex and HIM-6 is important for processing recombination intermediates, CO regulation and bivalent maturation. Careful examination of meiotic chromosomal morphology reveals an accumulation of inter-chromosomal bridges in smc-5; him-6 double mutants, leading to compromised chromosome segregation during meiotic cell divisions. Interestingly, we found that the lethality of smc-5; him-6 can be rescued by loss of the conserved BRCA1 ortholog BRC-1. Furthermore, the combined deletion of smc-5 and him-6 leads to an irregular distribution of condensin and to chromosome decondensation defects reminiscent of condensin depletion. Lethality conferred by condensin depletion can also be rescued by BRC-1 depletion. Our results suggest that SMC-5/6 and HIM-6 can synergistically regulate recombination intermediate metabolism and suppress ectopic recombination by controlling chromosome architecture during meiosis. PMID:27010650

  18. Mature Teachers Matter

    ERIC Educational Resources Information Center

    Berl, Patricia Scallan

    2005-01-01

    In this article, the author discusses the consequences of losing mature teachers due to voluntary separation or retirement and the mindset of a mature teacher that is different from younger teachers in a number of ways. Mature teachers are colleagues over 45 years of age possessing significant experience in the field. Future trends in teacher…

  19. Sexual maturation of female Saguinus oedipus oedipus

    SciTech Connect

    Tardif, S.D.

    1982-01-01

    This study is an examination of the process of female sexual maturation in the cotton-top tamarin, Saguinus oedipus oedipus, a South-American primate of the family, Callitrichidae. Two types of questions are addressed. The first question is whether the type of social grouping in which a young female lives affects the rate of her sexual maturation. Specifically, is there a difference between the maturation rate of a female housed with a strange adult male and a female housed with her natal group (i.e., her parents and various siblings). Second, the effect of sexual maturation on various social interactions is examined. Specifically are male-female interactions in mated pairs and mother-daughter interactions in natal groups changed by the sexual maturation of the young females. The mother's presence was not related to the daughter's maturation age. However, whether the natal group, as a whole, inhibited maturation, or unrelated males accelerated maturation, or both, remains unknown. Most of the behavioral interactions involving maturing females were unchanged by maturation. There was some indication that certain behaviors were affected by maturation, but only if a strange unrelated male was present.

  20. Low-affinity IgM antibodies lacking somatic hypermutations are produced in the secondary response of C57BL/6 mice to (4-hydroxy-3-nitrophenyl)acetyl hapten.

    PubMed

    Murakami, Akikazu; Moriyama, Hayato; Osako-Kabasawa, Mina; Endo, Kanako; Nishimura, Miyuki; Udaka, Keiko; Muramatsu, Masamichi; Honjo, Tasuku; Azuma, Takachika; Shimizu, Takeyuki

    2014-04-01

    Class-switched memory B cells, which are generated through the processes of somatic hypermutation (SHM) and affinity-based selection in germinal centers, contribute to the production of affinity-matured IgG antibodies in the secondary immune response. However, changes in the affinity of IgM antibodies during the immune response have not yet been studied, although IgM(+) memory B cells have been shown to be generated. In order to understand the relationship between IgM affinity and the recall immune response, we prepared hybridomas producing anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) IgM antibodies from C57BL/6 mice and from activation-induced cytidine deaminase (AID)-deficient mice. Binding analysis by ELISA showed that mAbs obtained from the secondary immune response contained IgM mAbs with affinity lower than the affinity of mAbs obtained from the primary response. By analyzing sequences of the IgM genes of hybridomas and plasma cells, we found many unmutated VH genes. VH genes that had neither tyrosine nor glycine at position 95 were frequent. The repertoire change may correlate with the lower affinity of IgM antibodies in the secondary response. The sequence and affinity changes in IgM antibodies were shown to be independent of SHM by analyzing hybridomas from AID-deficient mice. A functional assay revealed a reciprocal relationship between affinity and complement-dependent hemolytic activity toward NP-conjugated sheep RBCs; IgM antibodies with lower affinities had higher hemolytic activity. These findings indicate that lower affinity IgM antibodies with enhanced complement activation function are produced in the secondary immune response. PMID:24285827

  1. Local Affinity Release.

    PubMed

    Delplace, Vianney; Obermeyer, Jaclyn; Shoichet, Molly S

    2016-07-26

    The use of hydrogels for therapeutic delivery is a burgeoning area of investigation. These water-swollen polymer matrices are ideal platforms for localized drug delivery that can be further combined with specific ligands or nanotechnologies to advance the controlled release of small-molecule drugs and proteins. Due to the advantage of hydrophobic, electrostatic, or specific extracellular matrix interactions, affinity-based strategies can overcome burst release and challenges associated with encapsulation. Future studies will provide innovative binding tools, truly stimuli-responsive systems, and original combinations of emerging technologies to control the release of therapeutics spatially and temporally. Local drug delivery can be achieved by directly injecting a therapeutic to its site of action and is advantageous because off-target effects associated with systemic delivery can be minimized. For prolonged benefit, a vehicle that provides sustained drug release is required. Hydrogels are versatile platforms for localized drug release, owing to the large library of biocompatible building blocks from which they can be formed. Injectable hydrogel formulations that gel quickly in situ and provide sustained release of therapeutics are particularly advantageous to minimize invasiveness. The incorporation of polymers, ligands or nanoparticles that have an affinity for the therapeutic of interest improve control over the release of small-molecule drugs and proteins from hydrogels, enabling spatial and temporal control over the delivery. Such affinity-based strategies can overcome drug burst release and challenges associated with protein instability, allowing more effective therapeutic molecule delivery for a range of applications from therapeutic contact lenses to ischemic tissue regeneration. PMID:27403513

  2. Optimizing IV and V for Mature Organizations

    NASA Technical Reports Server (NTRS)

    Fuhman, Christopher

    2003-01-01

    NASA is intending for its future software development agencies to have at least a Level 3 rating in the Carnegie Mellon University Capability Maturity Model (CMM). The CMM has built-in Verification and Validation (V&V) processes that support higher software quality. Independent Verification and Validation (IV&V) of software developed by mature agencies can be therefore more effective than for software developed by less mature organizations. How is Independent V&V different with respect to the maturity of an organization? Knowing a priori the maturity of an organization's processes, how can IV&V planners better identify areas of need choose IV&V activities, etc? The objective of this research is to provide a complementary set of guidelines and criteria to assist the planning of IV&V activities on a project using a priori knowledge of the measurable levels of maturity of the organization developing the software.

  3. Affinity purification of aprotinin from bovine lung.

    PubMed

    Xin, Yu; Liu, Lanhua; Chen, Beizhan; Zhang, Ling; Tong, Yanjun

    2015-05-01

    An affinity protocol for the purification of aprotinin from bovine lung was developed. To simulate the structure of sucrose octasulfate, a natural specific probe for aprotinin, the affinity ligand was composed of an acidic head and a hydrophobic stick, and was then linked with Sepharose. The sorbent was then subjected to adsorption analysis with pure aprotinin. The purification process consisted of one step of affinity chromatography and another step of ultrafiltration. Then purified aprotinin was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, trypsin inhibitor activity, gel-filtration, and thin-layer chromatography analysis. As calculated, the theoretical maximum adsorption (Qmax ) of the affinity sorbent was 25,476.0 ± 184.8 kallikrein inactivator unit/g wet gel; the dissociation constant of the complex "immobilized ligand-aprotinin" (Kd ) was 4.6 ± 0.1 kallikrein inactivator unit/mL. After the affinity separation of bovine lung aprotinin, reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and gel-filtration chromatography revealed that the protein was a single polypeptide, and the purities were ∼ 97 and 100%, respectively; the purified peptide was also confirmed with aprotinin standard by gel-filtration chromatography and thin-layer chromatography. After the whole purification process, protein, and bioactivity recoveries were 2.2 and 92.6%, respectively; and the specific activity was up to 15,907.1 ± 10.2 kallikrein inactivator unit/mg. PMID:25677462

  4. Morphometric affinities of gigantopithecus.

    PubMed

    Gelvin, B R

    1980-11-01

    Multivariate analyses, supplemented by univariate statistical methods, of measurements from mandibular tooth crown dimensions and the mandible of Gigantopithecus blacki, G. bilaspurensis, Plio-Plelstocene hominids, Homo erectus, and seven Neogene ape species from the genera Proconsul, Sivapithecus, Ouranopithecus, and Dryopithecus were used to assess the morphometric affinities of Gigantopithecus. The results show that Gigantopithecus displays affinities to Ouranopithecus and to the hominids, particularly the Plio-Plelstocene hominids, rather than to the apes. Ouranopithecus demonstrated dental resemblances to G. bilaspurensis and the Plio-Pleistocene hominids but mandibular similarities to the apes. Results of analyses of tooth and mandibular shape indices, combined with multivariate distance and temporal relationships, suggest that Ouranopithecus is a more likely candidate for Gigantopithecus ancestry than is Silvapithecus indicus. Shape and allometric differences between G. bilaspurensis and the robust australopithecines weaken the argument for an ancestral-descendant relationship between these groups. The results support the hypothesis that Gigantopithecus is an extinct side branch of the Hominidae. PMID:7468790

  5. Age-Dependent Brain Gene Expression and Copy Number Anomalies in Autism Suggest Distinct Pathological Processes at Young Versus Mature Ages

    PubMed Central

    Winn, Mary E.; Barnes, Cynthia Carter; Li, Hai-Ri; Weiss, Lauren; Fan, Jian-Bing; Murray, Sarah; April, Craig; Belinson, Haim; Fu, Xiang-Dong; Wynshaw-Boris, Anthony; Schork, Nicholas J.; Courchesne, Eric

    2012-01-01

    Autism is a highly heritable neurodevelopmental disorder, yet the genetic underpinnings of the disorder are largely unknown. Aberrant brain overgrowth is a well-replicated observation in the autism literature; but association, linkage, and expression studies have not identified genetic factors that explain this trajectory. Few studies have had sufficient statistical power to investigate whole-genome gene expression and genotypic variation in the autistic brain, especially in regions that display the greatest growth abnormality. Previous functional genomic studies have identified possible alterations in transcript levels of genes related to neurodevelopment and immune function. Thus, there is a need for genetic studies involving key brain regions to replicate these findings and solidify the role of particular functional pathways in autism pathogenesis. We therefore sought to identify abnormal brain gene expression patterns via whole-genome analysis of mRNA levels and copy number variations (CNVs) in autistic and control postmortem brain samples. We focused on prefrontal cortex tissue where excess neuron numbers and cortical overgrowth are pronounced in the majority of autism cases. We found evidence for dysregulation in pathways governing cell number, cortical patterning, and differentiation in young autistic prefrontal cortex. In contrast, adult autistic prefrontal cortex showed dysregulation of signaling and repair pathways. Genes regulating cell cycle also exhibited autism-specific CNVs in DNA derived from prefrontal cortex, and these genes were significantly associated with autism in genome-wide association study datasets. Our results suggest that CNVs and age-dependent gene expression changes in autism may reflect distinct pathological processes in the developing versus the mature autistic prefrontal cortex. Our results raise the hypothesis that genetic dysregulation in the developing brain leads to abnormal regional patterning, excess prefrontal neurons

  6. Maturation processes in automatic change detection as revealed by event-related brain potentials and dipole source localization: significance for adult AD/HD.

    PubMed

    Wild-Wall, Nele; Oades, Robert D; Juran, Stephanie A

    2005-10-01

    Mismatch negativity (MMN) is an event-related potential reflecting automatic attention-related information processing marking the detection of auditory change. The bilateral scalp distribution develops by 14 years of age, and is elicited with adult latencies by 17 years. But consistent with reports of continued brain maturation after adolescence, we show here that features of the temporal and frontal lobe dipole sources also continue to develop in the third decade of life. This has consequences for studies of the developmental course of MMN anomalies, from childhood into adulthood, in attention-deficit/hyperactivity disorder. Two groups of healthy subjects with mean ages of 17 and 30 years were presented with a 3-tone auditory oddball. The duration-deviant MMN was recorded during attention to a visual discrimination (auditory-passive condition) and an active auditory discrimination. MMN amplitudes were smaller in the older subjects and the MMN lasted longer over the right hemisphere. Latencies and moments of the four dipoles in the temporal and frontal lobes did not distinguish the two subject-groups. But both temporal lobe sources were located significantly more ventrally and further left in the young adult than in the adolescent subjects. The left cingular source moved posteriorly and the right inferior frontal source moved antero-medially in the older subjects. Brain development in the third decade may cause the two frontal sources to move apart on the rostro-caudal axis but the temporal lobe sources to move left on the lateral and down on the dorsoventral axes. Thus special care is necessary in interpreting putative dysfunctional neurobiological changes in developmental attention-deficit disorders where as-yet-unspecified sub-groups may show a late developmental lag, partial lag, or no lag at all, associated with other impairments. PMID:15922470

  7. Adjoint affine fusion and tadpoles

    NASA Astrophysics Data System (ADS)

    Urichuk, Andrew; Walton, Mark A.

    2016-06-01

    We study affine fusion with the adjoint representation. For simple Lie algebras, elementary and universal formulas determine the decomposition of a tensor product of an integrable highest-weight representation with the adjoint representation. Using the (refined) affine depth rule, we prove that equally striking results apply to adjoint affine fusion. For diagonal fusion, a coefficient equals the number of nonzero Dynkin labels of the relevant affine highest weight, minus 1. A nice lattice-polytope interpretation follows and allows the straightforward calculation of the genus-1 1-point adjoint Verlinde dimension, the adjoint affine fusion tadpole. Explicit formulas, (piecewise) polynomial in the level, are written for the adjoint tadpoles of all classical Lie algebras. We show that off-diagonal adjoint affine fusion is obtained from the corresponding tensor product by simply dropping non-dominant representations.

  8. Maximum-Entropy Models of Sequenced Immune Repertoires Predict Antigen-Antibody Affinity.

    PubMed

    Asti, Lorenzo; Uguzzoni, Guido; Marcatili, Paolo; Pagnani, Andrea

    2016-04-01

    The immune system has developed a number of distinct complex mechanisms to shape and control the antibody repertoire. One of these mechanisms, the affinity maturation process, works in an evolutionary-like fashion: after binding to a foreign molecule, the antibody-producing B-cells exhibit a high-frequency mutation rate in the genome region that codes for the antibody active site. Eventually, cells that produce antibodies with higher affinity for their cognate antigen are selected and clonally expanded. Here, we propose a new statistical approach based on maximum entropy modeling in which a scoring function related to the binding affinity of antibodies against a specific antigen is inferred from a sample of sequences of the immune repertoire of an individual. We use our inference strategy to infer a statistical model on a data set obtained by sequencing a fairly large portion of the immune repertoire of an HIV-1 infected patient. The Pearson correlation coefficient between our scoring function and the IC50 neutralization titer measured on 30 different antibodies of known sequence is as high as 0.77 (p-value 10-6), outperforming other sequence- and structure-based models. PMID:27074145

  9. Maximum-Entropy Models of Sequenced Immune Repertoires Predict Antigen-Antibody Affinity

    PubMed Central

    Marcatili, Paolo; Pagnani, Andrea

    2016-01-01

    The immune system has developed a number of distinct complex mechanisms to shape and control the antibody repertoire. One of these mechanisms, the affinity maturation process, works in an evolutionary-like fashion: after binding to a foreign molecule, the antibody-producing B-cells exhibit a high-frequency mutation rate in the genome region that codes for the antibody active site. Eventually, cells that produce antibodies with higher affinity for their cognate antigen are selected and clonally expanded. Here, we propose a new statistical approach based on maximum entropy modeling in which a scoring function related to the binding affinity of antibodies against a specific antigen is inferred from a sample of sequences of the immune repertoire of an individual. We use our inference strategy to infer a statistical model on a data set obtained by sequencing a fairly large portion of the immune repertoire of an HIV-1 infected patient. The Pearson correlation coefficient between our scoring function and the IC50 neutralization titer measured on 30 different antibodies of known sequence is as high as 0.77 (p-value 10−6), outperforming other sequence- and structure-based models. PMID:27074145

  10. New definitions for cotton fiber maturity ratio

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cotton fiber maturity affects fiber physical, mechanical, and chemical properties, as well as the processability and qualities of yarn and fabrics. New definitions of cotton fiber maturity ratio are introduced. The influences of sampling, sample preparation, measurement method, and correlations am...

  11. 7 CFR 51.312 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Apples Definitions § 51.312 Mature. “Mature” means that the apples have reached the stage of development which will insure the proper completion of the ripening process. Before a mature apple becomes overripe it will show varying degrees of...

  12. 7 CFR 51.312 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Apples Definitions § 51.312 Mature. “Mature” means that the apples have reached the stage of development which will insure the proper completion of the ripening process. Before a mature apple becomes overripe it will show varying degrees of...

  13. Maturational characteristics of HIV-specific antibodies in viremic individuals

    PubMed Central

    Meffre, Eric; Louie, Aaron; Bannock, Jason; Kim, Leo J.Y.; Ho, Jason; Frear, Cody C.; Kardava, Lela; Wang, Wei; Buckner, Clarisa M.; Wang, Yimeng; Fankuchen, Olivia R.; Gittens, Kathleen R.; Chun, Tae-Wook; Li, Yuxing; Fauci, Anthony S.; Moir, Susan

    2016-01-01

    Despite the rare appearance of potent HIV-neutralizing mAbs in infected individuals requiring prolonged affinity maturation, little is known regarding this process in the majority of viremic individuals. HIV-infected individuals with chronic HIV viremia have elevated numbers of nonconventional tissue-like memory (TLM) B cells that predominate in blood over conventional resting memory (RM) B cells. Accordingly, we investigated affinity maturation in these 2 memory B cell populations. Analysis of IgG-expressing TLM B cells revealed a higher number of cell divisions compared with RM B cells; however, TLM B cells paradoxically displayed significantly lower frequencies of somatic hypermutation (SHM). To assess Ab reactivity in TLM and RM B cells, single-cell cloning was performed on HIV envelope CD4–binding site–sorted (CD4bs-sorted) B cells from 3 individuals with chronic HIV viremia. Several clonal families were present among the 127 cloned recombinant mAbs, with evidence of crosstalk between TLM and RM B cell populations that was largely restricted to non-VH4 families. Despite evidence of common origins, SHM frequencies were significantly decreased in TLM-derived mAbs compared with SHM frequencies in RM-derived mAbs. However, both cell populations had lower frequencies of SHMs than did broadly neutralizing CD4bs–specific mAbs. There was a significant correlation between SHM frequencies and the HIV-neutralizing capacities of the mAbs. Furthermore, HIV neutralization was significantly higher in the RM-derived mAbs compared with that seen in the TLM-derived mAbs, and both SHM frequencies and neutralizing capacity were lowest in TLM-derived mAbs with high polyreactivity. Thus, deficiencies in memory B cells that arise during chronic HIV viremia provide insight into the inadequacy of the Ab response in viremic individuals. PMID:27152362

  14. Inhibition of aggregation of amyloid peptides by beta-sheet breaker peptides and their binding affinity.

    PubMed

    Viet, Man Hoang; Ngo, Son Tung; Lam, Nguyen Sy; Li, Mai Suan

    2011-06-01

    The effects of beta-sheet breaker peptides KLVFF and LPFFD on the oligomerization of amyloid peptides were studied by all-atom simulations. It was found that LPFFD interferes the aggregation of Aβ(16-22) peptides to a greater extent than does KLVFF. Using the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method, we found that the former binds more strongly to Aβ(16-22). Therefore, by simulations, we have clarified the relationship between aggregation rates and binding affinity: the stronger the ligand binding, the slower the oligomerization process. The binding affinity of pentapeptides to full-length peptide Aβ(1-40) and its mature fibrils has been considered using the Autodock and MM-PBSA methods. The hydrophobic interaction between ligands and receptors plays a more important role for association than does hydrogen bonding. The influence of beta-sheet breaker peptides on the secondary structures of monomer Aβ(1-40) was studied in detail, and it turns out that, in their presence, the total beta-sheet content can be enhanced. However, the aggregation can be slowed because the beta-content is reduced in fibril-prone regions. Both pentapeptides strongly bind to monomer Aβ(1-40), as well as to mature fibrils, but KLVFF displays a lower binding affinity than LPFFD. Our findings are in accord with earlier experiments that both of these peptides can serve as prominent inhibitors. In addition, we predict that LPFFD inhibits/degrades the fibrillogenesis of full-length amyloid peptides better than KLVFF. This is probably related to a difference in their total hydrophobicities in that the higher the hydrophobicity, the lower the inhibitory capacity. The GROMOS96 43a1 force field with explicit water and the force field proposed by Morris et al. (Morris et al. J. Comput. Chem. 1998, 19, 1639 ) were employed for all-atom molecular dynamics simulations and Autodock experiments, respectively. PMID:21563780

  15. A minimax approach to spatial estimation using affinity matrices

    NASA Technical Reports Server (NTRS)

    Morris, C. N.

    1983-01-01

    Estimates made in the plane to improve on noisy unbiased estimates were combined. Only a small fraction of points in a giant grid were used to do this, those that are most like a given point. A component of this process defining an affinity matrix of values, indicating which points are relevant to others. Minimax rules are shown to be based on affinity matrices.

  16. The impact of water exchange rate and treatment processes on water-borne hormones in recirculation aquaculture systems containing sexually maturing Atlantic salmon Salmo salar

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A controlled seven-month study was conducted in six replicated water recirculation aquaculture systems (WRAS) to assess post-smolt Atlantic salmon (Salmo salar) performance in relation to WRAS water exchange rate. Unexpectedly high numbers of precocious sexually mature fish were observed in all WRAS...

  17. An asparagines residue at the N-terminus affects the maturation process of low molecular weight glutenin subunits of wheat endosperm

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Wheat (Triticum spp.) glutenin polymers are of two main types, high- (HMW-GS) and low- (LMW-GS) molecular weight subunits. The most common are the latter, based on the first amino acid of the mature sequence, are known as LMW-m and LMW-s types. They differ as a result of three extra amino acids (MET...

  18. Cone Early Maturity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hop cone early maturity is thought to be caused by diffuse infections of cone, just prior to harvest, by Podosphaera macularis. The disease is best managed by limiting the amount of leaf infection by P. macularis prior to bloom. The yield and quality reductions associated with Hop cone early matur...

  19. Development of a novel affinity chromatography resin for platform purification of lambda fabs.

    PubMed

    Eifler, Nora; Medaglia, Giovanni; Anderka, Oliver; Laurin, Linus; Hermans, Pim

    2014-01-01

    Antigen-binding fragments (Fabs) are novel formats in the growing pipeline of biotherapeutics. Sharing similar features to monoclonal antibodies (mAbs) with regard to expression, Fabs are considered as unchallenging for upstream development. Yet for downstream processing, the mature mAb downstream purification platform is not directly applicable. New approaches need to be found to achieve a lean purification process that maintains quality, productivity, and timelines while being generically applicable independent of the expression system. In a successful collaboration, BAC BV, GE Healthcare, and Novartis Pharma AG have developed a new affinity chromatography medium (resin) suitable to support cGMP manufacturing of lambda Fabs. We show that using this novel chromatography medium for the capture step, a purification platform for lambda Fabs can be established. PMID:25082738

  20. Affinity chromatography: a historical perspective.

    PubMed

    Hage, David S; Matsuda, Ryan

    2015-01-01

    Affinity chromatography is one of the most selective and versatile forms of liquid chromatography for the separation or analysis of chemicals in complex mixtures. This method makes use of a biologically related agent as the stationary phase, which provides an affinity column with the ability to bind selectively and reversibly to a given target in a sample. This review examines the early work in this method and various developments that have lead to the current status of this technique. The general principles of affinity chromatography are briefly described as part of this discussion. Past and recent efforts in the generation of new binding agents, supports, and immobilization methods for this method are considered. Various applications of affinity chromatography are also summarized, as well as the influence this field has played in the creation of other affinity-based separation or analysis methods. PMID:25749941

  1. Affinity+: Semi-Structured Brainstorming on Large Displays

    SciTech Connect

    Burtner, Edwin R.; May, Richard A.; Scarberry, Randall E.; LaMothe, Ryan R.; Endert, Alexander

    2013-04-27

    Affinity diagraming is a powerful method for encouraging and capturing lateral thinking in a group environment. The Affinity+ Concept was designed to improve the collaborative brainstorm process through the use of large display surfaces in conjunction with mobile devices like smart phones and tablets. The system works by capturing the ideas digitally and allowing users to sort and group them on a large touch screen manually. Additionally, Affinity+ incorporates theme detection, topic clustering, and other processing algorithms that help bring structured analytic techniques to the process without requiring explicit leadership roles and other overhead typically involved in these activities.

  2. The Purification of a Blood Group A Glycoprotein: An Affinity Chromatography Experiment.

    ERIC Educational Resources Information Center

    Estelrich, J.; Pouplana, R.

    1988-01-01

    Describes a purification process through affinity chromatography necessary to obtain specific blood group glycoproteins from erythrocytic membranes. Discusses the preparation of erythrocytic membranes, extraction of glycoprotein from membranes, affinity chromatography purification, determination of glycoproteins, and results. (CW)

  3. Data Product Maturity

    Atmospheric Science Data Center

    2013-03-25

    ... document, maturity levels are provided separately for each scientific data set (SDS) included with the data files. The data product ... indiscriminate use of these data products as the basis for research findings, journal publications, and/or presentations.   ...

  4. Maturation of the cellular and humoral immune responses to persistent infection in horses by equine infectious anemia virus is a complex and lengthy process.

    PubMed Central

    Hammond, S A; Cook, S J; Lichtenstein, D L; Issel, C J; Montelaro, R C

    1997-01-01

    Equine infectious anemia virus (EIAV) provides a natural model system by which immunological control of lentivirus infections may be studied. To date, no detailed study addressing in parallel both the humoral and cellular immune responses induced in horses upon infection by EIAV has been conducted. Therefore, we initiated the first comprehensive characterization of the cellular and humoral immune responses during clinical progression from chronic disease to inapparent stages of EIAV infection. Using new analyses of antibody avidity and antibody epitope conformation dependence that had not been previously employed in this system, we observed that the humoral immune response to EIAV required a 6- to 8-month period in which to fully mature. During this time frame, EIAV-specific antibody evolved gradually from a population characterized by low-avidity, nonneutralizing, and predominantly linear epitope specificity to an antibody population with an avidity of moderate to high levels, neutralizing activity, and predominantly conformational epitope specificity. Analyses of the cell-mediated immune response to EIAV revealed CD4+ and CD8+ major histocompatibility complex-restricted, EIAV-specific cytotoxic T-lymphocyte (CTL) activity apparent within 3 to 4 weeks postinfection, temporally correlating with the resolution of the primary viremia. After resolution of the initial viremia, EIAV-specific CTL activity differed greatly among the experimentally infected ponies, with some animals having readily detectable CTL activity while others had little measurable CTL activity. Thus, in contrast to the initial viremia, it appeared that no single immune parameter correlated with the resolution of further viremic episodes. Instead, immune control of EIAV infection during the clinically inapparent stage of infection appears to rely on a complex combination of immune system mechanisms to suppress viral replication that effectively functions only after the immune system has evolved to a

  5. A rapid measurement for cotton breeders of maturity and fineness from developing and mature fibers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The fiber’s maturity and fineness are important fiber properties that can impact the fiber’s downstream processing and the quality of yarn and fabric. The Cottonscope is a new instrument that simultaneously measures the fiber’s maturity and fineness using a very small amount of fiber sample. Previ...

  6. Aptamer-modified magnetic beads in affinity separation of proteins.

    PubMed

    Zhu, Guohong; Walter, Johanna-Gabriela

    2015-01-01

    Aptamers are valuable alternative ligands for affinity separations. Here, we describe the aptamer-based affinity separation of His-tagged proteins using an aptamer directed against the His-tag. The immobilization of the aptamer to magnetic beads is described as well as the aptamer-based purification and proper methods for the characterization of the process. Moreover, indications for the transfer of the process to other aptamers are given. PMID:25749947

  7. Color back projection for fruit maturity evaluation

    NASA Astrophysics Data System (ADS)

    Zhang, Dong; Lee, Dah-Jye; Desai, Alok

    2013-12-01

    In general, fruits and vegetables such as tomatoes and dates are harvested before they fully ripen. After harvesting, they continue to ripen and their color changes. Color is a good indicator of fruit maturity. For example, tomatoes change color from dark green to light green and then pink, light red, and dark red. Assessing tomato maturity helps maximize its shelf life. Color is used to determine the length of time the tomatoes can be transported. Medjool dates change color from green to yellow, and the orange, light red and dark red. Assessing date maturity helps determine the length of drying process to help ripen the dates. Color evaluation is an important step in the processing and inventory control of fruits and vegetables that directly affects profitability. This paper presents an efficient color back projection and image processing technique that is designed specifically for real-time maturity evaluation of fruits. This color processing method requires very simple training procedure to obtain the frequencies of colors that appear in each maturity stage. This color statistics is used to back project colors to predefined color indexes. Fruit maturity is then evaluated by analyzing the reprojected color indexes. This method has been implemented and used for commercial production.

  8. Improved native affinity purification of RNA.

    PubMed

    Batey, Robert T; Kieft, Jeffrey S

    2007-08-01

    RNA biochemical or structural studies often require an RNA sample that is chemically pure, and most protocols for its in vitro production use denaturing polyacrylamide gel electrophoresis to achieve this. Unfortunately, many RNAs do not quantitatively refold into an active conformation after denaturation, creating significant problems for downstream characterization or use. In addition, this traditional purification method is not amenable to studies demanding high-throughput RNA production. Recently, we presented the first general method for producing almost any RNA sequence that employs an affinity tag that is removed during the purification process. Because technical difficulties prevented application of this method to many RNAs, we have developed an improved version that utilizes a different activatable ribozyme and affinity tag that are considerably more robust, rapid, and broadly applicable. PMID:17548432

  9. Anti-HA Glycoforms Drive B Cell Affinity Selection and Determine Influenza Vaccine Efficacy.

    PubMed

    Wang, Taia T; Maamary, Jad; Tan, Gene S; Bournazos, Stylianos; Davis, Carl W; Krammer, Florian; Schlesinger, Sarah J; Palese, Peter; Ahmed, Rafi; Ravetch, Jeffrey V

    2015-07-01

    Protective vaccines elicit high-affinity, neutralizing antibodies by selection of somatically hypermutated B cell antigen receptors (BCR) on immune complexes (ICs). This implicates Fc-Fc receptor (FcR) interactions in affinity maturation, which, in turn, are determined by IgG subclass and Fc glycan composition within ICs. Trivalent influenza virus vaccination elicited regulation of anti-hemagglutinin (HA) IgG subclass and Fc glycans, with abundance of sialylated Fc glycans (sFc) predicting quality of vaccine response. We show that sFcs drive BCR affinity selection by binding the Type-II FcR CD23, thus upregulating the inhibitory FcγRIIB on activated B cells. This elevates the threshold requirement for BCR signaling, resulting in B cell selection for higher affinity BCR. Immunization with sFc HA ICs elicited protective, high-affinity IgGs against the conserved stalk of the HA. These results reveal a novel, endogenous pathway for affinity maturation that can be exploited for eliciting high-affinity, broadly neutralizing antibodies through immunization with sialylated immune complexes. PMID:26140596

  10. Affinity- and topology-dependent bound on current fluctuations

    NASA Astrophysics Data System (ADS)

    Pietzonka, Patrick; Barato, Andre C.; Seifert, Udo

    2016-08-01

    We provide a proof of a recently conjectured universal bound on current fluctuations in Markovian processes. This bound establishes a link between the fluctuations of an individual observable current, the cycle affinities driving the system into a non-equilibrium steady state, and the topology of the network. The proof is based on a decomposition of the network into independent cycles with both positive affinity and positive stationary cycle current. This formalism allows for a refinement of the bound for systems in equilibrium or with locally vanishing affinities.

  11. Need for speed: Sexual maturation precedes social maturation in gray mouse lemurs.

    PubMed

    Hohenbrink, Sarah; Zimmermann, Elke; Radespiel, Ute

    2015-10-01

    The life history of mammals underlies a fast-slow continuum, ranging from "slow" species with large body size, delayed sexual maturation, low fertility, and long lifespan, to "fast" species showing the opposite traits. Primates fall into the "slow" category, considering their relatively low offspring numbers and delayed juvenile development. However, social and sexual maturation processes do not necessarily have to be completed simultaneously. The comparison of the timeframes for sexual and social maturation is largely lacking for primates, with the prominent exception of humans. Here, we compare both maturation processes in a basal primate, the gray mouse lemur, which ranges in many aspects at the fast end of the slow-fast life history continuum among primates. We compared the patterns and frequencies of various social and solitary behaviors in young adults (YA, 12-13 months old) and older individuals (A, ≥2 years) of both sexes outside estrus. Observations were conducted during mix-sexed dyadic encounter experiments under controlled captive conditions (eight dyads per age class). Results indicate that although all young adults were sexually mature, social maturation was not yet completed in all behavioral domains: Age-dependent differences were found in the number of playing dyads, female marking behavior, female aggression, and social tolerance. Thus, this study provides a first indication that social maturation lags behind sexual maturation in an ancestral nocturnal primate model, indicating that these two developmental schemes may have been decoupled early and throughout the primate lineage. PMID:26119105

  12. Preselection Thymocytes Are More Sensitive to T Cell Receptor Stimulation Than Mature T Cells

    PubMed Central

    Davey, Gayle M.; Schober, Sonya L.; Endrizzi, Bart T.; Dutcher, Angela K.; Jameson, Stephen C.; Hogquist, Kristin A.

    1998-01-01

    During T cell development, thymocytes which are tolerant to self-peptides but reactive to foreign peptides are selected. The current model for thymocyte selection proposes that self-peptide–major histocompatibility complex (MHC) complexes that bind the T cell receptor with low affinity will promote positive selection while those with high affinity will result in negative selection. Upon thymocyte maturation, such low affinity self-peptide–MHC ligands no longer provoke a response, but foreign peptides can incidentally be high affinity ligands and can therefore stimulate T cells. For this model to work, thymocytes must be more sensitive to ligand than mature T cells. Contrary to this expectation, several groups have shown that thymocytes are less responsive than mature T cells to anti-T cell receptor for antigen (TCR)/CD3 mAb stimulation. Additionally, the lower TCR levels on thymocytes, compared with T cells, would potentially correlate with decreased thymocyte sensitivity. Here we compared preselection thymocytes and mature T cells for early activation events in response to peptide–MHC ligands. Remarkably, the preselection thymocytes were more responsive than mature T cells when stimulated with low affinity peptide variants, while both populations responded equally well to the antigenic peptide. This directly demonstrates the increased sensitivity of thymocytes compared with T cells for TCR engagement by peptide–MHC complexes. PMID:9815264

  13. Jealousy and Moral Maturity.

    ERIC Educational Resources Information Center

    Mathes, Eugene W.; Deuger, Donna J.

    Jealousy may be perceived as either good or bad depending upon the moral maturity of the individual. To investigate this conclusion, a study was conducted testing two hypothesis: a positive relationship exists between conventional moral reasoning (reference to norms and laws) and the endorsement and level of jealousy; and a negative relationship…

  14. Overview of affinity tags for protein purification.

    PubMed

    Kimple, Michelle E; Sondek, John

    2004-09-01

    Addition of an affinity tag is a useful method for differentiating recombinant proteins expressed in bacterial and eukaryotic expression systems from the background of total cellular proteins, and for detecting protein-protein interactions. This overview describes the historical basis for the development of affinity tags, affinity tags that are commonly used today, how to choose an appropriate affinity tag for a particular purpose, and several recently developed affinity tag technologies that may prove useful in the near future. PMID:18429272

  15. Overview of affinity tags for protein purification.

    PubMed

    Kimple, Michelle E; Brill, Allison L; Pasker, Renee L

    2013-01-01

    Addition of an affinity tag is a useful method for differentiating recombinant proteins expressed in bacterial and eukaryotic expression systems from the background of total cellular proteins, as well as for detecting protein-protein interactions. This overview describes the historical basis for the development of affinity tags, affinity tags that are commonly used today, how to choose an appropriate affinity tag for a particular purpose, and several recently developed affinity tag technologies that may prove useful in the near future. PMID:24510596

  16. Affine Contractions on the Plane

    ERIC Educational Resources Information Center

    Celik, D.; Ozdemir, Y.; Ureyen, M.

    2007-01-01

    Contractions play a considerable role in the theory of fractals. However, it is not easy to find contractions which are not similitudes. In this study, it is shown by counter examples that an affine transformation of the plane carrying a given triangle onto another triangle may not be a contraction even if it contracts edges, heights or medians.…

  17. Quantifying Affinity among Chinese Dialects.

    ERIC Educational Resources Information Center

    Cheng, Chin-Chuan

    A study of the relationships between Chinese dialects based on a quantitative measure of dialect affinity is summarized. First, tone values in all the dialect localities available in the early 1970s were used to calculate the dialectal differences in terms of tone height with respect to the "yin and yang" split. In the late 1970s, calculations of…

  18. Leveraging People-Related Maturity Issues for Achieving Higher Maturity and Capability Levels

    NASA Astrophysics Data System (ADS)

    Buglione, Luigi

    During the past 20 years Maturity Models (MM) become a buzzword in the ICT world. Since the initial Crosby's idea in 1979, plenty of models have been created in the Software & Systems Engineering domains, addressing various perspectives. By analyzing the content of the Process Reference Models (PRM) in many of them, it can be noticed that people-related issues have little weight in the appraisals of the capabilities of organizations while in practice they are considered as significant contributors in traditional process and organizational performance appraisals, as stressed instead in well-known Performance Management models such as MBQA, EFQM and BSC. This paper proposes some ways for leveraging people-related maturity issues merging HR practices from several types of maturity models into the organizational Business Process Model (BPM) in order to achieve higher organizational maturity and capability levels.

  19. DExD-box RNA-helicases in Listeria monocytogenes are important for growth, ribosomal maturation, rRNA processing and virulence factor expression

    PubMed Central

    Bäreclev, Caroline; Vaitkevicius, Karolis; Netterling, Sakura; Johansson, Jörgen

    2014-01-01

    RNA-helicases are proteins required for the unwinding of occluding secondary RNA structures, especially at low temperatures. In this work, we have deleted all 4 DExD-box RNA helicases in various combinations in the Gram-positive pathogen Listeria monocytogenes. Our results show that 3 out of 4 RNA-helicases were important for growth at low temperatures, whereas the effect was less prominent at 37°C. Over-expression of one RNA-helicase, Lmo1450, was able to overcome the reduced growth of the quadruple mutant strain at temperatures above 26°C, but not at lower temperatures. The maturation of ribosomes was affected in different degrees in the various strains at 20°C, whereas the effect was marginal at 37°C. This was accompanied by an increased level of immature 23S rRNA precursors in some of the RNA-helicase mutants at low temperatures. Although the expression of the PrfA regulated virulence factors ActA and LLO decreased in the quadruple mutant strain, this strain showed a slightly increased infection ability. Interestingly, even though the level of the virulence factor LLO was decreased in the quadruple mutant strain as compared with the wild-type strain, the hly-transcript (encoding LLO) was increased. Hence, our results could suggest a role for the RNA-helicases during translation. In this work, we show that DExD-box RNA-helicases are involved in bacterial virulence gene-expression and infection of eukaryotic cells. PMID:25590644

  20. Theoretical proton affinity and fluoride affinity of nerve agent VX.

    PubMed

    Bera, Narayan C; Maeda, Satoshi; Morokuma, Keiji; Viggiano, Al A

    2010-12-23

    Proton affinity and fluoride affinity of nerve agent VX at all of its possible sites were calculated at the RI-MP2/cc-pVTZ//B3LYP/6-31G* and RI-MP2/aug-cc-pVTZ//B3LYP/6-31+G* levels, respectively. The protonation leads to various unique structures, with H(+) attached to oxygen, nitrogen, and sulfur atoms; among which the nitrogen site possesses the highest proton affinity of -ΔE ∼ 251 kcal/mol, suggesting that this is likely to be the major product. In addition some H(2), CH(4) dissociation as well as destruction channels have been found, among which the CH(4) + [Et-O-P(═O)(Me)-S-(CH(2))(2)-N(+)(iPr)═CHMe] product and the destruction product forming Et-O-P(═O)(Me)-SMe + CH(2)═N(+)(iPr)(2) are only 9 kcal/mol less stable than the most stable N-protonated product. For fluoridization, the S-P destruction channel to give Et-O-P(═O)(Me)(F) + [S-(CH(2))(2)-N-(iPr)(2)](-) is energetically the most favorable, with a fluoride affinity of -ΔE ∼ 44 kcal. Various F(-) ion-molecule complexes are also found, with the one having F(-) interacting with two hydrogen atoms in different alkyl groups to be only 9 kcal/mol higher than the above destruction product. These results suggest VX behaves quite differently from surrogate systems. PMID:21117653

  1. Structure, Function and Dynamics in Adenovirus Maturation

    PubMed Central

    Mangel, Walter F.; San Martín, Carmen

    2014-01-01

    Here we review the current knowledge on maturation of adenovirus, a non-enveloped icosahedral eukaryotic virus. The adenovirus dsDNA genome fills the capsid in complex with a large amount of histone-like viral proteins, forming the core. Maturation involves proteolytic cleavage of several capsid and core precursor proteins by the viral protease (AVP). AVP uses a peptide cleaved from one of its targets as a “molecular sled” to slide on the viral genome and reach its substrates, in a remarkable example of one-dimensional chemistry. Immature adenovirus containing the precursor proteins lacks infectivity because of its inability to uncoat. The immature core is more compact and stable than the mature one, due to the condensing action of unprocessed core polypeptides; shell precursors underpin the vertex region and the connections between capsid and core. Maturation makes the virion metastable, priming it for stepwise uncoating by facilitating vertex release and loosening the condensed genome and its attachment to the icosahedral shell. The packaging scaffold protein L1 52/55k is also a substrate for AVP. Proteolytic processing of L1 52/55k disrupts its interactions with other virion components, providing a mechanism for its removal during maturation. Finally, possible roles for maturation of the terminal protein are discussed. PMID:25421887

  2. Structure, function and dynamics in adenovirus maturation

    SciTech Connect

    Mangel, Walter F.; San Martín, Carmen

    2014-11-21

    Here we review the current knowledge on maturation of adenovirus, a non-enveloped icosahedral eukaryotic virus. The adenovirus dsDNA genome fills the capsid in complex with a large amount of histone-like viral proteins, forming the core. Maturation involves proteolytic cleavage of several capsid and core precursor proteins by the viral protease (AVP). AVP uses a peptide cleaved from one of its targets as a “molecular sled” to slide on the viral genome and reach its substrates, in a remarkable example of one-dimensional chemistry. Immature adenovirus containing the precursor proteins lacks infectivity because of its inability to uncoat. The immature core is more compact and stable than the mature one, due to the condensing action of unprocessed core polypeptides; shell precursors underpin the vertex region and the connections between capsid and core. Maturation makes the virion metastable, priming it for stepwise uncoating by facilitating vertex release and loosening the condensed genome and its attachment to the icosahedral shell. The packaging scaffold protein L1 52/55k is also a substrate for AVP. Proteolytic processing of L1 52/55k disrupts its interactions with other virion components, providing a mechanism for its removal during maturation. In conclusion, possible roles for maturation of the terminal protein are discussed.

  3. Structure, function and dynamics in adenovirus maturation

    DOE PAGESBeta

    Mangel, Walter F.; San Martín, Carmen

    2014-11-21

    Here we review the current knowledge on maturation of adenovirus, a non-enveloped icosahedral eukaryotic virus. The adenovirus dsDNA genome fills the capsid in complex with a large amount of histone-like viral proteins, forming the core. Maturation involves proteolytic cleavage of several capsid and core precursor proteins by the viral protease (AVP). AVP uses a peptide cleaved from one of its targets as a “molecular sled” to slide on the viral genome and reach its substrates, in a remarkable example of one-dimensional chemistry. Immature adenovirus containing the precursor proteins lacks infectivity because of its inability to uncoat. The immature core ismore » more compact and stable than the mature one, due to the condensing action of unprocessed core polypeptides; shell precursors underpin the vertex region and the connections between capsid and core. Maturation makes the virion metastable, priming it for stepwise uncoating by facilitating vertex release and loosening the condensed genome and its attachment to the icosahedral shell. The packaging scaffold protein L1 52/55k is also a substrate for AVP. Proteolytic processing of L1 52/55k disrupts its interactions with other virion components, providing a mechanism for its removal during maturation. In conclusion, possible roles for maturation of the terminal protein are discussed.« less

  4. The effect of different composition of ternary mixtures of emulsifying salts on the consistency of processed cheese spreads manufactured from Swiss-type cheese with different degrees of maturity.

    PubMed

    Salek, R N; Černíková, M; Maděrová, S; Lapčík, L; Buňka, F

    2016-05-01

    The scope of this work was to investigate the dependence of selected textural (texture profile analysis, TPA) and viscoelastic properties of processed cheese on the composition of ternary mixtures of emulsifying salts [disodium hydrogenphosphate, DSP; tetrasodium diphosphate, TSPP; sodium salt of polyphosphate (with mean length n ≈ 20), P20; and trisodium citrate, TSC] during a 60-d storage period (6±2°C). The processed cheese samples [40% wt/wt dry matter (DM) content, 50% wt/wt fat in DM content] were manufactured using Swiss-type cheese (as the main raw material) with 4 different maturity degrees (4, 8, 12, and 16 wk of ripening). Moreover, the pH of the samples was adjusted (the target values within the range of 5.60-5.80), corresponding to the standard pH values of spreadable processed cheese. With respect to the individual application of emulsifying salts (regardless of the maturity degree of the Swiss-type cheese applied), the samples prepared with P20 were the hardest, followed by those prepared with TSPP, TSC, and DSP. Furthermore, a specific ratio of DSP:TSPP (1:1) led to a significant increase in the hardness of the samples. On the whole, the hardness of all processed cheese samples increased with the prolonging storage period, whereas their hardness significantly dropped with the rising ripening stage of the raw material utilized. In all of the cases, the trends of hardness development remained analogous, and only the absolute values differed significantly. Moreover, the findings of TPA were in accordance with those of the rheological analysis. In particular, the specific ratio of DSP:TSPP (1:1) resulted in the highest gel strength and interaction factor values, followed by P20, TSPP, TSC, and DSP (used individually), reporting the same trend which was demonstrated by TPA. The monitored values of the gel strength and interaction factor decreased with the increasing maturity degree of the Swiss-type cheese used. The intensity of the rigidity of the

  5. Enterotoxigenicity of Mature 45-Kilodalton and Processed 35-Kilodalton Forms of Hemagglutinin Protease Purified from a Cholera Toxin Gene-Negative Vibrio cholerae Non-O1, Non-O139 Strain

    PubMed Central

    Ghosh, A.; Saha, D. R.; Hoque, K. M.; Asakuna, M.; Yamasaki, S.; Koley, H.; Das, S. S.; Chakrabarti, M. K.; Pal, A.

    2006-01-01

    Cholera toxin gene-negative Vibrio cholerae non-O1, non-O139 strain PL-21 is the etiologic agent of cholera-like syndrome. Hemagglutinin protease (HAP) is one of the major secretory proteins of PL-21. The mature 45-kDa and processed 35-kDa forms of HAP were purified in the presence and absence of EDTA from culture supernatants of PL-21. Enterotoxigenicities of both forms of HAP were tested in rabbit ileal loop (RIL), Ussing chamber, and tissue culture assays. The 35-kDa HAP showed hemorrhagic fluid response in a dose-dependent manner in the RIL assay. Histopathological examination of 20 μg of purified protease-treated rabbit ileum showed the presence of erythrocytes and neutrophils in the upper part of the villous lamina propria. Treatment with 40 μg of protease resulted in gross damage of the villous epithelium with inflammation, hemorrhage, and necrosis. The 35-kDa form of HAP, when added to the lumenal surface of rat ileum loaded in an Ussing chamber, showed a decrease in the intestinal short-circuit current and a cell rounding effect on HeLa cells. The mature 45-kDa form of HAP showed an increase in intestinal short-circuit current in an Ussing chamber and a cell distending effect on HeLa cells. These results show that HAP may play a role in the pathogenesis of PL-21. PMID:16622232

  6. Processing of a composite large subunit rRNA. Studies with chlamydomonas mutants deficient in maturation of the 23s-like rrna.

    PubMed Central

    Holloway, S P; Herrin, D L

    1998-01-01

    (Cr.LSU). Little is known of the cis and trans requirements or of the processing pathway for this essential RNA. Previous work showed that the ribosome-deficient ac20 mutant overaccumulates an unspliced large subunit (LSU) RNA, suggesting that it might be a splicing mutant. To elucidate the molecular basis of the ac20 phenotype, a detailed analysis of the rrn transcripts in ac20 and wild-type cells was performed. The results indicate that processing of the ITSs, particularly ITS-1, is inefficient in ac20 and that ITS processing occurs after splicing. Deletion of the Cr.LSU intron from ac20 also did not alleviate the mutant phenotype. Thus, the primary defect in ac20 is not splicing but most likely is associated with ITS processing. A splicing deficiency was studied by transforming wild-type cells with rrnL genes containing point mutations in the intron core. Heteroplasmic transformants were obtained in most cases, except for P4 helix mutants; these strains grew slowly, were light sensitive, and had an RNA profile indicative of inefficient splicing. Transcript analysis in the P4 mutants also indicated that ITS processing can occur on an unspliced precursor, although with reduced efficiency. These latter results indicate that although there is not an absolutely required order for LSU processing, there does seem to be a preferred order that results in efficient processing in vivo. PMID:9668137

  7. Vocational Maturity and Self Concepts.

    ERIC Educational Resources Information Center

    Helbing, Hans

    The relationship between separate dimensions of vocational maturity and different self-concept and identity variables were examined. Subjects were Dutch students, age 14-18 years. The vocational maturity dimensions were measured by Dutch adaptations of American vocational maturity scales. Instruments for self-concept and identity measurement were…

  8. A Socioanalytic Model of Maturity

    ERIC Educational Resources Information Center

    Hogan, Robert; Roberts, Brent W.

    2004-01-01

    K0 describes a point of view on maturity that departs from earlier treatments in two ways. First, it rejects the popular assumption from humanistic psychology that maturity is a function of self-actualization and stipulates that maturity is related to certain performance capacities--namely, the ability to form lasting relationships and to achieve…

  9. Proline Substitutions and Threonine Pseudophosphorylation of the SH3 Ligand of 18.5-kDa Myelin Basic Protein Decrease Its Affinity for the Fyn-SH3 Domain and Alter Process Development and Protein Localization in Oligodendrocytes

    PubMed Central

    Smith, Graham S.T.; De Avila, Miguel; Paez, Pablo M.; Spreuer, Vilma; Wills, Melanie K.B.; Jones, Nina; Boggs, Joan M.; Harauz, George

    2012-01-01

    The developmentally regulated myelin basic proteins (MBPs), which arise from the golli (gene of oligodendrocyte lineage) complex, are highly positively charged, intrinsically disordered, multifunctional proteins having several alternatively spliced isoforms and posttranslational modifications, and they play key roles in myelin compaction. The classic 18.5-kDa MBP isoform has a proline-rich region comprising amino acids 92–99 (murine sequence –T92PRTPPPS99–) that contains a minimal SH3 ligand domain. We have previously shown that 18.5-kDa MBP binds to several SH3 domains, including that of Fyn, a member of the Src family of tyrosine kinases involved in a number of signaling pathways during CNS development. To determine the physiological role of this binding as well as the role of phosphorylation of Thr92 and Thr95, in the current study we have produced several MBP variants specifically targeting phosphorylation sites and key structural regions of MBP’s SH3 ligand domain. Using isothermal titration calorimetry, we have demonstrated that, compared with the wild-type protein, these variants have lower affinity for the SH3 domain of Fyn. Moreover, overexpression of N-terminal-tagged GFP versions in immortalized oligodendroglial N19 and N20.1 cell cultures results in aberrant elongation of membrane processes and increased branching complexity and inhibits the ability of MBP to decrease Ca2+ influx. Phosphorylation of Thr92 can also cause MBP to traffic to the nucleus, where it may participate in additional protein–protein interactions. Coexpression of MBP with a constitutively active form of Fyn kinase resulted in membrane process elaboration, a phenomenon that was abolished by point amino acid substitutions in MBP’s SH3 ligand domain. These results suggest that MBP’s SH3 ligand domain plays a key role in intracellular protein interactions in vivo and may be required for proper membrane elaboration of developing oligodendrocytes and, further, that

  10. Calcium ion currents mediating oocyte maturation events

    PubMed Central

    Tosti, Elisabetta

    2006-01-01

    During maturation, the last phase of oogenesis, the oocyte undergoes several changes which prepare it to be ovulated and fertilized. Immature oocytes are arrested in the first meiotic process prophase, that is morphologically identified by a germinal vesicle. The removal of the first meiotic block marks the initiation of maturation. Although a large number of molecules are involved in complex sequences of events, there is evidence that a calcium increase plays a pivotal role in meiosis re-initiation. It is well established that, during this process, calcium is released from the intracellular stores, whereas less is known on the role of external calcium entering the cell through the plasma membrane ion channels. This review is focused on the functional role of calcium currents during oocyte maturation in all the species, from invertebrates to mammals. The emerging role of specific L-type calcium channels will be discussed. PMID:16684344

  11. Viral and host control of cytomegalovirus maturation

    PubMed Central

    Tandon, Ritesh; Mocarski, Edward S.

    2012-01-01

    Maturation in herpesviruses initiates in the nucleus of the infected cell with encapsidation of viral DNA to form nucleocapsids and concludes with envelopment in the cytoplasm to form infectious virions that egress the cell. The entire process of virus maturation is orchestrated by protein-protein interactions and enzymatic activities of viral and host origin. Viral tegument proteins play important roles in maintaining the structural stability of capsids and directing the acquisition of virus envelope. Envelopment occurs at modified host membranes and exploits host vesicular trafficking. In this review, we summarize the current knowledge and concepts in human cytomegalovirus (HCMV) maturation and their parallels in other herpesviruses with an emphasis on viral and host factors regulating this process. PMID:22633075

  12. Lectin affinity chromatography of glycolipids

    SciTech Connect

    Torres, B.V.; Smith, D.F.

    1987-05-01

    Since glycolipids (GLs) are either insoluble or form mixed micelles in water, lectin affinity chromatography in aqueous systems has not been applied to their separation. They have overcome this problem by using tetrahydrofuran (THF) in the mobile phase during chromatography. Affinity columns prepared with the GalNAc-specific Helix pomatia agglutinin (HPA) and equilibrated in THF specifically bind the (/sup 3/H)oligosaccharide derived from Forssman GL indicating that the immobilized HPA retained its carbohydrate-binding specificity in this solvent. Intact Forssman GL was bound by the HPA-column equilibrated in THF and was specifically eluted with 0.1 mg/ml GalNAc in THF. Purification of the Forssman GL was achieved when a crude lipid extract of sheep erythrocyte membranes was applied to the HPA-column in THF. Non-specifically bound GLs were eluted from the column using a step gradient of aqueous buffer in THF, while the addition of GalNAc was required to elute the specifically bound GLs. Using this procedure the A-active GLs were purified from a crude lipid extract of type A human erythrocytes in a single chromatographic step. The use of solvents that maintain carbohydrate-binding specificity and lipid solubility will permit the application of affinity chromatography on immobilized carbohydrate-binding proteins to intact GLs.

  13. Flexible Linker Modulates Glycosaminoglycan Affinity of Decorin Binding Protein A.

    PubMed

    Morgan, Ashli; Sepuru, Krishna Mohan; Feng, Wei; Rajarathnam, Krishna; Wang, Xu

    2015-08-18

    Decorin binding protein A (DBPA) is a glycosaminoglycan (GAG)-binding adhesin found on the surface of the bacterium Borrelia burgdorferi (B. burgdorferi), the causative agent of Lyme disease. DBPA facilitates bacterial adherence to extracellular matrices of human tissues and is crucial during the early stage of the infection process. Interestingly, DBPA from different strains (B31, N40, and PBr) show significant differences in GAG affinities, but the structural basis for the differences is not clear. In this study, we show that GAG affinity of N40 DBPA is modulated in part by flexible segments that control access to the GAG binding site, such that shortening of the linker leads to higher GAG affinity when analyzed using ELISA, gel mobility shift assay, solution NMR, and isothermal titration calorimetry. Our observation that GAG affinity differences among different B. burgdorferi strains can be attributed to a flexible linker domain regulating access to the GAG-binding domain is novel. It also provides a rare example of how neutral amino acids and dynamic segments in GAG binding proteins can have a large influence on GAG affinity and provides insights into why the number of basic amino acids in the GAG-binding site may not be the only factor determining GAG affinity of proteins. PMID:26223367

  14. Teaching To Promote Intellectual and Personal Maturity: Incorporating Students' Worldviews and Identities into the Learning Process. The Jossey-Bass Higher and Adult Education Series.

    ERIC Educational Resources Information Center

    Magolda, Marcia B. Baxter, Ed.

    2000-01-01

    This volume offers an alternative diagnosis of the expectations gap that exists between students and professors. Professors' expectations often go far beyond students' assumptions about the nature of knowledge. It is not what students think but rather how they think that is important in the learning process. How students make meaning mediates how…

  15. Assessment of Solvated Interaction Energy Function for Ranking Antibody-Antigen Binding Affinities.

    PubMed

    Sulea, Traian; Vivcharuk, Victor; Corbeil, Christopher R; Deprez, Christophe; Purisima, Enrico O

    2016-07-25

    Affinity modulation of antibodies and antibody fragments of therapeutic value is often required in order to improve their clinical efficacies. Virtual affinity maturation has the potential to quickly focus on the critical hotspot residues without the combinatorial explosion problem of conventional display and library approaches. However, this requires a binding affinity scoring function that is capable of ranking single-point mutations of a starting antibody. We focus here on assessing the solvated interaction energy (SIE) function that was originally developed for and is widely applied to scoring of protein-ligand binding affinities. To this end, we assembled a structure-function data set called Single-Point Mutant Antibody Binding (SiPMAB) comprising several antibody-antigen systems suitable for this assessment, i.e., based on high-resolution crystal structures for the parent antibodies and coupled with high-quality binding affinity measurements for sets of single-point antibody mutants in each system. Using this data set, we tested the SIE function with several mutation protocols based on the popular methods SCWRL, Rosetta, and FoldX. We found that the SIE function coupled with a protocol limited to sampling only the mutated side chain can reasonably predict relative binding affinities with a Spearman rank-order correlation coefficient of about 0.6, outperforming more aggressive sampling protocols. Importantly, this performance is maintained for each of the seven system-specific component subsets as well as for other relevant subsets including non-alanine and charge-altering mutations. The transferability and enrichment in affinity-improving mutants can be further enhanced using consensus ranking over multiple methods, including the SIE, Talaris, and FOLDEF energy functions. The knowledge gained from this study can lead to successful prospective applications of virtual affinity maturation. PMID:27367467

  16. Relationship between DNA content and nuclear morphology of mature granulocytes.

    PubMed

    Berger, J; Kotelnikov, V M; Kozinec, G I

    1986-01-01

    Increased nuclear segmentation was experimentally induced in rat mature granulocytes. Some mature hypersegmented granulocytes were tetraploid, other hypersegmented granulocytes were diploid. The phenomenon of the formation of tetraploid mature granulocytes has not been observed till the present time. Our data suggest that the increase in nuclear segmentation of granulocytes can be stimulated by elevated DNA content, but the process of nuclear lobe formation is not dependent on cellular content of DNA. PMID:3803627

  17. Identification of proteins associated with the yeast mitochondrial RNA polymerase by tandem affinity purification

    PubMed Central

    Markov, Dmitriy A; Savkina, Maria; Anikin, Michael; Del Campo, Mark; Ecker, Karen; Lambowitz, Alan M; De Gnore, Jon P; McAllister, William T

    2009-01-01

    The abundance of mitochondrial (mt) transcripts varies under different conditions, and is thought to depend upon rates of transcription initiation, transcription termination/attenuation and RNA processing/degradation. The requirement to maintain the balance between RNA synthesis and processing may involve coordination between these processes; however, little is known about factors that regulate the activity of mtRNA polymerase (mtRNAP). Recent attempts to identify mtRNAP–protein interactions in yeast by means of a generalized tandem affinity purification (TAP) protocol were not successful, most likely because they involved a C-terminal mtRNAP–TAP fusion (which is incompatible with mtRNAP function) and because of the use of whole-cell solubilization protocols that did not preserve the integrity of mt protein complexes. Based upon the structure of T7 RNAP (to which mtRNAPs show high sequence similarity), we identified positions in yeast mtRNAP that allow insertion of a small affinity tag, confirmed the mature N-terminus, constructed a functional N-terminal TAP–mtRNAP fusion, pulled down associated proteins, and identified them by LC–MS–MS. Among the proteins found in the pull-down were a DEAD-box protein (Mss116p) and an RNA-binding protein (Pet127p). Previous genetic experiments suggested a role for these proteins in linking transcription and RNA degradation, in that a defect in the mt degradadosome could be suppressed by overexpression of either of these proteins or, independently, by mutations in either mtRNAP or its initiation factor Mtf1p. Further, we found that Mss116p inhibits transcription by mtRNAP in vitro in a steady-state reaction. Our results support the hypothesis that Mss116p and Pet127p are involved in modulation of mtRNAP activity. Copyright © 2009 John Wiley & Sons, Ltd. PMID:19536766

  18. Evaluating Hass Avocado Maturity Using Hyperspectral Imaging

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The maturity of avocado fruit is usually assessed by measuring its dry matter content(DM), which is a destructive and time consuming process. The aim of this study is tointroduce a non-destructive and quick technique that can estimate the DM content of an avocado fruit. 'Hass' avocado fruits at diff...

  19. 7 CFR 51.312 - Mature.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Standards for Grades of Apples Definitions § 51.312 Mature. “Mature” means that the apples have reached the... apple becomes overripe it will show varying degrees of firmness, depending upon the stage of the ripening process. The following terms are used for describing different stages of firmness of apples:...

  20. 7 CFR 51.312 - Mature.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Standards for Grades of Apples Definitions § 51.312 Mature. “Mature” means that the apples have reached the... apple becomes overripe it will show varying degrees of firmness, depending upon the stage of the ripening process. The following terms are used for describing different stages of firmness of apples:...

  1. 7 CFR 51.312 - Mature.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Standards for Grades of Apples Definitions § 51.312 Mature. “Mature” means that the apples have reached the... apple becomes overripe it will show varying degrees of firmness, depending upon the stage of the ripening process. The following terms are used for describing different stages of firmness of apples:...

  2. Immobilized metal ion affinity chromatography.

    PubMed

    Yip, T T; Hutchens, T W

    1992-01-01

    Immobilized metal ion affinity chromatography (IMAC) (1,2) is also referred to as metal chelate chromatography, metal ion interaction chromatography, and ligand-exchange chromatography. We view this affinity separation technique as an intermediate between highly specific, high-affinity bioaffinity separation methods, and wider spectrum, low-specificity adsorption methods, such as ion exchange. The IMAC stationary phases are designed to chelate certain metal ions that have selectivity for specific groups (e.g., His residues) in peptides (e.g., 3-7) and on protein surfaces (8-13). The number of stationary phases that can be synthesized for efficient chelation of metal ions is unlimited, but the critical consideration is that there must be enough exposure of the metal ion to interact with the proteins, preferably in a biospecific manner. Several examples are presented in Fig. 1. The challenge to produce new immobilized chelating groups, including protein surface metal-binding domains (14,15) is being explored continuously. Table 1 presents a list of published procedures for the synthesis and use of stationary phases with immobilized chelating groups. This is by no means exhaustive, and is intended only to give an idea of the scope and versatility of IMAC. Fig. 1 Schematic illustration of several types of immobilized metal-chelating groups, including, iminodiacetate (IDA), tris(carboxymethyl) ethylenediamine (TED), and the metal-binding peptides (GHHPH)(n)G (where n = 1,2,3, and 5) (14,15). Table 1 Immobilized Chelating Groups and Metal Ions Used for Immobilized Metal Ion Affinity Chromatography Chelating group Suitable metal ions Reference Commercial source Immodiacetate Transitional1,2 Pharmacia LKB Pierce Sigma Boehringer Mannheim TosoHaas 2-Hydroxy-3[N-(2- pyrtdylmethyl) glycme]propyl Transitional3 Not available ?-Alky1 mtrilo triacetic acid Transitional4 Not available Carboxymethylated asparhc acid Ca(II)13 Not available Tris (carboxy- methyl) ethylene Diamme

  3. Maturity Model for Advancing Smart Grid Interoperability

    SciTech Connect

    Knight, Mark; Widergren, Steven E.; Mater, J.; Montgomery, Austin

    2013-10-28

    Abstract—Interoperability is about the properties of devices and systems to connect and work properly. Advancing interoperability eases integration and maintenance of the resulting interconnection. This leads to faster integration, lower labor and component costs, predictability of projects and the resulting performance, and evolutionary paths for upgrade. When specifications are shared and standardized, competition and novel solutions can bring new value streams to the community of stakeholders involved. Advancing interoperability involves reaching agreement for how things join at their interfaces. The quality of the agreements and the alignment of parties involved in the agreement present challenges that are best met with process improvement techniques. The GridWise® Architecture Council (GWAC) sponsored by the United States Department of Energy is supporting an effort to use concepts from capability maturity models used in the software industry to advance interoperability of smart grid technology. An interoperability maturity model has been drafted and experience is being gained through trials on various types of projects and community efforts. This paper describes the value and objectives of maturity models, the nature of the interoperability maturity model and how it compares with other maturity models, and experiences gained with its use.

  4. Stacking the odds for Golgi cisternal maturation

    PubMed Central

    Mani, Somya; Thattai, Mukund

    2016-01-01

    What is the minimal set of cell-biological ingredients needed to generate a Golgi apparatus? The compositions of eukaryotic organelles arise through a process of molecular exchange via vesicle traffic. Here we statistically sample tens of thousands of homeostatic vesicle traffic networks generated by realistic molecular rules governing vesicle budding and fusion. Remarkably, the plurality of these networks contain chains of compartments that undergo creation, compositional maturation, and dissipation, coupled by molecular recycling along retrograde vesicles. This motif precisely matches the cisternal maturation model of the Golgi, which was developed to explain many observed aspects of the eukaryotic secretory pathway. In our analysis cisternal maturation is a robust consequence of vesicle traffic homeostasis, independent of the underlying details of molecular interactions or spatial stacking. This architecture may have been exapted rather than selected for its role in the secretion of large cargo. DOI: http://dx.doi.org/10.7554/eLife.16231.001 PMID:27542195

  5. Stacking the odds for Golgi cisternal maturation.

    PubMed

    Mani, Somya; Thattai, Mukund

    2016-01-01

    What is the minimal set of cell-biological ingredients needed to generate a Golgi apparatus? The compositions of eukaryotic organelles arise through a process of molecular exchange via vesicle traffic. Here we statistically sample tens of thousands of homeostatic vesicle traffic networks generated by realistic molecular rules governing vesicle budding and fusion. Remarkably, the plurality of these networks contain chains of compartments that undergo creation, compositional maturation, and dissipation, coupled by molecular recycling along retrograde vesicles. This motif precisely matches the cisternal maturation model of the Golgi, which was developed to explain many observed aspects of the eukaryotic secretory pathway. In our analysis cisternal maturation is a robust consequence of vesicle traffic homeostasis, independent of the underlying details of molecular interactions or spatial stacking. This architecture may have been exapted rather than selected for its role in the secretion of large cargo. PMID:27542195

  6. CFD - Mature Technology?

    NASA Technical Reports Server (NTRS)

    Kwak, Dochan

    2005-01-01

    Over the past 30 years, numerical methods and simulation tools for fluid dynamic problems have advanced as a new discipline, namely, computational fluid dynamics (CFD). Although a wide spectrum of flow regimes are encountered in many areas of science and engineering, simulation of compressible flow has been the major driver for developing computational algorithms and tools. This is probably due to a large demand for predicting the aerodynamic performance characteristics of flight vehicles, such as commercial, military, and space vehicles. As flow analysis is required to be more accurate and computationally efficient for both commercial and mission-oriented applications (such as those encountered in meteorology, aerospace vehicle development, general fluid engineering and biofluid analysis) CFD tools for engineering become increasingly important for predicting safety, performance and cost. This paper presents the author's perspective on the maturity of CFD, especially from an aerospace engineering point of view.

  7. An unexpected twist in viral capsid maturation

    SciTech Connect

    Gertsman, Ilya; Gan, Lu; Guttman, Miklos; Lee, Kelly; Speir, Jeffrey A.; Duda, Robert L.; Hendrix, Roger W.; Komives, Elizabeth A.; Johnson, John E.

    2009-04-14

    Lambda-like double-stranded (ds) DNA bacteriophage undergo massive conformational changes in their capsid shell during the packaging of their viral genomes. Capsid shells are complex organizations of hundreds of protein subunits that assemble into intricate quaternary complexes that ultimately are able to withstand over 50 atm of pressure during genome packaging. The extensive integration between subunits in capsids requires the formation of an intermediate complex, termed a procapsid, from which individual subunits can undergo the necessary refolding and structural rearrangements needed to transition to the more stable capsid. Although various mature capsids have been characterized at atomic resolution, no such procapsid structure is available for a dsDNA virus or bacteriophage. Here we present a procapsid X-ray structure at 3.65 {angstrom} resolution, termed prohead II, of the lambda-like bacteriophage HK97, the mature capsid structure of which was previously solved to 3.44 {angstrom}. A comparison of the two largely different capsid forms has unveiled an unprecedented expansion mechanism that describes the transition. Crystallographic and hydrogen/deuterium exchange data presented here demonstrate that the subunit tertiary structures are significantly different between the two states, with twisting and bending motions occurring in both helical and -sheet regions. We also identified subunit interactions at each three-fold axis of the capsid that are maintained throughout maturation. The interactions sustain capsid integrity during subunit refolding and provide a fixed hinge from which subunits undergo rotational and translational motions during maturation. Previously published calorimetric data of a closely related bacteriophage, P22, showed that capsid maturation was an exothermic process that resulted in a release of 90 kJ mol{sup -1} of energy. We propose that the major tertiary changes presented in this study reveal a structural basis for an exothermic

  8. Transcriptional Landscape of Cardiomyocyte Maturation

    PubMed Central

    Uosaki, Hideki; Cahan, Patrick; Lee, Dong I.; Wang, Songnan; Miyamoto, Matthew; Fernandez, Laviel; Kass, David A.; Kwon, Chulan

    2015-01-01

    SUMMARY Decades of progress in developmental cardiology has advanced our understanding of the early aspects of heart development, including cardiomyocyte (CM) differentiation. However, control of CM maturation which is subsequently required to generate adult myocytes, remains elusive. Here, we analyzed over 200 microarray datasets from early embryonic to adult hearts and identified a large number of genes whose expression shifts gradually and continuously during maturation. We generated an atlas of integrated gene expression, biological pathways, transcriptional regulators, and gene regulatory networks (GRNs), which show discrete sets of key transcriptional regulators and pathways activated or suppressed during CM maturation. We developed a GRN-based program named MatStatCM that indexes CM maturation status. MatStatCM reveals that pluripotent stem cell-derived CMs mature early in culture, but are arrested at the late embryonic stage with aberrant regulation of key transcription factors. Our study provides a foundation for understanding CM maturation. PMID:26586429

  9. The fsr Quorum-Sensing System and Cognate Gelatinase Orchestrate the Expression and Processing of Proprotein EF_1097 into the Mature Antimicrobial Peptide Enterocin O16

    PubMed Central

    Dundar, Halil; Brede, Dag A.; La Rosa, Sabina Leanti; El-Gendy, Ahmed Osama; Diep, Dzung B.

    2015-01-01

    ABSTRACT A novel antimicrobial peptide designated enterocin O16 was purified from Enterococcus faecalis. Mass spectrometry showed a monoisotopic mass of 7,231 Da, and N-terminal Edman degradation identified a 29-amino-acid sequence corresponding to residues 90 to 119 of the EF_1097 protein. Bioinformatic analysis showed that enterocin O16 is composed of the 68 most C-terminal residues of the EF_1097 protein. Introduction of an in-frame isogenic deletion in the ef1097 gene abolished the production of enterocin O16. Enterocin O16 has a narrow inhibitory spectrum, as it inhibits mostly lactobacilli. Apparently, E. faecalis is intrinsically resistant to the antimicrobial peptide, as no immunity connected to the production of enterocin O16 could be identified. ef1097 has previously been identified as one of three loci regulated by the fsr quorum-sensing system. The introduction of a nonsense mutation into fsrB consistently impaired enterocin O16 production, but externally added gelatinase biosynthesis-activating pheromone restored the antimicrobial activity. Functional genetic analysis showed that the EF_1097 proprotein is processed extracellularly into enterocin O16 by the metalloprotease GelE. Thus, it is evident that the fsr quorum-sensing system constitutes the regulatory unit that controls the expression of the EF_1097 precursor protein and the protease GelE and that the latter is required for the formation of enterocin O16. On the basis of these results, this study identified antibacterial antagonism as a novel aspect related to the function of fsr and provides a rationale for why ef1097 is part of the fsr regulon. IMPORTANCE The fsr quorum-sensing system modulates important physiological functions in E. faecalis via the activity of GelE. The present study presents a new facet of fsr signaling. The system controls the expression of three primary target operons (fsrABCD, gelE-sprE, and ef1097-ef1097b). We demonstrate that the concerted expression of these operons

  10. Indian craniometric variability and affinities.

    PubMed

    Raghavan, Pathmanathan; Bulbeck, David; Pathmanathan, Gayathiri; Rathee, Suresh Kanta

    2013-01-01

    Recently published craniometric and genetic studies indicate a predominantly indigenous ancestry of Indian populations. We address this issue with a fuller coverage of Indian craniometrics than any done before. We analyse metrical variability within Indian series, Indians' sexual dimorphism, differences between northern and southern Indians, index-based differences of Indian males from other series, and Indians' multivariate affinities. The relationship between a variable's magnitude and its variability is log-linear. This relationship is strengthened by excluding cranial fractions and series with a sample size less than 30. Male crania are typically larger than female crania, but there are also shape differences. Northern Indians differ from southern Indians in various features including narrower orbits and less pronounced medial protrusion of the orbits. Indians resemble Veddas in having small crania and similar cranial shape. Indians' wider geographic affinities lie with "Caucasoid" populations to the northwest, particularly affecting northern Indians. The latter finding is confirmed from shape-based Mahalanobis-D distances calculated for the best sampled male and female series. Demonstration of a distinctive South Asian craniometric profile and the intermediate status of northern Indians between southern Indians and populations northwest of India confirm the predominantly indigenous ancestry of northern and especially southern Indians. PMID:24455409

  11. Indian Craniometric Variability and Affinities

    PubMed Central

    Raghavan, Pathmanathan; Bulbeck, David; Pathmanathan, Gayathiri; Rathee, Suresh Kanta

    2013-01-01

    Recently published craniometric and genetic studies indicate a predominantly indigenous ancestry of Indian populations. We address this issue with a fuller coverage of Indian craniometrics than any done before. We analyse metrical variability within Indian series, Indians' sexual dimorphism, differences between northern and southern Indians, index-based differences of Indian males from other series, and Indians' multivariate affinities. The relationship between a variable's magnitude and its variability is log-linear. This relationship is strengthened by excluding cranial fractions and series with a sample size less than 30. Male crania are typically larger than female crania, but there are also shape differences. Northern Indians differ from southern Indians in various features including narrower orbits and less pronounced medial protrusion of the orbits. Indians resemble Veddas in having small crania and similar cranial shape. Indians' wider geographic affinities lie with “Caucasoid” populations to the northwest, particularly affecting northern Indians. The latter finding is confirmed from shape-based Mahalanobis-D distances calculated for the best sampled male and female series. Demonstration of a distinctive South Asian craniometric profile and the intermediate status of northern Indians between southern Indians and populations northwest of India confirm the predominantly indigenous ancestry of northern and especially southern Indians. PMID:24455409

  12. In vitro embryo production in llamas (Lama glama) from in vivo matured oocytes with raw semen processed with Androcoll-E using defined embryo culture media.

    PubMed

    Trasorras, V; Giuliano, S; Chaves, G; Neild, D; Agüero, A; Carretero, M; Pinto, M; Baca Castex, C; Alonso, A; Rodríguez, D; Morrell, J M; Miragaya, M

    2012-08-01

    The aim of this study was to carry out in vitro fertilization using spermatozoa selected with Androcoll-E™ and to evaluate the efficiency of the culture medium DMEM-F12 for in vitro embryo development in the llama. Twelve adult females from 18 superstimulated (67%) were used as oocyte donors. They were superstimulated with 1500 IU of eCG and after 5 days, received a single dose of buserelin. Twenty hours post-injection, follicular aspiration was conducted by flank laparotomy. Semen collections were performed under general anesthesia by electroejaculation of the male. The ejaculates were processed with a solution of collagenase (0.1%) and an Androcoll-E™ column was used to improve the sample. Sixty nine COCs were recovered from 79 aspirated follicles (87% recovery). Only expanded COCs were used (n = 67); they were randomly placed in groups of 1-5 in Fertil-TALP and the sperm suspension (20 × 10(6) live spermatozoa/ml) was added to each fertilization microdroplet. After 24 h, they were randomly placed in one of two culture media: SOF (n = 34) or DMEM-F12 (n = 33) and incubated for 6 days in humidified atmosphere of 5% CO(2) , 5% O(2) and 90% N(2) at 38°C. The blastocyst rate was 20% (7/34) in SOF medium (3 hatched, 2 expanded and 2 early blastocysts) and 15% (5/33) in DMEM medium (all expanded blastocysts). In conclusion, using Androcoll-E™ it is possible to select good quality spermatozoa from llama ejaculates for in vitro fertilization and to produce blastocysts in DMEM-F12 medium. This is also the first time that hatched llama blastocysts have been produced after culture in a defined medium such as SOFaa. PMID:21988486

  13. Increased hemoglobin O2 affinity protects during acute hypoxia.

    PubMed

    Yalcin, Ozlem; Cabrales, Pedro

    2012-08-01

    Acclimatization to hypoxia requires time to complete the adaptation mechanisms that influence oxygen (O(2)) transport and O(2) utilization. Although decreasing hemoglobin (Hb) O(2) affinity would favor the release of O(2) to the tissues, increasing Hb O(2) affinity would augment arterial O(2) saturation during hypoxia. This study was designed to test the hypothesis that pharmacologically increasing the Hb O(2) affinity will augment O(2) transport during severe hypoxia (10 and 5% inspired O(2)) compared with normal Hb O(2) affinity. RBC Hb O(2) affinity was increased by infusion of 20 mg/kg of 5-hydroxymethyl-2-furfural (5HMF). Control animals received only the vehicle. The effects of increasing Hb O(2) affinity were studied in the hamster window chamber model, in terms of systemic and microvascular hemodynamics and partial pressures of O(2) (Po(2)). Pimonidazole binding to hypoxic areas of mice heart and brain was also studied. 5HMF decreased the Po(2) at which the Hb is 50% saturated with O(2) by 12.6 mmHg. During 10 and 5% O(2) hypoxia, 5HMF increased arterial blood O(2) saturation by 35 and 48% from the vehicle group, respectively. During 5% O(2) hypoxia, blood pressure and heart rate were 58 and 30% higher for 5HMF compared with the vehicle. In addition, 5HMF preserved microvascular blood flow, whereas blood flow decreased to 40% of baseline in the vehicle group. Consequently, perivascular Po(2) was three times higher in the 5HMF group compared with the control group at 5% O(2) hypoxia. 5HMF also reduced heart and brain hypoxic areas in mice. Therefore, increased Hb O(2) affinity resulted in hemodynamics and oxygenation benefits during severe hypoxia. This acute acclimatization process may have implications in survival during severe environmental hypoxia when logistic constraints prevent chronic acclimatization. PMID:22636677

  14. Increased hemoglobin O2 affinity protects during acute hypoxia

    PubMed Central

    Yalcin, Ozlem

    2012-01-01

    Acclimatization to hypoxia requires time to complete the adaptation mechanisms that influence oxygen (O2) transport and O2 utilization. Although decreasing hemoglobin (Hb) O2 affinity would favor the release of O2 to the tissues, increasing Hb O2 affinity would augment arterial O2 saturation during hypoxia. This study was designed to test the hypothesis that pharmacologically increasing the Hb O2 affinity will augment O2 transport during severe hypoxia (10 and 5% inspired O2) compared with normal Hb O2 affinity. RBC Hb O2 affinity was increased by infusion of 20 mg/kg of 5-hydroxymethyl-2-furfural (5HMF). Control animals received only the vehicle. The effects of increasing Hb O2 affinity were studied in the hamster window chamber model, in terms of systemic and microvascular hemodynamics and partial pressures of O2 (Po2). Pimonidazole binding to hypoxic areas of mice heart and brain was also studied. 5HMF decreased the Po2 at which the Hb is 50% saturated with O2 by 12.6 mmHg. During 10 and 5% O2 hypoxia, 5HMF increased arterial blood O2 saturation by 35 and 48% from the vehicle group, respectively. During 5% O2 hypoxia, blood pressure and heart rate were 58 and 30% higher for 5HMF compared with the vehicle. In addition, 5HMF preserved microvascular blood flow, whereas blood flow decreased to 40% of baseline in the vehicle group. Consequently, perivascular Po2 was three times higher in the 5HMF group compared with the control group at 5% O2 hypoxia. 5HMF also reduced heart and brain hypoxic areas in mice. Therefore, increased Hb O2 affinity resulted in hemodynamics and oxygenation benefits during severe hypoxia. This acute acclimatization process may have implications in survival during severe environmental hypoxia when logistic constraints prevent chronic acclimatization. PMID:22636677

  15. Maturational and Non-Maturational Factors in Heritage Language Acquisition

    ERIC Educational Resources Information Center

    Moon, Ji Hye

    2012-01-01

    This dissertation aims to understand the maturational and non-maturational aspects of early bilingualism and language attrition in heritage speakers who have acquired their L1 incompletely in childhood. The study highlights the influential role of age and input dynamics in early L1 development, where the timing of reduction in L1 input and the…

  16. Affine hypersurfaces with parallel difference tensor relative to affine α-connection

    NASA Astrophysics Data System (ADS)

    Li, Cece

    2014-12-01

    Li and Zhang (2014) studied affine hypersurfaces of R n + 1 with parallel difference tensor relative to the affine α-connection ∇ (α), and characterized the generalized Cayley hypersurfaces by K n - 1 ≠ 0 and ∇ (α) K = 0 for some nonzero constant α, where the affine α-connection ∇ (α) of information geometry was introduced on affine hypersurface. In this paper, by a slightly different method we continue to study affine hypersurfaces with ∇ (α) K = 0, if α = 0 we further assume that the Pick invariant vanishes and affine metric is of constant sectional curvature. It is proved that they are either hyperquadrics or improper affine hypersphere with flat indefinite affine metric, the latter can be locally given as a graph of a polynomial of at most degree n + 1 with constant Hessian determinant. In particular, if the affine metric is definite, Lorentzian, or its negative index is 2, we complete the classification of such hypersurfaces.

  17. The maximal affinity of ligands

    PubMed Central

    Kuntz, I. D.; Chen, K.; Sharp, K. A.; Kollman, P. A.

    1999-01-01

    We explore the question of what are the best ligands for macromolecular targets. A survey of experimental data on a large number of the strongest-binding ligands indicates that the free energy of binding increases with the number of nonhydrogen atoms with an initial slope of ≈−1.5 kcal/mol (1 cal = 4.18 J) per atom. For ligands that contain more than 15 nonhydrogen atoms, the free energy of binding increases very little with relative molecular mass. This nonlinearity is largely ascribed to nonthermodynamic factors. An analysis of the dominant interactions suggests that van der Waals interactions and hydrophobic effects provide a reasonable basis for understanding binding affinities across the entire set of ligands. Interesting outliers that bind unusually strongly on a per atom basis include metal ions, covalently attached ligands, and a few well known complexes such as biotin–avidin. PMID:10468550

  18. The Mature Athlete

    PubMed Central

    McCarthy, Moira M.; Hannafin, Jo A.

    2014-01-01

    Context: Aging changes the biology, healing capacity, and biomechanical function of tendons and ligaments and results in common clinical pathologies that present to orthopedic surgeons, primary care physicians, physical therapists, and athletic trainers. A better understanding of the age-related changes in these connective tissues will allow better patient care. Evidence Acquisition: The PubMed database was searched in December 2012 for English-language articles pertaining to age-related changes in tendons and ligaments. Level of Evidence: Level 5. Results: The mature athlete faces challenges associated with age-dependent changes in the rotator cuff, Achilles tendon, lateral humeral epicondylar tendons, quadriceps tendon, and patellar tendon. The anterior cruciate ligament and the medial collateral ligament are the most studied intra-articular and extra-articular ligaments, and both are associated with age-dependent changes. Conclusion: Tendons and ligaments are highly arranged connective tissue structures that maintain joint motion and joint stability. These structures are subject to vascular and compositional changes with increasing age that alter their mechanotransduction, biology, healing capacity, and biomechanical function. Emerging research into the etiology of age-dependent changes will provide further information to help combat the age-related clinical complications associated with the injuries that occur to tendons and ligaments. PMID:24427441

  19. Career Maturity of Welfare Recipients.

    ERIC Educational Resources Information Center

    Beckman, Carol M.

    To investigate the career maturity of welfare recipients, this thesis examines six independent variables: (1) race; (2) sex; (3) age; (4) level of formal education; (5) general intelligence; and (6) locus of control. Scales taken from the Career Maturity Inventory served as the dependent variables. The sample consisted of 83 welfare recipients who…

  20. Histology of salmonid testes during maturation.

    PubMed

    Dziewulska, Katarzyna; Domagała, Józef

    2003-03-01

    The commonly applied classification systems of fish gonad maturity divide the maturation process into certain stages. However, the scales do not entirely reflect the continuity of the maturation process. Based on light microscope observations, the paper describes a comprehensive pattern of testicular transformations during maturation. The study was carried out on precocious underyearling and 1-year-old males of sea trout (Salmo trutta m. trutta L.), 1-year-old males of salmon (Salmo salar L.), and males of brown trout (Salmo trutta m. fario L.) aged from 7 months to 4 years. A total of 821 gonads collected during all seasons of the year were examined. The fish were fixed in Bouin's fluid. Histological slides of the mid-part of the gonad were made using the standard paraffin technique. The 3-6 microm sections were stained with Heidenhain haematoxylin. Histological changes of testes during maturation were similar in the three species studied. Immature and resting gonads contained type A spermatogonia in lobules only. The appearance of cystic structures containing type B spermatogonia in the lobules signalled the beginning of the sexual cycle in male gonads. Type B spermatogonia underwent synchronous mitotic divisions resulting in an increase in the total number of spermatogonia. As the spermatogenesis continued, the gonads showed a gradual increase in the number of cysts containing cells at all the spermatogenetic stages: type B spermatogonia, primary and secondary spermatocytes, spermatids, and spermatozoa. The well-formed spermatozoa were released to the lobule lumen once the Sertoli cells and spermatozoa connections broke up and the cyst disappeared. This was a continuous process observed throughout the spawning season. The spermatozoa were moved to the efferent duct. While some of the germ cells were completing spermatogenesis, the lobules contained less and less cysts with type B spermatogonia, primary and secondary spermatocytes, and spermatids; eventually all

  1. Protein Complex Purification by Affinity Capture.

    PubMed

    LaCava, John; Fernandez-Martinez, Javier; Hakhverdyan, Zhanna; Rout, Michael P

    2016-01-01

    Affinity capture has become a powerful technique for consistently purifying endogenous protein complexes, facilitating biochemical and biophysical assays on otherwise inaccessible biological assemblies, and enabling broader interactomic exploration. For this procedure, cells are broken and their contents separated and extracted into a solvent, permitting access to target macromolecular complexes thus released in solution. The complexes are specifically enriched from the extract onto a solid medium coupled with an affinity reagent-usually an antibody-that recognizes the target either directly or through an appended affinity tag, allowing subsequent characterization of the complex. Here, we discuss approaches and considerations for purifying endogenous yeast protein complexes by affinity capture. PMID:27371601

  2. A Novel Vertex Affinity for Community Detection

    SciTech Connect

    Yoo, Andy; Sanders, Geoffrey; Henson, Van; Vassilevski, Panayot

    2015-10-05

    We propose a novel vertex affinity measure in this paper. The new vertex affinity quantifies the proximity between two vertices in terms of their clustering strength and is ideal for such graph analytics applications as community detection. We also developed a framework that combines simple graph searches and resistance circuit formulas to compute the vertex affinity efficiently. We study the properties of the new affinity measure empirically in comparison to those of other popular vertex proximity metrics. Our results show that the existing metrics are ill-suited for community detection due to their lack of fundamental properties that are essential for correctly capturing inter- and intra-cluster vertex proximity.

  3. Structural determinants of sigma receptor affinity

    SciTech Connect

    Largent, B.L.; Wikstroem, H.G.; Gundlach, A.L.; Snyder, S.H.

    1987-12-01

    The structural determinants of sigma receptor affinity have been evaluated by examining a wide range of compounds related to opioids, neuroleptics, and phenylpiperidine dopaminergic structures for affinity at sigma receptor-binding sites labeled with (+)-(/sup 3/H)3-PPP. Among opioid compounds, requirements for sigma receptor affinity differ strikingly from the determinants of affinity for conventional opiate receptors. Sigma sites display reverse stereoselectivity to classical opiate receptors. Multi-ringed opiate-related compounds such as morphine and naloxone have negligible affinity for sigma sites, with the highest sigma receptor affinity apparent for benzomorphans which lack the C ring of opioids. Highest affinity among opioids and other compounds occurs with more lipophilic N-substituents. This feature is particularly striking among the 3-PPP derivatives as well as the opioids. The butyrophenone haloperidol is the most potent drug at sigma receptors we have detected. Among the series of butyrophenones, receptor affinity is primarily associated with the 4-phenylpiperidine moiety. Conformational calculations for various compounds indicate a fairly wide range of tolerance for distances between the aromatic ring and the amine nitrogen, which may account for the potency at sigma receptors of structures of considerable diversity. Among the wide range of structures that bind to sigma receptor-binding sites, the common pharmacophore associated with high receptor affinity is a phenylpiperidine with a lipophilic N-substituent.

  4. Compact noncontraction semigroups of affine operators

    NASA Astrophysics Data System (ADS)

    Voynov, A. S.; Protasov, V. Yu

    2015-07-01

    We analyze compact multiplicative semigroups of affine operators acting in a finite-dimensional space. The main result states that every such semigroup is either contracting, that is, contains elements of arbitrarily small operator norm, or all its operators share a common invariant affine subspace on which this semigroup is contracting. The proof uses functional difference equations with contraction of the argument. We look at applications to self-affine partitions of convex sets, the investigation of finite affine semigroups and the proof of a criterion of primitivity for nonnegative matrix families. Bibliography: 32 titles.

  5. Water channel in the binding site of a high affinity anti-methotrexate antibody.

    PubMed

    Gayda, Susan; Longenecker, Kenton L; Manoj, Sharmila; Judge, Russell A; Saldana, Sylvia C; Ruan, Qiaoqiao; Swift, Kerry M; Tetin, Sergey Y

    2014-06-17

    In the present study, we report the structure of the free and drug-bound Fab fragment of a high affinity anti-methotrexate antibody and perform a thermodynamic analysis of the binding process. The anti-methotrexate Fab fragment features a remarkably rigid tunnel-like binding site that extends into a water channel serving as a specialized route to move solvent out and into the site upon ligand binding and dissociation. This new finding in antibody structure-function relationships directly relates to the fast association (1 × 10⁷ M⁻¹ s⁻¹) and slow dissociation (4 × 10⁻⁵ s⁻¹) rates determined for mAb ADD056, resulting in a very strong binding with a K(D) ~ 3.6 pM at 20 °C. As follows from the X-ray data analysis, the methotrexate-antibody complex is stabilized by an extended network of hydrogen bonds and stacking interactions. The analysis also shows structural involvement of the CDR H3 in formation of the water channel revealing another important role of this hypervariable region. This suggests a new direction in natural affinity maturation and opens a new possibility in antibody engineering. Methotrexate is a widely used therapeutic agent for many malignant diseases and inflammatory disorders. Unfortunately, it may also interfere with central aspects of metabolism and thereby cause inevitable side effects. Therefore, methotrexate therapy requires careful monitoring of drug blood levels, which is traditionally done by immunoassays. An understanding of the structure-function properties of antibodies selected for drug monitoring substantiates the performance and robustness of such tests. PMID:24832237

  6. Improving Binding Affinity and Selectivity of Computationally Designed Ligand-Binding Proteins Using Experiments.

    PubMed

    Tinberg, Christine E; Khare, Sagar D

    2016-01-01

    The ability to de novo design proteins that can bind small molecules has wide implications for synthetic biology and medicine. Combining computational protein design with the high-throughput screening of mutagenic libraries of computationally designed proteins is emerging as a general approach for creating binding proteins with programmable binding modes, affinities, and selectivities. The computational step enables the creation of a binding site in a protein that otherwise does not (measurably) bind the intended ligand, and targeted mutagenic screening allows for validation and refinement of the computational model as well as provides orders-of-magnitude increases in the binding affinity. Deep sequencing of mutagenic libraries can provide insights into the mutagenic binding landscape and enable further affinity improvements. Moreover, in such a combined computational-experimental approach where the binding mode is preprogrammed and iteratively refined, selectivity can be achieved (and modulated) by the placement of specified amino acid side chain groups around the ligand in defined orientations. Here, we describe the experimental aspects of a combined computational-experimental approach for designing-using the software suite Rosetta-proteins that bind a small molecule of choice and engineering, using fluorescence-activated cell sorting and high-throughput yeast surface display, high affinity and ligand selectivity. We illustrated the utility of this approach by performing the design of a selective digoxigenin (DIG)-binding protein that, after affinity maturation, binds DIG with picomolar affinity and high selectivity over structurally related steroids. PMID:27094290

  7. An Unexpected Twist in Viral Capsid Maturation

    PubMed Central

    Gertsman, Ilya; Gan, Lu; Guttman, Miklos; Lee, Kelly; Speir, Jeffrey A.; Duda, Robert L.; Hendrix, Roger W.; Komives, Elizabeth A.; Johnson, John E.

    2009-01-01

    Lambda-like dsDNA bacteriophage undergo massive conformational changes in their capsid shell during the packaging of their viral genomes. Capsid shells are complex organizations of hundreds of protein subunits that assemble into intricate quaternary complexes that ultimately are able to withstand over 50 atm. of pressure during genome packaging1. The extensive integration between subunits in capsids is unlikely to form in a single assembly step, therefore requiring formation of an intermediate complex, termed a procapsid, from which individual subunits can undergo the necessary refolding and structural rearrangements needed to transition to the more stable capsid. Though various mature capsids have been characterized at atomic resolution, no such procapsid structure is available for a dsDNA virus or bacteriophage that undergoes large scale conformational changes. We present a procapsid x-ray structure at 3.65Å resolution, termed Prohead II, of the lambda like bacteriophage HK97, whose mature capsid structure was previously solved to 3.44 Å2. A comparison of the two largely different capsid forms has unveiled an unprecedented expansion mechanism that describes the transition. Crystallographic and Hydrogen/Deuterium exchange data presented here demonstrates that the subunit tertiary structures are significantly different between the two states, with twisting and bending motions occurring in both helical and β-sheet regions. We have also discovered conserved subunit interactions at each 3-fold of the virus capsid, from which capsid subunits maintain their integrity during refolding, facilitating the rotational and translational motions of maturation. Calormetric data of a closely related bacteriophage, P22, showed that capsid maturation was an exothermic process that resulted in a release of 90KJ/mol of energy3. We propose the major tertiary changes presented in this study reveal a structural basis for an exothermic maturation process likely present in many ds

  8. Maturation of Shark Single-Domain (IgNAR) Antibodies: Evidence for Induced-Fit Binding

    SciTech Connect

    Stanfield, R.L.; Dooley, H.; Verdino, P.; Flajnik, M.F.; Wilson, I.A.; /Scripps Res. Inst. /Maryland U.

    2007-07-13

    Sharks express an unusual heavy-chain isotype called IgNAR, whose variable regions bind antigen as independent soluble domains. To further probe affinity maturation of the IgNAR response, we structurally characterized the germline and somatically matured versions of a type II variable (V) region, both in the presence and absence of its antigen, hen egg-white lysozyme. Despite a disulfide bond linking complementarity determining regions (CDRs) 1 and 3, both germline and somatically matured V regions displayed significant structural changes in these CDRs upon complex formation with antigen. Somatic mutations in the IgNAR V region serve to increase the number of contacts with antigen, as reflected by a tenfold increase in affinity, and one of these mutations appears to stabilize the CDR3 region. In addition, a residue in the HV4 loop plays an important role in antibody-antigen interaction, consistent with the high rate of somatic mutations in this non-CDR loop.

  9. Immunoglobulin Gene Insertions and Deletions in the Affinity Maturation of HIV-1 Broadly Reactive Neutralizing Antibodies

    PubMed Central

    Kepler, Thomas B.; Liao, Hua-Xin; Alam, S. Munir; Bhaskarabhatla, Rekha; Zhang, Ruijun; Stewart, Shelley; Anasti, Kara; Kelsoe, Garnett; Parks, Robert; Lloyd, Krissey E.; Stolarchuk, Christina; Pritchett, Jamie; Solomon, Erika; Friberg, Emma; Morris, Lynn; Karim, Salim S. Abdool; Cohen, Myron S.; Walter, Emmanuel; Moody, M. Anthony; Wu, Xueling; Altae-Tran, Han R.; Georgiev, Ivelin S.; Kwong, Peter D.; Boyd, Scott D.; Fire, Andrew Z.; Mascola, John R.; Haynes, Barton F.

    2014-01-01

    Summary Induction of HIV-1 broad neutralizing antibodies (bnAbs) is a goal of HIV-1 vaccine development but has remained challenging partially due to unusual traits of bnAbs, including high somatic hypermutation (SHM) frequencies and in-frame insertions and deletions (indels). Here we examined the propensity and functional requirement for indels within HIV-1 bnAbs. High-throughput sequencing of the immunoglobulin (Ig) VHDJH genes in HIV-1 infected and uninfected individuals revealed that the indel frequency was elevated among HIV-1-infected subjects, with no unique properties attributable to bnAb-producing individuals. This increased indel occurrence depended only on the frequency of SHM point-mutations. Indel-encoded regions were generally proximal to antigen binding sites. Additionally, reconstruction of a HIV-1 CD4-binding site bnAb clonal lineage revealed that a large compound VHDJH indel was required for bnAb activity. Thus, vaccine development should focus on designing regimens targeted at sustained activation of bnAb lineages to achieve the required SHM and indel events. PMID:25211073

  10. Sperm Proteome Maturation in the Mouse Epididymis

    PubMed Central

    Skerget, Sheri; Rosenow, Matthew A.; Petritis, Konstantinos; Karr, Timothy L.

    2015-01-01

    In mammals, transit through the epididymis, which involves the acquisition, loss and modification of proteins, is required to confer motility and fertilization competency to sperm. The overall dynamics of maturation is poorly understood, and a systems level understanding of the complex maturation process will provide valuable new information about changes occurring during epididymal transport. We report the proteomes of sperm collected from the caput, corpus and cauda segments of the mouse epididymis, identifying 1536, 1720 and 1234 proteins respectively. This study identified 765 proteins that are present in sperm obtained from all three segments. We identified 1766 proteins that are potentially added (732) or removed (1034) from sperm during epididymal transit. Phenotypic analyses of the caput, corpus and cauda sperm proteomes identified 60 proteins that have known sperm phenotypes when mutated, or absent from sperm. Our analysis indicates that as much as one-third of proteins with known sperm phenotypes are added to sperm during epididymal transit. GO analyses revealed that cauda sperm are enriched for specific functions including sperm-egg recognition and motility, consistent with the observation that sperm acquire motility and fertilization competency during transit through the epididymis. In addition, GO analyses revealed that the immunity protein profile of sperm changes during sperm maturation. Finally, we identified components of the 26S proteasome, the immunoproteasome, and a proteasome activator in mature sperm. PMID:26556802

  11. Structure of classical affine and classical affine fractional W-algebras

    SciTech Connect

    Suh, Uhi Rinn

    2015-01-15

    We introduce a classical BRST complex (See Definition 3.2.) and show that one can construct a classical affine W-algebra via the complex. This definition clarifies that classical affine W-algebras can be considered as quasi-classical limits of quantum affine W-algebras. We also give a definition of a classical affine fractional W-algebra as a Poisson vertex algebra. As in the classical affine case, a classical affine fractional W-algebra has two compatible λ-brackets and is isomorphic to an algebra of differential polynomials as a differential algebra. When a classical affine fractional W-algebra is associated to a minimal nilpotent, we describe explicit forms of free generators and compute λ-brackets between them. Provided some assumptions on a classical affine fractional W-algebra, we find an infinite sequence of integrable systems related to the algebra, using the generalized Drinfel’d and Sokolov reduction.

  12. Predictive Capability Maturity Model (PCMM).

    SciTech Connect

    Swiler, Laura Painton; Knupp, Patrick Michael; Urbina, Angel

    2010-10-01

    Predictive Capability Maturity Model (PCMM) is a communication tool that must include a dicussion of the supporting evidence. PCMM is a tool for managing risk in the use of modeling and simulation. PCMM is in the service of organizing evidence to help tell the modeling and simulation (M&S) story. PCMM table describes what activities within each element are undertaken at each of the levels of maturity. Target levels of maturity can be established based on the intended application. The assessment is to inform what level has been achieved compared to the desired level, to help prioritize the VU activities & to allocate resources.

  13. Scaling analysis of affinity propagation.

    PubMed

    Furtlehner, Cyril; Sebag, Michèle; Zhang, Xiangliang

    2010-06-01

    We analyze and exploit some scaling properties of the affinity propagation (AP) clustering algorithm proposed by Frey and Dueck [Science 315, 972 (2007)]. Following a divide and conquer strategy we setup an exact renormalization-based approach to address the question of clustering consistency, in particular, how many cluster are present in a given data set. We first observe that the divide and conquer strategy, used on a large data set hierarchically reduces the complexity O(N2) to O(N((h+2)/(h+1))) , for a data set of size N and a depth h of the hierarchical strategy. For a data set embedded in a d -dimensional space, we show that this is obtained without notably damaging the precision except in dimension d=2 . In fact, for d larger than 2 the relative loss in precision scales such as N((2-d)/(h+1)d). Finally, under some conditions we observe that there is a value s* of the penalty coefficient, a free parameter used to fix the number of clusters, which separates a fragmentation phase (for ss*) of the underlying hidden cluster structure. At this precise point holds a self-similarity property which can be exploited by the hierarchical strategy to actually locate its position, as a result of an exact decimation procedure. From this observation, a strategy based on AP can be defined to find out how many clusters are present in a given data set. PMID:20866473

  14. Chemokines and the Signaling Modules Regulating Integrin Affinity

    PubMed Central

    Montresor, Alessio; Toffali, Lara; Constantin, Gabriela; Laudanna, Carlo

    2012-01-01

    Integrin-mediated adhesion is a general concept referring to a series of adhesive phenomena including tethering–rolling, affinity, valency, and binding stabilization altogether controlling cell avidity (adhesiveness) for the substrate. Arrest chemokines modulate each aspect of integrin activation, although integrin affinity regulation has been recognized as the prominent event in rapid leukocyte arrest induced by chemokines. A variety of inside-out and outside-in signaling mechanisms have been related to the process of integrin-mediated adhesion in different cellular models, but only few of them have been clearly contextualized to rapid integrin affinity modulation by arrest chemokines in primary leukocytes. Complex signaling processes triggered by arrest chemokines and controlling leukocyte integrin activation have been described for ras-related rap and for rho-related small GTPases. We summarize the role of rap and rho small GTPases in the regulation of rapid integrin affinity in primary leukocytes and provide a modular view of these pro-adhesive signaling events. A potential, albeit still speculative, mechanism of rho-mediated regulation of cytoskeletal proteins controlling the last step of integrin activation is also discussed. We also discuss data suggesting a functional integration between the rho- and rap-modules of integrin activation. Finally we examine the universality of signaling mechanisms regulating integrin triggering by arrest chemokines. PMID:22654882

  15. Peptides@mica: from affinity to adhesion mechanism.

    PubMed

    Gladytz, A; John, T; Gladytz, T; Hassert, R; Pagel, M; Risselada, H J; Naumov, S; Beck-Sickinger, A G; Abel, B

    2016-09-14

    Investigating the adsorption of peptides on inorganic surfaces, on the molecular level, is fundamental for medicinal and analytical applications. Peptides can be potent as linkers between surfaces and living cells in biochips or in implantation medicine. Here, we studied the adsorption process of the positively charged pentapeptide RTHRK, a recently identified binding sequence for surface oxidized silicon, and novel analogues thereof to negatively charged mica surfaces. Homogeneous formation of monolayers in the nano- and low micromolar peptide concentration range was observed. We propose an alternative and efficient method to both quantify binding affinity and follow adhesion behavior. This method makes use of the thermodynamic relationship between surface coverage, measured by atomic force microscopy (AFM), and the concomitant free energy of adhesion. A knowledge-based fit to the autocorrelation of the AFM images was used to correct for a biased surface coverage introduced by the finite lateral resolution of the AFM. Binding affinities and mechanisms were further explored by large scale molecular dynamics (MD) simulations. The combination of well validated MD simulations with topological data from AFM revealed a better understanding of peptide adsorption processes on the atomistic scale. We demonstrate that binding affinity is strongly determined by a peptide's ability to form salt bridges and hydrogen bonds with the surface lattice. Consequently, differences in hydrogen bond formation lead to substantial differences in binding affinity despite conservation of the peptide's overall charge. Further, MD simulations give access to relative changes in binding energy of peptide variations in comparison to a lead compound. PMID:27491508

  16. Methods for Improving Aptamer Binding Affinity.

    PubMed

    Hasegawa, Hijiri; Savory, Nasa; Abe, Koichi; Ikebukuro, Kazunori

    2016-01-01

    Aptamers are single stranded oligonucleotides that bind a wide range of biological targets. Although aptamers can be isolated from pools of random sequence oligonucleotides using affinity-based selection, aptamers with high affinities are not always obtained. Therefore, further refinement of aptamers is required to achieve desired binding affinities. The optimization of primary sequences and stabilization of aptamer conformations are the main approaches to refining the binding properties of aptamers. In particular, sequence optimization using combined in silico sequence recombinations and in vitro functional evaluations is effective for the improvement of binding affinities, however, the binding affinities of aptamers are limited by the low hydrophobicity of nucleic acids. Accordingly, introduction of hydrophobic moieties into aptamers expands the diversity of interactions between aptamers and targets. Moreover, construction of multivalent aptamers by connecting aptamers that recognize distinct epitopes is an attractive approach to substantial increases in binding affinity. In addition, binding affinities can be tuned by optimizing the scaffolds of multivalent constructs. In this review, we summarize the various techniques for improving the binding affinities of aptamers. PMID:27043498

  17. Affine root systems and dual numbers

    NASA Astrophysics Data System (ADS)

    Kostyakov, I. V.; Gromov, N. A.; Kuratov, V. V.

    The root systems in Carroll spaces with degenerate metric are defined. It is shown that their Cartan matrices and reflection groups are affine. Due to the geometric consideration the root system structure of affine algebras is determined by a sufficiently simple algorithm.

  18. Loop realizations of quantum affine algebras

    SciTech Connect

    Cautis, Sabin; Licata, Anthony

    2012-12-15

    We give a simplified description of quantum affine algebras in their loop presentation. This description is related to Drinfeld's new realization via halves of vertex operators. We also define an idempotent version of the quantum affine algebra which is suitable for categorification.

  19. The AGU Data Management Maturity Model Initiative

    NASA Astrophysics Data System (ADS)

    Bates, J. J.

    2015-12-01

    In September 2014, the AGU Board of Directors approved two initiatives to help the Earth and space sciences community address the growing challenges accompanying the increasing size and complexity of data. These initiatives are: 1) Data Science Credentialing: development of a continuing education and professional certification program to help scientists in their careers and to meet growing responsibilities and requirements around data science; and 2) Data Management Maturity (DMM) Model: development and implementation of a data management maturity model to assess process maturity against best practices, and to identify opportunities in organizational data management processes. Each of these has been organized within AGU as an Editorial Board and both Boards have held kick off meetings. The DMM model Editorial Board will recommend strategies for adapting and deploying a DMM model to the Earth and space sciences create guidance documents to assist in its implementation, and provide input on a pilot appraisal process. This presentation will provide an overview of progress to date in the DMM model Editorial Board and plans for work to be done over the upcoming year.

  20. Improving image segmentation by learning region affinities

    SciTech Connect

    Prasad, Lakshman; Yang, Xingwei; Latecki, Longin J

    2010-11-03

    We utilize the context information of other regions in hierarchical image segmentation to learn new regions affinities. It is well known that a single choice of quantization of an image space is highly unlikely to be a common optimal quantization level for all categories. Each level of quantization has its own benefits. Therefore, we utilize the hierarchical information among different quantizations as well as spatial proximity of their regions. The proposed affinity learning takes into account higher order relations among image regions, both local and long range relations, making it robust to instabilities and errors of the original, pairwise region affinities. Once the learnt affinities are obtained, we use a standard image segmentation algorithm to get the final segmentation. Moreover, the learnt affinities can be naturally unutilized in interactive segmentation. Experimental results on Berkeley Segmentation Dataset and MSRC Object Recognition Dataset are comparable and in some aspects better than the state-of-art methods.

  1. A Drosophila model to image phagosome maturation.

    PubMed

    Shandala, Tetyana; Lim, Chiaoxin; Sorvina, Alexandra; Brooks, Douglas A

    2013-01-01

    Phagocytosis involves the internalization of extracellular material by invagination of the plasma membrane to form intracellular vesicles called phagosomes, which have functions that include pathogen degradation. The degradative properties of phagosomes are thought to be conferred by sequential fusion with endosomes and lysosomes; however, this maturation process has not been studied in vivo. We employed Drosophila hemocytes, which are similar to mammalian professional macrophages, to establish a model of phagosome maturation. Adult Drosophila females, carrying transgenic Rab7-GFP endosome and Lamp1-GFP lysosome markers, were injected with E. coli DH5α and the hemocytes were collected at 15, 30, 45 and 60 minutes after infection. In wild-type females, E. coli were detected within enlarged Rab7-GFP positive phagosomes at 15 to 45 minutes after infection; and were also observed in enlarged Lamp1-GFP positive phagolysosomes at 45 minutes. Two-photon imaging of hemocytes in vivo confirmed this vesicle morphology, including enlargement of Rab7-GFP and Lamp1-GFP structures that often appeared to protrude from hemocytes. The interaction of endosomes and lysosomes with E. coli phagosomes observed in Drosophila hemocytes was consistent with that previously described for phagosome maturation in human ex vivo macrophages. We also tested our model as a tool for genetic analysis using 14-3-3e mutants, and demonstrated altered phagosome maturation with delayed E. coli internalization, trafficking and/or degradation. These findings demonstrate that Drosophila hemocytes provide an appropriate, genetically amenable, model for analyzing phagosome maturation ex vivo and in vivo. PMID:24709696

  2. Cerebellar mature teratoma in adulthood.

    PubMed

    Zavanone, M; Alimehmeti, R; Campanella, R; Ram-Pini, P; Locatelli, M; Egidi, M; Righini, A; Bauer, D

    2002-03-01

    Mature teratoma of the posterior cranial fossa in adults is extremely rare. We report a particularly rare case of medio-lateral cerebellar mature teratoma that became symptomatic in a middle-aged man. The CT revealed the lesion of heterogeneous density with calcifications in the solid medial portion. Only the MRI could reliably define the borders of the cystic component extending into the left cerebellar lobe. Histologically the presence of fully matured representative tissues of the 3 germ layers ensured the diagnosis of mature teratoma. We suggest that the cyst formation from progressive latent hemorrhage and/or secretion from the gland cells of the tumor, may be responsible for the clinical decompensation even in adulthood. PMID:12118223

  3. Mechanical Properties of Murine Leukemia Virus Particles: Effect of Maturation

    PubMed Central

    Kol, Nitzan; Gladnikoff, Micha; Barlam, David; Shneck, Roni Z.; Rein, Alan; Rousso, Itay

    2006-01-01

    After budding from the host cell, retroviruses undergo a process of internal reorganization called maturation, which is prerequisite to infectivity. Viral maturation is accompanied by dramatic morphological changes, which are poorly understood in physical/mechanistic terms. Here, we study the mechanical properties of live mature and immature murine leukemia virus particles by indentation-type experiments conducted with an atomic force microscope tip. We find that both mature and immature particles have an elastic shell. Strikingly, the virus shell is twofold stiffer in the immature (0.68 N/m) than the mature (0.31 N/m) form. However, finite-element simulation shows that the average Young's modulus of the immature form is more than fourfold lower than that of the mature form. This finding suggests that per length unit, the protein-protein interactions in the mature shell are stronger than those in the immature shell. We also show that the mature virus shell is brittle, since it can be broken by application of large loading forces, by firm attachment to a substrate, or by repeated application of force. Our results are the first analysis of the mechanical properties of an animal virus, and demonstrate a linkage between virus morphology and mechanical properties. PMID:16632508

  4. Kinetics of mature T-cell development in the thymus

    SciTech Connect

    Egerton, M.; Scollay, R.; Shortman, K. )

    1990-04-01

    We have reexamined the balance between cell birth, cell maturation, and cell death in the thymus by labeling dividing thymocytes and their progeny in vivo with (3H)-thymidine, isolating clearly defined subpopulations by fluorescence-activated cell sorting, and determining the distribution of label by autoradiography. When mature thymocytes were precisely defined (as CD4+CD8- CD3+ or CD4-CD8+ CD3+) and separated from immature single positives (CD4+CD8- CD3- and CD4-CD8+ CD3-), a lag was observed in the rate of entry of (3H)thymidine into mature cells. Thus, many of the mature thymocytes appear to derive from a small nondividing cortical thymocyte pool, rather than originating directly from the earliest dividing CD4+CD8+ blasts. There was little evidence for cell division during or after mature thymocyte formation, suggesting a one-for-one differentiation from cortical cells rather than selective clonal expansion. The rate of production of mature single positive thymocytes agreed closely with estimates of the rate of export of mature T cells from the thymus and was only 3% of the rate of production of double-positive cortical thymocytes. This was compatible with a stringent selection process and extensive intrathymic cell death and suggested that no extensive negative selection occurred after the mature cells were formed.

  5. 7 CFR 51.3746 - Mature.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Standards for Grades of Honey Dew and Honey Ball Type Melons Definitions § 51.3746 Mature. Mature means that the melon has reached the stage of maturity which will insure the proper completion of the...

  6. 7 CFR 51.3746 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Honey Dew and Honey Ball Type Melons Definitions § 51.3746 Mature. Mature means that the melon has reached the stage of maturity which will...

  7. 7 CFR 51.3746 - Mature.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Standards for Grades of Honey Dew and Honey Ball Type Melons Definitions § 51.3746 Mature. Mature means that the melon has reached the stage of maturity which will insure the proper completion of the...

  8. 7 CFR 51.3746 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Honey Dew and Honey Ball Type Melons Definitions § 51.3746 Mature. Mature means that the melon has reached the stage of maturity which will...

  9. 7 CFR 51.3746 - Mature.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Standards for Grades of Honey Dew and Honey Ball Type Melons Definitions § 51.3746 Mature. Mature means that the melon has reached the stage of maturity which will insure the proper completion of the...

  10. Identification of Candidate Angiogenic Inhibitors Processed by Matrix Metalloproteinase 2 (MMP-2) in Cell-Based Proteomic Screens: Disruption of Vascular Endothelial Growth Factor (VEGF)/Heparin Affin Regulatory Peptide (Pleiotrophin) and VEGF/Connective Tissue Growth Factor Angiogenic Inhibitory Complexes by MMP-2 Proteolysis▿ †

    PubMed Central

    Dean, Richard A.; Butler, Georgina S.; Hamma-Kourbali, Yamina; Delbé, Jean; Brigstock, David R.; Courty, José; Overall, Christopher M.

    2007-01-01

    Matrix metalloproteinases (MMPs) exert both pro- and antiangiogenic functions by the release of cytokines or proteolytically generated angiogenic inhibitors from extracellular matrix and basement membrane remodeling. In the Mmp2−/− mouse neovascularization is greatly reduced, but the mechanistic aspects of this remain unclear. Using isotope-coded affinity tag labeling of proteins analyzed by multidimensional liquid chromatography and tandem mass spectrometry we explored proteome differences between Mmp2−/− cells and those rescued by MMP-2 transfection. Proteome signatures that are hallmarks of proteolysis revealed cleavage of many known MMP-2 substrates in the cellular context. Proteomic evidence of MMP-2 processing of novel substrates was found. Insulin-like growth factor binding protein 6, follistatin-like 1, and cystatin C protein cleavage by MMP-2 was biochemically confirmed, and the cleavage sites in heparin affin regulatory peptide (HARP; pleiotrophin) and connective tissue growth factor (CTGF) were sequenced by matrix-assisted laser desorption ionization-time of flight mass spectrometry. MMP-2 processing of HARP and CTGF released vascular endothelial growth factor (VEGF) from angiogenic inhibitory complexes. The cleaved HARP N-terminal domain increased HARP-induced cell proliferation, whereas the HARP C-terminal domain was antagonistic and decreased cell proliferation and migration. Hence the unmasking of cytokines, such as VEGF, by metalloproteinase processing of their binding proteins is a new mechanism in the control of cytokine activation and angiogenesis. PMID:17908800

  11. Identification of small molecule compounds with higher binding affinity to guanine deaminase (cypin) than guanine.

    PubMed

    Fernández, José R; Sweet, Eric S; Welsh, William J; Firestein, Bonnie L

    2010-09-15

    Guanine deaminase (GDA; cypin) is an important metalloenzyme that processes the first step in purine catabolism, converting guanine to xanthine by hydrolytic deamination. In higher eukaryotes, GDA also plays an important role in the development of neuronal morphology by regulating dendritic arborization. In addition to its role in the maturing brain, GDA is thought to be involved in proper liver function since increased levels of GDA activity have been correlated with liver disease and transplant rejection. Although mammalian GDA is an attractive and potential drug target for treatment of both liver diseases and cognitive disorders, prospective novel inhibitors and/or activators of this enzyme have not been actively pursued. In this study, we employed the combination of protein structure analysis and experimental kinetic studies to seek novel potential ligands for human guanine deaminase. Using virtual screening and biochemical analysis, we identified common small molecule compounds that demonstrate a higher binding affinity to GDA than does guanine. In vitro analysis demonstrates that these compounds inhibit guanine deamination, and more surprisingly, affect GDA (cypin)-mediated microtubule assembly. The results in this study provide evidence that an in silico drug discovery strategy coupled with in vitro validation assays can be successfully implemented to discover compounds that may possess therapeutic value for the treatment of diseases and disorders where GDA activity is abnormal. PMID:20716488

  12. Comparison of nir methods for measuring cotton fiber maturity and fineness

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maturity and fineness are important cotton fiber properties, as they can impact the fiber’s downstream processability and dye performance. Maturity and fineness are often indicated indirectly by the fiber’s micronaire. Maturity and fineness can be measured directly, but most available methods are ...

  13. Epigenetic mechanisms in pubertal brain maturation

    PubMed Central

    Morrison, Kathleen E.; Rodgers, Ali B.; Morgan, Christopher P.; Bale, Tracy L.

    2014-01-01

    Puberty is a critical period of development during which the reemergence of gonadotropin releasing hormone secretion from the hypothalamus triggers a cascade of hormone-dependent processes. Maturation of specific brain regions including the prefrontal cortex occurs during this window, but the complex mechanisms underlying these dynamic changes are not well understood. Particularly, the potential involvement of epigenetics in this programming has been under-examined. The epigenome is known to guide earlier stages of development, and it is similarly poised to regulate vital pubertal-driven brain maturation. Further, as epigenetic machinery is highly environmentally responsive, its involvement may also lend this period of growth to greater vulnerability to external insults, resulting in reprogramming and increased disease risk. Importantly, neuropsychiatric diseases commonly present in individuals during or immediately following puberty, and environmental perturbations including stress may precipitate disease onset by disrupting the normal trajectory of pubertal brain development via epigenetic mechanisms. In this review, we discuss epigenetic processes involved in pubertal brain maturation, the potential points of derailment, and the importance of future studies for understanding this dynamic developmental window and gaining a better understanding of neuropsychiatric disease risk. PMID:24239720

  14. A molecular determinant of phosphoinositide affinity in mammalian TRPV channels

    PubMed Central

    Velisetty, Phanindra; Borbiro, Istvan; Kasimova, Marina A.; Liu, Luyu; Badheka, Doreen; Carnevale, Vincenzo; Rohacs, Tibor

    2016-01-01

    Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is an important cofactor for ion channels. Affinity for this lipid is a major determinant of channel inhibition by depletion of PI(4,5)P2 upon phospholipase C (PLC) activation. Little is known about what determines PI(4,5)P2 affinity in mammalian ion channels. Here we report that two members of the Transient Receptor Potential Vanilloid (TRPV) ion channel family, TRPV5 and TRPV6 lack a positively charged residue in the TM4-TM5 loop that was shown to interact with PI(4,5)P2 in TRPV1, which shows high affinity for this lipid. When this positively charged residue was introduced to either TRPV6 or TRPV5, they displayed markedly higher affinities for PI(4,5)P2, and were largely resistant to inhibition by PI(4,5)P2 depletion. Furthermore, Ca2+-induced inactivation of TRPV6 was essentially eliminated in the G488R mutant, showing the importance of PLC-mediated PI(4,5)P2 depletion in this process. Computational modeling shows that the introduced positive charge interacts with PI(4,5)P2 in TRPV6. PMID:27291418

  15. Affinity and Avidity in Antibody-Based Tumor Targeting

    PubMed Central

    Rudnick, Stephen I.

    2009-01-01

    Summation Many factors contribute to successful tumor targeting by antibodies. Besides properties of the tumor tissue and general antibody pharmacology, a relationship exists between an antibody and its antigen that can shape penetration, catabolism, specificity, and efficacy. The affinity and avidity of the binding interactions play critical roles in these dynamics. In this work, we review the principles that guide models predicting tumor penetration and cellular internalization while providing a critical overview of studies aimed at experimentally determining the specific role of affinity and avidity in these processes. One should gain the perspective that binding affinity can, in part, dictate the localization of antibodies in tumors, leading to high concentrations in the perivascular space or low concentrations diffused throughout the tumor. These patterns can be simply due to the diminution of available dose by binding antigen and are complicated by internalization and degradation stemming from slow rates of dissociation. As opposed to the trend of simply increasing affinity to increase efficacy, novel strategies that increase avidity and broaden specificity have made significant progress in tumor targeting. PMID:19409036

  16. A molecular determinant of phosphoinositide affinity in mammalian TRPV channels.

    PubMed

    Velisetty, Phanindra; Borbiro, Istvan; Kasimova, Marina A; Liu, Luyu; Badheka, Doreen; Carnevale, Vincenzo; Rohacs, Tibor

    2016-01-01

    Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is an important cofactor for ion channels. Affinity for this lipid is a major determinant of channel inhibition by depletion of PI(4,5)P2 upon phospholipase C (PLC) activation. Little is known about what determines PI(4,5)P2 affinity in mammalian ion channels. Here we report that two members of the Transient Receptor Potential Vanilloid (TRPV) ion channel family, TRPV5 and TRPV6 lack a positively charged residue in the TM4-TM5 loop that was shown to interact with PI(4,5)P2 in TRPV1, which shows high affinity for this lipid. When this positively charged residue was introduced to either TRPV6 or TRPV5, they displayed markedly higher affinities for PI(4,5)P2, and were largely resistant to inhibition by PI(4,5)P2 depletion. Furthermore, Ca(2+)-induced inactivation of TRPV6 was essentially eliminated in the G488R mutant, showing the importance of PLC-mediated PI(4,5)P2 depletion in this process. Computational modeling shows that the introduced positive charge interacts with PI(4,5)P2 in TRPV6. PMID:27291418

  17. Kinetic Studies of Biological Interactions By Affinity Chromatography

    PubMed Central

    Schiel, John E.; Hage, David S.

    2009-01-01

    The rates at which biological interactions occur can provide important information on the mechanism and behavior of such processes in living systems. This review will discuss how affinity chromatography can be used as a tool to examine the kinetics of biological interactions. This approach, referred to here as biointeraction chromatography, uses a column with an immobilized binding agent to examine the association or dissociation of this agent with other compounds. The use of HPLC-based affinity columns in kinetic studies has received particular attention in recent years. Advantages of using HPLC with affinity chromatography for this purpose include the ability to reuse the same ligand within a column for a large number of experiments, and the good precision and accuracy of this approach. A number of techniques are available for kinetic studies through the use of affinity columns and biointeraction chromatography. These approaches include plate height measurements, peak profiling, peak fitting, split-peak measurements, and peak decay analysis. The general principles for each of these methods are discussed in this review and some recent applications of these techniques are presented. The advantages and potential limitations of each approach are also considered. PMID:19391173

  18. Oocyte Maturation and Development

    PubMed Central

    Verlhac, Marie-Hélène; Terret, Marie-Emilie

    2016-01-01

    Sexual reproduction is essential for many organisms to propagate themselves. It requires the formation of haploid female and male gametes: oocytes and sperms. These specialized cells are generated through meiosis, a particular type of cell division that produces cells with recombined genomes that differ from their parental origin. In this review, we highlight the end process of female meiosis, the divisions per se, and how they can give rise to a functional female gamete preparing itself for the ensuing zygotic development. In particular, we discuss why such an essential process in the propagation of species is so poorly controlled, producing a strong percentage of abnormal female gametes in the end. Eventually, we examine aspects related to the lack of centrosomes in female oocytes, the asymmetry in size of the mammalian oocyte upon division, and in mammals the direct consequences of these long-lived cells in the ovary. PMID:26998245

  19. Affinity Proteomics in the mountains: Alpbach 2015.

    PubMed

    Taussig, Michael J

    2016-09-25

    The 2015 Alpbach Workshop on Affinity Proteomics, organised by the EU AFFINOMICS consortium, was the 7th workshop in this series. As in previous years, the focus of the event was the current state of affinity methods for proteome analysis, including complementarity with mass spectrometry, progress in recombinant binder production methods, alternatives to classical antibodies as affinity reagents, analysis of proteome targets, industry focus on biomarkers, and diagnostic and clinical applications. The combination of excellent science with Austrian mountain scenery and winter sports engender an atmosphere that makes this series of workshops exceptional. The articles in this Special Issue represent a cross-section of the presentations at the 2015 meeting. PMID:27118167

  20. Optimized Affinity Capture of Yeast Protein Complexes.

    PubMed

    LaCava, John; Fernandez-Martinez, Javier; Hakhverdyan, Zhanna; Rout, Michael P

    2016-01-01

    Here, we describe an affinity isolation protocol. It uses cryomilled yeast cell powder for producing cell extracts and antibody-conjugated paramagnetic beads for affinity capture. Guidelines for determining the optimal extraction solvent composition are provided. Captured proteins are eluted in a denaturing solvent (sodium dodecyl sulfate polyacrylamide gel electrophoresis sample buffer) for gel-based proteomic analyses. Although the procedures can be modified to use other sources of cell extract and other forms of affinity media, to date we have consistently obtained the best results with the method presented. PMID:27371596

  1. Aptamers in Affinity Separations: Stationary Separation

    NASA Astrophysics Data System (ADS)

    Ravelet, Corinne; Peyrin, Eric

    The use of DNA or RNA aptamers as tools in analytical chemistry is a very promising field of research because of their capabilities to bind specifically the target molecules with an affinity similar to that of antibodies. Notably, they appear to be of great interest as target-specific ligands for the separation and capture of various analytes in affinity chromatography and related affinity-based methods such as magnetic bead technology. In this chapter, the recent developments of these aptamer-based separation/capture approaches are addressed.

  2. Affinity purification of heme-tagged proteins.

    PubMed

    Asher, Wesley B; Bren, Kara L

    2014-01-01

    Protein affinity purification techniques are widely used for isolating pure target proteins for biochemical and structural characterization. Herein, we describe the protocol for affinity-based purification of proteins expressed in Escherichia coli that uses the coordination of a peptide tag covalently modified with heme c, known as a heme-tag, to an L-histidine immobilized Sepharose resin. This approach provides an affinity purification tag visible to the eye, facilitating tracking of the protein. In addition, we describe methods for specifically detecting heme-tagged proteins in SDS-PAGE gels using a heme-staining procedure and for quantifying the proteins using a pyridine hemochrome assay. PMID:24943311

  3. Multi-parametric evaluation of the white matter maturation.

    PubMed

    Kulikova, S; Hertz-Pannier, L; Dehaene-Lambertz, G; Buzmakov, A; Poupon, C; Dubois, J

    2015-11-01

    In vivo evaluation of the brain white matter maturation is still a challenging task with no existing gold standards. In this article we propose an original approach to evaluate the early maturation of the white matter bundles, which is based on comparison of infant and adult groups using the Mahalanobis distance computed from four complementary MRI parameters: quantitative qT1 and qT2 relaxation times, longitudinal λ║ and transverse λ⊥ diffusivities from diffusion tensor imaging. Such multi-parametric approach is expected to better describe maturational asynchrony than conventional univariate approaches because it takes into account complementary dependencies of the parameters on different maturational processes, notably the decrease in water content and the myelination. Our approach was tested on 17 healthy infants (aged 3- to 21-week old) for 18 different bundles. It finely confirmed maturational asynchrony across the bundles: the spino-thalamic tract, the optic radiations, the cortico-spinal tract and the fornix have the most advanced maturation, while the superior longitudinal and arcuate fasciculi, the anterior limb of the internal capsule and the external capsule have the most delayed maturation. Furthermore, this approach was more reliable than univariate approaches as it revealed more maturational relationships between the bundles and did not violate a priori assumptions on the temporal order of the bundle maturation. Mahalanobis distances decreased exponentially with age in all bundles, with the only difference between them explained by different onsets of maturation. Estimation of these relative delays confirmed that the most dramatic changes occur during the first post-natal year. PMID:25183543

  4. Affinity improvement by fine tuning of single-chain variable fragment against aflatoxin B1.

    PubMed

    Min, Won-Ki; Na, Kang-In; Yoon, Jung-Hyun; Heo, Yoon-Jee; Lee, Daesang; Kim, Sung-Gun; Seo, Jin-Ho

    2016-10-15

    Aflatoxin B1 (AFB1) produced in Aspergillus flavus is a major hepatocarcinogen found in foods and feed. For effective immunological detection of AFB1 at low concentrations, the development of high affinity antibody for AFB1 is required. Previously, an affinity-maturated single-chain variable fragment containing 6 mutations (scFv-M37) was isolated from an artificial mutagenic library, which showed a 9-fold higher affinity than its wild type scFv. In this study, the effect of the 6 mutated residues on the affinity improvement was characterized using surface plasmon resonance analysis, which identified a deleterious mutation (VH-A110T) located on a framework region of the scFv-M37. The back mutation of VH-A110T resulted in a 3.2-fold affinity improvement, which was attributed to decrease of dissociation rate constant (kd) in interaction between AFB1 and the back mutant scFv. The biophysical analyses using circular dichroism and gel filtration revealed that the back mutation of VH-A110T caused a subtle conformational change of the scFv toward tighter binding to AFB1. PMID:27173568

  5. Selection and maturation of antibodies by phage display through fusion to pIX.

    PubMed

    Tornetta, Mark; Reddy, Ramachandra; Wheeler, John C

    2012-09-01

    Antibody discovery and optimization by M13 phage display have evolved significantly over the past twenty years. Multiple methods of antibody display and selection have been developed - direct display on pIII or indirect display through a Cysteine disulfide linkage or a coiled-coil adapter protein. Here we describe display of Fab libraries on the smaller pIX protein at the opposite end of the virion and its application to discovery of novel antibodies from naive libraries. Antibody selection based on pIX-mediated display produces results comparable to other in vitro methods and uses an efficient direct infection of antigen-bound phages, eliminating any chemical dissociation step(s). Additionally, some evidence suggests that pIX-mediated display can be more efficient than pIII-mediated display in affinity selections. Functional assessment of phage-derived antibodies can be hindered by insufficient affinities or lack of epitopic diversity. Here we describe an approach to managing primary hits from our Fab phage libraries into epitope bins and subsequent high-throughput maturation of clones to isolate epitope- and sequence-diverse panels of high affinity binders. Use of the Octet biosensor was done to examine Fab binding in a facile label-free method and determine epitope competition groups. A receptor extracellular domain and chemokine were subjected to this method of binning and affinity maturation. Parental clones demonstrated improvement in affinity from 1-100nM to 10-500pM. PMID:22841960

  6. Peloid Mud: a multi-step maturation analysis

    NASA Astrophysics Data System (ADS)

    Redolfi, M.

    2013-12-01

    The aim of this work is understanding the process involved in the maturation of artificial peloid mud commonly use in thermal spa. I prepare a standard protocol for analysis: XRD, chemical, heat capacity and heavy metal sequential extraction . I also prepare 12 artificial peloid mud following the procedure describe in Veniale et al. (2004) mixing natural thermal waters form the Lazio region with a common clay also collected in Lazio Region with a ration of 1:1 in weight and put this mud in a sealed box at 40 °C for all the maturation process without remixing. Each peloid mud was sampled at one, three and six month of maturation, dried at 60 °C degree and milled for analysis. Each mud was compared with the result at one, three and six month to identify the major different of parameters at different time of maturation.

  7. Transcriptional Programs Controlling Perinatal Lung Maturation

    PubMed Central

    Xu, Yan; Wang, Yanhua; Besnard, Valérie; Ikegami, Machiko; Wert, Susan E.; Heffner, Caleb; Murray, Stephen A.; Donahue, Leah Rae; Whitsett, Jeffrey A.

    2012-01-01

    The timing of lung maturation is controlled precisely by complex genetic and cellular programs. Lung immaturity following preterm birth frequently results in Respiratory Distress Syndrome (RDS) and Broncho-Pulmonary Dysplasia (BPD), which are leading causes of mortality and morbidity in preterm infants. Mechanisms synchronizing gestational length and lung maturation remain to be elucidated. In this study, we designed a genome-wide mRNA expression time-course study from E15.5 to Postnatal Day 0 (PN0) using lung RNAs from C57BL/6J (B6) and A/J mice that differ in gestational length by ∼30 hr (B6maturation. We identified both temporal and strain dependent gene expression patterns during lung maturation. For time dependent changes, cell adhesion, vasculature development, and lipid metabolism/transport were major bioprocesses induced during the saccular stage of lung development at E16.5–E17.5. CEBPA, PPARG, VEGFA, CAV1 and CDH1 were found to be key signaling and transcriptional regulators of these processes. Innate defense/immune responses were induced at later gestational ages (E18.5–20.5), STAT1, AP1, and EGFR being important regulators of these responses. Expression of RNAs associated with the cell cycle and chromatin assembly was repressed during prenatal lung maturation and was regulated by FOXM1, PLK1, chromobox, and high mobility group families of transcription factors. Strain dependent lung mRNA expression differences peaked at E18.5. At this time, mRNAs regulating surfactant and innate immunity were more abundantly expressed in lungs of B6 (short gestation) than in A/J (long gestation) mice, while expression of genes involved in chromatin assembly and histone modification were expressed at lower levels in B6 than in A/J mice. The present study systemically mapped key regulators, bioprocesses

  8. Development of the affinity materials for phosphorylated proteins/peptides enrichment in phosphoproteomics analysis.

    PubMed

    Wang, Zhi-Gang; Lv, Nan; Bi, Wen-Zhi; Zhang, Ji-Lin; Ni, Jia-Zuan

    2015-04-29

    Reversible protein phosphorylation is a key event in numerous biological processes. Mass spectrometry (MS) is the most powerful analysis tool in modern phosphoproteomics. However, the direct MS analysis of phosphorylated proteins/peptides is still a big challenge because of the low abundance and insufficient ionization of phosphorylated proteins/peptides as well as the suppression effects of nontargets. Enrichment of phosphorylated proteins/peptides by affinity materials from complex biosamples is the most widely used strategy to enhance the MS detection. The demand of efficiently enriching phosphorylated proteins/peptides has spawned diverse affinity materials based on different enrichment principles (e.g., electronic attraction, chelating). In this review, we summarize the recent development of various affinity materials for phosphorylated proteins/peptides enrichment. We will highlight the design and fabrication of these affinity materials, discuss the enrichment mechanisms involved in different affinity materials, and suggest the future challenges and research directions in this field. PMID:25845677

  9. PRINCIPLES OF AFFINITY-BASED BIOSENSORS

    EPA Science Inventory

    Despite the amount of resources that have been invested by national and international academic, government, and commercial sectors to develop affinity-based biosensor products, little obvious success has been realized through commercialization of these devices for specific applic...

  10. Minimal information to determine affine shape equivalence.

    PubMed

    Wagemans, J; Van Gool, L; Lamote, C; Foster, D H

    2000-04-01

    Participants judged the affine equivalence of 2 simultaneously presented 4-point patterns. Performance level (d') varied between 1.5 and 2.7, depending on the information available for solving the correspondence problem (insufficient in Experiment 1a, superfluous in Experiment 1b, and minimal in Experiments 1c, 2a, 2b) and on the exposure time (unlimited in Experiments 1 and 2a and 500 ms in Experiment 2b), but it did not vary much with the complexity of the affine transformation (rotation and slant in Experiment 1 and same plus tilt in Experiment 2). Performance in Experiment 3 was lower with 3-point patterns than with 4-point patterns, whereas blocking the trials according to the affine transformation parameters had little effect. Determining affine shape equivalence with minimal-information displays is based on a fast assessment of qualitatively or quasi-invariant properties such as convexity/ concavity, parallelism, and collinearity. PMID:10811156

  11. Protein purification using PDZ affinity chromatography.

    PubMed

    Walkup, Ward G; Kennedy, Mary B

    2015-01-01

    PDZ domains function in nature as protein-binding domains within scaffold and membrane-associated proteins. They comprise approximately 90 residues and undergo specific, high-affinity interactions with complementary C-terminal peptide sequences, other PDZ domains, and/or phospholipids. We have previously shown that the specific, strong interactions of PDZ domains with their ligands make them well suited for use in affinity chromatography. This unit provides protocols for the PDZ affinity chromatography procedure that are applicable for the purification of proteins that contain PDZ domains or PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We detail the preparation of affinity resins composed of PDZ domains or PDZ domain peptide ligands coupled to solid supports. These resins can be used to purify proteins containing endogenous or genetically introduced PDZ domains or ligands, eluting the proteins with free PDZ domain peptide ligands. PMID:25829303

  12. Rational development of high-affinity T-cell receptor-like antibodies.

    PubMed

    Stewart-Jones, Guillaume; Wadle, Andreas; Hombach, Anja; Shenderov, Eugene; Held, Gerhard; Fischer, Eliane; Kleber, Sascha; Nuber, Natko; Stenner-Liewen, Frank; Bauer, Stefan; McMichael, Andrew; Knuth, Alexander; Abken, Hinrich; Hombach, Andreas A; Cerundolo, Vincenzo; Jones, E Yvonne; Renner, Christoph

    2009-04-01

    T-cell interaction with a target cell is a key event in the adaptive immune response and primarily driven by T-cell receptor (TCR) recognition of peptide-MHC (pMHC) complexes. TCR avidity for a given pMHC is determined by number of MHC molecules, availability of coreceptors, and TCR affinity for MHC or peptide, respectively, with peptide recognition being the most important factor to confer target specificity. Here we present high-resolution crystal structures of 2 Fab antibodies in complex with the immunodominant NY-ESO-1(157-165) peptide analogue (SLLMWITQV) presented by HLA-A*0201 and compare them with a TCR recognizing the same pMHC. Binding to the central methionine-tryptophan peptide motif and orientation of binding were almost identical for Fabs and TCR. As the MW "peg" dominates the contacts between Fab and peptide, we estimated the contributions of individual amino acids between the Fab and peptide to provide the rational basis for a peptide-focused second-generation, high-affinity antibody library. The final Fab candidate achieved better peptide binding by 2 light-chain mutations, giving a 20-fold affinity improvement to 2-4 nM, exceeding the affinity of the TCR by 1,000-fold. The high-affinity Fab when grafted as recombinant TCR on T cells conferred specific killing of HLA-A*0201/NY-ESO-1(157-165) target cells. In summary, we prove that affinity maturation of antibodies mimicking a TCR is possible and provide a strategy for engineering high-affinity antibodies that can be used in targeting specific pMHC complexes for diagnostic and therapeutic purposes. PMID:19307587

  13. Rational development of high-affinity T-cell receptor-like antibodies

    PubMed Central

    Stewart-Jones, Guillaume; Wadle, Andreas; Hombach, Anja; Shenderov, Eugene; Held, Gerhard; Fischer, Eliane; Kleber, Sascha; Nuber, Natko; Stenner-Liewen, Frank; Bauer, Stefan; McMichael, Andrew; Knuth, Alexander; Abken, Hinrich; Hombach, Andreas A.; Cerundolo, Vincenzo; Jones, E. Yvonne; Renner, Christoph

    2009-01-01

    T-cell interaction with a target cell is a key event in the adaptive immune response and primarily driven by T-cell receptor (TCR) recognition of peptide-MHC (pMHC) complexes. TCR avidity for a given pMHC is determined by number of MHC molecules, availability of coreceptors, and TCR affinity for MHC or peptide, respectively, with peptide recognition being the most important factor to confer target specificity. Here we present high-resolution crystal structures of 2 Fab antibodies in complex with the immunodominant NY-ESO-1157–165 peptide analogue (SLLMWITQV) presented by HLA-A*0201 and compare them with a TCR recognizing the same pMHC. Binding to the central methionine-tryptophan peptide motif and orientation of binding were almost identical for Fabs and TCR. As the MW “peg” dominates the contacts between Fab and peptide, we estimated the contributions of individual amino acids between the Fab and peptide to provide the rational basis for a peptide-focused second-generation, high-affinity antibody library. The final Fab candidate achieved better peptide binding by 2 light-chain mutations, giving a 20-fold affinity improvement to 2–4 nM, exceeding the affinity of the TCR by 1,000-fold. The high-affinity Fab when grafted as recombinant TCR on T cells conferred specific killing of HLA-A*0201/NY-ESO-1157–165 target cells. In summary, we prove that affinity maturation of antibodies mimicking a TCR is possible and provide a strategy for engineering high-affinity antibodies that can be used in targeting specific pMHC complexes for diagnostic and therapeutic purposes. PMID:19307587

  14. Novel trends in affinity biosensors: current challenges and perspectives

    NASA Astrophysics Data System (ADS)

    Arugula, Mary A.; Simonian, Aleksandr

    2014-03-01

    Molecular biorecognition processes facilitate physical and biochemical interactions between molecules in all crucial metabolic pathways. Perhaps the target analyte and the biorecognition element interactions have the most impactful use in biosensing applications. Traditional analytical sensing systems offer excellent biorecognition elements with the ability to detect and determine the presence of analytes. High affinity antibodies and DNA play an important role in the development of affinity biosensors based on electrochemical, optical and mass sensitive approaches. Advancements in this area routinely employ labels, label free, nanoparticles, multifunctional matrices, carbon nanotubes and other methods to meet the requirements of its own application. However, despite increasing affinity ceilings for conventional biosensors, the field draws back in meeting specifically important demands, such as long-term stability, ultrasensitivity, rapid detection, extreme selectivity, strong biological base, calibration, in vivo measurements, regeneration, satisfactory performance and ease of production. Nevertheless, recent efforts through this line have produced novel high-tech nanosensing systems such as ‘aptamers’ and ‘phages’ which exhibit high-throughput sensing. Aptamers and phages are powerful tools that excel over antibodies in sensibility, stability, multi-detection, in vivo measurements and regeneration. Phages are superior in stability, screening for affinity-based target molecules ranging from small to proteins and even cells, and easy production. In this review, we focus mainly on recent developments in affinity-based biosensors such as immunosensors, DNA sensors, emphasizing aptasensors and phage-based biosensors basing on novel electrochemical, optical and mass sensitive detection techniques. We also address enzyme inhibition-based biosensors and the current problems associated with the above sensors and their future perspectives.

  15. Cheese maturity assessment using ultrasonics.

    PubMed

    Benedito, J; Carcel, J; Clemente, G; Mulet, A

    2000-02-01

    The relationship between Mahon cheese maturity and ultrasonic velocity was examined. Moisture and textural properties were used as maturity indicators. The ultrasonic velocity of the cheese varied between 1630 and 1740 m/s, increasing with the curing time mainly because of loss of water, which also produced an increase of the textural properties. Because of the nature of low-intensity ultrasonics, velocity was better related to those textural parameters that involved small displacements. Ultrasonic velocity decreased with increasing temperature because of the negative temperature coefficient of the ultrasonic velocity of fat and the melting of fat. These results highlight the potential use of ultrasonic velocity measurements to rapidly and nondestructively assess cheese maturity. PMID:10714857

  16. Technology Maturation of Integrated System Health Management

    NASA Astrophysics Data System (ADS)

    Feather, Martin S.; Uckun, Serdar; Hicks, Kenneth A.

    2008-01-01

    Despite two decades of significant investments in R&D of Integrated System Health Management (ISHM), mission-critical applications of it in aerospace are few and far between. ISHM is subject to the general difficulty of transitioning technologies out of R&D labs and into practical applications. New and unproven methods such as ISHM introduce multiple mission risks (technology, schedule, cost), and may require a transition to unconventional and as-yet-unproven operations concepts in order to be effective. Laboratory and flight demonstrations are necessary but insufficient to adequately reduce those risks. What is needed is a solid business case before a new technology can be considered for fleetwide deployment. To address these problems, we recently applied a technology maturation assessment process developed at NASA's Jet Propulsion Laboratory to study the challenges of ISHM technology maturation. This application resulted in identification of the technologies (and technology maturation activities) that would result in the greatest risk reduction per investment dollar. Our approach and its results are described herein.

  17. Decelerating Mature Adipocyte Dedifferentiation by Media Composition.

    PubMed

    Huber, Birgit; Kluger, Petra J

    2015-12-01

    The establishment of adipose tissue test systems is still a major challenge in the investigation of cellular and molecular interactions responsible for the pathogenesis of inflammatory diseases involving adipose tissue. Mature adipocytes are mainly involved in these pathologies, but rarely used in vitro, due to the lack of an appropriate culture medium which inhibits dedifferentiation and maintains adipocyte functionality. In our study, we showed that Dulbecco's Modified Eagle's Medium/Ham's F-12 with 10% fetal calf serum (FCS) reported for the culture of mature adipocytes favors dedifferentiation, which was accompanied by a high glycerol release, a decreasing release of leptin, and a low expression of the adipocyte marker perilipin A, but high expression of CD73 after 21 days. Optimized media containing FCS, biotin, pantothenate, insulin, and dexamethasone decelerated the dedifferentiation process. These cells showed a lower lipolysis rate, a high level of leptin release, as well as a high expression of perilipin A. CD73-positive dedifferentiated fat cells were only found in low quantity. In this work, we showed that mature adipocytes when cultured under optimized conditions could be highly valuable for adipose tissue engineering in vitro. PMID:26228997

  18. Femtomolar Fab binding affinities to a protein target by alternative CDR residue co-optimization strategies without phage or cell surface display

    PubMed Central

    Plittersdorf, Hanna; Hesse, Oliver; Scheidig, Andreas; Strerath, Michael; Gritzan, Uwe; Pellengahr, Klaus; Scholz, Peter; Eicker, Andrea; Myszka, David; Haupts, Ulrich

    2012-01-01

    In therapeutic or diagnostic antibody discovery, affinity maturation is frequently required to optimize binding properties. In some cases, achieving very high affinity is challenging using the display-based optimization technologies. Here we present an approach that begins with the creation and clonal, quantitative analysis of soluble Fab libraries with complete diversification in adjacent residue pairs encompassing every complementarity-determining region position. This was followed by alternative recombination approaches and high throughput screening to co-optimize large sets of the found improving mutations. We applied this approach to the affinity maturation of the anti-tumor necrosis factor antibody adalimumab and achieved ~500-fold affinity improvement, resulting in femtomolar binding. To our knowledge, this is the first report of the in vitro engineering of a femtomolar affinity antibody against a protein target without display screening. We compare our findings to a previous report that employed extensive mutagenesis and recombination libraries with yeast display screening. The present approach is widely applicable to the most challenging of affinity maturation efforts. PMID:22531438

  19. In vitro growth factor-induced bio engineering of mature articular cartilage

    PubMed Central

    Khan, Ilyas M.; Francis, Lewis; Theobald, Peter S.; Perni, Stefano; Young, Robert D.; Prokopovich, Polina; Conlan, R. Steven; Archer, Charles W.

    2013-01-01

    Articular cartilage maturation is the postnatal development process that adapts joint surfaces to their site-specific biomechanical demands. Maturation involves gross morphological changes that occur through a process of synchronised growth and resorption of cartilage and generally ends at sexual maturity. The inability to induce maturation in biomaterial constructs designed for cartilage repair has been cited as a major cause for their failure in producing persistent cell-based repair of joint lesions. The combination of growth factors FGF2 and TGFβ1 induces accelerated articular cartilage maturation in vitro such that many molecular and morphological characteristics of tissue maturation are observable. We hypothesised that experimental growth factor-induced maturation of immature cartilage would result in a biophysical and biochemical composition consistent with a mature phenotype. Using native immature and mature cartilage as reference, we observed that growth factor-treated immature cartilages displayed increased nano-compressive stiffness, decreased surface adhesion, decreased water content, increased collagen content and smoother surfaces, correlating with a convergence to the mature cartilage phenotype. Furthermore, increased gene expression of surface structural protein collagen type I in growth factor-treated explants compared to reference cartilages demonstrates that they are still in the dynamic phase of the postnatal developmental transition. These data provide a basis for understanding the regulation of postnatal maturation of articular cartilage and the application of growth factor-induced maturation in vitro and in vivo in order to repair and regenerate cartilage defects. PMID:23182922

  20. Improving affinity chromatography resin efficiency using semi-continuous chromatography.

    PubMed

    Mahajan, Ekta; George, Anupa; Wolk, Bradley

    2012-03-01

    Protein A affinity chromatography is widely used for purification of monoclonal antibodies (MAbs) from harvested cell culture fluid (HCCF). At the manufacturing scale, the HCCF is typically loaded on a single Protein A affinity chromatography column in cycles until all of the HCCF is processed. Protein A resin costs are significant, comprising a substantial portion of the raw material costs in MAb manufacturing. Cost can be reduced by operating the process continuously using multiple smaller columns to a higher binding capacity in lieu of one industrial scale column. In this study, a series of experiments were performed using three 1-ml Hi-Trap™ MabSelect SuRe™ columns on a modified ÄKTA™ system operated according to the three Column Periodic Counter Current Chromatography (3C PCC) principle. The columns were loaded individually at different times until the 70% breakthrough point was achieved. The HCCF with unbound protein from the column was then loaded onto the next column to capture the MAb, preventing any protein loss. At any given point, all three columns were in operation, either loading or washing, enabling a reduction in processing time. The product yield and quality were evaluated and compared with a batch process to determine the effect of using the three column continuous process. The continuous operation shows the potential to reduce both resin volume and buffer consumption by ∼40%, however the system hardware and the process is more complex than the batch process. Alternative methods using a single standard affinity column, such as recycling load effluent back to the tank or increasing residence time, were also evaluated to improve Protein A resin efficiency. These alternative methods showed similar cost benefits but required longer processing time. PMID:22265178

  1. Novel maturity parameters for mature to over-mature source rocks and oils based on the distribution of phenanthrene series compounds.

    PubMed

    Wang, Zixiang; Wang, Yongli; Wu, Baoxiang; Wang, Gen; Sun, Zepeng; Xu, Liang; Zhu, Shenzhen; Sun, Lina; Wei, Zhifu

    2016-03-01

    Pyrolysis experiments of a low-mature bitumen sample originated from Cambrian was conducted in gold capsules. Abundance and distribution of phenanthrene series compounds in pyrolysis products were measured by GC-MS to investigate their changes with thermal maturity. Several maturity parameters based on the distribution of phenanthrene series compounds have been discussed. The results indicate that the distribution changes of phenanthrene series compounds are complex, and cannot be explained by individual reaction process during thermal evolution. The dealkylation cannot explain the increase of phenanthrene within the EasyRo range of 0.9% ∼ 2.1%. Adding of phenanthrene into maturity parameters based on the methylphenanthrene isomerization is unreasonable, even though MPI 1 and MPI 2 could be used to some extent. Two additional novel and an optimized maturation parameters based on the distribution of phenanthrene series compounds are proposed and their relationships to EasyRo% (x) are established: log(MPs/P) = 0.19x + 0.08 (0.9% < EasyRo% < 2.1%); log(MPs/P) = 0.64x - 0.86 (2.1% < EasyRo% < 3.4%); log(DMPs/TMPs) = 0.71x - 0.55 (0.9% < EasyRo% < 3.4%); log(MTR) = 0.84x - 0.75 (0.9% < EasyRo% < 3.4%). These significant positive correlations are strong argument for using log(MPs/P), log(DMPs/TMPs) and log(MTR) as maturity parameters, especially for mature to over-mature source rocks. PMID:27441263

  2. Affinity engineering of maltoporin: variants with enhanced affinity for particular ligands.

    PubMed

    Clune, A; Lee, K S; Ferenci, T

    1984-05-31

    Affinity-chromatographic selection on immobilized starch was used to selectively enhance the affinity of the maltodextrin-specific pore protein ( maltoporin , LamB protein, or lambda receptor protein) in the outer membrane of E. coli. Selection strategies were established for rare bacteria in large populations producing maltoporin variants with enhanced affinities for both starch and maltose, for starch but not maltose and for maltose but not starch. Three classes of lamB mutants with up to eight-fold increase in affinity for particular ligands were isolated. These mutants provide a unique range of modifications in the specificity of a transport protein. PMID:6375667

  3. Toward the Decision Tree for Inferring Requirements Maturation Types

    NASA Astrophysics Data System (ADS)

    Nakatani, Takako; Kondo, Narihito; Shirogane, Junko; Kaiya, Haruhiko; Hori, Shozo; Katamine, Keiichi

    Requirements are elicited step by step during the requirements engineering (RE) process. However, some types of requirements are elicited completely after the scheduled requirements elicitation process is finished. Such a situation is regarded as problematic situation. In our study, the difficulties of eliciting various kinds of requirements is observed by components. We refer to the components as observation targets (OTs) and introduce the word “Requirements maturation.” It means when and how requirements are elicited completely in the project. The requirements maturation is discussed on physical and logical OTs. OTs Viewed from a logical viewpoint are called logical OTs, e.g. quality requirements. The requirements of physical OTs, e.g., modules, components, subsystems, etc., includes functional and non-functional requirements. They are influenced by their requesters' environmental changes, as well as developers' technical changes. In order to infer the requirements maturation period of each OT, we need to know how much these factors influence the OTs' requirements maturation. According to the observation of actual past projects, we defined the PRINCE (Pre Requirements Intelligence Net Consideration and Evaluation) model. It aims to guide developers in their observation of the requirements maturation of OTs. We quantitatively analyzed the actual cases with their requirements elicitation process and extracted essential factors that influence the requirements maturation. The results of interviews of project managers are analyzed by WEKA, a data mining system, from which the decision tree was derived. This paper introduces the PRINCE model and the category of logical OTs to be observed. The decision tree that helps developers infer the maturation type of an OT is also described. We evaluate the tree through real projects and discuss its ability to infer the requirements maturation types.

  4. The People Capability Maturity Model

    ERIC Educational Resources Information Center

    Wademan, Mark R.; Spuches, Charles M.; Doughty, Philip L.

    2007-01-01

    The People Capability Maturity Model[R] (People CMM[R]) advocates a staged approach to organizational change. Developed by the Carnegie Mellon University Software Engineering Institute, this model seeks to bring discipline to the people side of management by promoting a structured, repeatable, and predictable approach for improving an…

  5. Enticing Mature Females into College.

    ERIC Educational Resources Information Center

    Loseth, Lexie; Moreau, Linda

    Following a review of the literature on mature female students, this paper examines enrollment trends in a selection of colleges in Alberta (Canada) and presents the findings of a survey of returning women students at Red Deer College. The literature review highlights factors related to the personal and professional development of women graduates…

  6. Adolescent Maturation in Transitioning Cultures.

    ERIC Educational Resources Information Center

    Mulroy, Kevin; Palacios, Anna; Reid, Robert E.

    This is a theoretical study of adolescent maturation within a cultural context. Personality development and disintegration due to the pressure of a dominant culture on a minority culture is considered. An attempt is made to understand how teachers might assist students to work out their psychological growth by story telling. The need for cultural…

  7. Motivational Maturity and Helping Behavior

    ERIC Educational Resources Information Center

    Haymes, Michael; Green, Logan

    1977-01-01

    Maturity in conative development (type of motivation included in Maslow's needs hierarchy) was found to be predictive of helping behavior in middle class white male college students. The effects of safety and esteem needs were compared, and the acceptance of responsibility was also investigated. (GDC)

  8. Chyloabdomen in a mature cat.

    PubMed Central

    Nelson, K L

    2001-01-01

    A mature, castrated male cat presented with progressive lethargy and a severely distended abdomen. Abdominal radiographs, abdominocentesis, and evaluation of the fluid obtained led to a diagnosis of chyloabdomen. The underlying pathology, etiology, diagnosis, and treatment associated with this disease are discussed. PMID:11360862

  9. Mature Cystic Teratoma of Liver

    PubMed Central

    Gupta, Richa; Bansal, Kalpana; Manchanda, Vivek

    2013-01-01

    A four-year-old boy presented with constipation and mild abdominal distention for one year. Radiologic investigations showed a multiloculated cystic lesion in the caudate lobe of liver with focal calcification in the wall. The child underwent laparotomy with marsupialization of the cystic lesion. Histopathologic examination showed mature teratoma of liver. PMID:24040591

  10. Affinity-Driven Immobilization of Proteins to Hematite Nanoparticles.

    PubMed

    Zare-Eelanjegh, Elaheh; Bora, Debajeet K; Rupper, Patrick; Schrantz, Krisztina; Thöny-Meyer, Linda; Maniura-Weber, Katharina; Richter, Michael; Faccio, Greta

    2016-08-10

    Functional nanoparticles are valuable materials for energy production, bioelectronics, and diagnostic devices. The combination of biomolecules with nanosized material produces a new hybrid material with properties that can exceed the ones of the single components. Hematite is a widely available material that has found application in various sectors such as in sensing and solar energy production. We report a single-step immobilization process based on affinity and achieved by genetically engineering the protein of interest to carry a hematite-binding peptide. Fabricated hematite nanoparticles were then investigated for the immobilization of the two biomolecules C-phycocyanin (CPC) and laccase from Bacillus pumilus (LACC) under mild conditions. Genetic engineering of biomolecules with a hematite-affinity peptide led to a higher extent of protein immobilization and enhanced the catalytic activity of the enzyme. PMID:27429157

  11. Evolution based on chromosome affinity from a network perspective

    NASA Astrophysics Data System (ADS)

    Monteiro, R. L. S.; Fontoura, J. R. A.; Carneiro, T. K. G.; Moret, M. A.; Pereira, H. B. B.

    2014-06-01

    Recent studies have focused on models to simulate the complex phenomenon of evolution of species. Several studies have been performed with theoretical models based on Darwin's theories to associate them with the actual evolution of species. However, none of the existing models include the affinity between individuals using network properties. In this paper, we present a new model based on the concept of affinity. The model is used to simulate the evolution of species in an ecosystem composed of individuals and their relationships. We propose an evolutive algorithm that incorporates the degree centrality and efficiency network properties to perform the crossover process and to obtain the network topology objective, respectively. Using a real network as a starting point, we simulate its evolution and compare its results with the results of 5788 computer-generated networks.

  12. Molecular modeling of the affinity chromatography of monoclonal antibodies.

    PubMed

    Paloni, Matteo; Cavallotti, Carlo

    2015-01-01

    Molecular modeling is a methodology that offers the possibility of studying complex systems such as protein-ligand complexes from an atomistic point of view, making available information that can be difficultly obtained from experimental studies. Here, a protocol for the construction of molecular models of the interaction between antibodies and ligands that can be used for an affinity chromatography process is presented. The outlined methodology focuses mostly on the description of a procedure that may be adopted to determine the structure and free energy of interaction between the antibody and the affinity ligand. A procedure to extend the proposed methodology to include the effect of the environment (buffer solution, spacer, support matrix) is also briefly outlined. PMID:25749965

  13. The full amino acid repertoire is superior to serine/tyrosine for selection of high affinity immunoglobulin G binders from the fibronectin scaffold.

    PubMed

    Hackel, Benjamin J; Wittrup, K Dane

    2010-04-01

    The design of combinatorial libraries for molecular recognition requires extensive diversity to provide high affinity binding to myriad epitopes while maintaining a high degree of functionality to enable inclusion of binders in the limited screenable library size. In the current work, we directly compare minimal and maximal amino acid diversity libraries in the context of the 10th type III domain of human fibronectin. Libraries with either serine/tyrosine or full 20 amino acid diversity were created, pooled and screened for binding to rabbit and goat immunoglobulin G (IgG), and affinity matured by directed evolution. Multiple picomolar binders to rabbit IgG and nanomolar binders to goat IgG were engineered with peak affinities of 51 +/- 4 pM and 1.2 +/- 0.4 nM, respectively. Sequence analysis reveals that 93% of the selected BC and FG loops, including those from the highest affinity clones, originate from the full diversity library. Thus, with a modest initial library size (approximately 1 x 10(8)) and an efficient affinity maturation scheme, more extensive diversity is superior to a binary serine/tyrosine code for the generation of picomolar to low nanomolar binders in the fibronectin domain. The highest affinity binders demonstrated utility in affinity purification of IgG from serum and as detection reagents in flow cytometry. PMID:20067921

  14. T cell adolescence: maturation events beyond positive selection1

    PubMed Central

    Hogquist, Kristin A.; Xing, Yan; Hsu, Fan-Chi; Shapiro, Virginia Smith

    2015-01-01

    Single positive (SP) thymocytes that successfully complete positive and negative selection must still undergo one final step, generally termed T cell maturation, before they gain functional competency and enter the long-lived T cell pool. Maturation initiates after positive selection in SP thymocytes, and continues in the periphery in recent thymic emigrants (RTEs), before these newly produced T cells gain functional competency and are ready to participate in the immune response as peripheral naïve T cells. Recent work using genetically altered mice demonstrates that T cell maturation is not a single process, but a series of steps that occur independently and sequentially after positive selection. This review will focus on the changes that occur during T maturation, and the molecules and pathways that are critical at each step. PMID:26254267

  15. T Cell Adolescence: Maturation Events Beyond Positive Selection.

    PubMed

    Hogquist, Kristin A; Xing, Yan; Hsu, Fan-Chi; Shapiro, Virginia Smith

    2015-08-15

    Single-positive thymocytes that successfully complete positive and negative selection must still undergo one final step, generally termed T cell maturation, before they gain functional competency and enter the long-lived T cell pool. Maturation initiates after positive selection in single-positive thymocytes and continues in the periphery in recent thymic emigrants, before these newly produced T cells gain functional competency and are ready to participate in the immune response as peripheral naive T cells. Recent work using genetically altered mice demonstrates that T cell maturation is not a single process, but a series of steps that occur independently and sequentially after positive selection. This review focuses on the changes that occur during T cell maturation, as well as the molecules and pathways that are critical at each step. PMID:26254267

  16. Characterizing informative sequence descriptors and predicting binding affinities of heterodimeric protein complexes

    PubMed Central

    2015-01-01

    Background Protein-protein interactions (PPIs) are involved in various biological processes, and underlying mechanism of the interactions plays a crucial role in therapeutics and protein engineering. Most machine learning approaches have been developed for predicting the binding affinity of protein-protein complexes based on structure and functional information. This work aims to predict the binding affinity of heterodimeric protein complexes from sequences only. Results This work proposes a support vector machine (SVM) based binding affinity classifier, called SVM-BAC, to classify heterodimeric protein complexes based on the prediction of their binding affinity. SVM-BAC identified 14 of 580 sequence descriptors (physicochemical, energetic and conformational properties of the 20 amino acids) to classify 216 heterodimeric protein complexes into low and high binding affinity. SVM-BAC yielded the training accuracy, sensitivity, specificity, AUC and test accuracy of 85.80%, 0.89, 0.83, 0.86 and 83.33%, respectively, better than existing machine learning algorithms. The 14 features and support vector regression were further used to estimate the binding affinities (Pkd) of 200 heterodimeric protein complexes. Prediction performance of a Jackknife test was the correlation coefficient of 0.34 and mean absolute error of 1.4. We further analyze three informative physicochemical properties according to their contribution to prediction performance. Results reveal that the following properties are effective in predicting the binding affinity of heterodimeric protein complexes: apparent partition energy based on buried molar fractions, relations between chemical structure and biological activity in principal component analysis IV, and normalized frequency of beta turn. Conclusions The proposed sequence-based prediction method SVM-BAC uses an optimal feature selection method to identify 14 informative features to classify and predict binding affinity of heterodimeric protein

  17. Biological maturation of youth athletes: assessment and implications.

    PubMed

    Malina, Robert M; Rogol, Alan D; Cumming, Sean P; Coelho e Silva, Manuel J; Figueiredo, Antonio J

    2015-07-01

    The search for talent is pervasive in youth sports. Selection/exclusion in many sports follows a maturity-related gradient largely during the interval of puberty and growth spurt. As such, there is emphasis on methods for assessing maturation. Commonly used methods for assessing status (skeletal age, secondary sex characteristics) and estimating timing (ages at peak height velocity (PHV) and menarche) in youth athletes and two relatively recent anthropometric (non-invasive) methods (status-percentage of predicted near adult height attained at observation, timing-predicted maturity offset/age at PHV) are described and evaluated. The latter methods need further validation with athletes. Currently available data on the maturity status and timing of youth athletes are subsequently summarised. Selection for sport and potential maturity-related correlates are then discussed in the context of talent development and associated models. Talent development from novice to elite is superimposed on a constantly changing base-the processes of physical growth, biological maturation and behavioural development, which occur simultaneously and interact with each other. The processes which are highly individualised also interact with the demands of a sport per se and with involved adults (coaches, trainers, administrators, parents/guardians). PMID:26084525

  18. Molecular modeling of oscillating GHz electric field influence on the kinesin affinity to microtubule

    NASA Astrophysics Data System (ADS)

    R. Saeidi, H.; S. Setayandeh, S.; Lohrasebi, A.

    2015-08-01

    Kinesin is a microtubule-associated motor protein which can respond to the external electric field due to its polarity. Using a molecular dynamics simulation method, the effect of such a field on the affinity of kinesin to the αβ-tubulin is investigated in this study. To consider kinesin affinity, the system is exposed to an electric field of 0.03 V/nm with frequency values of 1, 2, …, 9, and 10 GHz. It is found that the applied electric field can change kinesin affinity to the microtubule. These changes could perturb the normal operation of kinesin, such as the processive motility of kinesin on the microtubule.

  19. Classification of neocortical interneurons using affinity propagation

    PubMed Central

    Santana, Roberto; McGarry, Laura M.; Bielza, Concha; Larrañaga, Pedro; Yuste, Rafael

    2013-01-01

    In spite of over a century of research on cortical circuits, it is still unknown how many classes of cortical neurons exist. In fact, neuronal classification is a difficult problem because it is unclear how to designate a neuronal cell class and what are the best characteristics to define them. Recently, unsupervised classifications using cluster analysis based on morphological, physiological, or molecular characteristics, have provided quantitative and unbiased identification of distinct neuronal subtypes, when applied to selected datasets. However, better and more robust classification methods are needed for increasingly complex and larger datasets. Here, we explored the use of affinity propagation, a recently developed unsupervised classification algorithm imported from machine learning, which gives a representative example or exemplar for each cluster. As a case study, we applied affinity propagation to a test dataset of 337 interneurons belonging to four subtypes, previously identified based on morphological and physiological characteristics. We found that affinity propagation correctly classified most of the neurons in a blind, non-supervised manner. Affinity propagation outperformed Ward's method, a current standard clustering approach, in classifying the neurons into 4 subtypes. Affinity propagation could therefore be used in future studies to validly classify neurons, as a first step to help reverse engineer neural circuits. PMID:24348339

  20. Classification of neocortical interneurons using affinity propagation.

    PubMed

    Santana, Roberto; McGarry, Laura M; Bielza, Concha; Larrañaga, Pedro; Yuste, Rafael

    2013-01-01

    In spite of over a century of research on cortical circuits, it is still unknown how many classes of cortical neurons exist. In fact, neuronal classification is a difficult problem because it is unclear how to designate a neuronal cell class and what are the best characteristics to define them. Recently, unsupervised classifications using cluster analysis based on morphological, physiological, or molecular characteristics, have provided quantitative and unbiased identification of distinct neuronal subtypes, when applied to selected datasets. However, better and more robust classification methods are needed for increasingly complex and larger datasets. Here, we explored the use of affinity propagation, a recently developed unsupervised classification algorithm imported from machine learning, which gives a representative example or exemplar for each cluster. As a case study, we applied affinity propagation to a test dataset of 337 interneurons belonging to four subtypes, previously identified based on morphological and physiological characteristics. We found that affinity propagation correctly classified most of the neurons in a blind, non-supervised manner. Affinity propagation outperformed Ward's method, a current standard clustering approach, in classifying the neurons into 4 subtypes. Affinity propagation could therefore be used in future studies to validly classify neurons, as a first step to help reverse engineer neural circuits. PMID:24348339

  1. Affinity learning with diffusion on tensor product graph.

    PubMed

    Yang, Xingwei; Prasad, Lakshman; Latecki, Longin Jan

    2013-01-01

    In many applications, we are given a finite set of data points sampled from a data manifold and represented as a graph with edge weights determined by pairwise similarities of the samples. Often the pairwise similarities (which are also called affinities) are unreliable due to noise or due to intrinsic difficulties in estimating similarity values of the samples. As observed in several recent approaches, more reliable similarities can be obtained if the original similarities are diffused in the context of other data points, where the context of each point is a set of points most similar to it. Compared to the existing methods, our approach differs in two main aspects. First, instead of diffusing the similarity information on the original graph, we propose to utilize the tensor product graph (TPG) obtained by the tensor product of the original graph with itself. Since TPG takes into account higher order information, it is not a surprise that we obtain more reliable similarities. However, it comes at the price of higher order computational complexity and storage requirement. The key contribution of the proposed approach is that the information propagation on TPG can be computed with the same computational complexity and the same amount of storage as the propagation on the original graph. We prove that a graph diffusion process on TPG is equivalent to a novel iterative algorithm on the original graph, which is guaranteed to converge. After its convergence we obtain new edge weights that can be interpreted as new, learned affinities. We stress that the affinities are learned in an unsupervised setting. We illustrate the benefits of the proposed approach for data manifolds composed of shapes, images, and image patches on two very different tasks of image retrieval and image segmentation. With learned affinities, we achieve the bull's eye retrieval score of 99.99 percent on the MPEG-7 shape dataset, which is much higher than the state-of-the-art algorithms. When the data

  2. On Affine Fusion and the Phase Model

    NASA Astrophysics Data System (ADS)

    Walton, Mark A.

    2012-11-01

    A brief review is given of the integrable realization of affine fusion discovered recently by Korff and Stroppel. They showed that the affine fusion of the su(n) Wess-Zumino-Novikov-Witten (WZNW) conformal field theories appears in a simple integrable system known as the phase model. The Yang-Baxter equation leads to the construction of commuting operators as Schur polynomials, with noncommuting hopping operators as arguments. The algebraic Bethe ansatz diagonalizes them, revealing a connection to the modular S matrix and fusion of the su(n) WZNW model. The noncommutative Schur polynomials play roles similar to those of the primary field operators in the corresponding WZNW model. In particular, their 3-point functions are the su(n) fusion multiplicities. We show here how the new phase model realization of affine fusion makes obvious the existence of threshold levels, and how it accommodates higher-genus fusion.

  3. The dynamics of metric-affine gravity

    SciTech Connect

    Vitagliano, Vincenzo; Sotiriou, Thomas P.; Liberati, Stefano

    2011-05-15

    Highlights: > The role and the dynamics of the connection in metric-affine theories is explored. > The most general second order action does not lead to a dynamical connection. > Including higher order invariants excites new degrees of freedom in the connection. > f(R) actions are also discussed and shown to be a non- representative class. - Abstract: Metric-affine theories of gravity provide an interesting alternative to general relativity: in such an approach, the metric and the affine (not necessarily symmetric) connection are independent quantities. Furthermore, the action should include covariant derivatives of the matter fields, with the covariant derivative naturally defined using the independent connection. As a result, in metric-affine theories a direct coupling involving matter and connection is also present. The role and the dynamics of the connection in such theories is explored. We employ power counting in order to construct the action and search for the minimal requirements it should satisfy for the connection to be dynamical. We find that for the most general action containing lower order invariants of the curvature and the torsion the independent connection does not carry any dynamics. It actually reduces to the role of an auxiliary field and can be completely eliminated algebraically in favour of the metric and the matter field, introducing extra interactions with respect to general relativity. However, we also show that including higher order terms in the action radically changes this picture and excites new degrees of freedom in the connection, making it (or parts of it) dynamical. Constructing actions that constitute exceptions to this rule requires significant fine tuned and/or extra a priori constraints on the connection. We also consider f(R) actions as a particular example in order to show that they constitute a distinct class of metric-affine theories with special properties, and as such they cannot be used as representative toy theories to

  4. Displacement phenomena in lectin affinity chromatography.

    PubMed

    Cho, Wonryeon

    2015-10-01

    The work described here examines displacement phenomena that play a role in lectin affinity chromatography and their potential to impact reproducibility. This was achieved using Lycopersicon esculentum lectin (LEL), a lectin widely used in monitoring cancer. Four small identical LEL columns were coupled in series to form a single affinity chromatography system with the last in the series connected to an absorbance detector. The serial affinity column set (SACS) was then loaded with human plasma proteins. At the completion of loading, the column set was disassembled, the four columns were eluted individually, the captured proteins were trypsin digested, the peptides were deglycosylated with PNGase F, and the parent proteins were identified through mass spectral analyses. Significantly different sets of glycoproteins were selected by each column, some proteins appearing to be exclusively bound to the first column while others were bound further along in the series. Clearly, sample displacement chromatography (SDC) occurs. Glycoproteins were bound at different places in the column train, identifying the presence of glycoforms with different affinity on a single glycoprotein. It is not possible to see these phenomena in the single column mode of chromatography. Moreover, low abundance proteins were enriched, which facilitates detection. The great advantage of this method is that it differentiates between glycoproteins on the basis of their binding affinity. Displacement phenomena are concluded to be a significant component of the separation mechanism in heavily loaded lectin affinity chromatography columns. This further suggests that care must be exercised in sample loading of lectin columns to prevent analyte displacement with nonretained proteins. PMID:26348026

  5. Surface affinity role in graphoepitaxy of lamellar block copolymers

    NASA Astrophysics Data System (ADS)

    Claveau, G.; Quemere, P.; Argoud, M.; Hazart, J.; Pimenta Barros, P.; Sarrazin, A.; Posseme, N.; Tiron, R.; Chevalier, X.; Nicolet, C.; Navarro, C.

    2016-03-01

    Overcoming the optical limitations of 193nm immersion lithography can be achieved using Directed Self Assembly (DSA) of block-copolymers (BCPs) as a low-cost and versatile complementary technique. The goal of this paper is to investigate the potential of DSA to address line and space (L/S) high resolution patterning by performing the density multiplication of lines with the graphoepitaxy approach. As surface affinity is a key parameter in self-assembly, three variations, or "flavors", of DSA template affinity are investigated regarding several success criteria such as morphology control or defectivity. More precisely, both the methodology to register DSA defects and the impact of process parameters on defectivity are detailed. Using the 300mm pilot line available in LETI and Arkema's advanced materials, we investigate process optimization of DSA line/space patterning of a 38nm period lamellar PS-b-PMMA BCP (L38). For this study, our integration scheme, depicted in figure 2-1, is based on BCP self-assembly inside organic hard mask guiding patterns obtained using 193i nm lithography. Defect analysis coupled with the fine tuning of process parameters (annealing, brush material) provided the optimum conditions for the L38 self-assembly. Using such conditions, DSA using the three affinity flavors is investigated by means of SEM top-view and cross-section review. Lithographic performances of one selected flavor are then evaluated with the comparison of Process Windows (PWs) function of either commensurability, morphology or LWR. This work is a first step in finding the best process for an industrial graphoepitaxy approach.

  6. Negative Electron Affinity Mechanism for Diamond Surfaces

    NASA Technical Reports Server (NTRS)

    Krainsky, I. L.; Asnin, V. M.

    1998-01-01

    The energy distribution of the secondary electrons for chemical vacuum deposited diamond films with Negative Electron Affinity (NEA) was investigated. It was found that while for completely hydrogenated diamond surfaces the negative electron affinity peak in the energy spectrum of the secondary electrons is present for any energy of the primary electrons, for partially hydrogenated diamond surfaces there is a critical energy above which the peak is present in the spectrum. This critical energy increases sharply when hydrogen coverage of the diamond surface diminishes. This effect was explained by the change of the NEA from the true type for the completely hydrogenated surface to the effective type for the partially hydrogenated surfaces.

  7. New unitary affine-Virasoro constructions

    SciTech Connect

    Halpern, M.B.; Kiritsis, E.; Obers, N.A.; Poratti, M. ); Yamron, J.P. )

    1990-06-20

    This paper reports on a quasi-systematic investigation of the Virasoro master equation. The space of all affine-Virasoro constructions is organized by K-conjugation into affine-Virasoro nests, and an estimate of the dimension of the space shows that most solutions await discovery. With consistent ansatze for the master equation, large classes of new unitary nests are constructed, including quadratic deformation nests with continuous conformal weights, and unitary irrational central charge nests, which may dominate unitary rational central charge on compact g.

  8. Adsorption affinity of anions on metal oxyhydroxides

    NASA Astrophysics Data System (ADS)

    Pechenyuk, S. I.; Semushina, Yu. P.; Kuz'mich, L. F.

    2013-03-01

    The dependences of anion (phosphate, carbonate, sulfate, chromate, oxalate, tartrate, and citrate) adsorption affinity anions from geometric characteristics, acid-base properties, and complex forming ability are generalized. It is shown that adsorption depends on the nature of both the anions and the ionic medium and adsorbent. It is established that anions are generally grouped into the following series of adsorption affinity reduction: PO{4/3-}, CO{3/2-} > C2O{4/2-}, C(OH)(CH2)2(COO){3/3-}, (CHOH)2(COO){2/2-} > CrO{4/2-} ≫ SO{4/2-}.

  9. Virtual-real spatial information visualization registration using affine representations

    NASA Astrophysics Data System (ADS)

    Wu, Xueling; Ren, Fu; Du, Qingyun

    2009-10-01

    Virtual-real registration in Outdoor Augmented Reality is committed to enhance user's spatial cognition by overlaying virtual geographical objects on real scene. According to analyze fiducial detection registration method in indoor AR, for the purpose of avoiding complex and tedious process of position tracking and camera calibration in traditional registration methods, it puts forward and practices a virtual-real spatial information visualization registration method using affine representations. Based on the observation from Koenderink and van Doorn, Ullman and Basri in 1991 which is given a set of four or more non-coplanar 3D points, the projection of all points in the set can be computed as a linear combination of the projection of just four of the points, it sets up global affine coordinate system in light of world coordinates, camera coordinates and virtual coordinates and extracts four feature points from scene image and calculates the global affine coordinates of key points of virtual objects. Then according to a linear homogeneous coordinates of the four feature point's projection, it calculates projection pixel coordinates of key points of virtual objects. In addition, it proposes an approach to obtain pixel relative depth for hidden surface removal. Finally, by a case study, it verifies the feasibility and efficiency of the registration methods. The method would not only explore a new research direction for Geographical Information Science, but also would provide location-based information and services for outdoor AR.

  10. Affitins for protein purification by affinity magnetic fishing.

    PubMed

    Fernandes, Cláudia S M; Dos Santos, Raquel; Ottengy, Stella; Viecinski, Aline Canani; Béhar, Ghislaine; Mouratou, Barbara; Pecorari, Frédéric; Roque, A Cecília A

    2016-07-29

    Currently most economical and technological bottlenecks in protein production are placed in the downstream processes. With the aim of increasing the efficiency and reducing the associated costs, various affinity ligands have been developed. Affitins are small, yet robust and easy to produce, proteins derived from the archaeal extremophilic "7kDa DNA-binding" protein family. By means of combinatorial protein engineering and ribosome display selection techniques, Affitins have shown to bind a diversity of targets. In this work, two previously developed Affitins (anti-lysozyme and anti-IgG) were immobilized onto magnetic particles to assess their potential for protein purification by magnetic fishing. The optimal lysozyme and human IgG binding conditions yielded 58mg lysozyme/g support and 165mgIgG/g support, respectively. The recovery of proteins was possible in high yield (≥95%) and with high purity, namely ≥95% and 81%, when recovering lysozyme from Escherichia coli supernatant and IgG from human plasma, respectively. Static binding studies indicated affinity constants of 5.0×10(4)M(-1) and 9.3×10(5)M(-1) for the anti-lysozyme and anti-IgG magnetic supports. This work demonstrated that Affitins, which can be virtually evolved for any protein of interest, can be coupled onto magnetic particles creating novel affinity adsorbents for purification by magnetic fishing. PMID:27342136

  11. Negative Enrichment of Target Cells by Microfluidic Affinity Chromatography

    PubMed Central

    Li, Peng; Gao, Yan; Pappas, Dimitri

    2011-01-01

    A three-dimensional microfluidic channel was developed for high purity cell separations. This system featured high capture affinity using multiple vertical inlets to an affinity surface. In cell separations, positive selection (capture of the target cell) is usually employed. Negative enrichment, the capture of non-target cells and elution of target cells, has distinct advantages over positive selection. In negative enrichment, target cells are not labeled, and are not subjected to strenuous elution conditions or dilution. As a result, negative enrichment systems are amenable to multi-step processes in microfluidic systems. In previous work, we reported cell capture enhancement effects at vertical inlets to the affinity surface. In this study, we designed a chip that has multiple vertical and horizontal channels, forming a three-dimensional separation system. Enrichment of target cells showed separation purities of 92-96%, compared with straight-channel systems (77% purity). A parallelized chip was also developed for increased sample throughput. A two-channel showed similar separation purity with twice the sample flow rate. This microfluidic system, featuring high separation purity, ease of fabrication and use, is suitable for cell separations when subsequent analysis of target cells is required. PMID:21939198

  12. Maturation of the adolescent brain

    PubMed Central

    Arain, Mariam; Haque, Maliha; Johal, Lina; Mathur, Puja; Nel, Wynand; Rais, Afsha; Sandhu, Ranbir; Sharma, Sushil

    2013-01-01

    Adolescence is the developmental epoch during which children become adults – intellectually, physically, hormonally, and socially. Adolescence is a tumultuous time, full of changes and transformations. The pubertal transition to adulthood involves both gonadal and behavioral maturation. Magnetic resonance imaging studies have discovered that myelinogenesis, required for proper insulation and efficient neurocybernetics, continues from childhood and the brain’s region-specific neurocircuitry remains structurally and functionally vulnerable to impulsive sex, food, and sleep habits. The maturation of the adolescent brain is also influenced by heredity, environment, and sex hormones (estrogen, progesterone, and testosterone), which play a crucial role in myelination. Furthermore, glutamatergic neurotransmission predominates, whereas gamma-aminobutyric acid neurotransmission remains under construction, and this might be responsible for immature and impulsive behavior and neurobehavioral excitement during adolescent life. The adolescent population is highly vulnerable to driving under the influence of alcohol and social maladjustments due to an immature limbic system and prefrontal cortex. Synaptic plasticity and the release of neurotransmitters may also be influenced by environmental neurotoxins and drugs of abuse including cigarettes, caffeine, and alcohol during adolescence. Adolescents may become involved with offensive crimes, irresponsible behavior, unprotected sex, juvenile courts, or even prison. According to a report by the Centers for Disease Control and Prevention, the major cause of death among the teenage population is due to injury and violence related to sex and substance abuse. Prenatal neglect, cigarette smoking, and alcohol consumption may also significantly impact maturation of the adolescent brain. Pharmacological interventions to regulate adolescent behavior have been attempted with limited success. Since several factors, including age, sex

  13. Structural correlates of affinity in fetal versus adult endplate nicotinic receptors

    NASA Astrophysics Data System (ADS)

    Nayak, Tapan Kumar; Chakraborty, Srirupa; Zheng, Wenjun; Auerbach, Anthony

    2016-04-01

    Adult-type nicotinic acetylcholine receptors (AChRs) mediate signalling at mature neuromuscular junctions and fetal-type AChRs are necessary for proper synapse development. Each AChR has two neurotransmitter binding sites located at the interface of a principal and a complementary subunit. Although all agonist binding sites have the same core of five aromatic amino acids, the fetal site has ~30-fold higher affinity for the neurotransmitter ACh. Here we use molecular dynamics simulations of adult versus fetal homology models to identify complementary-subunit residues near the core that influence affinity, and use single-channel electrophysiology to corroborate the results. Four residues in combination determine adult versus fetal affinity. Simulations suggest that at lower-affinity sites, one of these unsettles the core directly and the others (in loop E) increase backbone flexibility to unlock a key, complementary tryptophan from the core. Swapping only four amino acids is necessary and sufficient to exchange function between adult and fetal AChRs.

  14. Structural correlates of affinity in fetal versus adult endplate nicotinic receptors

    PubMed Central

    Nayak, Tapan Kumar; Chakraborty, Srirupa; Zheng, Wenjun; Auerbach, Anthony

    2016-01-01

    Adult-type nicotinic acetylcholine receptors (AChRs) mediate signalling at mature neuromuscular junctions and fetal-type AChRs are necessary for proper synapse development. Each AChR has two neurotransmitter binding sites located at the interface of a principal and a complementary subunit. Although all agonist binding sites have the same core of five aromatic amino acids, the fetal site has ∼30-fold higher affinity for the neurotransmitter ACh. Here we use molecular dynamics simulations of adult versus fetal homology models to identify complementary-subunit residues near the core that influence affinity, and use single-channel electrophysiology to corroborate the results. Four residues in combination determine adult versus fetal affinity. Simulations suggest that at lower-affinity sites, one of these unsettles the core directly and the others (in loop E) increase backbone flexibility to unlock a key, complementary tryptophan from the core. Swapping only four amino acids is necessary and sufficient to exchange function between adult and fetal AChRs. PMID:27101778

  15. Structural correlates of affinity in fetal versus adult endplate nicotinic receptors.

    PubMed

    Nayak, Tapan Kumar; Chakraborty, Srirupa; Zheng, Wenjun; Auerbach, Anthony

    2016-01-01

    Adult-type nicotinic acetylcholine receptors (AChRs) mediate signalling at mature neuromuscular junctions and fetal-type AChRs are necessary for proper synapse development. Each AChR has two neurotransmitter binding sites located at the interface of a principal and a complementary subunit. Although all agonist binding sites have the same core of five aromatic amino acids, the fetal site has ∼30-fold higher affinity for the neurotransmitter ACh. Here we use molecular dynamics simulations of adult versus fetal homology models to identify complementary-subunit residues near the core that influence affinity, and use single-channel electrophysiology to corroborate the results. Four residues in combination determine adult versus fetal affinity. Simulations suggest that at lower-affinity sites, one of these unsettles the core directly and the others (in loop E) increase backbone flexibility to unlock a key, complementary tryptophan from the core. Swapping only four amino acids is necessary and sufficient to exchange function between adult and fetal AChRs. PMID:27101778

  16. ESCRT-III-Associated Protein ALIX Mediates High-Affinity Phosphate Transporter Trafficking to Maintain Phosphate Homeostasis in Arabidopsis

    PubMed Central

    Cardona-López, Ximena; Cuyas, Laura; Marín, Elena; Irigoyen, María Luisa; Gil, Erica; Puga, María Isabel; Bligny, Richard; Nussaume, Laurent; Geldner, Niko; Paz-Ares, Javier

    2015-01-01

    Prior to the release of their cargoes into the vacuolar lumen, sorting endosomes mature into multivesicular bodies (MVBs) through the action of ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT (ESCRT) protein complexes. MVB-mediated sorting of high-affinity phosphate transporters (PHT1) to the vacuole limits their plasma membrane levels under phosphate-sufficient conditions, a process that allows plants to maintain phosphate homeostasis. Here, we describe ALIX, a cytosolic protein that associates with MVB by interacting with ESCRT-III subunit SNF7 and mediates PHT1;1 trafficking to the vacuole in Arabidopsis thaliana. We show that the partial loss-of-function mutant alix-1 displays reduced vacuolar degradation of PHT1;1. ALIX derivatives containing the alix-1 mutation showed reduced interaction with SNF7, providing a simple molecular explanation for impaired cargo trafficking in alix-1 mutants. In fact, the alix-1 mutation also hampered vacuolar sorting of the brassinosteroid receptor BRI1. We also show that alix-1 displays altered vacuole morphogenesis, implying a new role for ALIX proteins in vacuolar biogenesis, likely acting as part of ESCRT-III complexes. In line with a presumed broad target spectrum, the alix-1 mutation is pleiotropic, leading to reduced plant growth and late flowering, with stronger alix mutations being lethal, indicating that ALIX participates in diverse processes in plants essential for their life. PMID:26342016

  17. Contributions of proteomics to understanding phagosome maturation

    PubMed Central

    Rogers, Lindsay D; Foster, Leonard J

    2008-01-01

    In metazoans macrophage cells use phagocytosis, the process of engulfing large particles, to control the spread of pathogens in the body, to clear dead or dying cells, and to aid in tissue remodelling, while the same process is also used by unicellular eukaryotes to ingest food. Phagocytosing cells essentially swallow the particles, trapping them in vacuoles called phagosomes that go through a series of maturation steps, culminating in the destruction of the internalized cargo. Because of their central role in innate immunity and their relatively simple structure (one membrane bilayer surrounding a single particle), phagosomes have been a popular subject for organelle proteomics studies. Qualitative proteomic technologies are now very sensitive so hundreds of different proteins have been identified in phagosomes from several species, revealing new properties of these intriguing compartments. More recently, quantitative proteomic approaches have also been applied, shedding new light on the dynamics and composition of maturing phagosomes. In this review we summarize the studies that have applied proteomic technologies to phagosomes and how they have changed our understanding of phagosome biology. PMID:18331591

  18. Maturity model for enterprise interoperability

    NASA Astrophysics Data System (ADS)

    Guédria, Wided; Naudet, Yannick; Chen, David

    2015-01-01

    Historically, progress occurs when entities communicate, share information and together create something that no one individually could do alone. Moving beyond people to machines and systems, interoperability is becoming a key factor of success in all domains. In particular, interoperability has become a challenge for enterprises, to exploit market opportunities, to meet their own objectives of cooperation or simply to survive in a growing competitive world where the networked enterprise is becoming a standard. Within this context, many research works have been conducted over the past few years and enterprise interoperability has become an important area of research, ensuring the competitiveness and growth of European enterprises. Among others, enterprises have to control their interoperability strategy and enhance their ability to interoperate. This is the purpose of the interoperability assessment. Assessing interoperability maturity allows a company to know its strengths and weaknesses in terms of interoperability with its current and potential partners, and to prioritise actions for improvement. The objective of this paper is to define a maturity model for enterprise interoperability that takes into account existing maturity models while extending the coverage of the interoperability domain. The assessment methodology is also presented. Both are demonstrated with a real case study.

  19. Roles of the signal peptide and mature domains in the secretion and maturation of the neutral metalloprotease from Streptomyces cacaoi.

    PubMed Central

    Chang, S C; Su, M H; Lee, Y H

    1997-01-01

    The neutral metalloprotease (Npr) of Streptomyces cacaoi is synthesized as a prepro-Npr precursor form consisting of a secretory signal peptide, a propeptide and the mature metalloprotease. The maturation of Npr occurs extracellularly via an autoproteolytic processing of the secreted pro-Npr. The integrity of the propeptide is essential for the formation of mature active Npr but not for its secretion [Chang, Chang and Lee (1994) J. Biol. Chem. 269, 3548-3554]. In this study we investigated whether the secretion and maturation of Npr require the integrity of its signal peptide region and mature protease domain. Five signal peptide mutants were generated, including the substitution mutations at the positively charged region (mutant IR6LE), the central hydrophobic region (mutants GI19EL and G19N), the boundary of the hydrophobic core-cleavage region (mutant P30L) and at the residues adjacent to the signal peptidase cleavage site (mutant YA33SM). All these lesions delayed the export of Npr to the growth medium and also resulted in a 2-10-fold decrease in Npr export. The most severe effect was noted in mutants GI19EL and P30L. When these signal peptide mutations were fused separately with the propeptide lacking the Npr mature domain, the secretory defect on the propeptide was also observed, and this impairment was again more severely expressed in mutants GI19EL and P30L. Thus the Npr signal peptide seems to have more constraints on the hydrophobic core region and at the proline residue within the boundary of the hydrophobic core-cleavage site. Deletion mutations within the C-terminal mature protease domain that left its active site intact still blocked the proteolytic processing of mutant precursor forms of pro-Npr, although their secretions were unaffected. These results, together with our previous findings, strongly suggest that the signal peptide of Npr plays a pivotal role in the secretion of both Npr and the propeptide, but not in the maturation of Npr. On the

  20. Optimization of affinity, specificity and function of designed influenza inhibitors using deep sequencing

    PubMed Central

    Whitehead, Timothy A; Chevalier, Aaron; Song, Yifan; Dreyfus, Cyrille; Fleishman, Sarel J; De Mattos, Cecilia; Myers, Chris A; Kamisetty, Hetunandan; Blair, Patrick; Wilson, Ian A; Baker, David

    2013-01-01

    We show that comprehensive sequence-function maps obtained by deep sequencing can be used to reprogram interaction specificity and to leapfrog over bottlenecks in affinity maturation by combining many individually small contributions not detectable in conventional approaches. We use this approach to optimize two computationally designed inhibitors against H1N1 influenza hemagglutinin and, in both cases, obtain variants with subnanomolar binding affinity. The most potent of these, a 51-residue protein, is broadly cross-reactive against all influenza group 1 hemagglutinins, including human H2, and neutralizes H1N1 viruses with a potency that rivals that of several human monoclonal antibodies, demonstrating that computational design followed by comprehensive energy landscape mapping can generate proteins with potential therapeutic utility. PMID:22634563

  1. Optimization of affinity, specificity and function of designed influenza inhibitors using deep sequencing

    SciTech Connect

    Whitehead, Timothy A.; Chevalier, Aaron; Song, Yifan; Dreyfus, Cyrille; Fleishman, Sarel J.; De Mattos, Cecilia; Myers, Chris A.; Kamisetty, Hetunandan; Blair, Patrick; Wilson, Ian A.; Baker, David

    2012-06-19

    We show that comprehensive sequence-function maps obtained by deep sequencing can be used to reprogram interaction specificity and to leapfrog over bottlenecks in affinity maturation by combining many individually small contributions not detectable in conventional approaches. We use this approach to optimize two computationally designed inhibitors against H1N1 influenza hemagglutinin and, in both cases, obtain variants with subnanomolar binding affinity. The most potent of these, a 51-residue protein, is broadly cross-reactive against all influenza group 1 hemagglutinins, including human H2, and neutralizes H1N1 viruses with a potency that rivals that of several human monoclonal antibodies, demonstrating that computational design followed by comprehensive energy landscape mapping can generate proteins with potential therapeutic utility.

  2. AIRE-Deficient Patients Harbor Unique High-Affinity Disease-Ameliorating Autoantibodies.

    PubMed

    Meyer, Steffen; Woodward, Martin; Hertel, Christina; Vlaicu, Philip; Haque, Yasmin; Kärner, Jaanika; Macagno, Annalisa; Onuoha, Shimobi C; Fishman, Dmytro; Peterson, Hedi; Metsküla, Kaja; Uibo, Raivo; Jäntti, Kirsi; Hokynar, Kati; Wolff, Anette S B; Krohn, Kai; Ranki, Annamari; Peterson, Pärt; Kisand, Kai; Hayday, Adrian

    2016-07-28

    APS1/APECED patients are defined by defects in the autoimmune regulator (AIRE) that mediates central T cell tolerance to many self-antigens. AIRE deficiency also affects B cell tolerance, but this is incompletely understood. Here we show that most APS1/APECED patients displayed B cell autoreactivity toward unique sets of approximately 100 self-proteins. Thereby, autoantibodies from 81 patients collectively detected many thousands of human proteins. The loss of B cell tolerance seemingly occurred during antibody affinity maturation, an obligatorily T cell-dependent step. Consistent with this, many APS1/APECED patients harbored extremely high-affinity, neutralizing autoantibodies, particularly against specific cytokines. Such antibodies were biologically active in vitro and in vivo, and those neutralizing type I interferons (IFNs) showed a striking inverse correlation with type I diabetes, not shown by other anti-cytokine antibodies. Thus, naturally occurring human autoantibodies may actively limit disease and be of therapeutic utility. PMID:27426947

  3. XGef Mediates Early CPEB Phosphorylation during Xenopus Oocyte Meiotic Maturation

    PubMed Central

    Martínez, Susana E.; Yuan, Lei; Lacza, Charlemagne; Ransom, Heather; Mahon, Gwendolyn M.; Whitehead, Ian P.; Hake, Laura E.

    2005-01-01

    Polyadenylation-induced translation is an important regulatory mechanism during metazoan development. During Xenopus oocyte meiotic progression, polyadenylation-induced translation is regulated by CPEB, which is activated by phosphorylation. XGef, a guanine exchange factor, is a CPEB-interacting protein involved in the early steps of progesterone-stimulated oocyte maturation. We find that XGef influences early oocyte maturation by directly influencing CPEB function. XGef and CPEB interact during oogenesis and oocyte maturation and are present in a c-mos messenger ribonucleoprotein (mRNP). Both proteins also interact directly in vitro. XGef overexpression increases the level of CPEB phosphorylated early during oocyte maturation, and this directly correlates with increased Mos protein accumulation and acceleration of meiotic resumption. To exert this effect, XGef must retain guanine exchange activity and the interaction with CPEB. Overexpression of a guanine exchange deficient version of XGef, which interacts with CPEB, does not enhance early CPEB phosphorylation. Overexpression of a version of XGef that has significantly reduced interaction with CPEB, but retains guanine exchange activity, decreases early CPEB phosphorylation and delays oocyte maturation. Injection of XGef antibodies into oocytes blocks progesterone-induced oocyte maturation and early CPEB phosphorylation. These findings indicate that XGef is involved in early CPEB activation and implicate GTPase signaling in this process. PMID:15635100

  4. Ghrelin accelerates in vitro maturation of bovine oocytes.

    PubMed

    Dovolou, E; Messinis, I E; Periquesta, E; Dafopoulos, K; Gutierrez-Adan, A; Amiridis, G S

    2014-08-01

    Ghrelin, apart from its metabolic role, is nowadays considered as a basic regulator of reproductive functions of mammals, acting at central and gonadal levels. Here, we investigated for possible direct actions of ghrelin on in vitro maturation of bovine oocytes and for its effects on blastocyst yield and quality. In experiment 1, cumulus oocyte complexes (COCs) were matured in the presence of four different concentrations of ghrelin (0, 200, 800 and 2000 pg/ml). In vitro fertilization and embryo culture were carried out in the absence of ghrelin, and blastocyst formation rates were examined on days 7, 8 and 9. In experiment 2, only the 800 pg/ml dose of ghrelin was used. Four groups of COCs were matured for 18 or 24 h (C18, Ghr18, C24 and Ghr24), and subsequently, they were examined for oocyte nuclear maturation and cumulus layer expansion; blastocysts were produced as in experiment 1. The relative mRNA abundance of various genes related to metabolism, oxidation, developmental competence and apoptosis was examined in snap-frozen cumulus cells, oocytes and day-7 blastocysts. In experiment 1, ghrelin significantly suppressed blastocyst formation rates. In experiment 2, more ghrelin-treated oocytes matured for 18 h reached MII compared with controls, while no difference was observed when maturation lasted for 24 h. At 18 and 24 h, the cumulus layer was more expanded in ghrelin-treated COCs than in the controls. The blastocyst formation rate was higher in Ghr18 (27.7 ± 2.4%) compared with Ghr24 (17.5 ± 2.4%). Differences were detected in various genes' expression, indicating that in the presence of ghrelin, incubation of COCs for 24 h caused over-maturation (induced ageing) of oocytes, but formed blastocysts had a higher hatching rate compared with the controls. We infer that ghrelin exerts a specific and direct role on the oocyte, accelerating its maturational process. PMID:24889518

  5. 7 CFR 51.2651 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ..., CERTIFICATION, AND STANDARDS) United States Standards for Grades for Sweet Cherries 1 Definitions § 51.2651 Mature. Mature means that the cherries have reached the stage of growth which will insure the...

  6. Young Children's Social Maturity and Their Drawings

    ERIC Educational Resources Information Center

    Hendrickson, Norejane J.; And Others

    1974-01-01

    The relationship between preschool children's level of social maturity and their drawing expressions was tested; findings stregthened the contention that the drawings of preschoolers are not appropriate indicators of their social maturity. (SDH)

  7. High affinity of lead for fetal haemoglobin.

    PubMed Central

    Ong, C N; Lee, W R

    1980-01-01

    In-vitro experiments using 203Pb were performed to identify lead-binding components in human haemoglobin. Sephadex A-50 ion-exchange chromatography of haemolysate showed that different types of haemoglobin had different affinities for lead. For the haemolysate from adults, lead was present in both Hb A (alpha 2 beta 2) and Hb A2 (alpha 2 delta 2), whereas, in the haemolysate from new-born infants, the haemoglobin of fetal origin, Hb F (alpha 2 gamma 2) showed a much greater affinity for 203Pb than the adult haemoglobin Hb A (alpha 2 beta 2), obtained from maternal blood. Analysis of the 203 Pb-labelled haemoglobin suggested that about 82% of 203Pb was in the globin polypeptide. Further analysis with carboxylmethyl (CM) cellulose chromatography indicated that the gamma globin of fetal origin had a higher affinity for 203Pb than the beta globin, whereas alpha globin appeared to be unimportant in lead binding. The results of the different affinities for lead of different Hb types are discussed with regard to the effect of lead upon haemoglobin synthesis. PMID:6158989

  8. Vygotsky's and Buber's Pedagogical Perspectives: Some Affinities

    ERIC Educational Resources Information Center

    Bartholo, Roberto; Tunes, Elizabeth; Tacca, Maria Carmen Villela Rosa

    2010-01-01

    The purpose of this paper is to examine the dialogical and creative character of pedagogic work by analyzing the affinities between Martin Buber's "I-Thou relation" and Lev Semenovich Vygotsky's "Zone of Proximal Development". Backed up by empirical studies on the teacher-student relation, we understand that education can only result in students'…

  9. Fan Affinity Laws from a Collision Model

    ERIC Educational Resources Information Center

    Bhattacharjee, Shayak

    2012-01-01

    The performance of a fan is usually estimated using hydrodynamical considerations. The calculations are long and involved and the results are expressed in terms of three affinity laws. In this paper we use kinetic theory to attack this problem. A hard sphere collision model is used, and subsequently a correction to account for the flow behaviour…

  10. Induction of Bulb Maturity of Ornithogalum Thyrsoides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The influence of bulb maturity at bulb harvest on growth and flowering response of Ornithogalum thyrsoides Jacq. ‘Chesapeake Starlight’ was investigated. Experiments were designed to determine if bulb maturity can be induced by bulb storage temperatures and whether bulb maturity can be evaluated by...

  11. Thermal Maturation of Gas Shale Systems

    NASA Astrophysics Data System (ADS)

    Bernard, Sylvain; Horsfield, Brian

    2014-05-01

    Shale gas systems serve as sources, reservoirs, and seals for unconventional natural gas accumulations. These reservoirs bring numerous challenges to geologists and petroleum engineers in reservoir characterization, most notably because of their heterogeneous character due to depositional and diagenetic processes but also because of their constituent rocks' fine-grained nature and small pore size -- much smaller than in conventional sandstone and carbonate reservoirs. Significant advances have recently been achieved in unraveling the gaseous hydrocarbon generation and retention processes that occur within these complex systems. In addition, cutting-edge characterization technologies have allowed precise documentation of the spatial variability in chemistry and structure of thermally mature organic-rich shales at the submicrometer scale, revealing the presence of geochemical heterogeneities within overmature gas shale samples and, notably, the presence of nanoporous pyrobitumen. Such research advances will undoubtedly lead to improved performance, producibility, and modeling of such strategic resources at the reservoir scale.

  12. Epididymal protein Rnase10 is required for post-testicular sperm maturation and male fertility.

    PubMed

    Krutskikh, Anton; Poliandri, Ariel; Cabrera-Sharp, Victoria; Dacheux, Jean Louis; Poutanen, Matti; Huhtaniemi, Ilpo

    2012-10-01

    Eutherian spermatozoa are dependent on the environment of the proximal epididymis to complete their maturation; however, no specific epididymal factors that mediate this process have so far been identified. Here, we show that targeted disruption of the novel gene Rnase10 encoding a secreted proximal epididymal protein in the mouse results in a binding defect in spermatozoa and their inability to pass through the uterotubal junction in the female. The failure to gain the site of fertilization in the knockout spermatozoa is associated with a gradual loss of ADAM3 and ADAM6 proteins during epididymal transit. In the distal epididymis, these spermatozoa appear to lack calcium-dependent associations with the immobilizing glutinous extracellular material and are released as single, vigorously motile cells that display no tendency for head-to-head agglutination and lack affinity to the oviductal epithelium. In sperm-egg binding assay, they are unable to establish a tenacious association with the zona pellucida, yet they are capable of fertilization. Furthermore, these sperm show accelerated capacitation resulting in an overall in vitro fertilizing ability superior to that of wild-type sperm. We conclude that the physiological role of sperm adhesiveness is in the mechanism of restricted sperm entry into the oviduct rather than in sperm-egg interaction. PMID:22750516

  13. Targeted inhibition of oncogenic miR-21 maturation with designed RNA-binding proteins.

    PubMed

    Chen, Yu; Yang, Fan; Zubovic, Lorena; Pavelitz, Tom; Yang, Wen; Godin, Katherine; Walker, Matthew; Zheng, Suxin; Macchi, Paolo; Varani, Gabriele

    2016-09-01

    The RNA recognition motif (RRM) is the largest family of eukaryotic RNA-binding proteins. Engineered RRMs with well-defined specificity would provide valuable tools and an exacting test of the current understanding of specificity. We have redesigned the specificity of an RRM using rational methods and demonstrated retargeting of its activity in cells. We engineered the conserved RRM of human Rbfox proteins to specifically bind to the terminal loop of a microRNA precursor (pre-miR-21) with high affinity and inhibit its processing by Drosha and Dicer. We further engineered Giardia Dicer by replacing its PAZ domain with the designed RRM. The reprogrammed enzyme degrades pre-miR-21 specifically in vitro and suppresses mature miR-21 levels in cells, which results in increased expression of the tumor suppressor PDCD4 and significantly decreased viability for cancer cells. The results demonstrate the feasibility of rationally engineering the sequence-specificity of RRMs and of using this ubiquitous platform for diverse biological applications. PMID:27428511

  14. T47D Cells Expressing Myeloperoxidase Are Able to Process, Traffic and Store the Mature Protein in Lysosomes: Studies in T47D Cells Reveal a Role for Cys319 in MPO Biosynthesis that Precedes Its Known Role in Inter-Molecular Disulfide Bond Formation

    PubMed Central

    Laura, Richard P.; Dong, David; Reynolds, Wanda F.; Maki, Richard A.

    2016-01-01

    Among the human heme-peroxidase family, myeloperoxidase (MPO) has a unique disulfide-linked oligomeric structure resulting from multi-step processing of the pro-protein monomer (proMPO) after it exits the endoplasmic reticulum (ER). Related family members undergo some, but not all, of the processing steps involved with formation of mature MPO. Lactoperoxidase has its pro-domain proteolytically removed and is a monomer in its mature form. Eosinophil peroxidase undergoes proteolytic removal of its pro-domain followed by proteolytic separation into heavy and light chains and is a heterodimer. However, only MPO undergoes both these proteolytic modifications and then is further oligomerized into a heterotetramer by a single inter-molecular disulfide bond. The details of how and where the post-ER processing steps of MPO occur are incompletely understood. We report here that T47D breast cancer cells stably transfected with an MPO expression plasmid are able to efficiently replicate all of the processing steps that lead to formation of the mature MPO heterotetramer. MPO also traffics to the lysosome granules of T47D cells where it accumulates, allowing in-depth immunofluorescent microscopy studies of MPO trafficking and storage for the first time. Using this novel cell model we show that formation of MPO’s single inter-molecular disulfide bond can occur normally in the absence of the proteolytic events that lead to separation of the MPO heavy and light chains. We further demonstrate that Cys319, which forms MPO’s unique inter-molecular disulfide bond, is important for events that precede this step. Mutation of this residue alters the glycosylation and catalytic activity of MPO and blocks its entry into the endocytic pathway where proteolytic processing and disulfide bonding occur. Finally, using the endocytic trafficking of lysosomal hydrolases as a guide, we investigate the role of candidate receptors in the endocytic trafficking of MPO. PMID:26890638

  15. T47D Cells Expressing Myeloperoxidase Are Able to Process, Traffic and Store the Mature Protein in Lysosomes: Studies in T47D Cells Reveal a Role for Cys319 in MPO Biosynthesis that Precedes Its Known Role in Inter-Molecular Disulfide Bond Formation.

    PubMed

    Laura, Richard P; Dong, David; Reynolds, Wanda F; Maki, Richard A

    2016-01-01

    Among the human heme-peroxidase family, myeloperoxidase (MPO) has a unique disulfide-linked oligomeric structure resulting from multi-step processing of the pro-protein monomer (proMPO) after it exits the endoplasmic reticulum (ER). Related family members undergo some, but not all, of the processing steps involved with formation of mature MPO. Lactoperoxidase has its pro-domain proteolytically removed and is a monomer in its mature form. Eosinophil peroxidase undergoes proteolytic removal of its pro-domain followed by proteolytic separation into heavy and light chains and is a heterodimer. However, only MPO undergoes both these proteolytic modifications and then is further oligomerized into a heterotetramer by a single inter-molecular disulfide bond. The details of how and where the post-ER processing steps of MPO occur are incompletely understood. We report here that T47D breast cancer cells stably transfected with an MPO expression plasmid are able to efficiently replicate all of the processing steps that lead to formation of the mature MPO heterotetramer. MPO also traffics to the lysosome granules of T47D cells where it accumulates, allowing in-depth immunofluorescent microscopy studies of MPO trafficking and storage for the first time. Using this novel cell model we show that formation of MPO's single inter-molecular disulfide bond can occur normally in the absence of the proteolytic events that lead to separation of the MPO heavy and light chains. We further demonstrate that Cys319, which forms MPO's unique inter-molecular disulfide bond, is important for events that precede this step. Mutation of this residue alters the glycosylation and catalytic activity of MPO and blocks its entry into the endocytic pathway where proteolytic processing and disulfide bonding occur. Finally, using the endocytic trafficking of lysosomal hydrolases as a guide, we investigate the role of candidate receptors in the endocytic trafficking of MPO. PMID:26890638

  16. Designer protein delivery: From natural to engineered affinity-controlled release systems.

    PubMed

    Pakulska, Malgosia M; Miersch, Shane; Shoichet, Molly S

    2016-03-18

    Exploiting binding affinities between molecules is an established practice in many fields, including biochemical separations, diagnostics, and drug development; however, using these affinities to control biomolecule release is a more recent strategy. Affinity-controlled release takes advantage of the reversible nature of noncovalent interactions between a therapeutic protein and a binding partner to slow the diffusive release of the protein from a vehicle. This process, in contrast to degradation-controlled sustained-release formulations such as poly(lactic-co-glycolic acid) microspheres, is controlled through the strength of the binding interaction, the binding kinetics, and the concentration of binding partners. In the context of affinity-controlled release--and specifically the discovery or design of binding partners--we review advances in in vitro selection and directed evolution of proteins, peptides, and oligonucleotides (aptamers), aided by computational design. PMID:26989257

  17. Affinity measurement of single chain antibodies: a mathematical method facilitated by statistical software SigmaPlot.

    PubMed

    Safdari, Yaghoub; Farajnia, Safar; Asgharzadeh, Mohammad; Khalili, Masoumeh; Jaliani, Hossein Zarei

    2014-02-01

    Because they are monovalent for antigen, single chain antibodies display a different antibody-antigen interaction pattern from that of full-length antibodies. Using the law of mass action and considering the antibody-antigen binding pattern at OD-100% and OD-50% points, we introduced a formula for estimating single chain antibody affinity. Sigmoid curves of optical density values versus antibody concentrations were drawn and used to determine antibody concentrations at OD-50% points using statistical software SigmaPlot. The OD-50% points were then used to calculate the affinity via the mathematical formula. A software-adapted format of the equation is also presented for further facilitation of the calculation process. The accuracy of this method for affinity calculation was proved by surface plasma resonance. This method offers a precise evaluation of antibody affinity without requiring special material or apparatus, making it possible to be performed in any biological laboratory with minimum facilities. PMID:24555931

  18. Tandem Affinity Purification Combined with Mass Spectrometry to Identify Components of Protein Complexes

    PubMed Central

    Kaiser, Peter; Meierhofer, David; Wang, Xiaorong; Huang, Lan

    2011-01-01

    Most biological processes are governed by multiprotein complexes rather than individual proteins. Identification of protein complexes therefore is becoming increasingly important to gain a molecular understanding of cells and organisms. Mass spectrometry–based proteomics combined with affinity-tag-based protein purification is one of the most effective strategies to isolate and identify protein complexes. The development of tandem-affinity purification approaches has revolutionized proteomics experiments. These two-step affinity purification strategies allow rapid, effective purification of protein complexes and, at the same time, minimize background. Identification of even very low-abundant protein complexes with modern sensitive mass spectrometers has become routine. Here, we describe two general strategies for tandem-affinity purification followed by mass spectrometric identification of protein complexes. PMID:18370112

  19. miRNA Tagging and Affinity-purification (miRAP)

    PubMed Central

    He, Miao

    2016-01-01

    MicroRNAs(miRNAs) are a group of endogenously expressed 20~23 nt small noncoding RNAs, which can directly regulate mRNA stability or translation in a sequence specific manner by incomplete base pairing at the 3′UTR of target mRNA, or indirectly affect transcriptional network by regulating transcription factors. As key regulators of gene expression, miRNAs are involved in the control of diverse developmental and physiological processes, including embryogenesis, differentiation, developmental timing, organogenesis, growth control, and programmed cell death. Aberrant miRNA expression profiles have been observed in many pathological conditions, including cancers, psychiatric diseases, virus infection, etc. However, the underlying mechanisms have been difficult to study in part due to the cellular heterogeneity of complex tissue. To systematically analyze miRNA expression in complex tissue, we present here a novel miRNA tagging and Affinity Purification method, miRAP, which can be applied to genetically defined cell types in any complex tissues in mice. This method is based on the fact that mature miRNAs are incorporated into RNA-induced silencing complex (RISC), in which the Argonaute protein AGO2 directly binds miRNAs and their mRNA targets. We demonstrate that epitope tagging of AGO2 protein allows direct purification of miRNAs from tissue homogenates using antibodies against the engineered molecular tag. We further established a Cre-loxP binary expression system to deliver epitope-tagged AGO2 (tAGO2) to genetically defined cell types.

  20. Oocyte Maturation: The Coming of Age of a Germ Cell

    PubMed Central

    Jamnongjit, Michelle; Hammes, Stephen R.

    2006-01-01

    Normal female fertility relies on proper development of the oocyte. This growth culminates just prior to ovulation, when oocyte maturation occurs. Oocyte maturation refers to a release of meiotic arrest that allows oocytes to advance from prophase I to metaphase II of meiosis. This precisely regulated meiotic progression is essential for normal ovulation and subsequent fertilization, and involves changes in the delicate balance between factors promoting meiotic arrest and others that are stimulating maturation. Most of the inhibitory mechanisms appear to involve the upregulation of intracellular cyclic adenosine monophosphate levels. These processes may include direct transport of the nucleotide into oocytes via gap junctions, G protein-mediated stimulation of adenylyl cyclase, and inhibition of intracellular phosphodiesterases. In contrast, potential factors that play roles in triggering oocyte maturation include gonadotropins (e.g., follicle-stimulating factor and luteinizing hormone), growth factors (e.g., amphiregulin and epiregulin), sterols (e.g., follicular fluid-derived meiosis-activating sterol), and steroids (e.g., testosterone progesterone, and estradiol). Delineating the complex interactions between these positive and negative components is critical for determining the role that oocyte maturation plays in regulating follicle development and ovulation, and may lead to novel methods that can be used to modulate these processes in women with both normal and aberrant fertility. PMID:16059829

  1. Kinetic analysis of drug-protein interactions by affinity chromatography.

    PubMed

    Bi, Cong; Beeram, Sandya; Li, Zhao; Zheng, Xiwei; Hage, David S

    2015-10-01

    Information on the kinetics of drug-protein interactions is of crucial importance in drug discovery and development. Several methods based on affinity chromatography have been developed in recent years to examine the association and dissociation rates of these processes. These techniques include band-broadening measurements, the peak decay method, peak fitting methods, the split-peak method, and free fraction analysis. This review will examine the general principles and applications of these approaches and discuss their use in the characterization, screening and analysis of drug-protein interactions in the body. PMID:26724332

  2. New alignment marks for improved measurement maturity

    NASA Astrophysics Data System (ADS)

    Weidenmueller, U.; Alves, H.; Schnabel, B.; Icard, B.; Pain, L.; Le Denmat, J.-C.; Manakli, S.; Pradelles, J.

    2008-04-01

    With shrinking dimensions in the semiconductor industry the lithographic demands are exceeding the parameters of the standard optical lithography. Electron beam direct write (EBDW) presents a good solution to overcome these limits and to successfully use this technology in R&D as well as in prototyping and some niche applications. For the industrial application of EBDW an alignment strategy adapted to the industrial standards is required to be compatible with optical lithography. In this context the crucial factor is the overlay performance, i.e. the maturity of the alignment strategy under different process conditions. New alignment marks improve the alignment repeatability and increase the window of the signal-to-noise ratio towards smaller or noisier signals. Particularly the latter has proved to be a major contribution to a higher maturity of the alignment. A comparison between the double cross and the new Barker mark type is presented in this paper. Furthermore, the mark reading repeatability and the final overlay results achieved are discussed.

  3. Pitch perception prior to cortical maturation

    NASA Astrophysics Data System (ADS)

    Lau, Bonnie K.

    Pitch perception plays an important role in many complex auditory tasks including speech perception, music perception, and sound source segregation. Because of the protracted and extensive development of the human auditory cortex, pitch perception might be expected to mature, at least over the first few months of life. This dissertation investigates complex pitch perception in 3-month-olds, 7-month-olds and adults -- time points when the organization of the auditory pathway is distinctly different. Using an observer-based psychophysical procedure, a series of four studies were conducted to determine whether infants (1) discriminate the pitch of harmonic complex tones, (2) discriminate the pitch of unresolved harmonics, (3) discriminate the pitch of missing fundamental melodies, and (4) have comparable sensitivity to pitch and spectral changes as adult listeners. The stimuli used in these studies were harmonic complex tones, with energy missing at the fundamental frequency. Infants at both three and seven months of age discriminated the pitch of missing fundamental complexes composed of resolved and unresolved harmonics as well as missing fundamental melodies, demonstrating perception of complex pitch by three months of age. More surprisingly, infants in both age groups had lower pitch and spectral discrimination thresholds than adult listeners. Furthermore, no differences in performance on any of the tasks presented were observed between infants at three and seven months of age. These results suggest that subcortical processing is not only sufficient to support pitch perception prior to cortical maturation, but provides adult-like sensitivity to pitch by three months.

  4. Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding affibody molecule.

    PubMed

    Friedman, Mikaela; Orlova, Anna; Johansson, Eva; Eriksson, Tove L J; Höidén-Guthenberg, Ingmarie; Tolmachev, Vladimir; Nilsson, Fredrik Y; Ståhl, Stefan

    2008-03-01

    The epidermal growth factor receptor 1 (EGFR) is overexpressed in various malignancies and is associated with a poor patient prognosis. A small, receptor-specific, high-affinity imaging agent would be a useful tool in diagnosing malignant tumors and in deciding upon treatment and assessing the response to treatment. We describe here the affinity maturation procedure for the generation of Affibody molecules binding with high affinity and specificity to EGFR. A library for affinity maturation was constructed by rerandomization of selected positions after the alignment of first-generation binding variants. New binders were selected with phage display technology, using a single oligonucleotide in a single-library effort, and the best second-generation binders had an approximately 30-fold improvement in affinity (K(d)=5-10 nM) for the soluble extracellular domain of EGFR in biospecific interaction analysis using Biacore. The dissociation equilibrium constant, K(d), was also determined for the Affibody with highest affinity using EGFR-expressing A431 cells in flow cytometric analysis (K(d)=2.8 nM). A retained high specificity for EGFR was verified by a dot blot assay showing staining only of EGFR proteins among a panel of serum proteins and other EGFR family member proteins (HER2, HER3, and HER4). The EGFR-binding Affibody molecules were radiolabeled with indium-111, showing specific binding to EGFR-expressing A431 cells and successful targeting of the A431 tumor xenografts with 4-6% injected activity per gram accumulated in the tumor 4 h postinjection. PMID:18207161

  5. Fluorescent measurement of affinity binding between thrombin and its aptamers using on-chip affinity monoliths.

    PubMed

    Gao, Changlu; Sun, Xiuhua; Woolley, Adam T

    2013-05-24

    A microfluidic chip with integrated 2mm long monoliths incorporated with poly(ethylene glycol) (PEG) groups was developed for thrombin-aptamer interaction study. The non-G quartet forming oligonucleotide coated monoliths was compared to a 15 mer thrombin-binding aptamer, in which affinity binding and elution processes were real-time monitored fluorescently. The results showed that the fluorescence intensity of aptamer stationary phase is approximately 10 times higher than that of the control column, which is probably due to the successful suppression of nonspecific adsorption between thrombin and aptamers/monoliths by using PEG-monolith. The experiment was repeated using human serum albumin (HSA) and green fluorescence protein (GFP) as interferences, it was double confirmed that thrombin was selectively retained by PEG-monolith. An elution efficiency of 75% was achieved with an elute of 200mM acetic acid and 2M NaCI, and the eluted thrombin was successfully separated in an ionic buffer system of 20mM NaHCO3 (pH 9.5) with 3% PEG. The hydrophilic and antifouling properties of PEG-monolith greatly decrease nonspecific adsorption and enhance detection sensitivity, which provided an alternative method to perform on-chip fluorescent measurement of bioaffinity binding. PMID:23587316

  6. Affinity purification of metalloprotease from marine bacterium using immobilized metal affinity chromatography.

    PubMed

    Li, Shangyong; Wang, Linna; Yang, Juan; Bao, Jing; Liu, Junzhong; Lin, Shengxiang; Hao, Jianhua; Sun, Mi

    2016-06-01

    In this study, an efficient affinity purification protocol for an alkaline metalloprotease from marine bacterium was developed using immobilized metal affinity chromatography. After screening and optimization of the affinity ligands and spacer arm lengths, Cu-iminmodiacetic acid was chosen as the optimal affinity ligand, which was coupled to Sepharose 6B via a 14-atom spacer arm. The absorption analysis of this medium revealed a desorption constant Kd of 21.5 μg/mL and a theoretical maximum absorption Qmax of 24.9 mg/g. Thanks to this affinity medium, the enzyme could be purified by only one affinity purification step with a purity of approximately 95% pure when analyzed by high-performance liquid chromatography and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recovery of the protease activity reached 74.6%, which is much higher than the value obtained by traditional protocols (8.9%). These results contribute to the industrial purifications and contribute a significant reference for the purification of other metalloproteases. PMID:27058973

  7. Smooth big bounce from affine quantization

    NASA Astrophysics Data System (ADS)

    Bergeron, Hervé; Dapor, Andrea; Gazeau, Jean Pierre; Małkiewicz, Przemysław

    2014-04-01

    We examine the possibility of dealing with gravitational singularities on a quantum level through the use of coherent state or wavelet quantization instead of canonical quantization. We consider the Robertson-Walker metric coupled to a perfect fluid. It is the simplest model of a gravitational collapse, and the results obtained here may serve as a useful starting point for more complex investigations in the future. We follow a quantization procedure based on affine coherent states or wavelets built from the unitary irreducible representation of the affine group of the real line with positive dilation. The main issue of our approach is the appearance of a quantum centrifugal potential allowing for regularization of the singularity, essential self-adjointness of the Hamiltonian, and unambiguous quantum dynamical evolution.

  8. Protein affinity map of chemical space.

    PubMed

    Kauvar, L M; Villar, H O; Sportsman, J R; Higgins, D L; Schmidt, D E

    1998-09-11

    Affinity fingerprinting is a quantitative method for mapping chemical space based on binding preferences of compounds for a reference panel of proteins. An effective reference panel of <20 proteins can be empirically selected which shows differential interaction with nearly all compounds. By using this map to iteratively sample the chemical space, identification of active ligands from a library of 30,000 candidate compounds has been accomplished for a wide spectrum of specific protein targets. In each case, <200 compounds were directly assayed against the target. Further, analysis of the fingerprint database suggests a strategy for effective selection of affinity chromatography ligands and scaffolds for combinatorial chemistry. With such a system, the large numbers of potential therapeutic targets emerging from genome research can be categorized according to ligand binding properties, complementing sequence based classification. PMID:9792501

  9. Affinity Chromatography in Nonionic Detergent Solutions

    NASA Astrophysics Data System (ADS)

    Robinson, Jack B.; Strottmann, James M.; Wick, Donald G.; Stellwagen, Earle

    1980-10-01

    Anionic dye affinity chromatography is commonly unproductive in the presence of nonionic detergents used to extract particulate proteins. Using lactate dehydrogenase as a model protein, Cibacron blue F3GA as a model dye, and Triton X-100 as a model detergent, we find that the dye is encapsulated in nonionic detergent micelles, rendering the dye incapable of ligation with the enzyme. However, the dye can be liberated from the micelles without altering the nonionic detergent concentration by addition of an anionic detergent, such as deoxycholate or sodium dodecyl sulfate, forming mixed anionic/nonionic micelles that displace the anionic dye. Encapsulation of the anionic detergents prevents their activity as protein denaturants. These observations have been successfully translated to the dye affinity chromatography of a detergent extract of brain particulate cyclic nucleotide phosphodiesterase.

  10. A MEMS Dielectric Affinity Glucose Biosensor.

    PubMed

    Huang, Xian; Li, Siqi; Davis, Erin; Li, Dachao; Wang, Qian; Lin, Qiao

    2013-06-20

    Continuous glucose monitoring (CGM) sensors based on affinity detection are desirable for long-term and stable glucose management. However, most affinity sensors contain mechanical moving structures and complex design in sensor actuation and signal readout, limiting their reliability in subcutaneously implantable glucose detection. We have previously demonstrated a proof-of-concept dielectric glucose sensor that measured pre-mixed glucose-sensitive polymer solutions at various glucose concentrations. This sensor features simplicity in sensor design, and possesses high specificity and accuracy in glucose detection. However, lack of glucose diffusion passage, this device is unable to fulfill real-time in-vivo monitoring. As a major improvement to this device, we present in this paper a fully implantable MEMS dielectric affinity glucose biosensor that contains a perforated electrode embedded in a suspended diaphragm. This capacitive-based sensor contains no moving parts, and enables glucose diffusion and real-time monitoring. The experimental results indicate that this sensor can detect glucose solutions at physiological concentrations and possesses good reversibility and reliability. This sensor has a time constant to glucose concentration change at approximately 3 min, which is comparable to commercial systems. The sensor has potential applications in fully implantable CGM that require excellent long-term stability and reliability. PMID:24511215

  11. On constructing purely affine theories with matter

    NASA Astrophysics Data System (ADS)

    Cervantes-Cota, Jorge L.; Liebscher, D.-E.

    2016-08-01

    We explore ways to obtain the very existence of a space-time metric from an action principle that does not refer to it a priori. Although there are reasons to believe that only a non-local theory can viably achieve this goal, we investigate here local theories that start with Schrödinger's purely affine theory (Schrödinger in Space-time structure. Cambridge UP, Cambridge, 1950), where he gave reasons to set the metric proportional to the Ricci curvature aposteriori. When we leave the context of unified field theory, and we couple the non-gravitational matter using some weak equivalence principle, we can show that the propagation of shock waves does not define a lightcone when the purely affine theory is local and avoids the explicit use of the Ricci tensor in realizing the weak equivalence principle. When the Ricci tensor is substituted for the metric, the equations seem to have only a very limited set of solutions. This backs the conviction that viable purely affine theories have to be non-local.

  12. Phosphopeptide Enrichment by Immobilized Metal Affinity Chromatography.

    PubMed

    Thingholm, Tine E; Larsen, Martin R

    2016-01-01

    Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively charged metal ions such as Fe(3+), Ga(3+), Al(3+), Zr(4+), and Ti(4+) has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from nonspecific binding of non-phosphorylated peptides. This problem is mainly caused by highly acidic peptides that also share high binding affinity towards these metal ions. By lowering the pH of the loading buffer nonspecific binding can be reduced significantly, however with the risk of reducing specific binding capacity. After binding, the enriched phosphopeptides are released from the metal ions using alkaline buffers of pH 10-11, EDTA, or phosphate-containing buffers. Here we describe a protocol for IMAC using Fe(3+) for phosphopeptide enrichment. The principles are illustrated on a semi-complex peptide mixture. PMID:26584922

  13. PERSISTENT WRIST PAIN IN A MATURE GOLFER

    PubMed Central

    Hazle, Charles

    2012-01-01

    Clients presenting with ulnar-sided wrist pain can provide diagnostic and management challenges for physical therapists. Symptoms in this region may originate from multiple structures. Integration of clinical examination and diagnostic imaging results is often required for optimal decision-making and patient management. To obtain the most informative imaging results, practitioners need an understanding of injury patterns and their detection by various imaging modalities. This case describes a mature golfer who presented with persistent ulnar-sided wrist pain and was eventually determined to have a fracture of the hook of the hamate accompanied by neighboring soft tissue involvement also contributing to his symptom complex. His history and the diagnostic process are detailed along with a brief discussion of his subsequent management post-operatively. Level of Evidence: 5 (Single Case Report) PMID:22893862

  14. Maturation Pathways of Cross-Reactive HIV-1 Neutralizing Antibodies

    PubMed Central

    Xiao, Xiaodong; Chen, Weizao; Feng, Yang; Dimitrov, Dimiter S.

    2009-01-01

    Several human monoclonal antibodies (hmAbs) and antibody fragments, including the best characterized in terms of structure-function b12 and Fab X5, exhibit relatively potent and broad HIV-1 neutralizing activity. However, the elicitation of b12 or b12-like antibodies in vivo by vaccine immunogens based on the HIV-1 envelope glycoprotein (Env) has not been successful. B12 is highly divergent from the closest corresponding germline antibody while X5 is less divergent. We have hypothesized that the relatively high degree of specific somatic hypermutations may preclude binding of the HIV-1 envelope glycoprotein (Env) to closest germline antibodies, and that identifying antibodies that are intermediates in the pathways to maturation could help design novel vaccine immunogens to guide the immune system for their enhanced elicitation. In support of this hypothesis we have previously found that a germline-like b12 (monovalent and bivalent scFv as an Fc fusion protein or IgG) lacks measurable binding to an Env as measured by ELISA with a sensitivity in the μM range [1]; here we present evidence confirming and expanding these findings for a panel of Envs. In contrast, a germline-like scFv X5 bound Env with high (nM) affinity. To begin to explore the maturation pathways of these antibodies we identified several possible b12 intermediate antibodies and tested their neutralizing activity. These intermediate antibodies neutralized only some HIV-1 isolates and with relatively weak potency. In contrast, germline-like scFv X5 neutralized a subset of the tested HIV-1 isolates with comparable efficiencies to that of the mature X5. These results could help explain the relatively high immunogenicity of the coreceptor binding site on gp120 and the abundance of CD4-induced (CD4i) antibodies in HIV-1-infected patients (X5 is a CD4i antibody) as well as the maturation pathway of X5. They also can help identify antigens that can bind specifically to b12 germline and intermediate antibodies

  15. Integrated microfluidic approach for quantitative high-throughput measurements of transcription factor binding affinities

    PubMed Central

    Glick, Yair; Orenstein, Yaron; Chen, Dana; Avrahami, Dorit; Zor, Tsaffrir; Shamir, Ron; Gerber, Doron

    2016-01-01

    Protein binding to DNA is a fundamental process in gene regulation. Methodologies such as ChIP-Seq and mapping of DNase I hypersensitive sites provide global information on this regulation in vivo. In vitro methodologies provide valuable complementary information on protein–DNA specificities. However, current methods still do not measure absolute binding affinities. There is a real need for large-scale quantitative protein–DNA affinity measurements. We developed QPID, a microfluidic application for measuring protein–DNA affinities. A single run is equivalent to 4096 gel-shift experiments. Using QPID, we characterized the different affinities of ATF1, c-Jun, c-Fos and AP-1 to the CRE consensus motif and CRE half-site in two different genomic sequences on a single device. We discovered that binding of ATF1, but not of AP-1, to the CRE half-site is highly affected by its genomic context. This effect was highly correlated with ATF1 ChIP-seq and PBM experiments. Next, we characterized the affinities of ATF1 and ATF3 to 128 genomic CRE and CRE half-site sequences. Our affinity measurements explained that in vivo binding differences between ATF1 and ATF3 to CRE and CRE half-sites are partially mediated by differences in the minor groove width. We believe that QPID would become a central tool for quantitative characterization of biophysical aspects affecting protein–DNA binding. PMID:26635393

  16. Affinity Enhancement by Ligand Clustering Effect Inspired by Peptide Dendrimers−Shank PDZ Proteins Interactions

    PubMed Central

    Liu, Jiahui; Liu, Miao; Zheng, Bo; Yao, Zhongping; Xia, Jiang

    2016-01-01

    High-affinity binders are desirable tools to probe the function that specific protein−protein interactions play in cell. In the process of seeking a general strategy to design high-affinity binders, we found a clue from the βPIX (p21-activated kinase interacting exchange factor)−Shank PDZ interaction in synaptic assembly: three PDZ-binding sites are clustered by a parallel coiled-coil trimer but bind to Shank PDZ protein with 1:1 stoichiometry (1 trimer/1 PDZ). Inspired by this architecture, we proposed that peptide dendrimer, mimicking the ligand clustering in βPIX, will also show enhanced binding affinity, yet with 1:1 stoichiometry. This postulation has been proven here, as we synthesized a set of monomeric, dimeric and trimeric peptides and measured their binding affinity and stoichiometry with Shank PDZ domains by isothermal titration calorimetry, native mass spectrometry and surface plasmon resonance. This affinity enhancement, best explained by proximity effect, will be useful to guide the design of high-affinity blockers for protein−protein interactions. PMID:26918521

  17. ELISA-mimic screen for synthetic polymer nanoparticles with high affinity to target proteins.

    PubMed

    Yonamine, Yusuke; Hoshino, Yu; Shea, Kenneth J

    2012-09-10

    Synthetic polymer nanoparticles (NPs) that display high affinity to protein targets have significant potential for medical and biotechnological applications as protein capture agents or functional replacements of antibodies ("plastic antibodies"). In this study, we modified an immunological assay (enzyme-linked immunosorbent assay: ELISA) into a high-throughput screening method to select nanoparticles with high affinity to target proteins. Histone and fibrinogen were chosen as target proteins to demonstrate this concept. The selection process utilized a biotinylated NP library constructed with combinations of functional monomers. The screen identified NPs with distinctive functional group compositions that exhibited high affinity to either histone or fibrinogen. The variation of protein affinity with changes in the nature and amount of functional groups in the NP provided chemical insight into the principle determinants of protein-NP binding. The NP affinity was semiquantified using the ELISA-mimic assay by varying the NP concentrations. The screening results were found to correlate with solution-based assay results. This screening system utilizing a biotinylated NP is a general approach to optimize functional monomer compositions and can be used to rapidly search for synthetic polymers with high (or low) affinity for target biological macromolecules. PMID:22813352

  18. Localization of Free Field Realizations of Affine Lie Algebras

    NASA Astrophysics Data System (ADS)

    Futorny, Vyacheslav; Grantcharov, Dimitar; Martins, Renato A.

    2015-04-01

    We use localization technique to construct new families of irreducible modules of affine Kac-Moody algebras. In particular, localization is applied to the first free field realization of the affine Lie algebra or, equivalently, to imaginary Verma modules.

  19. EF5 Is the High-Affinity Mg(2+) Site in ALG-2.

    PubMed

    Tanner, John J; Frey, Benjamin B; Pemberton, Travis; Henzl, Michael T

    2016-09-13

    The penta-EF-hand (PEF) protein ALG-2 (apoptosis-linked gene 2) has been implicated in several important physiological processes, including endoplasmic reticulum-Golgi vesicular transport and endosomal biogenesis/transport. ALG-2 was recently shown to harbor a metal ion-binding site with a high affinity for Mg(2+) and a low affinity for Ca(2+). We herein present the X-ray structure of Mg(2+)-bound ALG-2des23(wt). Although the C(α) trace is nearly indistinguishable from that of the Ca(2+)-free protein, the orientation of the C-terminal helix differs in the two structures. Consistent with that observation, replacement of the +x ligand in EF5, D169, with alanine eliminates high-affinity Mg(2+) binding. It also eliminates the low-affinity Ca(2+) site and lowers the affinity of the remaining Ca(2+)-binding sites, EF3 and EF1. The coordination environment in EF5 approaches ideal Mg(2+) octahedral geometry. The ligand array, consisting of three carboxylates (+x, +y, +z), a backbone carbonyl (-y), and two water molecules (-x, -z), may offer a recipe for a high-affinity, high-selectivity Mg(2+)-binding site. Sequence data for other PEF proteins indicate that select calpain large subunits, notably CAPN1 and CAPN8, may also possess a high-affinity Mg(2+)-binding site. In Mg(2+)-bound ALG-2, the carbonyl of F188 and the C-terminal carboxylate of V191 interact with the ε-ammonium group of K137 in the opposing subunit, suggesting that Mg(2+) binding could have an impact on dimerization. Interestingly, EF1 and EF3 are also occupied in the crystal, despite having modest affinity for Mg(2+). The results of a calorimetry-based analysis indicate that their Mg(2+) binding constants are 2 orders of magnitude lower than that determined for EF5. PMID:27541325

  20. Ecological and evolutionary patterns of freshwater maturation in Pacific and Atlantic salmonines

    USGS Publications Warehouse

    Sloat, Matthew R.; Fraser, Dylan J.; Dunham, Jason B.; Falke, Jeffery A.; Jordan, Chris E.; McMillan, John R.; Ohms, Haley A.

    2014-01-01

    Reproductive tactics and migratory strategies in Pacific and Atlantic salmonines are inextricably linked through the effects of migration (or lack thereof) on age and size at maturity. In this review, we focus on the ecological and evolutionary patterns of freshwater maturation in salmonines, a key process resulting in the diversification of their life histories. We demonstrate that the energetics of maturation and reproduction provides a unifying theme for understanding both the proximate and ultimate causes of variation in reproductive schedules among species, populations, and the sexes. We use probabilistic maturation reaction norms to illustrate how variation in individual condition, in terms of body size, growth rate, and lipid storage, influences the timing of maturation. This useful framework integrates both genetic and environmental contributions to conditional strategies for maturation and, in doing so, demonstrates how flexible life histories can be both heritable and subject to strong environmental influences. We review evidence that the propensity for freshwater maturation in partially anadromous species is predictable across environmental gradients at geographic and local spatial scales. We note that growth is commonly associated with the propensity for freshwater maturation, but that life-history responses to changes in growth caused by temperature may be strikingly different than changes caused by differences in food availability. We conclude by exploring how contemporary management actions can constrain or promote the diversity of maturation phenotypes in Pacific and Atlantic salmonines and caution against underestimating the role of freshwater maturing forms in maintaining the resiliency of these iconic species.

  1. High-affinity binding sites involved in the import of porin into mitochondria.

    PubMed Central

    Pfaller, R; Neupert, W

    1987-01-01

    The specific recognition by mitochondria of the precursor of porin and the insertion into the outer membrane were studied with a radiolabeled water-soluble form of porin derived from the mature protein. High-affinity binding sites had a number of 5-10 pmol/mg mitochondrial protein and a ka of 1-5 X 10(8) M-1. Binding was abolished after trypsin pretreatment of mitochondria indicating that binding sites were of protein-aceous nature. Specifically bound porin could be extracted at alkaline pH but not by high salt and was protected against low concentrations of proteinase K. It could be chased to a highly protease resistant form corresponding to mature porin. High-affinity binding sites could be extracted from mitochondria with detergent and reconstituted in asolectin-ergosterol liposomes. Water-soluble porin competed for the specific binding and import of the precursor of the ADP/ATP carrier, an inner membrane protein. We suggest that (i) binding of precursors to proteinaceous receptors serves as an initial step for recognition, (ii) the receptor for porin may also be involved in the import of precursors of inner membrane proteins, and (iii) interaction with the receptor triggers partial insertion of the precursor into the outer membrane. Images Fig. 4. PMID:2960520

  2. Pain perception development and maturation.

    PubMed

    Simons, Sinno H P; Tibboel, Dick

    2006-08-01

    Newborn infants are not small adults. The pharmacokinetics and dynamics of analgesic drugs are immature at birth. Volumes of distribution, drug clearances, side-effects and drug efficacy all differ in newborns as compared to adults. Interestingly, these parameters develop before birth and during the postnatal period, reaching adult values after a period of months or years. This means that clinicians should anticipate on pharmacokinetic/pharmacodynamic (PK/PD) changes in newborns with increasing post-conceptual age. The ability to perceive pain might also be immature at birth. Lower pain thresholds due to the absence of inhibitory descending spinothalamic fibers and a not yet fully developed cortical pain memory system are points of interest for our understanding of differences in pain perception in the newborn infant. Although this is a relatively unexplored area of research in humans, we will discuss the maturation and development of neonatal pain experience and perception in this paper. PMID:16621747

  3. Maturation of the MOUTh Intervention

    PubMed Central

    Jablonski-Jaudon, Rita A.; Kolanowski, Ann M.; Winstead, Vicki; Jones-Townsend, Corteza; Azuero, Andres

    2016-01-01

    The purpose of the current article is to describe a personalized practice originally conceived as a way to prevent and minimize care-resistant behavior to provide mouth care to older adult with dementia. The original intervention, Managing Oral Hygiene Using Threat Reduction Strategies (MOUTh), matured during the clinical trial study into a relationship-centered intervention with emphasis on developing strategies that support residents behavioral health and staff involved in care. Relationships that were initially pragmatic (i.e., focused on the task of completing mouth care) developed into more personal and responsive relationships that involved deeper engagement between mouth care providers and nursing home (NH) residents. Mouth care was accomplished and completed in a manner enjoyable to NH residents and mouth care providers. The MOUTh intervention may also concurrently affirm the dignity and personhood of the care recipient because of its emphasis on connecting with older adults. PMID:26934969

  4. Effects of inhibitors on 1-methyladenine induced maturation of starfish oocytes

    NASA Astrophysics Data System (ADS)

    Lee, Harold H.; Xu, Quanhan

    1986-12-01

    1-methladenine (1-MA) induces starfish oocytes maturation via surface reaction followed by the appearance of a cytoplasmic maturation factor which in turn induces germinal vesicle breakdown (GVBD) to resume meiosis. Cellular mechanisms involved in GVBD were investigated by microinjection of metabolic inhibitors. Colchicine (Co) inhibited maturation, cytochalasin-B (CB) delayed GVBD and actinomycin-D-(Act-D) and puromycin (Pu) had no effect. It appears that the microtubule and the microfilament systems are associated with the nuclear membrane dissolution during the process of oocyte maturation of starfish.

  5. A Novel Mechanism of Lysosomal Acid Sphingomyelinase Maturation

    PubMed Central

    Jenkins, Russell W.; Idkowiak-Baldys, Jolanta; Simbari, Fabio; Canals, Daniel; Roddy, Patrick; Riner, Clarke D.; Clarke, Christopher J.; Hannun, Yusuf A.

    2011-01-01

    Acid sphingomyelinase (aSMase) catalyzes the hydrolysis of sphingomyelin (SM) to form the bioactive lipid ceramide (Cer). Notably, aSMase exists in two forms: a zinc (Zn2+)-independent lysosomal aSMase (L-SMase) and a Zn2+-dependent secreted aSMase (S-SMase) that arise from alternative trafficking of a single protein precursor. Despite extensive investigation into the maturation and trafficking of aSMase, the exact identity of mature L-SMase has remained unclear. Here, we describe a novel mechanism of aSMase maturation involving C-terminal proteolytic processing within, or in close proximity to, endolysosomes. Using two different C-terminal-tagged constructs of aSMase (V5, DsRed), we demonstrate that aSMase is processed from a 75-kDa, Zn2+-activated proenzyme to a mature 65 kDa, Zn2+-independent L-SMase. L-SMase is recognized by a polyclonal Ab to aSMase, but not by anti-V5 or anti-DsRed antibodies, suggesting that the C-terminal tag is lost during maturation. Furthermore, indirect immunofluorescence staining demonstrated that mature L-SMase colocalized with the lysosomal marker LAMP1, whereas V5-aSMase localized to the Golgi secretory pathway. Moreover, V5-aSMase possessed Zn2+-dependent activity suggesting it may represent the common protein precursor of S-SMase and L-SMase. Importantly, the 65-kDa L-SMase, but not V5-aSMase, was sensitive to the lysosomotropic inhibitor desipramine, co-fractionated with lysosomes, and migrated at the same Mr as partially purified human aSMase. Finally, three aSMase mutants containing C-terminal Niemann-Pick mutations (R600H, R600P, ΔR608) exhibited defective proteolytic maturation. Taken together, these results demonstrate that mature L-SMase arises from C-terminal proteolytic processing of pro-aSMase and suggest that impaired C-terminal proteolysis may lead to severe defects in L-SMase function. PMID:21098024

  6. Dehydrin expression in seeds and maturation drying: a paradigm change.

    PubMed

    Radwan, A; Hara, M; Kleinwächter, M; Selmar, D

    2014-09-01

    Dehydrins are well known for being expressed in leaves during the course of developmental processes as well as under drought stress, being part of the protective machinery. Moreover, in seed physiology, dehydrins are classified as late embryogenesis-related proteins (LEA protein), where they are thought to be responsible for persistence and longevity of seeds. Although both topics are a focus of modern plant biology, a direct linkage between these both areas is generally lacking. Based on an alignment of the chain of events, this paper will help to generate understanding that the occurrence of dehydrins in maturing seeds and leaves suffering drought stress is part of the same basic principle: basic principle: dehydrins are expressed in response to water shortage. Unfortunately, the related developmental process in seeds, i.e. maturation drying, has not been adequately considered as a part of this process. As a corresponding implication, the chain of events must be adjusted: the differences in dehydrin expression in orthodox, intermediate and recalcitrant seeds could be directly attributed to the occurrence or absence of maturation drying. The differences in dehydrin expression in orthodox, intermediate and recalcitrant seeds, and thus the differences in longevity, could be attributed to the occurrence or absence of a maturation drying. PMID:25040649

  7. Gene expression analysis of early and late maturation stage rat enamel organ

    PubMed Central

    LACRUZ, RODRIGO S.; SMITH, CHARLES E.; CHEN, YI-BU; HUBBARD, MICHAEL J.; HACIA, JOSEPH G.; PAINE, MICHAEL L.

    2011-01-01

    Enamel maturation is a dynamic process that involves high rates of mineral acquisition, associated fluctuations in extracellular pH and resorption of extracellular enamel proteins. During maturation, ameloblasts change from a tall, thin and highly polarized organization characteristic of the secretory stage, to a low columnar and widened morphology in the maturation stage. To identify potential differences in gene expression throughout maturation, we obtained enamel organ epithelial cells derived from the early and late maturation stages from rat incisor and analyzed global gene expression profiles at each stage. Sixty three candidate genes were identified with potential roles in the maturation process. qPCR was used to confirm results from this genome-wide analysis in a subset of genes. Enriched transcripts in late maturation (n= 38) included those associated with lysosomal activity, solute carrier transport and calcium signaling. Cellular responses to oxidative stress, proton transport, cell death and immune system-related transcripts were also up-regulated. Transcripts down-regulated in the late maturation stage (n= 25) included those with functions related to cell adhesion, cell signaling, and T-cell activation. These results indicate that ameloblasts undergo widespread molecular changes during the maturation stage of amelogenesis and so provide the bases for future functional investigations into the mechanistic basis of enamel mineralization. PMID:22243241

  8. Cell-type-specific responses of RT4 neural cell lines to dibutyryl-cAMP: branch determination versus maturation

    SciTech Connect

    Droms, K.; Sueoka, N.

    1987-03-01

    This report describes the induction of cell-type-specific maturation, by dibutyryl-cAMP and testololactone, of neuronal and glial properties in a family of cell lines derived from a rat peripheral neurotumor, RT4. This maturation allows further understanding of the process of determination because of the close lineage relationship between the cell types of the RT4 family. The RT4 family is characterized by the spontaneous conversion of one of the cell types, RT4-AC (stem-cell type), to any of three derivative cell types, RT4-B, RT4-D, or RT4-E, with a frequency of about 10(-5). The RT4-AC cells express some properties characteristic of both neuronal and glial cells. Of these neural properties expressed by RT4-AC cells, only the neuronal properties are expressed by the RT4-B and RT4-E cells, and only the glial properties are expressed by the RT4-D cells. This in vitro cell-type conversion of RT4-AC to three derivative cell types is a branch point for the coordinate regulation of several properties and seems to resemble determination in vivo. In our standard culture conditions, several other neuronal and glial properties are not expressed by these cell types. However, addition of dibutyryl-cAMP induces expression of additional properties, in a cell-type-specific manner: formation of long cellular processes in the RT4-B8 and RT4-E5 cell lines and expression of high-affinity uptake of gamma-aminobutyric acid, by a glial-cell-specific mechanism, in the RT4-D6-2 cell line. These new properties are maximally expressed 2-3 days after addition of dibutyryl-cAMP.

  9. A Maturity Model for Assessing the Use of ICT in School Education

    ERIC Educational Resources Information Center

    Solar, Mauricio; Sabattin, Jorge; Parada, Victor

    2013-01-01

    This article describes an ICT-based and capability-driven model for assessing ICT in education capabilities and maturity of schools. The proposed model, called ICTE-MM (ICT in School Education Maturity Model), has three elements supporting educational processes: information criteria, ICT resources, and leverage domains. Changing the traditional…

  10. Simultaneous measurements of Cotton fiber maturity, fineness, ribbon width, and micronaire

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maturity (degree of secondary wall development) and fineness (linear density) are important cotton quality and processing properties, but their direct measurement is often difficult and/or expensive to perform. An indirect but critical measurement of maturity and fineness is micronaire, which is on...

  11. Near infrared measurement of cotton fiber micronaire, maturity and fineness--a comparative investigation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A key cotton fiber quality property is micronaire, which is an air flow resistance measurement that is highly correlated to the fiber’s maturity and fineness. Fiber maturity and fineness can impact the downstream processing of the fiber into yarn and fabric and the fiber’s dyeability. Previous studi...

  12. Measurements of cotton fiber maturity and fineness: method comparisons and fiber growth analyses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maturity (degree of secondary wall development) and fineness (linear density) are important cotton fiber quality and processing properties. Most maturity and fineness measurements are slow, laborious, require chemicals, and/or require expensive instruments. The Cottonscope is a small-footprint instr...

  13. The Cytochrome c Maturation Components CcmF, CcmH, and CcmI Form a Membrane-integral Multisubunit Heme Ligation Complex*

    PubMed Central

    Sanders, Carsten; Turkarslan, Serdar; Lee, Dong-Woo; Onder, Ozlem; Kranz, Robert G.; Daldal, Fevzi

    2008-01-01

    Cytochrome c maturation (Ccm) is a post-translational and post-export protein modification process that involves ten (CcmABCDEFGHI and CcdA or DsbD) components in most Gram-negative bacteria. The absence of any of these components abolishes the ability of cells to form cytochrome c, leading in the case of Rhodobacter capsulatus to the loss of photosynthetic proficiency and respiratory cytochrome oxidase activity. Based on earlier molecular genetic studies, we inferred that R. capsulatus CcmF, CcmH, and CcmI interact with each other to perform heme-apocytochrome c ligation. Here, using functional epitope-tagged derivatives of these components coproduced in appropriate mutant strains, we determined protein-protein interactions between them in detergent-dispersed membranes. Reciprocal affinity purification as well as tandem size exclusion and affinity chromatography analyses provided the first biochemical evidence that CcmF, CcmH, and CcmI associate stably with each other, indicating that these Ccm components form a membrane-integral complex. Under the conditions used, the CcmFHI complex does not contain CcmG, suggesting that the latter thio-reduction component is not always associated with the heme ligation components. The findings are discussed with respect to defining the obligatory components of a minimalistic heme-apocytochrome c ligation complex in R. capsulatus. PMID:18753134

  14. High-Affinity Fc Receptor Expression Indicates Relative Immaturity in Human Monocytes.

    PubMed

    Clanchy, Felix I L

    2016-05-01

    Within monocyte heterogeneity, subsets represent discrete, well-characterized phenotypes. Although many studies have highlighted differences between subsets, there is evidence that subpopulations represent contiguous stages in a maturational series. As CD14(hi)CD64(hi) monocytes have higher proliferative potential than CD14(hi)CD64(lo) monocytes, the surface marker profile on 4 subsets defined by CD14 and CD64 was measured. The profiles were compared to that of subsets defined by the high-affinity IgE receptor (FcɛRIα), CD16, and CD14; further differences in size, granularity, and buoyancy were measured in subsets delineated by these markers. There was a positive correlation between proliferative monocyte (PM) prevalence and CD64 expression on the classical monocyte subset, and also between PM prevalence and circulating FcɛRIα(+) monocytes. The expression of CD64, the high-affinity IgG receptor, on canonical human monocyte subsets was determined before and after short-term culture, and in response to interleukin (IL)-6, IL-10, macrophage colony-stimulating factor, granulocyte/macrophage colony-stimulating factor and interferon-γ; the influence of these cytokines on monocyte subset transition was also measured. The loss of FcɛRIα expression preceded an increase in CD16 expression in whole blood cultures. These data indicate that high-affinity Fc receptors are expressed on less mature monocytes and that FcɛRIα(+) monocytes are developmentally antecedent to the canonical classical and intermediate monocyte subsets. PMID:26714112

  15. Measuring an antibody affinity distribution molecule by molecule

    SciTech Connect

    Bradbury, Andrew M; Werner, James H; Temirov, Jamshid

    2008-01-01

    Single molecule fluorescence mIcroscopy was used to observe the binding and unbinding of hapten decorated quantum dots with individual surface immobilized antibodies. The fluorescence time history from an individual antibody site can be used to calculate its binding affinity. While quantum dot blinking occurs during these measurements, we describe a simple empirical method to correct the apparent/observed affinity to account for the blinking contribution. The combination of many single molecule affinity measurements from different antibodies yields not only the average affinity, it directly measures the full shape and character of the surface affinity distribution function.

  16. On the structure of self-affine convex bodies

    SciTech Connect

    Voynov, A S

    2013-08-31

    We study the structure of convex bodies in R{sup d} that can be represented as a union of their affine images with no common interior points. Such bodies are called self-affine. Vallet's conjecture on the structure of self-affine bodies was proved for d = 2 by Richter in 2011. In the present paper we disprove the conjecture for all d≥3 and derive a detailed description of self-affine bodies in R{sup 3}. Also we consider the relation between properties of self-affine bodies and functional equations with a contraction of an argument. Bibliography: 10 titles.

  17. Avoiding degenerate coframes in an affine gauge approach to quantum gravity

    SciTech Connect

    Mielke, E.W.; McCrea, J.D.; Ne`eman, Y.; Hehl, F.W.

    1993-04-01

    This report discusses the following concepts on quantum gravity: The affine gauge approach; affine gauge transformations versus active differomorphisms; affine gauge approach to quantum gravity with topology change.

  18. Aluminum monocation basicity and affinity scales.

    PubMed

    Gal, Jean-François; Yáñez, Manuel; Mó, Otilia

    2015-01-01

    The experimental aspects of the determination of thermochemical data for the attachment of the aluminum monocation Al(+) to neutral atoms and molecules are reviewed. Literature aluminum cation affinities (enthalpy scale) and basicities (Gibbs energy scale) are tabulated and discussed. Ab initio quantum chemical calculations at the G4 level on 43 adducts provide a consistent picture of the energetics of the adducts and their structures. The Al(+)-ligand bonding is analyzed in terms of natural bond orbital and atom-in molecule analyses. A brief comparison of the Al(+) basicity scales and other gas- phase cation basicities is presented. PMID:26307732

  19. Contractions of affine Kac-Moody algebras

    NASA Astrophysics Data System (ADS)

    Daboul, J.; Daboul, C.; de Montigny, M.

    2008-08-01

    I review our recent work on contractions of affine Kac-Moody algebras (KMA) and present new results. We study generalized contractions of KMA with respect to their twisted and untwisted KM subalgebras. As a concrete example, we discuss contraction of D(1)4 and D(3)4, based on Z3-grading. We also describe examples of 'level-dependent' contractions, which are based on Z-gradings of KMA. Our work generalizes the Inönü-Wigner contraction of P. Majumdar in several directions. We also give an algorithm for constructing Kac-Moody-like algebras hat g for any Lie algebra g.

  20. Flexible Molybdenum Electrodes towards Designing Affinity Based Protein Biosensors.

    PubMed

    Kamakoti, Vikramshankar; Panneer Selvam, Anjan; Radha Shanmugam, Nandhinee; Muthukumar, Sriram; Prasad, Shalini

    2016-01-01

    Molybdenum electrode based flexible biosensor on porous polyamide substrates has been fabricated and tested for its functionality as a protein affinity based biosensor. The biosensor performance was evaluated using a key cardiac biomarker; cardiac Troponin-I (cTnI). Molybdenum is a transition metal and demonstrates electrochemical behavior upon interaction with an electrolyte. We have leveraged this property of molybdenum for designing an affinity based biosensor using electrochemical impedance spectroscopy. We have evaluated the feasibility of detection of cTnI in phosphate-buffered saline (PBS) and human serum (HS) by measuring impedance changes over a frequency window from 100 mHz to 1 MHz. Increasing changes to the measured impedance was correlated to the increased dose of cTnI molecules binding to the cTnI antibody functionalized molybdenum surface. We achieved cTnI detection limit of 10 pg/mL in PBS and 1 ng/mL in HS medium. The use of flexible substrates for designing the biosensor demonstrates promise for integration with a large-scale batch manufacturing process. PMID:27438863

  1. Reflection symmetry detection using locally affine invariant edge correspondence.

    PubMed

    Wang, Zhaozhong; Tang, Zesheng; Zhang, Xiao

    2015-04-01

    Reflection symmetry detection receives increasing attentions in recent years. The state-of-the-art algorithms mainly use the matching of intensity-based features (such as the SIFT) within a single image to find symmetry axes. This paper proposes a novel approach by establishing the correspondence of locally affine invariant edge-based features, which are superior to the intensity based in the aspects that it is insensitive to illumination variations, and applicable to textureless objects. The locally affine invariance is achieved by simple linear algebra for efficient and robust computations, making the algorithm suitable for detections under object distortions like perspective projection. Commonly used edge detectors and a voting process are, respectively, used before and after the edge description and matching steps to form a complete reflection detection pipeline. Experiments are performed using synthetic and real-world images with both multiple and single reflection symmetry axis. The test results are compared with existing algorithms to validate the proposed method. PMID:25608306

  2. Robust activation method for negative electron affinity photocathodes

    DOEpatents

    Mulhollan, Gregory A.; Bierman, John C.

    2011-09-13

    A method by which photocathodes(201), single crystal, amorphous, or otherwise ordered, can be surface modified to a robust state of lowered and in best cases negative, electron affinity has been discovered. Conventional methods employ the use of Cs(203) and an oxidizing agent(207), typically carried by diatomic oxygen or by more complex molecules, for example nitrogen trifluoride, to achieve a lowered electron affinity(404). In the improved activation method, a second alkali, other than Cs(205), is introduced onto the surface during the activation process, either by co-deposition, yo-yo, or sporadic or intermittent application. Best effect for GaAs photocathodes has been found through the use of Li(402) as the second alkali, though nearly the same effect can be found by employing Na(406). Suitable photocathodes are those which are grown, cut from boules, implanted, rolled, deposited or otherwise fabricated in a fashion and shape desired for test or manufacture independently supported or atop a support structure or within a framework or otherwise affixed or suspended in the place and position required for use.

  3. Boronate affinity adsorption of RNA: possible role of conformational changes

    NASA Technical Reports Server (NTRS)

    Singh, N.; Willson, R. C.; Fox, G. E. (Principal Investigator)

    1999-01-01

    Batch equilibrium adsorption isotherm determination is used to characterize the adsorption of mixed yeast RNA on agarose-immobilized m-aminophenylboronic acid. It is shown that the affinity-enhancing influence of divalent cations depends strongly on the precise nature of the cation used, with barium being far more effective than the conventionally-used magnesium. This adsorption-promoting influence of barium is suggested to arise primarily from ionic influences on the structure and rigidity of the RNA molecule, as the adsorption of ribose-based small molecules is not similarly affected. The substitution of barium for the standard magnesium counterion does not greatly promote the adsorption of DNA, implying that the effect is specific to RNA and may be useful in boronate-based RNA separations. RNA adsorption isotherms exhibit sharp transitions as functions of temperature, and these transitions occur at different temperatures with Mg2+ and with Ba2+. Adsorption affinity and capacity were found to increase markedly at lower temperatures, suggestive of an enthalpically favored interaction process. The stoichiometric displacement parameter, Z, in Ba2+ buffer is three times the value in Mg2+ buffer, and is close to unity.

  4. Binding Affinity of Glycoconjugates to BACILLUS Spores and Toxins

    NASA Astrophysics Data System (ADS)

    Rasol, Aveen; Eassa, Souzan; Tarasenko, Olga

    2010-04-01

    Early recognition of Bacillus cereus group species is important since they can cause food-borne illnesses and deadly diseases in humans. Glycoconjugates (GCs) are carbohydrates covalently linked to non-sugar moieties including lipids, proteins or other entities. GCs are involved in recognition and signaling processes intrinsic to biochemical functions in cells. They also stimulate cell-cell adhesion and subsequent recognition and activation of receptors. We have demonstrated that GCs are involved in Bacillus cereus spore recognition. In the present study, we have investigated whether GCs possess the ability to bind and recognize B. cereus spores and Bacillus anthracis recombinant single toxins (sTX) and complex toxins (cTX). The affinity of GCs to spores + sTX and spores + cTX toxins was studied in the binding essay. Our results demonstrated that GC9 and GC10 were able to selectively bind to B. cereus spores and B. anthracis toxins. Different binding affinities for GCs were found toward Bacillus cereus spores + sTX and spores + cTX. Dilution of GCs does not impede the recognition and binding. Developed method provides a tool for simultaneous recognition and targeting of spores, bacteria toxins, and/or other entities.

  5. Embryonic Cardiac Chamber Maturation: Trabeculation, Conduction and Cardiomyocyte Proliferation

    PubMed Central

    Samsa, Leigh Ann; Yang, Betsy; Liu, Jiandong

    2013-01-01

    Congenital heart diseases are one of the most common human birth defects. Though some congenital heart defects can be surgically corrected, treatment options for other congenital heart diseases are very limited. In many congenital heart diseases, genetic defects lead to impaired embryonic heart development or growth. One of the key development processes in cardiac development is chamber maturation, and alterations in this maturation process can manifest as a variety of congenital defects including noncompaction, systolic dysfunction, diastolic dysfunction, and arrhythmia. During development, to meet the increasing metabolic demands of the developing embryo, the myocardial wall undergoes extensive remodeling characterized by the formation of muscular luminal protrusions called cardiac trabeculae, increased cardiomyocyte mass, and development of the ventricular conduction system. Though the basic morphological and cytological changes involved in early heart development are clear, much remains unknown about the complex biomolecular mechanisms governing chamber maturation. In this review, we highlight evidence suggesting that a wide variety of basic signaling pathways and biomechanical forces are involved in cardiac wall maturation. PMID:23720419

  6. Top 10 Research Questions Related to Growth and Maturation of Relevance to Physical Activity, Performance, and Fitness

    ERIC Educational Resources Information Center

    Malina, Robert M.

    2014-01-01

    Growth, maturation, and development dominate the daily lives of children and adolescents for approximately the first 2 decades of life. Growth and maturation are biological processes, while development is largely a behavioral process. The 3 processes occur simultaneously and interact. They can be influenced by physical activity and also can…

  7. TECHNOLOGY MATURATION PLAN FOR ALUMINUM REMOVAL AND SODIUM HYDROXIDE REGENERATION FROM HANFORD WASTE BY LITHIUM HYDROTALCITE PRECIPITATION

    SciTech Connect

    SAMS TL; GUILLOT S

    2011-01-27

    This Technology Maturation Plan schedules the development process that will bring the Lithium Hydrotalcite waste pretreatment process from its current estimated Technology Readiness Level of 3, to a level of 6. This maturation approach involves chemical and engineering research and development work, from laboratory scale to pilot scale testing, to incrementally make the process progress towards its integration in a fully qualified industrial system.

  8. Common and novel transcriptional routes to behavioral maturation in worker and male honey bees.

    PubMed

    Zayed, A; Naeger, N L; Rodriguez-Zas, S L; Robinson, G E

    2012-04-01

    Worker honey bees (Apis mellifera) undergo a process of behavioral maturation leading to their transition from in-hive tasks to foraging--a process which is associated with profound transcriptional changes in the brain. Changes in brain gene expression observed during worker behavioral maturation could represent either a derived program underlying division of labor or a general program unrelated to sociality. Male bees (drones) undergo a process of behavioral maturation associated with the onset of mating flights, but do not partake in division of labor. Drones thus provide an excellent reference point for polarizing transcriptional changes associated with behavioral maturation in honey bees. We assayed the brain transcriptomes of adult drones and workers to compare and contrast differences associated with behavioral maturation in the two sexes. Both behavioral maturation and sex were associated with changes in expression of thousands of genes in the brain. Many genes involved in neuronal development, behavior, and the biosynthesis of neurotransmitters regulating the perception of reward showed sex-biased gene expression. Furthermore, most of the transcriptional changes associated with behavioral maturation were common to drones and workers, consistent with common genetic and physiological regulation. Our study suggests that there is a common behavioral maturation program that has been co-opted and modified to yield the different behavioral and cognitive phenotypes of worker and drone bees. PMID:22050787

  9. Motivational maturity and helping behavior.

    PubMed

    Haymes, M; Green, L

    1977-12-01

    This study was undertaken to examine the independent influences of conative development (the Maslow needs hierarchy) upon behavioral aspects of prosocial orientations. It provides a behavioral demonstration of conative effects in a helping paradigm, among college-age men. A comparison of the conative data across the ages of 15-22 provided a cross-sectional view of conative development itself. Conative maturity was found to be predictive of greater helping among college-age men. Situational demands were demonstrated which tended to mask, but not override, these predispositional influences on helping. The cross-sectional data on conative development point to probable movement to early esteem concerns among high school men who have reached the conative level of love and belonging. On the other hand, the stability across the years of 15-22 of proportion of safety concerns suggests fixation of such concerns in those exhibiting them in high school. Results are discussed in terms of conative growth for development of prosocial orientations. PMID:24408562

  10. Smart Grid Interoperability Maturity Model

    SciTech Connect

    Widergren, Steven E.; Levinson, Alex; Mater, J.; Drummond, R.

    2010-04-28

    The integration of automation associated with electricity resources (including transmission and distribution automation and demand-side resources operated by end-users) is key to supporting greater efficiencies and incorporating variable renewable resources and electric vehicles into the power system. The integration problems faced by this community are analogous to those faced in the health industry, emergency services, and other complex communities with many stakeholders. To highlight this issue and encourage communication and the development of a smart grid interoperability community, the GridWise Architecture Council (GWAC) created an Interoperability Context-Setting Framework. This "conceptual model" has been helpful to explain the importance of organizational alignment in addition to technical and informational interface specifications for "smart grid" devices and systems. As a next step to building a community sensitive to interoperability, the GWAC is investigating an interoperability maturity model (IMM) based on work done by others to address similar circumstances. The objective is to create a tool or set of tools that encourages a culture of interoperability in this emerging community. The tools would measure status and progress, analyze gaps, and prioritize efforts to improve the situation.

  11. High-affinity Cyclic Peptide Matriptase Inhibitors*

    PubMed Central

    Quimbar, Pedro; Malik, Uru; Sommerhoff, Christian P.; Kaas, Quentin; Chan, Lai Y.; Huang, Yen-Hua; Grundhuber, Maresa; Dunse, Kerry; Craik, David J.; Anderson, Marilyn A.; Daly, Norelle L.

    2013-01-01

    The type II transmembrane serine protease matriptase is a key activator of multiple signaling pathways associated with cell proliferation and modification of the extracellular matrix. Deregulated matriptase activity correlates with a number of diseases, including cancer and hence highly selective matriptase inhibitors may have therapeutic potential. The plant-derived cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1), is a promising drug scaffold with potent matriptase inhibitory activity. In the current study we have analyzed the structure-activity relationships of SFTI-1 and Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II), a structurally divergent trypsin inhibitor from Momordica cochinchinensis that also contains a cyclic backbone. We show that MCoTI-II is a significantly more potent matriptase inhibitor than SFTI-1 and that all alanine mutants of both peptides, generated using positional scanning mutagenesis, have decreased trypsin affinity, whereas several mutations either maintain or result in enhanced matriptase inhibitory activity. These intriguing results were used to design one of the most potent matriptase inhibitors known to date with a 290 pm equilibrium dissociation constant, and provide the first indication on how to modulate affinity for matriptase over trypsin in cyclic peptides. This information might be useful for the design of more selective and therapeutically relevant inhibitors of matriptase. PMID:23548907

  12. Heparin affinity purification of extracellular vesicles

    PubMed Central

    Balaj, Leonora; Atai, Nadia A.; Chen, Weilin; Mu, Dakai; Tannous, Bakhos A.; Breakefield, Xandra O.; Skog, Johan; Maguire, Casey A.

    2015-01-01

    Extracellular vesicles (EVs) are lipid membrane vesicles released by cells. They carry active biomolecules including DNA, RNA, and protein which can be transferred to recipient cells. Isolation and purification of EVs from culture cell media and biofluids is still a major challenge. The most widely used isolation method is ultracentrifugation (UC) which requires expensive equipment and only partially purifies EVs. Previously we have shown that heparin blocks EV uptake in cells, supporting a direct EV-heparin interaction. Here we show that EVs can be purified from cell culture media and human plasma using ultrafiltration (UF) followed by heparin-affinity beads. UF/heparin-purified EVs from cell culture displayed the EV marker Alix, contained a diverse RNA profile, had lower levels of protein contamination, and were functional at binding to and uptake into cells. RNA yield was similar for EVs isolated by UC. We were able to detect mRNAs in plasma samples with comparable levels to UC samples. In conclusion, we have discovered a simple, scalable, and effective method to purify EVs taking advantage of their heparin affinity. PMID:25988257

  13. Affine conformal vectors in space-time

    NASA Astrophysics Data System (ADS)

    Coley, A. A.; Tupper, B. O. J.

    1992-05-01

    All space-times admitting a proper affine conformal vector (ACV) are found. By using a theorem of Hall and da Costa, it is shown that such space-times either (i) admit a covariantly constant vector (timelike, spacelike, or null) and the ACV is the sum of a proper affine vector and a conformal Killing vector or (ii) the space-time is 2+2 decomposable, in which case it is shown that no ACV can exist (unless the space-time decomposes further). Furthermore, it is proved that all space-times admitting an ACV and a null covariantly constant vector (which are necessarily generalized pp-wave space-times) must have Ricci tensor of Segré type {2,(1,1)}. It follows that, among space-times admitting proper ACV, the Einstein static universe is the only perfect fluid space-time, there are no non-null Einstein-Maxwell space-times, and only the pp-wave space-times are representative of null Einstein-Maxwell solutions. Otherwise, the space-times can represent anisotropic fluids and viscous heat-conducting fluids, but only with restricted equations of state in each case.

  14. Fatigue damage prognosis using affine arithmetic

    NASA Astrophysics Data System (ADS)

    Gbaguidi, Audrey; Kim, Daewon

    2014-02-01

    Among the essential steps to be taken in structural health monitoring systems, damage prognosis would be the field that is least investigated due to the complexity of the uncertainties. This paper presents the possibility of using Affine Arithmetic for uncertainty propagation of crack damage in damage prognosis. The structures examined are thin rectangular plates made of titanium alloys with central mode I cracks and a composite plate with an internal delamination caused by mixed mode I and II fracture modes, under a harmonic uniaxial loading condition. The model-based method for crack growth rates are considered using the Paris Erdogan law model for the isotropic plates and the delamination growth law model proposed by Kardomateas for the composite plate. The parameters for both models are randomly taken and their uncertainties are considered as defined by an interval instead of a probability distribution. A Monte Carlo method is also applied to check whether Affine Arithmetic (AA) leads to tight bounds on the lifetime of the structure.

  15. Exploring Fluorous Affinity by Liquid Chromatography.

    PubMed

    Catani, Martina; Guzzinati, Roberta; Marchetti, Nicola; Pasti, Luisa; Cavazzini, Alberto

    2015-07-01

    Terms such as "fluorous affinity" and "fluorophilicity" have been used to describe the unique partition and sorption properties often exhibited by highly fluorinated organic compounds, that is molecules rich in sp(3) carbon-fluorine bonds. In this work, we made use of a highly fluorinated stationary phase and a series of benzene derivatives to study the effect of one single perfluorinated carbon on the chromatographic behavior and adsorption properties of molecules. For this purpose, the adsorption equilibria of α,α,α-trifluorotoluene, toluene, and other alkylbenzenes have been studied by means of nonlinear chromatography in a variety of acetonitrile/water eluents. Our results reveal that one single perfluorinated carbon is already enough to induce a drastic change in the adsorption properties of molecules on the perfluorinated stationary phase. In particular, it has been found that adsorption is monolayer if the perfluoroalkyl carbon is present but that, when this unit is missing, molecules arrange as multilayer stack structures. These findings can contribute to the understanding of molecular mechanisms of fluorous affinity. PMID:26047527

  16. Quantification of hydrophobic interaction affinity of colloids

    NASA Astrophysics Data System (ADS)

    Saini, G.; Nasholm, N.; Wood, B. D.

    2009-12-01

    Colloids play an important role in a wide variety of disciplines, including water and wastewater treatment, subsurface transport of metals and organic contaminants, migration of fines in oil reservoirs, biocolloid (virus and bacteria) transport in subsurface, and are integral to laboratory transport studies. Although the role of hydrophobicity in adhesion and transport of colloids, particularly bacteria, is well known; there is scarcity of literature regarding hydrophobicity measurement of non-bacterial colloids and other micron-sized particles. Here we detail an experimental approach based on differential partitioning of colloids between two liquid phases (hydrocarbon and buffer) as a measure of the hydrophobic interaction affinity of colloids. This assay, known as Microbial adhesion to hydrocarbons or MATH, is frequently used in microbiology and bacteriology for quantifying the hydrophobicity of microbes. Monodispersed colloids and particles, with sizes ranging from 1 micron to 33 micron, were used for the experiments. A range of hydrophobicity values were observed for different particles. The hydrophobicity results are also verified against water contact angle measurements of these particles. This liquid-liquid partitioning assay is quick, easy-to-perform and requires minimal instrumentation. Estimation of the hydrophobic interaction affinity of colloids would lead to a better understanding of their adhesion to different surfaces and subsequent transport in porous media.

  17. Laboratory and outside the laboratory measurements of ginned and ot ginned cotton for fiber micronaire and maturity by portable Near Infrared (NIR) Instruments

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Micronaire is a key cotton quality assessment property, impacting downstream fiber processing and dye consistency. A component of micronaire is fiber maturity (degree of secondary wall development). Historically, micronaire and maturity are measured in a laboratory under tight environmental condit...

  18. Egg maturation dynamics of Homalodisca vitripennis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The egg maturation dynamics of holometabolous insects are particularly well studied compared to those of hemimetabolous insects. The wealth of knowledge produced from studies on holometabolous insects has allowed researchers to test for correlations between egg maturation schedule and specific life...

  19. Biased experimental fineness and maturity results

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In Part I of this series, models were developed and computer simulations were performed to understand the variability in coefficients of determination (R2) between fineness and maturity, micronaire and fineness, and micronaire and maturity of cotton. Part II concentrated on derivation and testing of...

  20. 7 CFR 51.767 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies may be obtained from, Florida Department of Citrus... Mature. Mature shall have the same meaning assigned the term in the Florida Citrus Code, Chapter 601, 1995 Edition, and the Official Rules Affecting the Florida Citrus Industry, in effect as of February...

  1. 7 CFR 51.1823 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ...(a) and 1 CFR part 51. Copies may be obtained from, Florida Department of Citrus, Post Office Box 148... Mature. Mature shall have the same meaning assigned the term in the Florida Citrus Code, Chapter 601, 1995 Edition, and the Official Rules Affecting the Florida Citrus Industry, in effect as of February...

  2. 7 CFR 51.767 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies may be obtained from, Florida Department of Citrus... Mature. Mature shall have the same meaning assigned the term in the Florida Citrus Code, Chapter 601, 1995 Edition, and the Official Rules Affecting the Florida Citrus Industry, in effect as of February...

  3. 7 CFR 51.1158 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ...(a) and 1 CFR part 51. Copies may be obtained from, Florida Department of Citrus, Post Office Box 148... § 51.1158 Mature. Mature shall have the same meaning assigned the term in the Florida Citrus Code, Chapter 601, 1995 Edition, and the Official Rules Affecting the Florida Citrus Industry, in effect as...

  4. 7 CFR 51.1158 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ...(a) and 1 CFR part 51. Copies may be obtained from, Florida Department of Citrus, Post Office Box 148... § 51.1158 Mature. Mature shall have the same meaning assigned the term in the Florida Citrus Code, Chapter 601, 1995 Edition, and the Official Rules Affecting the Florida Citrus Industry, in effect as...

  5. 7 CFR 51.1823 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ...(a) and 1 CFR part 51. Copies may be obtained from, Florida Department of Citrus, Post Office Box 148... Mature. Mature shall have the same meaning assigned the term in the Florida Citrus Code, Chapter 601, 1995 Edition, and the Official Rules Affecting the Florida Citrus Industry, in effect as of February...

  6. The FMI: Dimensions of Follower Maturity

    ERIC Educational Resources Information Center

    Moore, Loren I.

    1976-01-01

    The Follower Maturity Index (FMI) is an instrument derived from leadership theory and based on observations of verbal and nonverbal behavior of followers in task groups. Dimensions of follower maturity--achievement, responsibility, experience, activity, dependence, variety, interests, perspective, position, and awareness--are discussed. For…

  7. Canopy temperature and maturity in cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Heat units are a widely used indicator of maturity in cotton. It is generally assumed that it takes approximately 2200°F (1222°C) heat units for a cotton plant on the South High Plains of Texas to mature. This value is based on a typical planting date of May 15 with ample irrigation. As water for c...

  8. Motivation and Maturity Patterns in Marital Success.

    ERIC Educational Resources Information Center

    McClelland, David C.; And Others

    1978-01-01

    Married couples rated their marital satisfaction and played interpersonal competitive games which revealed the success with which they interacted. Younger husbands who scored more maturely on the Stewart measure of psychosocial maturity belonged to more successful marriages, as did college-educated wives who showed less immaturity and more phallic…

  9. Thinned Mature Deciduous Forest Silvopastures for Appalachia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Little information is available on effective management and utilization of silvopastures developed from the ubiquitous mature woodlots which comprise 40-50% of small Appalachian farm acreage. We thinned a white oak dominated mature second growth forested area establishing two 0.5 ha, eight-paddock,...

  10. Effects of glucose metabolism during in vitro maturation on cytoplasmic maturation of mouse oocytes.

    PubMed

    Xie, Hong-Li; Wang, Yan-Bo; Jiao, Guang-Zhong; Kong, De-Ling; Li, Qing; Li, Hong; Zheng, Liang-Liang; Tan, Jing-He

    2016-01-01

    Although there are many reports on the effect of glucose metabolism on oocyte nuclear maturation, there are few studies on its effect on ooplasmic maturation. By manipulating glucose metabolism pathways using a maturation medium that could support oocyte nuclear maturation but only a limited blastocyst formation without glucose, this study determined effects of glucose metabolism pathways on ooplasmic maturation. During maturation of cumulus-oocyte-complexes (COCs) with glucose, the presence of PPP inhibitor, DHEA or glycolysis inhibitor, iodoacetate significantly decreased blastocyst rates, intraoocyte glutathione and ATP. While blastocyst rates, GSH/GSSG ratio and NADPH were higher, ROS was lower significantly in COCs matured with iodoacetate than with DHEA. Fructose-6-phosphate overcame the inhibitory effect of DHEA on PPP. During maturation of COCs with pyruvate, electron transport inhibitor, rotenone or monocarboxylate transfer inhibitor, 4-CIN significantly decreased blastocyst rates. Cumulus-denuded oocytes had a limited capacity to use glucose or lactate, but they could use pyruvate to support maturation. In conclusion, whereas glycolysis promoted ooplasmic maturation mainly by supplying energy, PPP facilitated ooplasmic maturation to a greater extent by both reducing oxidative stress and supplying energy through providing fructose-6-phosphate for glycolysis. Pyruvate was transferred by monocarboxylate transporters and utilized through mitochondrial electron transport to sustain ooplasmic maturation. PMID:26857840

  11. Effects of glucose metabolism during in vitro maturation on cytoplasmic maturation of mouse oocytes

    PubMed Central

    Xie, Hong-Li; Wang, Yan-Bo; Jiao, Guang-Zhong; Kong, De-Ling; Li, Qing; Li, Hong; Zheng, Liang-Liang; Tan, Jing-He

    2016-01-01

    Although there are many reports on the effect of glucose metabolism on oocyte nuclear maturation, there are few studies on its effect on ooplasmic maturation. By manipulating glucose metabolism pathways using a maturation medium that could support oocyte nuclear maturation but only a limited blastocyst formation without glucose, this study determined effects of glucose metabolism pathways on ooplasmic maturation. During maturation of cumulus-oocyte-complexes (COCs) with glucose, the presence of PPP inhibitor, DHEA or glycolysis inhibitor, iodoacetate significantly decreased blastocyst rates, intraoocyte glutathione and ATP. While blastocyst rates, GSH/GSSG ratio and NADPH were higher, ROS was lower significantly in COCs matured with iodoacetate than with DHEA. Fructose-6-phosphate overcame the inhibitory effect of DHEA on PPP. During maturation of COCs with pyruvate, electron transport inhibitor, rotenone or monocarboxylate transfer inhibitor, 4-CIN significantly decreased blastocyst rates. Cumulus-denuded oocytes had a limited capacity to use glucose or lactate, but they could use pyruvate to support maturation. In conclusion, whereas glycolysis promoted ooplasmic maturation mainly by supplying energy, PPP facilitated ooplasmic maturation to a greater extent by both reducing oxidative stress and supplying energy through providing fructose-6-phosphate for glycolysis. Pyruvate was transferred by monocarboxylate transporters and utilized through mitochondrial electron transport to sustain ooplasmic maturation. PMID:26857840

  12. [Eco-physiology and regulation of leaf senescence and maturity performance in cotton: A review].

    PubMed

    Chen, Yi-zhen; Dong, He-zhong

    2016-02-01

    Senescence is a natural termination process at the plant or organ level of cotton, leading to the inevitable end of the growth and development process. Maturity performance is termed as senescence performance and results of a cotton plant during boll opening, including normal maturity, premature senescence and late-maturity in cotton. Senescence and maturity performance are influenced by genotype and environment interactions. This paper summarized and reviewed the research progress in eco-physiology and molecular biology of cotton leaf senescence. Strategies were proposed to regulate cotton growth and aging through breeding of stably-developed varieties, rational application of plant growth regulators and agronomic cultivation measures, to realize normal maturity and improve yield and quality of cotton. PMID:27396141

  13. Influence of hyaluronan accumulation during cumulus expansion on in vitro porcine oocyte maturation.

    PubMed

    Yokoo, Masaki; Kimura, Naoko; Abe, Hiroyuki; Sato, Eimei

    2008-11-01

    During oocyte maturation, the cumulus-oocyte complexes (COCs) expand dramatically. This phenomenon, which is known as cumulus expansion, is the result of the synthesis and accumulation of hyaluronan in the extracellular space between cumulus cells. The purpose of this study was to investigate the effect of 6-diazo-5-oxo-l-norleucine (DON), an inhibitor of hyaluronan synthesis, on cumulus expansion during in vitro porcine oocyte maturation and hyaluronan accumulation within COCs. Further, this study aimed to examine the influence of hyaluronan accumulation within COCs on the rate of oocyte maturation. Cumulus expansion was observed during in vitro maturation. However, the addition of DON to the maturation medium significantly inhibited cumulus expansion. The total inhibition of hyaluronan accumulation within COCs was observed with the use of confocal microscopy. Moreover, a positive correlation between the area of cumulus expansion and the rate of oocyte maturation was observed. These results demonstrate that the hyaluronan accumulation within the COCs during oocyte maturation affects oocyte maturation. On the basis of these results, we propose that hyaluronan accumulation within the COCs during cumulus expansion is a necessary step in the porcine oocyte maturation process. PMID:18838022

  14. High-affinity FRβ-specific CAR T cells eradicate AML and normal myeloid lineage without HSC toxicity.

    PubMed

    Lynn, R C; Feng, Y; Schutsky, K; Poussin, M; Kalota, A; Dimitrov, D S; Powell, D J

    2016-06-01

    Acute myeloid leukemia (AML) is an aggressive malignancy, and development of new treatments to prolong remissions is warranted. Chimeric antigen receptor (CAR) T-cell therapies appear promising but on-target, off-tumor recognition of antigen in healthy tissues remains a concern. Here we isolated a high-affinity (HA) folate receptor beta (FRβ)-specific single-chain variable fragment (2.48 nm KD) for optimization of FRβ-redirected CAR T-cell therapy for AML. T cells stably expressing the HA-FRβ CAR exhibited greatly enhanced antitumor activity against FRβ(+) AML in vitro and in vivo compared with a low-affinity FRβ CAR (54.3 nm KD). Using the HA-FRβ immunoglobulin G, FRβ expression was detectable in myeloid-lineage hematopoietic cells; however, expression in CD34(+) hematopoietic stem cells (HSCs) was nearly undetectable. Accordingly, HA-FRβ CAR T cells lysed mature CD14(+) monocytes, while HSC colony formation was unaffected. Because of the potential for elimination of mature myeloid lineage, mRNA CAR electroporation for transient CAR expression was evaluated. mRNA-electroporated HA-FRβ CAR T cells retained effective antitumor activity in vitro and in vivo. Together, our results highlight the importance of antibody affinity in target protein detection and CAR development and suggest that transient delivery of potent HA-FRβ CAR T cells is highly effective against AML and reduces the risk for long-term myeloid toxicity. PMID:26898190

  15. Affinity Crystallography: A New Approach to Extracting High-Affinity Enzyme Inhibitors from Natural Extracts.

    PubMed

    Aguda, Adeleke H; Lavallee, Vincent; Cheng, Ping; Bott, Tina M; Meimetis, Labros G; Law, Simon; Nguyen, Nham T; Williams, David E; Kaleta, Jadwiga; Villanueva, Ivan; Davies, Julian; Andersen, Raymond J; Brayer, Gary D; Brömme, Dieter

    2016-08-26

    Natural products are an important source of novel drug scaffolds. The highly variable and unpredictable timelines associated with isolating novel compounds and elucidating their structures have led to the demise of exploring natural product extract libraries in drug discovery programs. Here we introduce affinity crystallography as a new methodology that significantly shortens the time of the hit to active structure cycle in bioactive natural product discovery research. This affinity crystallography approach is illustrated by using semipure fractions of an actinomycetes culture extract to isolate and identify a cathepsin K inhibitor and to compare the outcome with the traditional assay-guided purification/structural analysis approach. The traditional approach resulted in the identification of the known inhibitor antipain (1) and its new but lower potency dehydration product 2, while the affinity crystallography approach led to the identification of a new high-affinity inhibitor named lichostatinal (3). The structure and potency of lichostatinal (3) was verified by total synthesis and kinetic characterization. To the best of our knowledge, this is the first example of isolating and characterizing a potent enzyme inhibitor from a partially purified crude natural product extract using a protein crystallographic approach. PMID:27498895

  16. Arnold Gesell and the maturation controversy.

    PubMed

    Dalton, Thomas C

    2005-01-01

    This article examines the work of Arnold Lucius Gesell and argues that he not only paved the way for contemporary research in motor development, but that he and colleagues anticipated fundamental issues about growth that must be addressed by psychologists and neuroscientists who are committed to the advancement of developmental science. Arnold Lucius Gesell was a pioneer in developmental psychology when the field was in its infancy. He worked diligently for the rights of physically and mentally handicapped children to receive special education that would enable them to find gainful employment. Gesell's writings in books and popular magazines increased public awareness of and support for preschool education and better foster care for orphans. Despite these achievements, many of his successors have questioned his views about infant development. Developmental psychologists have criticized Gesell for proposing a stage theory of infant growth that has fallen into disfavor among contemporary researchers. His conception of development as a maturational process has been challenged for allegedly reducing complex behavioral, perceptual, and learning processes to genetic factors. The author rejects this overly simplistic interpretation and contends that Gesell's work continues to stand the test of time. PMID:17549936

  17. MicroRNA Expression during Bovine Oocyte Maturation and Fertilization

    PubMed Central

    Gilchrist, Graham C.; Tscherner, Allison; Nalpathamkalam, Thomas; Merico, Daniele; LaMarre, Jonathan

    2016-01-01

    Successful fertilization and subsequent embryo development rely on complex molecular processes starting with the development of oocyte competence through maturation. MicroRNAs (miRNAs) are small non-coding RNA molecules that function as gene regulators in many biological systems, including the oocyte and embryo. In order to further explore the roles of miRNAs in oocyte maturation, we employed small RNA sequencing as a screening tool to identify and characterize miRNA populations present in pools of bovine germinal vesicle (GV) oocytes, metaphase II (MII) oocytes, and presumptive zygotes (PZ). Each stage contained a defined miRNA population, some of which showed stable expression while others showed progressive changes between stages that were subsequently confirmed by quantitative reverse transcription polymerase chain reaction (RT-PCR). Bta-miR-155, bta-miR-222, bta-miR-21, bta-let-7d, bta-let-7i, and bta-miR-190a were among the statistically significant differentially expressed miRNAs (p < 0.05). To determine whether changes in specific primary miRNA (pri-miRNA) transcripts were responsible for the observed miRNA changes, we evaluated pri-miR-155, -222 and let-7d expression. Pri-miR-155 and -222 were not detected in GV oocytes but pri-miR-155 was present in MII oocytes, indicating transcription during maturation. In contrast, levels of pri-let-7d decreased during maturation, suggesting that the observed increase in let-7d expression was likely due to processing of the primary transcript. This study demonstrates that both dynamic and stable populations of miRNAs are present in bovine oocytes and zygotes and extend previous studies supporting the importance of the small RNA landscape in the maturing bovine oocyte and early embryo. PMID:26999121

  18. MicroRNA Expression during Bovine Oocyte Maturation and Fertilization.

    PubMed

    Gilchrist, Graham C; Tscherner, Allison; Nalpathamkalam, Thomas; Merico, Daniele; LaMarre, Jonathan

    2016-01-01

    Successful fertilization and subsequent embryo development rely on complex molecular processes starting with the development of oocyte competence through maturation. MicroRNAs (miRNAs) are small non-coding RNA molecules that function as gene regulators in many biological systems, including the oocyte and embryo. In order to further explore the roles of miRNAs in oocyte maturation, we employed small RNA sequencing as a screening tool to identify and characterize miRNA populations present in pools of bovine germinal vesicle (GV) oocytes, metaphase II (MII) oocytes, and presumptive zygotes (PZ). Each stage contained a defined miRNA population, some of which showed stable expression while others showed progressive changes between stages that were subsequently confirmed by quantitative reverse transcription polymerase chain reaction (RT-PCR). Bta-miR-155, bta-miR-222, bta-miR-21, bta-let-7d, bta-let-7i, and bta-miR-190a were among the statistically significant differentially expressed miRNAs (p < 0.05). To determine whether changes in specific primary miRNA (pri-miRNA) transcripts were responsible for the observed miRNA changes, we evaluated pri-miR-155, -222 and let-7d expression. Pri-miR-155 and -222 were not detected in GV oocytes but pri-miR-155 was present in MII oocytes, indicating transcription during maturation. In contrast, levels of pri-let-7d decreased during maturation, suggesting that the observed increase in let-7d expression was likely due to processing of the primary transcript. This study demonstrates that both dynamic and stable populations of miRNAs are present in bovine oocytes and zygotes and extend previous studies supporting the importance of the small RNA landscape in the maturing bovine oocyte and early embryo. PMID:26999121

  19. Structure of a High-Affinity

    SciTech Connect

    Saphire, E.O.; Montero, M.; Menendez, A.; Houten, N.E.van; Irving, M.B.; Pantophlet, R.; Swick, M.B.; Parren, P.W.H.I.; Burton, D.R.; Scott, J.K.; Wilson, I.A.; /Scripps Res. Inst. /Simon Fraser U. /British Columbia U.

    2007-07-13

    The human antibody b12 recognizes a discontinuous epitope on gp120 and is one of the rare monoclonal antibodies that neutralize a broad range of primary human immunodeficiency virus type 1 (HIV-1) isolates. We previously reported the isolation of B2.1, a dimeric peptide that binds with high specificity to b12 and competes with gp120 for b12 antibody binding. Here, we show that the affinity of B2.1 was improved 60-fold over its synthetic-peptide counterpart by fusing it to the N terminus of a soluble protein. This affinity, which is within an order of magnitude of that of gp120, probably more closely reflects the affinity of the phage-borne peptide. The crystal structure of a complex between Fab of b12 and B2.1 was determined at 1.8 Angstrom resolution. The structural data allowed the differentiation of residues that form critical contacts with b12 from those required for maintenance of the antigenic structure of the peptide, and revealed that three contiguous residues mediate B2.1's critical contacts with b12. This single region of critical contact between the B2.1 peptide and the b12 paratope is unlikely to mimic the discontinuous key binding residues involved in the full b12 epitope for gp120, as previously identified by alanine scanning substitutions on the gp120 surface. These structural observations are supported by experiments that demonstrate that B2.1 is an ineffective immunogenic mimic of the b12 epitope on gp120. Indeed, an extensive series of immunizations with B2.1 in various forms failed to produce gp120 cross-reactive sera. The functional and structural data presented here, however, suggest that the mechanism by which b12 recognizes the two antigens is very different. Here, we present the first crystal structure of peptide bound to an antibody that was originally raised against a discontinuous protein epitope. Our results highlight the challenge of producing immunogens that mimic discontinuous protein epitopes, and the necessity of combining

  20. Isolation and optimization for affinity and biophysical characteristics of anti-CCL17 antibodies from the VH1-69 germline gene.

    PubMed

    Kehoe, John W; Whitaker, Brian; Bethea, Deidra; Lacy, Eilyn R; Boakye, Ken; Santulli-Marotto, Sandra; Ryan, Mary H; Feng, Yiqing; Wheeler, John C

    2014-06-01

    CCL17 is a homeostatic chemokine associated with several human inflammatory pathologies. This makes CCL17 a potential point of intervention in inflammatory diseases. Using a Fab-pIX phage display system we were able to select antibodies that specifically bind to CCL17 and neutralize CCL17-mediated signaling. Many of the selected antibodies belong to the VH1-69 germline gene family. The VH1-69 germline gene is represented at a high frequency in the human antibody repertoire and is seen in the early immune response to a variety of pathogens. The heavy chain CDR2 of this germline gene is notably hydrophobic and can insert into hydrophobic pockets of antigens, providing much of the binding energy for these antibodies. Affinity maturation of our primary binders by light chain mutagenesis produced antibodies with sub-nanomolar affinities, with affinity improvements up to 100-fold. These were screened for non-specific protein-protein interactions as a filter for solubility. All of our high affinity antibodies were found to have high levels of non-specific protein-protein interactions. We speculated that this was due to the hydrophobicity within the germline heavy chain CDR1 and CDR2. To ameliorate this problem, we generated a phage display library for one of the clones, where the surface-exposed residues within H-CDR1 and H-CDR2 were randomized. High stringency panning of this library against human CCL17 resulted in further affinity improvement, along with reduction in protein-protein interaction in some new variants. In addition, we improved the cross-reactivity to cynomolgus CCL17. We demonstrate that affinity maturation through targeted libraries in the VH1-69 germline gene can improve both affinity and biophysical characteristics of antibodies derived from this gene scaffold. PMID:24742503

  1. A novel 2,5-diaminopyrimidine-based affinity probe for Bruton’s tyrosine kinase

    PubMed Central

    Zuo, Yingying; Shi, Yanxia; Li, Xitao; Teng, Yingqi; Pan, Zhengying

    2015-01-01

    As a critical regulator of the B-cell receptor signaling pathway, Bruton’s tyrosine kinase (Btk) has attracted intensive drug discovery efforts for treating B-cell lineage cancers and autoimmune disorders. In particular, covalent inhibitors targeting Cys481 in Btk have demonstrated impressive clinical benefits, and their companion affinity probes have been crucial in the drug development process. Recently, we have discovered a novel series of 2,5-diaminopyrimidine-based covalent irreversible inhibitors of Btk. Here, we present the discovery of a novel affinity Btk probe based on the aforementioned scaffold and demonstrate its usage in evaluating the target engagement of Btk inhibitors in live cells. PMID:26531233

  2. A diffusion model for picosecond electron bunches from negative electron affinity GaAs photo cathodes

    SciTech Connect

    P. Hartmann; J. Bermuth; D. v. Harrach; J. Hoffmann; S. Kobis; E. Reichert; K. Aulenbacher; J. Schuler; M. Steigerwald

    1998-10-27

    Even though theoretical estimates predict response times for the photo emission process of electrons from a negative electron affinity GaAs photo emitter in excess of hundreds of picoseconds, recent measurements found electron bunch durations of 40 ps or less. This work presents precise measurements of picosecond electron bunches from a negative affinity bulk GaAs photo cathode and develops a model which explains the measured bunch durations as well as the observed bunch shapes. The bunch shape turns out to be independent from the quantum efficiency of the photo emitter.

  3. A novel 2,5-diaminopyrimidine-based affinity probe for Bruton’s tyrosine kinase

    NASA Astrophysics Data System (ADS)

    Zuo, Yingying; Shi, Yanxia; Li, Xitao; Teng, Yingqi; Pan, Zhengying

    2015-11-01

    As a critical regulator of the B-cell receptor signaling pathway, Bruton’s tyrosine kinase (Btk) has attracted intensive drug discovery efforts for treating B-cell lineage cancers and autoimmune disorders. In particular, covalent inhibitors targeting Cys481 in Btk have demonstrated impressive clinical benefits, and their companion affinity probes have been crucial in the drug development process. Recently, we have discovered a novel series of 2,5-diaminopyrimidine-based covalent irreversible inhibitors of Btk. Here, we present the discovery of a novel affinity Btk probe based on the aforementioned scaffold and demonstrate its usage in evaluating the target engagement of Btk inhibitors in live cells.

  4. Temperature dependence of high-affinity CCK receptor binding and CCK internalization in rat pancreatic acini

    SciTech Connect

    Williams, J.A.; Bailey, A.C.; Roach, E. Univ. of California, San Francisco )

    1988-04-01

    {sup 125}I-labeled cholecystokinin (CCK) binding and internalization were studied as a function of temperatures in isolated rat pancreatic acini. At 37{degree}C, acini readily bound and degraded {sup 125}I-CCK. When labeled hormone binding was inhibited by increasing amounts of unlabeled CCK, competition-inhibition curves were biphasic, consistent with both high- (K{sub d}, 18 pM) and low-affinity (K{sub d}, 13 nM) binding sites. At 4{degree}C, acini bound only one-third as much {sup 125}I-CCK and degradation was essentially abolished. At 4{degree}C, CCK competition curves were consistent with a single class of low-affinity binding sites (K{sub d}, 19 nM). Internalization of {sup 125}I-CCK was evaluated by three washing procedures utilizing acid, base, and trypsin. All were shown to remove membrane-bound {sup 125}I-CCK, and this finding was validated for trypsin by electron microscope autotradiography. When internalization of {sup 125}I-CCK was evaluated as a function of the medium concentration of CCK, both high- and low-affinity components were observed. These results suggest that high-affinity CCK binding and CCK internalization are separate temperature-sensitive processes. Moreover, internalization is not uniquely associated with high-affinity binding.

  5. [Estrogens and feminine brain maturation during adolescence: emergency contraceptive pill].

    PubMed

    López Moratalla, Natalia; Errasti Alcalá, Tania; Santiago, Esteban

    2011-01-01

    In the period between puberty and maturity takes place the process of brain maturation. Hormone levels induce changes in neurons and direct the architecture and structural functionality thus affecting patterns of development of different brain areas. The onset of puberty brings with it the invasion of the female brain by high levels of hormones, cyclic surges of estrogen and progesterone in addition to steroids produced in situ. Control centers of emotions (amygdala), memory and learning (hippocampus) and sexual activity (hypothalamus) are modified according to the cyclical concentrations of both hormones. Sex hormones stimulate multimodal actions, both short and longer terms, because neurons in various brain areas have different types of receptors, membrane, cytoplasmic and nuclear. The composition of emergency contraceptive pill (postcoital pill) with high hormonal content raises the urgency of a thorough knowledge about the possible effect that the lack of control of the menstrual cycle in a time of consolidation of brain maturation, can bring in structuring and development of brain circuitry. Changes in the availability of sex steroids during puberty and adolescence underlie psychiatric disorders whose prevalence is typically feminine, such as depression, anxiety disorders. It is a fundamental ethical duty to present scientific data about the influence of estrogen in young female brain maturation, both for full information to potential users, and also to induce the appropriate public health measures. PMID:22040134

  6. Identification of potent maturation inhibitors against HIV-1 clade C.

    PubMed

    Timilsina, Uddhav; Ghimire, Dibya; Timalsina, Bivek; Nitz, Theodore J; Wild, Carl T; Freed, Eric O; Gaur, Ritu

    2016-01-01

    Antiretroviral therapy has led to a profound improvement in the clinical care of HIV-infected patients. However, drug tolerability and the evolution of drug resistance have limited treatment options for many patients. Maturation inhibitors are a new class of antiretroviral agents for treatment of HIV-1. They act by interfering with the maturation of the virus by blocking the last step in Gag processing: the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA by the viral protease (PR). The first-in-class maturation inhibitor bevirimat (BVM) failed against a subset of HIV-1 isolates in clinical trials due to polymorphisms present in the CA-SP1 region of the Gag protein. Sequence analysis indicated that these polymorphisms are more common in non-clade B strains of HIV-1 such as HIV-1 clade C. Indeed, BVM was found to be ineffective against HIV-1 clade C molecular clones tested in this study. A number of BVM analogs were synthesized by chemical modifications at the C-28 position to improve its activity. The new BVM analogs displayed potent activity against HIV-1 clade B and C and also reduced infectivity of the virus. This study identifies novel and broadly active BVM analogs that may ultimately demonstrate efficacy in the clinic. PMID:27264714

  7. CCN1 Regulates Chondrocyte Maturation and Cartilage Development

    PubMed Central

    Zhang, Yongchun; Sheu, Tzong-jen; Hoak, Donna; Shen, Jie; Hilton, Matthew J; Zuscik, Michael J; Jonason, Jennifer H; O’Keefe, Regis J

    2016-01-01

    WNT/β-CATENIN signaling is involved in multiple aspects of skeletal development, including chondrocyte differentiation and maturation. Although the functions of β-CATENIN in chondrocytes have been extensively investigated through gain-of-function and loss-of-function mouse models, the precise downstream effectors through which β-CATENIN regulates these processes are not well defined. Here, we report that the matricellular protein, CCN1, is induced by WNT/β-CATENIN signaling in chondrocytes. Specifically, we found that β-CATENIN signaling promotes CCN1 expression in isolated primary sternal chondrocytes and both embryonic and postnatal cartilage. Additionally, we show that, in vitro, CCN1 overexpression promotes chondrocyte maturation, whereas inhibition of endogenous CCN1 function inhibits maturation. To explore the role of CCN1 on cartilage development and homeostasis in vivo, we generated a novel transgenic mouse model for conditional Ccn1 overexpression and show that cartilage-specific CCN1 overexpression leads to chondrodysplasia during development and cartilage degeneration in adult mice. Finally, we demonstrate that CCN1 expression increases in mouse knee joint tissues after meniscal/ligamentous injury (MLI) and in human cartilage after meniscal tear. Collectively, our data suggest that CCN1 is an important regulator of chondrocyte maturation during cartilage development and homeostasis. PMID:26363286

  8. CCN1 Regulates Chondrocyte Maturation and Cartilage Development.

    PubMed

    Zhang, Yongchun; Sheu, Tzong-Jen; Hoak, Donna; Shen, Jie; Hilton, Matthew J; Zuscik, Michael J; Jonason, Jennifer H; O'Keefe, Regis J

    2016-03-01

    WNT/β-CATENIN signaling is involved in multiple aspects of skeletal development, including chondrocyte differentiation and maturation. Although the functions of β-CATENIN in chondrocytes have been extensively investigated through gain-of-function and loss-of-function mouse models, the precise downstream effectors through which β-CATENIN regulates these processes are not well defined. Here, we report that the matricellular protein, CCN1, is induced by WNT/β-CATENIN signaling in chondrocytes. Specifically, we found that β-CATENIN signaling promotes CCN1 expression in isolated primary sternal chondrocytes and both embryonic and postnatal cartilage. Additionally, we show that, in vitro, CCN1 overexpression promotes chondrocyte maturation, whereas inhibition of endogenous CCN1 function inhibits maturation. To explore the role of CCN1 on cartilage development and homeostasis in vivo, we generated a novel transgenic mouse model for conditional Ccn1 overexpression and show that cartilage-specific CCN1 overexpression leads to chondrodysplasia during development and cartilage degeneration in adult mice. Finally, we demonstrate that CCN1 expression increases in mouse knee joint tissues after meniscal/ligamentous injury (MLI) and in human cartilage after meniscal tear. Collectively, our data suggest that CCN1 is an important regulator of chondrocyte maturation during cartilage development and homeostasis. © 2015 American Society for Bone and Mineral Research. PMID:26363286

  9. Control of Oocyte Growth and Meiotic Maturation in C. elegans

    PubMed Central

    Kim, Seongseop; Spike, Caroline; Greenstein, David

    2013-01-01

    In sexually reproducing animals, oocytes arrest at diplotene or diakinesis and resume meiosis (meiotic maturation) in response to hormones. Chromosome segregation errors in female meiosis I are the leading cause of human birth defects, and age-related changes in the hormonal environment of the ovary are a suggested cause. C. elegans is emerging as a genetic paradigm for studying hormonal control of meiotic maturation. The meiotic maturation processes in C. elegans and mammals share a number of biological and molecular similarities. Major sperm protein (MSP) and luteinizing hormone (LH), though unrelated in sequence, both trigger meiotic resumption using somatic Gαs-adenylate cyclase pathways and soma-germline gap-junctional communication. At a molecular level, the oocyte responses apparently involve the control of conserved protein kinase pathways and post-transcriptional gene regulation in the oocyte. At a cellular level, the responses include cortical cytoskeletal rearrangement, nuclear envelope breakdown, assembly of the acentriolar meiotic spindle, chromosome segregation, and likely changes important for fertilization and the oocyte-to-embryo transition. This chapter focuses on signaling mechanisms required for oocyte growth and meiotic maturation in C. elegans and discusses how these mechanisms coordinate the completion of meiosis and the oocyte-to-embryo transition. PMID:22872481

  10. Identification of potent maturation inhibitors against HIV-1 clade C

    PubMed Central

    Timilsina, Uddhav; Ghimire, Dibya; Timalsina, Bivek; Nitz, Theodore J.; Wild, Carl T.; Freed, Eric O.; Gaur, Ritu

    2016-01-01

    Antiretroviral therapy has led to a profound improvement in the clinical care of HIV-infected patients. However, drug tolerability and the evolution of drug resistance have limited treatment options for many patients. Maturation inhibitors are a new class of antiretroviral agents for treatment of HIV-1. They act by interfering with the maturation of the virus by blocking the last step in Gag processing: the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA by the viral protease (PR). The first-in-class maturation inhibitor bevirimat (BVM) failed against a subset of HIV-1 isolates in clinical trials due to polymorphisms present in the CA-SP1 region of the Gag protein. Sequence analysis indicated that these polymorphisms are more common in non-clade B strains of HIV-1 such as HIV-1 clade C. Indeed, BVM was found to be ineffective against HIV-1 clade C molecular clones tested in this study. A number of BVM analogs were synthesized by chemical modifications at the C-28 position to improve its activity. The new BVM analogs displayed potent activity against HIV-1 clade B and C and also reduced infectivity of the virus. This study identifies novel and broadly active BVM analogs that may ultimately demonstrate efficacy in the clinic. PMID:27264714

  11. Robust template matching for affine resistant image watermarks.

    PubMed

    Pereira, S; Pun, T

    2000-01-01

    Digital watermarks have been proposed as a method for discouraging illicit copying and distribution of copyrighted material. This paper describes a method for the secure and robust copyright protection of digital images. We present an approach for embedding a digital watermark into an image using the Fourier transform. To this watermark is added a template in the Fourier transform domain to render the method robust against general linear transformations. We detail a new algorithm based on polar maps for the accurate and efficient recovery of the template in an image which has undergone a general affine transformation. We also present results which demonstrate the robustness of the method against some common image processing operations such as compression, rotation, scaling, and aspect ratio changes. PMID:18255481

  12. 3D affine registration using teaching-learning based optimization

    NASA Astrophysics Data System (ADS)

    Jani, Ashish; Savsani, Vimal; Pandya, Abhijit

    2013-09-01

    3D image registration is an emerging research field in the study of computer vision. In this paper, two effective global optimization methods are considered for the 3D registration of point clouds. Experiments were conducted by applying each algorithm and their performance was evaluated with respect to rigidity, similarity and affine transformations. Comparison of algorithms and its effectiveness was tested for the average performance to find the global solution for minimizing the error in the terms of distance between the model cloud and the data cloud. The parameters for the transformation matrix were considered as the design variables. Further comparisons of the considered methods were done for the computational effort, computational time and the convergence of the algorithm. The results reveal that the use of TLBO was outstanding for image processing application involving 3D registration. [Figure not available: see fulltext.

  13. Advances in affinity ligand-functionalized nanomaterials for biomagnetic separation.

    PubMed

    Fields, Conor; Li, Peng; O'Mahony, James J; Lee, Gil U

    2016-01-01

    The downstream processing of proteins remains the most significant cost in protein production, and is largely attributed to rigorous chromatographic purification protocols, where the stringency of purity for biopharmaceutical products sometimes exceeds 99%. With an ever burgeoning biotechnology market, there is a constant demand for alternative purification methodologies, to ameliorate the dependence on chromatography, while still adhering to regulatory concerns over product purity and safety. In this article, we present an up-to-date view of bioseparation, with emphasis on magnetic separation and its potential application in the field. Additionally, we discuss the economic and performance benefits of synthetic ligands, in the form of peptides and miniaturized antibody fragments, compared to full-length antibodies. We propose that adoption of synthetic affinity ligands coupled with magnetic adsorbents, will play an important role in enabling sustainable bioprocessing in the future. PMID:26032605

  14. Political ideology: its structure, functions, and elective affinities.

    PubMed

    Jost, John T; Federico, Christopher M; Napier, Jaime L

    2009-01-01

    Ideology has re-emerged as an important topic of inquiry among social, personality, and political psychologists. In this review, we examine recent theory and research concerning the structure, contents, and functions of ideological belief systems. We begin by defining the construct and placing it in historical and philosophical context. We then examine different perspectives on how many (and what types of) dimensions individuals use to organize their political opinions. We investigate (a) how and to what extent individuals acquire the discursive contents associated with various ideologies, and (b) the social-psychological functions that these ideologies serve for those who adopt them. Our review highlights "elective affinities" between situational and dispositional needs of individuals and groups and the structure and contents of specific ideologies. Finally, we consider the consequences of ideology, especially with respect to attitudes, evaluations, and processes of system justification. PMID:19035826

  15. Automatic gesture analysis using constant affine velocity.

    PubMed

    Cifuentes, Jenny; Boulanger, Pierre; Pham, Minh Tu; Moreau, Richard; Prieto, Flavio

    2014-01-01

    Hand human gesture recognition has been an important research topic widely studied around the world, as this field offers the ability to identify, recognize, and analyze human gestures in order to control devices or to interact with computer interfaces. In particular, in medical training, this approach is an important tool that can be used to obtain an objective evaluation of a procedure performance. In this paper, some obstetrical gestures, acquired by a forceps, were studied with the hypothesis that, as the scribbling and drawing movements, they obey the one-sixth power law, an empirical relationship which connects path curvature, torsion, and euclidean velocity. Our results show that obstetrical gestures have a constant affine velocity, which is different for each type of gesture and based on this idea this quantity is proposed as an appropriate classification feature in the hand human gesture recognition field. PMID:25570332

  16. Effectively nonlocal metric-affine gravity

    NASA Astrophysics Data System (ADS)

    Golovnev, Alexey; Koivisto, Tomi; Sandstad, Marit

    2016-03-01

    In metric-affine theories of gravity such as the C-theories, the spacetime connection is associated to a metric that is nontrivially related to the physical metric. In this article, such theories are rewritten in terms of a single metric, and it is shown that they can be recast as effectively nonlocal gravity. With some assumptions, known ghost-free theories with nonsingular and cosmologically interesting properties may be recovered. Relations between different formulations are analyzed at both perturbative and nonperturbative levels, taking carefully into account subtleties with boundary conditions in the presence of integral operators in the action, and equivalences between theories related by nonlocal redefinitions of the fields are verified at the level of equations of motion. This suggests a possible geometrical interpretation of nonlocal gravity as an emergent property of non-Riemannian spacetime structure.

  17. Affinities of the Swartkrans early Homo mandibles.

    PubMed

    Curnoe, Darren

    2008-01-01

    The southern African early Homo assemblage continues to make important contributions to understanding the systematics, adaptations and evolutionary history of the human genus. However, the taxonomy of this sample is in a state of flux. This study examines the size and shape of the mandibular bodies of Swartkrans SK 15 and SK 45 comparing them with variation in two early Homo taxa (H. habilis sensu lato and H. sapiens erectus). The research aims to clarify their phenetic affinities and systematics through univariate statistics, inferential testing and multivariate analysis employing size (Log-transformed) and shape (Mosimann variables). Neither of them strongly resembles H. habilis sensu lato or H. sapiens erectus, rather, they probably sample a novel species of Homo not seen in East Africa. Moreover, there is considerable morphological variability within the Swartkrans sample and the possibility of more than one novel species being sampled at this site cannot be excluded. PMID:18402959

  18. Wetting on rough self-affine surfaces

    NASA Astrophysics Data System (ADS)

    Palasantzas, George

    1995-05-01

    In this paper, we present a general investigation of the effective potential for complete wetting on self-affine rough surfaces. The roughness effect is investigated by means of the height-height correlation model in Fourier space ~(1+aξ2q2)-1-H. The parameters H and ξ are, respectively, the roughness exponent and the substrate in-plane correlation length. It is observed that the effect of H on the free interface profile is significant for ξ>ξ) regime is characterized by a power-law scaling ~Y-2.

  19. Novel thermo-responsive fucose binding ligands for glycoprotein purification by affinity precipitation.

    PubMed

    Arnold, Lindsay; Chen, Rachel

    2014-02-01

    Novel thermo-responsive affinity sugar binders were developed by fusing a bacterial fucose lectin with a thermo-responsive polypeptide. These designer affinity ligand fusions were produced using an Escherichia coli system capable of extracellular secretion of recombinant proteins and were isolated with a high recovery yield (95%) directly from growth medium by Inverse Temperature Cycling (ITC). With horse radish peroxidase (HRP) as a model protein, we demonstrate here that the designer thermo-responsive ligands are capable of interacting with glycans on a glycoprotein, a property that was used to develop a novel affinity precipitation method for glycoprotein purification. The method, requiring only simple process steps, affords full recovery of a target glycoprotein, and is effective at a target glycoprotein concentration as low as 1.4 pM in the presence of large amounts of contaminants. By developing other sugar binders in the similar fashion, the method should be highly useful for glycoprotein purification and detection. PMID:25271333

  20. Dye affinity cryogels for plasmid DNA purification.

    PubMed

    Çimen, Duygu; Yılmaz, Fatma; Perçin, Işık; Türkmen, Deniz; Denizli, Adil

    2015-11-01

    The aim of this study is to prepare megaporous dye-affinity cryogel discs for the purification of plasmid DNA (pDNA) from bacterial lysate. Poly(hydroxyethyl methacrylate) [PHEMA] cryogel discs were produced by free radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) redox pair in an ice bath. Cibacron Blue F3GA was used as an affinity ligand (loading amount: 68.9μmol/g polymer). The amount of pDNA adsorbed onto the PHEMA-Cibacron Blue F3GA cryogel discs first increased and then reached a plateau value (i.e., 32.5mg/g cryogel) at 3.0mg/mL pDNA concentration. Compared with the PHEMA cryogel (0.11mg/g cryogel), the pDNA adsorption capacity of the PHEMA-Cibacron Blue F3GA cryogel (32.4mg/g polymer) was improved significantly due to the Cibacron Blue 3GA immobilization onto the polymeric matrix. pDNA adsorption amount decreased from 11.7mg/g to 1.1mg/g with the increasing of NaCl concentration. The maximum pDNA adsorption was achieved at 4°C. The overall recovery of pDNA was calculated as 90%. The PHEMA-Cibacron Blue F3GA cryogel discs could be used five times without decreasing the pDNA adsorption capacity significantly. The results show that the PHEMA-Cibacron Blue F3GA cryogel discs promise high selectivity for pDNA. PMID:26249596

  1. Salt restrains maturation in subsalt plays

    SciTech Connect

    Mello, U.T. ); Anderson, R.N.; Karner, G.D. . Lamont-Doherty Earth Observatory)

    1994-01-31

    The thermal positive anomaly associated with the top of salt diapirs has attracted significant attention in modifying the temperature structure and history of a sedimentary basin. Here the authors explore the role of the negative thermal anomaly beneath salt in modifying the maturation history of the source rocks in subsalt sediments. Organic matter maturation is believed to follow temperature dependent chemical reactions. Therefore, any temperature anomaly associated with salt masses affects the nearby maturation of potential source rocks. The level of maturity of source rocks close to salt diapirs will differ from that predicted based on regional trends. The impact of the thermal anomaly on a given point will depend on the duration and distance of the thermal anomaly to this particular point. Consequently, the maturation history of source rocks in salt basins is closely related to the salt motion history, implying that a transient thermal analysis is necessary to evaluate the sure impact on maturation of the thermal anomalies associated with salt diapirism. The paper describes vitrinite kinetics, salt in evolving basins, correlation of salt and temperature, salt dome heat drains, and restrained maturation.

  2. Protein syntehsis during soybean seed maturation

    SciTech Connect

    Rosenberg, L.A.; Rinne, R.W.

    1987-04-01

    The authors previous work has demonstrated that physiological and biochemical changes specifically associated with soybean seed maturation can be separated from events associated with seed development. The objective of this study was to determine if soybean seed metabolism is altered during maturation drying at the level of protein synthesis. Seed harvested 35 days after flowering (0% seedling growth) were induced to mature (100% seedling growth) through controlled dehydration. Proteins labeled with (/sup 35/S)-methionine were extracted and analyzed by 1-D PAGE coupled with autoradiography and densitometry. Results show a 31 kD and 128 kD polypeptide synthesized de novo during dehydration and precocious maturation. The same two polypeptides are synthesized during natural dehydration and maturation (>60 days after flowering). Furthermore, these polypeptides persist during rehydration and germination of both precociously and naturally matured seed, but specifically disappear during early seedling growth. The authors are currently investigating the role of protein synthesis during soybean seed maturation and if it is required for establishment of a soybean seedling.

  3. High-affinity uptake of noradrenaline in postsynaptic neurones.

    PubMed Central

    al-Damluji, S.; Krsmanovic, L. Z.; Catt, K. J.

    1993-01-01

    1. Neurotransmitters released from nerve endings are inactivated by re-uptake into the presynaptic nerve terminals and possibly into neighbouring glial cells. While analysing the functional properties of alpha 1-adrenoceptors in the hypothalamus, we observed a high-affinity uptake process for noradrenaline in postsynaptic peptidergic neurones. 2. In primary hypothalamic cell cultures and in a hypothalamic neuronal cell line, [3H]-prazosin bound with high affinity and was displaced by unlabelled prazosin in concentrations of 10(-10) to 10(-7) M. However, at concentrations of unlabelled prazosin above 10(-7) M, there was a paradoxical increase in apparent [3H]-prazosin binding. 3. Methoxamine, an alpha 1-adrenoceptor ligand that is not subject to significant neuronal uptake, displaced [3H]-prazosin but did not cause the paradoxical increase in the apparent binding of [3H]-prazosin. Cooling the cells to 4 degrees C reduced the total amount of prazosin associated with the cells; under these conditions, methoxamine almost completely inhibited [3H]-prazosin binding to the cells. 4. In the presence of desipramine (DMI), unlabelled prazosin displaced [3H]-prazosin as before, but no paradoxical increase in apparent binding was seen above 10(-7) M. 5. The paradoxical increase of [3H]-prazosin binding was not observed in membrane preparations of hypothalamic neurones. These findings indicated that the paradoxical increase in apparent [3H]-prazosin binding was due to a cellular uptake process that becomes evident at high concentrations of the ligand. 6. DMI (10(-5) M) had no effect on the specific binding of [3H]-prazosin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8358534

  4. Prediction of Neutral Salt Elution Profiles for Affinity Chromatography

    NASA Astrophysics Data System (ADS)

    Robinson, Jack B.; Strottmann, James M.; Stellwagen, Earle

    1981-04-01

    Neutral salts exhibit very marked differences as eluants of proteins from affinity columns. We observe: (i) that the relative potencies of neutral salts as eluants are independent of the protein or the affinity ligand in the systems studied, (ii) that the absolute salt concentration necessary to elute any given protein bound to the affinity matrix is proportional to the algebraic sum of a set of elution coefficients defined herein for the separate ions present in the solution, and (iii) that the proportionality between elution potency and elution coefficient is a function of the affinity of the protein for the immobilized ligand. Given the concentration of one neutral salt required for elution of a protein of interest from an affinity column, the elution capability of any neutral salt at any temperature can be quantitatively predicted for that protein. Accordingly, application and elution protocols for affinity chromatography can be designed to optimize the yield and fold purification of proteins.

  5. Paraoxonase 1 and HDL maturation.

    PubMed

    Gugliucci, Alejandro; Menini, Teresita

    2015-01-15

    Understanding the kinetics and function of paraoxonase 1 (PON1) is becoming an important issue in atherosclerosis. Low PON1 activity has been consistently linked with an increased risk of major cardiovascular events in the setting of secondary prevention of coronary artery disease. Recent studies have shown that there is a specific interaction of myeloperoxidase (MPO)-apoAI-PON1 on HDL surface that seems to be germane to atherogenesis. MPO specifically inhibits PON1 and PON1 mitigates MPO effects. Surprisingly, very little is known about the routes by which PON1 gets integrated into HDL or its fate during HDL remodeling in the intravascular space. We have developed a method that assesses PON1 activity in the individual HDL subclasses with the aid of which we have shown that PON1 is present across the HDL particle range and preferentially in HDL3, confirming data from ultracentrifugation (UC) studies. Upon HDL maturation ex vivo PON1 is activated and it shows a flux to both smaller and larger HDL particles as well as to VLDL and sdLDL. At the same time apoE, AI and AII are shifted across particle sizes. PON1 activation and flux across HDL particles are blocked by CETP and LCAT inhibitors. In a group of particles with such a complex biology as HDL, knowledge of the interaction between apo-lipoproteins, lipids and enzymes is key for an increased understanding of the yet multiple unknown features of its function. Solving the HDL paradox will necessitate the development of techniques to explore HDL function that are practical and well adapted to clinical studies and eventually become useful in patient monitoring. The confluence of proteomic, functional studies, HDL subclasses, PON1 assays and zymogram will yield data to draw a more elaborate and comprehensive picture of the function of HDL. It must be noted that all these studies are static and conducted in the fasting state. The crucial phase will be achieved when human kinetic studies (both in the fasting and post

  6. Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.

    PubMed

    Morris, Jacqueline; Jayanthi, Srinivas; Langston, Rebekah; Daily, Anna; Kight, Alicia; McNabb, David S; Henry, Ralph; Kumar, Thallapuranam Krishnaswamy Suresh

    2016-10-01

    Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a

  7. Sexual maturity in western Atlantic bluefin tuna.

    PubMed

    Heinisch, Gilad; Rosenfeld, Hanna; Knapp, Jessica M; Gordin, Hillel; Lutcavage, Molly E

    2014-01-01

    We introduce a novel endocrine approach for assessing the unresolved matter of the timing of sexual maturation in western Atlantic bluefin tuna (ABFT), a highly migratory population whose status remains uncertain. Ratios of follicle stimulating hormone to luteinizing hormone, a sexual maturity indicator, in all ABFT ≥ 134 cm curved fork length (CFL) were <0.4, similar to Mediterranean spawners, indicating that western ABFT mature at considerably smaller sizes and at a much younger age than currently assumed (≥ 185 cm CFL). PMID:25431301

  8. Delayed visual maturation and lead pollution.

    PubMed

    Gulson, B L; Yui, L A; Howarth, D

    1998-12-11

    Three children were born in the Broken Hill Australia lead mining community with delayed visual maturation of the optic nerve (blindness) within a period of 19 months. Because of the association with the lead pollution, the delayed visual maturation was attributed to lead exposure of the fetus during pregnancy. Lead isotopic analyses of the shed deciduous teeth from the three children demonstrate that they were not exposed to increased levels of lead from a mining or any other source during pregnancy and the etiology of the delayed visual maturation must be sought elsewhere. PMID:9926437

  9. The function and affinity maturation of HIV-1 gp120-specific monoclonal antibodies derived from colostral B cells

    PubMed Central

    Jeffries, Thomas L; Sacha, CR; Pollara, Justin; Himes, Jon; Jaeger, Frederick H; Dennison, S Moses; McGuire, Erin; Kunz, Erika; Eudailey, Joshua A; Trama, Ashley M; LaBranche, Celia; Fouda, Genevieve G; Wiehe, Kevin; Montefiori, David C; Haynes, Barton F; Liao, Hua-Xin; Ferrari, Guido; Alam, S Munir; Moody, M Anthony; Permar, Sallie R

    2015-01-01

    Despite the risk of transmitting HIV-1, mothers in resource-poor areas are encouraged to breastfeed their infants due to beneficial immunologic and nutritional factors in milk. Interestingly, in the absence of antiretroviral prophylaxis, the overwhelming majority of HIV-1-exposed, breastfeeding infants are naturally protected from infection. To understand the role of HIV-1 Envelope (Env)-specific antibodies in breast milk in natural protection against infant virus transmission, we produced 19 HIV-1 Env-specific monoclonal antibodies (mAbs) isolated from colostrum B cells of HIV-1-infected mothers and investigated their specificity, evolution and anti-HIV-1 functions. Despite the previously reported genetic compartmentalization and gp120-specific bias of colostrum HIV Env-specific B cells, the colostrum Env-specific mAbs described here demonstrated a broad range of gp120 epitope specificities and functions, including inhibition of epithelial cell binding and dendritic cell mediated virus transfer, neutralization, and antibody-dependent cellular cytotoxicity. Interestingly, we also identified divergent patterns of colostrum Env-specific B cell lineage evolution with respect to cross-reactivity to gastrointestinal commensal bacteria, indicating that commensal bacterial antigens play a role in shaping the local breast milk IgG repertoire. Maternal vaccine strategies to specifically target this breast milk B cell population may be necessary to achieve safe breastfeeding for all HIV-1-exposed infants. PMID:26242599

  10. The function and affinity maturation of HIV-1 gp120-specific monoclonal antibodies derived from colostral B cells.

    PubMed

    Jeffries, T L; Sacha, C R; Pollara, J; Himes, J; Jaeger, F H; Dennison, S M; McGuire, E; Kunz, E; Eudailey, J A; Trama, A M; LaBranche, C; Fouda, G G; Wiehe, K; Montefiori, D C; Haynes, B F; Liao, H-X; Ferrari, G; Alam, S M; Moody, M A; Permar, S R

    2016-03-01

    Despite the risk of transmitting HIV-1, mothers in resource-poor areas are encouraged to breastfeed their infants because of beneficial immunologic and nutritional factors in milk. Interestingly, in the absence of antiretroviral prophylaxis, the overwhelming majority of HIV-1-exposed, breastfeeding infants are naturally protected from infection. To understand the role of HIV-1 envelope (Env)-specific antibodies in breast milk in natural protection against infant virus transmission, we produced 19 HIV-1 Env-specific monoclonal antibodies (mAbs) isolated from colostrum B cells of HIV-1-infected mothers and investigated their specificity, evolution, and anti-HIV-1 functions. Despite the previously reported genetic compartmentalization and gp120-specific bias of colostrum HIV Env-specific B cells, the colostrum Env-specific mAbs described here demonstrated a broad range of gp120 epitope specificities and functions, including inhibition of epithelial cell binding and dendritic cell-mediated virus transfer, neutralization, and antibody-dependent cellular cytotoxicity. We also identified divergent patterns of colostrum Env-specific B-cell lineage evolution with respect to crossreactivity to gastrointestinal commensal bacteria, indicating that commensal bacterial antigens play a role in shaping the local breast milk immunoglobulin G (IgG) repertoire. Maternal vaccine strategies to specifically target this breast milk B-cell population may be necessary to achieve safe breastfeeding for all HIV-1-exposed infants. PMID:26242599

  11. Affine Vertex Operator Algebras and Modular Linear Differential Equations

    NASA Astrophysics Data System (ADS)

    Arike, Yusuke; Kaneko, Masanobu; Nagatomo, Kiyokazu; Sakai, Yuichi

    2016-05-01

    In this paper, we list all affine vertex operator algebras of positive integral levels whose dimensions of spaces of characters are at most 5 and show that a basis of the space of characters of each affine vertex operator algebra in the list gives a fundamental system of solutions of a modular linear differential equation. Further, we determine the dimensions of the spaces of characters of affine vertex operator algebras whose numbers of inequivalent simple modules are not exceeding 20.

  12. Assessing Technology Maturity for ST-9 Technology Validation Experiments

    NASA Technical Reports Server (NTRS)

    Stocky, John F.

    2005-01-01

    This viewgraph presentation reviews the process of assessing the maturity of a technology for use in NASA missions. The NASA New Millennium Program (NMP) identifies and flight validates breakthrough technologi es that will significantly benefit future operational Space and Earth Science missions NMP conducts two types of flight validation projec ts for the Space Science Enterprise: (1) Integrated System Validatio n Projects, and (2) Stand-Alone Subsystem Validation Projects.

  13. Methylation changes associated with early maturation stages in the Atlantic salmon

    PubMed Central

    2011-01-01

    Background Early maturation in the Atlantic salmon is an interesting subject for numerous research lines. Prior to sea migration, parr can reach sexual maturation and successfully fertilize adult female eggs during the reproductive season. These individuals are known as precocious parr, mature parr or "sneakers". Reasons for early maturation are unknown and this transitory stage is usually considered to be a threshold trait. Here, we compare methylation patterns between mature and immature salmon parr from two different rivers in order to infer if such methylation differences may be related to their maturation condition. First we analyzed genetic differences between rivers by means of AFLPs. Then, we compared the DNA methylation differences between mature and immature parrs, using a Methylation-Sensitive Amplified Polymorphism (MSAP), which is a modification of the AFLPs method by making use of the differential sensitivity of a pair of restriction enzymes isoschizomeres to cytosine methylation. The tissues essayed included brain, liver and gonads. Results AFLPs statistical analysis showed that there was no significant differentiation between rivers or a significant differentiation between maturation states in each river. MSAP statistical analysis showed that among the three tissues sampled, the gonads had the highest number of significant single-locus variation among populations with 74 loci followed by brain with 70 and finally liver with only 12. Principal components analysis (PCA) of the MSAP profiles revealed different profiles among different tissues (liver, brain and testis) clearly separating maturation states in the testis tissue when compared to the liver. Conclusions Our results reveal that genetically-similar mature and immature salmon parr present high levels of DNA methylation variation in two of the three analyzed tissues. We hypothesize that early maturation may be mostly mediated by epigenetic processes rather than by genetic differences between

  14. Involvement of PLA2, COX and LOX in Rhinella arenarum oocyte maturation.

    PubMed

    Ortiz, Maria Eugenia; Bühler, Marta Inés; Zelarayán, Liliana Isabel

    2014-11-01

    In Rhinella arenarum, progesterone is the physiological nuclear maturation inducer that interacts with the oocyte surface and starts a cascade of events that leads to germinal vesicle breakdown (GVBD). Polyunsaturated fatty acids and their metabolites produced through cyclooxygenase (COX) and lipoxygenase (LOX) pathways play an important role in reproductive processes. In amphibians, to date, the role of arachidonic acid (AA) metabolites in progesterone (P4)-induced oocyte maturation has not been clarified. In this work we studied the participation of three enzymes involved in AA metabolism - phospholipase A2 (PLA2), COX and LOX in Rhinella arenarum oocyte maturation. PLA2 activation induced maturation in Rhinella arenarum oocytes in a dose-dependent manner. Oocytes when treated with 0.08 μM melittin showed the highest response (78 ± 6% GVBD). In follicles, PLA2 activation did not significantly induce maturation at the assayed doses (12 ± 3% GVBD). PLA2 inhibition with quinacrine prevented melittin-induced GVBD in a dose-dependent manner, however PLA2 inactivation did not affect P4-induced maturation. This finding suggests that PLA2 is not the only phospholipase involved in P4-induced maturation in this species. P4-induced oocyte maturation was inhibited by the COX inhibitors indomethacin and rofecoxib (65 ± 3% and 63 ± 3% GVBD, respectively), although COX activity was never blocked by their addition. Follicles showed a similar response following the addition of these inhibitors. Participation of LOX metabolites in maturation seems to be correlated with seasonal variation in ovarian response to P4. During the February to June period (low P4 response), LOX inhibition by nordihydroguaiaretic acid or lysine clonixinate increased maturation by up to 70%. In contrast, during the July to January period (high P4 response), LOX inhibition had no effect on hormone-induced maturation. PMID:23443017

  15. Stretched Lens Array Squarerigger (SLASR) Technology Maturation

    NASA Technical Reports Server (NTRS)

    O'Neill, Mark; McDanal, A.J.; Howell, Joe; Lollar, Louis; Carrington, Connie; Hoppe, David; Piszczor, Michael; Suszuki, Nantel; Eskenazi, Michael; Aiken, Dan; Fulton, Michael; Brandhorst, Henry; Schuller, Michael

    2007-01-01

    Since April 2005, our team has been underway on a competitively awarded program sponsored by NASA s Exploration Systems Mission Directorate to develop, refine, and mature the unique solar array technology known as Stretched Lens Array SquareRigger (SLASR). SLASR offers an unprecedented portfolio of performance metrics, SLASR offers an unprecedented portfolio of performance metrics, including the following: Areal Power Density = 300 W/m2 (2005) - 400 W/m2 (2008 Target) Specific Power = 300 W/kg (2005) - 500 W/kg (2008 Target) for a Full 100 kW Solar Array Stowed Power = 80 kW/cu m (2005) - 120 kW/m3 (2008 Target) for a Full 100 kW Solar Array Scalable Array Capacity = 100 s of W s to 100 s of kW s Super-Insulated Small Cell Circuit = High-Voltage (300-600 V) Operation at Low Mass Penalty Super-Shielded Small Cell Circuit = Excellent Radiation Hardness at Low Mass Penalty 85% Cell Area Savings = 75% Lower Array Cost per Watt than One-Sun Array Modular, Scalable, & Mass-Producible at MW s per Year Using Existing Processes and Capacities

  16. Maintaining Ancient Organelles: Mitochondrial Biogenesis and Maturation

    PubMed Central

    Vega, Rick B.; Horton, Julie L.; Kelly, Daniel P.

    2015-01-01

    The ultrastructure of the cardiac myocyte is remarkable for the high density of mitochondria tightly packed between sarcomeres. This structural organization is designed to provide energy in the form of ATP to fuel normal pump function of the heart. A complex system comprised of regulatory factors and energy metabolic machinery, encoded by both mitochondrial and nuclear genomes, is required for the coordinate control of cardiac mitochondrial biogenesis, maturation, and high-capacity function. This process involves the action of a transcriptional regulatory network that builds and maintains the mitochondrial genome, and to drive the expression of the energy transduction machinery. This finely tuned system is responsive to developmental and physiological cues as well as changes in fuel substrate availability. Deficiency of components critical for mitochondrial energy production frequently manifests as a cardiomyopathic phenotype, underscoring the requirement to maintain high respiration rates in the heart. Although a precise causative role is not clear, there is increasing evidence that perturbations in this regulatory system occur in the hypertrophied and failing heart. This review summarizes current knowledge and highlights recent advances in our understanding of the transcriptional regulatory factors and signaling networks that serve to regulate mitochondrial biogenesis and function in the mammalian heart. PMID:25999422

  17. Smart Grid Interoperability Maturity Model Beta Version

    SciTech Connect

    Widergren, Steven E.; Drummond, R.; Giroti, Tony; Houseman, Doug; Knight, Mark; Levinson, Alex; longcore, Wayne; Lowe, Randy; Mater, J.; Oliver, Terry V.; Slack, Phil; Tolk, Andreas; Montgomery, Austin

    2011-12-02

    The GridWise Architecture Council was formed by the U.S. Department of Energy to promote and enable interoperability among the many entities that interact with the electric power system. This balanced team of industry representatives proposes principles for the development of interoperability concepts and standards. The Council provides industry guidance and tools that make it an available resource for smart grid implementations. In the spirit of advancing interoperability of an ecosystem of smart grid devices and systems, this document presents a model for evaluating the maturity of the artifacts and processes that specify the agreement of parties to collaborate across an information exchange interface. You are expected to have a solid understanding of large, complex system integration concepts and experience in dealing with software component interoperation. Those without this technical background should read the Executive Summary for a description of the purpose and contents of the document. Other documents, such as checklists, guides, and whitepapers, exist for targeted purposes and audiences. Please see the www.gridwiseac.org website for more products of the Council that may be of interest to you.

  18. Asymmetric conformational maturation of HIV-1 reverse transcriptase

    PubMed Central

    Zheng, Xunhai; Perera, Lalith; Mueller, Geoffrey A; DeRose, Eugene F; London, Robert E

    2015-01-01

    HIV-1 reverse transcriptase utilizes a metamorphic polymerase domain that is able to adopt two alternate structures that fulfill catalytic and structural roles, thereby minimizing its coding requirements. This ambiguity introduces folding challenges that are met by a complex maturation process. We have investigated this conformational maturation using NMR studies of methyl-labeled RT for the slower processes in combination with molecular dynamics simulations for rapid processes. Starting from an inactive conformation, the p66 precursor undergoes a unimolecular isomerization to a structure similar to its active form, exposing a large hydrophobic surface that facilitates initial homodimer formation. The resulting p66/p66' homodimer exists as a conformational heterodimer, after which a series of conformational adjustments on different time scales can be observed. Formation of the inter-subunit RH:thumb' interface occurs at an early stage, while maturation of the connection' and unfolding of the RH' domains are linked and occur on a much slower time scale. DOI: http://dx.doi.org/10.7554/eLife.06359.001 PMID:26037594

  19. Plasticity of intrinsic excitability in mature granule cells of the dentate gyrus

    PubMed Central

    Lopez-Rojas, Jeffrey; Heine, Martin; Kreutz, Michael R.

    2016-01-01

    The dentate gyrus is the main entry gate for cortical input to the hippocampus and one of the few brain areas where adult neurogenesis occurs. Several studies have shown that it is relatively difficult to induce synaptic plasticity in mature but not in newborn dentate granule cells. In the present work we have systematically addressed how classical protocols to induce synaptic plasticity affect action potential firing and intrinsic excitability in mature granule cells. We found that stimulation paradigms considered to be relevant for learning processes consistently modified the probability to generate action potentials in response to a given synaptic input in mature cells, in some paradigms even without any modification of synaptic strength. Collectively the results suggest that plasticity of intrinsic dendritic excitability has a lower induction-threshold than synaptic plasticity in mature granule cells and that this form of plasticity might be an important mechanism by which mature granule cells contribute to hippocampal function. PMID:26857841

  20. Physical fitness for the mature woman.

    PubMed

    Idiculla, A A; Goldberg, G

    1987-01-01

    The ability to perform physical activity, whether at work or as recreation, is an important aspect of human experience for men and women of all ages. The extension of the range of physical power output that characterizes improvement in "physical fitness" is a desirable goal for all. It helps to make physical activity more enjoyable and less fatiguing, it can facilitate the learning of new skills and may actually improve cognitive functions, and it decreases the likelihood of physical injury and helps to speed recovery from injury. A sound mind in a sound body has been a medical ideal since Greek and Roman times. For a continuing state of good health, all systems--physical, mental, and spiritual--should be exercised in harmony. At the beginning of the twentieth century, the average life span for women was barely 5 years beyond the menopause. Menopause heralded the twilight years of life and enhanced susceptibility to disease and deterioration of health. Today, women look forward to 25 additional years, a full one third of a lifetime; menopause now heralds potential promise, adventure, and exploration, an exciting new phase of the life cycle. Exercise represents a vital part of a prescription to maximize quality of life throughout this period. Physical conditioning cannot be expected to complete reverse the unrelenting effects of passing time, but it appears that it can substantially modify this process. Functional losses that in the past have been attributed only to age may, in fact, represent a combined effect of biologic aging and physical inactivity. Exercise helps to maintain the physiologic adaptations that support an enhanced biologic reserve in body systems that tend to lose reserve capacity with advancing age. "If you don't use it, you lose it!," and its positive corollary, "If you use it, you enthuse it!," may be maxims that imply a natural law of deep consequence. An optimal diet and suitably vigorous activity are advisable for all women as they enter the

  1. Towards a Regolith Maturity Index for Howardites

    NASA Technical Reports Server (NTRS)

    Mittlefehldt, David W.; Cartwright, J. A.; Herrin, J. S.; Johnson, K. N.

    2011-01-01

    The Dawn spacecraft has just arrived at asteroid 4 Vesta, parent of the howardite, eucrite and diogenite (HED) meteorites [1], to begin a yearlong surface study from orbit [2]. As Dawn will view a debris-covered surface, understanding the formation and mixing processes for the debris layer will strongly aid surface data interpretations. Howardites are polymict breccias mainly composed of clasts derived from basaltic (eucritic) and orthopy-roxenitic (diogenitic) parent materials [3]. Some howardites are poorly reworked (fragmental howardites) whilst others have been extensively gardened in an active regolith (regolithic howardites) [4]. The latter may represent an ancient, well-mixed regolith, whilst the former may be from more recent ejecta deposits [4]. Due to environmental differences, regolith development on Vesta differs in detail from that on the Moon [4-6]. We have been developing petrological criteria to apply to howardite thin sections to determine their relative regolithic maturity, which we are fine-tuning with comparison to noble gas data [7, 8]. Whilst we previously emphasized the abundance of reworked clasts (fragmental and impact-melt breccia clasts), this is an imperfect criterion: one howardite with abundant re-worked clasts (EET 99408) shows no evidence of solar wind Ne (SW-Ne), yet, two of our alleged fragmental howardites have clear SW-Ne signatures (LEW 85313, MET 00423) [7, 8]. We are now investigating the diversity in minor and trace element contents of low-Ca pyroxene clasts in howardites as a measure of regolith grade, and will begin analyses of such grains within reworked clasts. Our hypothesis is that regolithic howardites (and the breccia clasts they contain) will show greater diversity because they sampled more diverse diogenitic plutons than fragmental howardites, which formed from ejecta from only a few impacts [e.g. 4]. Our initial LA-ICP-MS work showed ranges in trace element diversity in low-Ca pyroxenes (estimated from the standard

  2. G-protein coupled estrogen receptor (GPER) inhibits final oocyte maturation in common carp, Cyprinus carpio.

    PubMed

    Majumder, Suravi; Das, Sumana; Moulik, Sujata Roy; Mallick, Buddhadev; Pal, Puja; Mukherjee, Dilip

    2015-01-15

    GPR-30, now named as GPER (G protein-coupled estrogen receptor) was first identified as an orphan receptor and subsequently shown to be required for estrogen-mediated signaling in certain cancer cells. Later studies demonstrated that GPER has the characteristics of a high affinity estrogen membrane receptor on Atlantic croaker and zebra fish oocytes and mediates estrogen inhibition of oocyte maturation in these two distantly related teleost. To determine the broad application of these findings to other teleost, expression of GPER mRNA and its involvement in 17β-estradiol mediated inhibition of oocyte maturation in other cyprinid, Cyprinus carpio was investigated. Carp oocytes at pre-vitellogenic, late-vitellogenic and post-vitellogenic stages of development contained GPER mRNA and its transcribed protein with a maximum at late-vitellogenic oocytes. Ovarian follicular cells did not express GPER mRNA. Carp oocytes GPER mRNA was essentially identical to that found in other perciformes and cyprinid fish oocytes. Both spontaneous and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P)-induced oocyte maturation in carp was significantly decreased when they were incubated with either E2, or GPER agonist G-1. On the other hand spontaneous oocyte maturation was significantly increased when carp ovarian follicles were incubated with an aromatase inhibitor, fadrozole, GPER antagonist, G-15 and enzymatic removal of the ovarian follicle cell layers. This increase in oocyte maturation was partially reversed by co-treatment with E2. Consistent with previous findings with human and fish GPR30, E2 treatment in carp oocytes caused increase in cAMP production and simultaneously decrease in oocyte maturation, which was inhibited by the addition of 17,20β-P. The results suggest that E2 and GPER play a critical role in regulating re-entry in to meiotic cell cycle in carp oocytes. PMID:25485460

  3. Physical and chemical characteristics of pitaya fruits at physiological maturity.

    PubMed

    Ortiz, T A; Takahashi, L S A

    2015-01-01

    The aim of this study was to analyze the physical and chemical characteristics of the maturation process of pitaya fruit (Hylocereus undatus) to identify indicators that can be used to determine the point of physiological maturity and establish the optimal timing of physiological maturity for harvesting the fruit. A completely randomized experimental design was employed and four biological repeats were performed. Physiological maturity was assessed using various physical characteristics: longitudinal length (LL), equatorial diameter (ED), pericarp thickness (PeT), pulp thickness (PuT), fruit mass (FM), pulp mass (PuM), pericarp mass (PeM), pericarp percentage (%Pe), pulp percentage (%Pu), pulp/pericarp ratio (Pu/Pe), pericarp color index (CI), hue color angle (h°), lightness index (L*), chroma (C*), blue-yellow variation (b*), and green-red variation (a*). Additionally, chemical characteristics such as soluble solid content (SS), titratable acidity (TA), SS/TA ratio, and pH were screened. The data were statistically analyzed by fitting regression models and computing Pearson's correlation coefficients (P < 0.05). Physiological maturity in pitaya fruits occurred between the 30th and 32nd days after anthesis, and this proved to be the optimal period for harvest. At this time, the fruit was completely red with high SS, and had the recommended values of TA, pH, and SS/TA ratio. During this period, ED, PuT, FM, PuM, %Pu, and Pu/Pe increased while PeT, PeM, and %Pe fell; these changes are considered desirable by producers and/or consumers. PuM was the variable that displayed more strong's association with other variables in the analysis. PMID:26600501

  4. 7 CFR 51.1218 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Peaches Definitions § 51.1218 Mature. “Mature” means that the peach has reached the stage of growth which will ensure a proper completion of...

  5. 7 CFR 51.1218 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Peaches Definitions § 51.1218 Mature. “Mature” means that the peach has reached the stage of growth which will ensure a proper completion of...

  6. Quantifying Semantic Linguistic Maturity in Children.

    PubMed

    Hansson, Kristina; Bååth, Rasmus; Löhndorf, Simone; Sahlén, Birgitta; Sikström, Sverker

    2016-10-01

    We propose a method to quantify semantic linguistic maturity (SELMA) based on a high dimensional semantic representation of words created from the co-occurrence of words in a large text corpus. The method was applied to oral narratives from 108 children aged 4;0-12;10. By comparing the SELMA measure with maturity ratings made by human raters we found that SELMA predicted the rating of semantic maturity made by human raters over and above the prediction made using a child's age and number of words produced. We conclude that the semantic content of narratives changes in a predictable pattern with children's age and argue that SELMA is a measure quantifying semantic linguistic maturity. The study opens up the possibility of using quantitative measures for studying the development of semantic representation in children's narratives, and emphasizes the importance of word co-occurrences for understanding the development of meaning. PMID:26440529

  7. The effects analysis of two neonicotinoid insecticides on in vitro maturation of porcine oocytes using hanging drop monoculture method

    PubMed Central

    ISHIKAWA, Sadamasa; HIRAGA, Kou; HIRADATE, Yuuki; TANEMURA, Kentaro

    2015-01-01

    Acetamiprid (ACE) and imidacroprid (IMI) are known neonicotinoid insecticides with strong affinities for the insect-selective nicotinic acetylcholine receptor. These provide insect control by hyperstimulating insect nerves and are used for agricultural pest management. However, it has also been reported that ACE and IMI affect mammalian reproductive function. We determined the effects of ACE and IMI on the in vitro maturation of porcine oocytes. Significant decreases in nuclear maturation rates were observed in the ACE or IMI-exposed groups. Also, in matured oocytes from the ACE or IMI-exposed groups, irregular chromosomes were observed. Our results suggest that ACE and IMI exposure was detrimental to porcine oocytes and the extent of the effects depends on the concentration of exposure. PMID:25715671

  8. The effects analysis of two neonicotinoid insecticides on in vitro maturation of porcine oocytes using hanging drop monoculture method.

    PubMed

    Ishikawa, Sadamasa; Hiraga, Kou; Hiradate, Yuuki; Tanemura, Kentaro

    2015-06-01

    Acetamiprid (ACE) and imidacroprid (IMI) are known neonicotinoid insecticides with strong affinities for the insect-selective nicotinic acetylcholine receptor. These provide insect control by hyperstimulating insect nerves and are used for agricultural pest management. However, it has also been reported that ACE and IMI affect mammalian reproductive function. We determined the effects of ACE and IMI on the in vitro maturation of porcine oocytes. Significant decreases in nuclear maturation rates were observed in the ACE or IMI-exposed groups. Also, in matured oocytes from the ACE or IMI-exposed groups, irregular chromosomes were observed. Our results suggest that ACE and IMI exposure was detrimental to porcine oocytes and the extent of the effects depends on the concentration of exposure. PMID:25715671

  9. Evolution of microbial dynamics during the maturation phase of the composting of different types of waste.

    PubMed

    Villar, Iria; Alves, David; Garrido, Josefina; Mato, Salustiano

    2016-08-01

    During composting, facilities usually exert greater control over the bio-oxidative phase of the process, which uses a specific technology and generally has a fixed duration. After this phase, the material is deposited to mature, with less monitoring during the maturation phase. While there has been considerable study of biological parameters during the thermophilic phase, there is less research on the stabilization and maturation phase. This study evaluates the effects of the type of starting material on the evolution of microbial dynamics during the maturation phase of composting. Three waste types were used: sludge from the fish processing industry, municipal sewage sludge and pig manure, each independently mixed with shredded pine wood as bulking agent. The composting system for each waste type comprised a static reactor with capacity of 600L for the bio-oxidative phase followed by stabilization and maturation phase in triplicate 200L boxes for 112days. Phospholipid fatty acids, enzyme activities and physico-chemical parameters were measured throughout the maturation phase. The evolution of the total microbial biomass, Gram + bacteria, Gram - bacteria, fungi and enzymatic activities (β-glucosidase, cellulase, protease, acid and alkaline phosphatase) depended significantly on the waste type (p<0.001). The predominant microbial community for each waste type remained present throughout the maturation process, indicating that the waste type determines the microorganisms that are able to develop at this stage. While fungi predominated during fish sludge maturation, manure and municipal sludge were characterized by a greater proportion of bacteria. Both the structure of the microbial community and enzymatic activities provided important information for monitoring the composting process. More attention should be paid to the maturation phase in order to optimize composting. PMID:27236404

  10. Inhibiting HER3-Mediated Tumor Cell Growth with Affibody Molecules Engineered to Low Picomolar Affinity by Position-Directed Error-Prone PCR-Like Diversification

    PubMed Central

    Malm, Magdalena; Kronqvist, Nina; Lindberg, Hanna; Gudmundsdotter, Lindvi; Bass, Tarek; Frejd, Fredrik Y.; Höidén-Guthenberg, Ingmarie; Varasteh, Zohreh; Orlova, Anna; Tolmachev, Vladimir; Ståhl, Stefan; Löfblom, John

    2013-01-01

    The HER3 receptor is implicated in the progression of various cancers as well as in resistance to several currently used drugs, and is hence a potential target for development of new therapies. We have previously generated Affibody molecules that inhibit heregulin-induced signaling of the HER3 pathways. The aim of this study was to improve the affinity of the binders to hopefully increase receptor inhibition efficacy and enable a high receptor-mediated uptake in tumors. We explored a novel strategy for affinity maturation of Affibody molecules that is based on alanine scanning followed by design of library diversification to mimic the result from an error-prone PCR reaction, but with full control over mutated positions and thus less biases. Using bacterial surface display and flow-cytometric sorting of the maturation library, the affinity for HER3 was improved more than 30-fold down to 21 pM. The affinity is among the higher that has been reported for Affibody molecules and we believe that the maturation strategy should be generally applicable for improvement of affinity proteins. The new binders also demonstrated an improved thermal stability as well as complete refolding after denaturation. Moreover, inhibition of ligand-induced proliferation of HER3-positive breast cancer cells was improved more than two orders of magnitude compared to the previously best-performing clone. Radiolabeled Affibody molecules showed specific targeting of a number of HER3-positive cell lines in vitro as well as targeting of HER3 in in vivo mouse models and represent promising candidates for future development of targeted therapies and diagnostics. PMID:23675426

  11. Effect of hemiplegia on skeletal maturation.

    PubMed

    Roberts, C D; Vogtle, L; Stevenson, R D

    1994-11-01

    Children with cerebral palsy have been reported to have poor growth and delayed skeletal maturation, but it is unclear whether these effects are related to the underlying brain injury or to concomitant malnutrition. This study was designed to evaluate the effects of hemiplegic cerebral palsy on skeletal maturation and growth, with the unaffected side used as each subject's control. Bilateral hand-wrist radiographs were obtained for 19 children with spastic hemiplegia. Skeletal maturation was determined in a blinded fashion with the Fels method. The skeletal age of the affected (hemiplegic) side was less than that of the unaffected (control) side in all 19 subjects; the mean difference in skeletal age was 7.3 months (p < 0.001). The delay in skeletal maturation of the affected side correlated linearly with age and upper extremity function. These findings show that brain injury results in delayed skeletal maturation independent of malnutrition. This effect on skeletal maturation may explain, in part, the reason that some children with cerebral palsy grow poorly. PMID:7965443

  12. Review of Sexual Maturity in the Minipig.

    PubMed

    Howroyd, Paul C; Peter, Birgit; de Rijk, Eveline

    2016-06-01

    It is important to know whether the animals used in toxicology studies are sexually mature. As minipigs are being used increasingly in toxicity studies, we reviewed published data on the age of sexual maturity in the minipig. Maturity in females was assessed on the basis either of normal cycles of progesterone secretion or of the histological presence of corpora lutea and, in males, was assessed on the histological appearance of the seminiferous tubules and epididymides. In female Göttingen minipigs, the first progesterone peak was at 3.7 to 4.2 or 6.1 to 6.5 months of age. These animals were in the presence of a boar. In female Göttingen minipigs in toxicology studies, which were not in the presence of a boar, at least 1 corpus luteum in the ovaries was present in only 50% of the females by 6.5 months of age, while all were mature by 7.7 months of age. Histological maturity in the male Yucatan minipig is reported to be attained at about 4.4 months old, but in male Göttingen minipigs at about 2 months old, although the definition of maturity may have been different in the 2 studies. PMID:27102651

  13. Capturing Complexity through Maturity Modelling

    ERIC Educational Resources Information Center

    Underwood, Jean; Dillon, Gayle

    2004-01-01

    The impact of information and communication technologies (ICT) on the process and products of education is difficult to assess for a number of reasons. In brief, education is a complex system of interrelationships, of checks and balances. This context is not a neutral backdrop on which teaching and learning are played out. Rather, it may help, or…

  14. Biomimetic design of affinity peptide ligand for capsomere of virus-like particle.

    PubMed

    Li, Yanying; Liu, Xiaodan; Dong, Xiaoyan; Zhang, Lin; Sun, Yan

    2014-07-22

    Virus-like particle (VLP) of murine polyomavirus (MPV) is a T = 7d icosahedral capsid that self-assembles from 72 capsomeres (Caps), each of which is a pentamer of major coat protein VP1. VLP has great potential in vaccinology, gene therapy, drug delivery, and materials science. However, its application is hindered by high cost downstream processes, leading to an urgent demand of a highly efficient affinity ligand for the separation and purification of Cap by affinity chromatography. Herein a biomimetic design strategy of an affinity peptide ligand of Cap has been developed on the basis of the binding structure of the C-terminus of minor coat protein (VP2-C) on the inner surface of Cap. The molecular interactions between VP2-C and Cap were first examined using all-atom molecular dynamics (MD) simulations coupled with the molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) method, where V283, P285, D286, W287, L289, and Y296 of VP2-C were identified as the hot spots. An affinity peptide library (DWXLXLXY, X denotes arbitrary amino acids except cysteine) was then constructed for virtual screening sequently by docking with AUTODOCK VINA, binding structure comparison, and final docking with ROSETTA FlexPepDock. Ten peptide candidates were selected and further confirmed by MD simulations and MM/PBSA, where DWDLRLLY was found to have the highest affinity to Cap. In DWDLRLLY, six residues are favorable for the binding, including W2, L4, L6 and Y8 inheriting from VP2-C, and R5 and L7 selected in the virtual screening. This confirms the high efficiency and accuracy of the biomimetic design strategy. DWDLRLLY was then experimentally validated by a one-step purification of Cap from crude cell lysate using affinity chromatography with the octapeptide immobilized on Sepharose gel. The purified Caps were observed to self-assemble into VLP with consistent structure of authentic MPV. PMID:24976378

  15. Development and maturation of norovirus antibodies in childhood.

    PubMed

    Blazevic, Vesna; Malm, Maria; Honkanen, Hanna; Knip, Mikael; Hyöty, Heikki; Vesikari, Timo

    2016-04-01

    The burden of norovirus (NoV) gastroenteritis is substantial in young children. Maternal antibodies are thought to protect a child from NoV infection in early infancy but subsequent development of NoV-specific protective immunity in children is still largely unexplored. We have determined NoV-specific antibody seroconversion to GII.4 virus-like particles as an indicator of NoV infection in two children prospectively followed from birth to eight years of age. Blocking activity and affinity maturation of maternal and serum IgG antibodies were evaluated. Our results show that multiple infections occur in children up to eight years of age. The titer, blocking activity and avidity of maternal antibodies determined susceptibility of an infant to NoV infection. NoV GII.4-specific antibodies with high blocking potential and avidity were developed at two to three years of age and were retained throughout the follow-up. Subsequent NoV infections may have contributed to the duration of protective NoV-specific immune responses that lasted for several years. This study adds to current understanding of the duration of passive protection by maternal antibodies and the duration and quality of acquired immunity following primary and subsequent NoV infections in infants and young children, who are the main target group for NoV vaccine development. PMID:26724451

  16. A Differential Dielectric Affinity Glucose Sensor

    PubMed Central

    Huang, Xian; Leduc, Charles; Ravussin, Yann; Li, Siqi; Davis, Erin; Song, Bing; Li, Dachao; Xu, Kexin; Accili, Domenico; Wang, Qian; Leibel, Rudolph; Lin, Qiao

    2013-01-01

    A continuous glucose monitor with a differential dielectric sensor implanted within the subcutaneous tissue that determines the glucose in the interstitial fluid is presented. The device, created using microelectromechanical systems (MEMS) technology, consists of sensing and reference modules that are identical in design and placed in close proximity. Each module contains a microchamber housing a pair of capacitive electrodes residing on the device substrate and embedded in a suspended, perforated polymer diaphragm. The microchambers, enclosed in semi-permeable membranes, are filled with either a polymer solution that has specific affinity to glucose or a glucose-insensitive reference solution. To accurately determine the glucose concentration, changes in the permittivity of the sensing and the reference solutions induced by changes in glucose concentration are measured differentially. In vitro characterization demonstrated the sensor capable of measuring glucose concentrations from 0 to 500 mg/dL with resolution and accuracy of ∼1.7 μg/dL and ∼1.74 mg/dL, respectively. In addition, device drift was reduced to 1.4% (uncontrolled environment) and 11% (5 °C of temperature variation) of that from non-differential measurements, indicating significant stability improvements. Preliminary animal testing demonstrated that the differential sensor accurately tracks glucose concentration in blood. This sensor can potentially be used clinically as a subcutaneously implanted continuous monitoring device in diabetic patients. PMID:24220675

  17. Affine gravity, Palatini formalism and charges

    NASA Astrophysics Data System (ADS)

    Katz, Joseph; Livshits, Gideon I.

    2011-12-01

    Affine gravity and the Palatini formalism contribute both to produce a simple and unique formula for calculating charges at spatial and null infinity for Lovelock type Lagrangians whose variational derivatives do not depend on second-order derivatives of the field components. The method is based on the covariant generalization due to Julia and Silva of the Regge-Teitelboim procedure that was used to define properly the mass in the classical formulation of Einstein's theory of gravity. Numerous applications reproduce standard results obtained by other secure but mostly specialized method like in ADM energy for asymptotically flat spacetimes and in Abbot and Deser for asymptotically de Sitter and anti-de Sitter spacetimes, both at spatial infinity. As a novel application we calculate the Bondi energy loss in five dimensional gravity, based on the asymptotic solution given by Tanabe et al. and obtain, as expected, the same result. We also give the for Einstein-Gauss-Bonnet gravity and find the superpotential for Lovelock theories of gravity when the number of dimensions tends to infinity with maximally symmetrical boundaries. The paper is written in standard component formalism.

  18. Ligand Affinities Estimated by Quantum Chemical Calculations.

    PubMed

    Söderhjelm, Pär; Kongsted, Jacob; Ryde, Ulf

    2010-05-11

    We present quantum chemical estimates of ligand-binding affinities performed, for the first time, at a level of theory for which there is a hope that dispersion and polarization effects are properly accounted for (MP2/cc-pVTZ) and at the same time effects of solvation, entropy, and sampling are included. We have studied the binding of seven biotin analogues to the avidin tetramer. The calculations have been performed by the recently developed PMISP approach (polarizable multipole interactions with supermolecular pairs), which treats electrostatic interactions by multipoles up to quadrupoles, induction by anisotropic polarizabilities, and nonclassical interactions (dispersion, exchange repulsion, etc.) by explicit quantum chemical calculations, using a fragmentation approach, except for long-range interactions that are treated by standard molecular-mechanics Lennard-Jones terms. In order to include effects of sampling, 10 snapshots from a molecular dynamics simulation are studied for each biotin analogue. Solvation energies are estimated by the polarized continuum model (PCM), coupled to the multipole-polarizability model. Entropy effects are estimated from vibrational frequencies, calculated at the molecular mechanics level. We encounter several problems, not previously discussed, illustrating that we are first to apply such a method. For example, the PCM model is, in the present implementation, questionable for large molecules, owing to the use of a surface definition that gives numerous small cavities in a protein. PMID:26615702

  19. Divalent cation affinity sites in Paramecium aurelia.

    PubMed

    Fisher, G; Kaneshiro, E S; Peters, P D

    1976-05-01

    Sites with high calcium affinity in Paramecium aurelia were identified by high calcium (5 mM) fixation and electron microscope methods. Electron-opaque deposits were observed on the cytoplasmic side of surface membranes, particularly at the basal regions of cilia and trichocyst-pellicle fusion sites. Deposits were also observed on some smooth cytomembranes, within the axoneme of cilia, and on basal bodies. The divalent cations, Mg2+, Mn2+, Sr2+, Ni2+, Ba2+, and Zn2+, could be substituted for Ca2+ in the procedure. Deposits were larger with 5 mM Sr2+. Ba2+, and Mn2+ at ciliary transverse plates and the terminal plate of basal bodies. Microprobe analysis showed that Ca and C1 were concentrated within deposits. In some analyses, S and P were detected in deposits. Also, microprobe analysis of 5 mM Mn2+-fixed P. aurelia showed that those deposits were enriched in Mn and C1 and sometimes enriched in P. Deposits were seen only when the ciliates were actively swimming at the time of fixation. Locomotory mutants having defective membrane Ca-gating mechanisms and ciliates fixed while exhibiting ciliary reversal showed no obvious differences in deposition pattern and intensity. Possible correlations between electron-opaque deposits and the locations of intramembranous particles seen by freeze-fracture studied, as well as sites where fibrillar material associate with membranes are considered. The possibility that the action sites of calcium and other divalent cations were identified is discussed. PMID:1262398

  20. Multiplexed protein profiling by sequential affinity capture.

    PubMed

    Ayoglu, Burcu; Birgersson, Elin; Mezger, Anja; Nilsson, Mats; Uhlén, Mathias; Nilsson, Peter; Schwenk, Jochen M

    2016-04-01

    Antibody microarrays enable parallelized and miniaturized analysis of clinical samples, and have proven to provide novel insights for the analysis of different proteomes. However, there are concerns that the performance of such direct labeling and single antibody assays are prone to off-target binding due to the sample context. To improve selectivity and sensitivity while maintaining the possibility to conduct multiplexed protein profiling, we developed a multiplexed and semi-automated sequential capture assay. This novel bead-based procedure encompasses a first antigen capture, labeling of captured protein targets on magnetic particles, combinatorial target elution and a read-out by a secondary capture bead array. We demonstrate in a proof-of-concept setting that target detection via two sequential affinity interactions reduced off-target contribution, while lowered background and noise levels, improved correlation to clinical values compared to single binder assays. We also compared sensitivity levels with single binder and classical sandwich assays, explored the possibility for DNA-based signal amplification, and demonstrate the applicability of the dual capture bead-based antibody microarray for biomarker analysis. Hence, the described concept enhances the possibilities for antibody array assays to be utilized for protein profiling in body fluids and beyond. PMID:26935855